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WO2011011061A2 - Method of regulating angiogenesis and lymphangiogenesis, and a pharmaceutical composition for effecting anti-angiogenic and anti-lymphangiogenic cancer therapy - Google Patents

Method of regulating angiogenesis and lymphangiogenesis, and a pharmaceutical composition for effecting anti-angiogenic and anti-lymphangiogenic cancer therapy Download PDF

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WO2011011061A2
WO2011011061A2 PCT/US2010/002045 US2010002045W WO2011011061A2 WO 2011011061 A2 WO2011011061 A2 WO 2011011061A2 US 2010002045 W US2010002045 W US 2010002045W WO 2011011061 A2 WO2011011061 A2 WO 2011011061A2
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mir
tumor
pharmaceutical composition
cancer
egfl7
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WO2011011061A3 (en
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Calvin Kuo
Frank Kuhnert
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C12N15/09—Recombinant DNA-technology
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    • C12N2310/321—2'-O-R Modification
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    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention provides a method of regulating angiogenesis or neovascularization and lymphangiogenesis using an antagonist against miR-126, as well as a composition for effecting both anti-angiogenic and lymphangiogenic cancer therapy.
  • the present invention also provides a methods of analyzing the effects of global miR-126 deletion on mammary tumor progression, evaluating the efficacy of miR- 126- inhibition against pre-established tumors, and also benchmarking such inhibition against pharmacological inhibition with m/R-726-targeted anti-miRs.
  • Tumors produce some angiogenic or neovascular substances directly, or may induce the body to increase production of these factors that stimulate vascular endothelial cells.
  • Angiogenic factors that have been identified include Vascular
  • VEGF Endothelial Growth Factor
  • basic and acidic fibroblast growth factor and TNF- ⁇ , for example.
  • TNF- ⁇ Endothelial Growth Factor
  • VEGF appears to be the most important as it induces vascular endothelial cell mitosis, and also increases vascular permeability, which is critical for angiogenesis.
  • Protease inhibitors such as cartilage-derived inhibitor (CDI), angiostatic steroids such as tetrahydrocortisol, fungus-derived prothelial proliferatives, and others.
  • CDI cartilage-derived inhibitor
  • angiostatic steroids such as tetrahydrocortisol
  • angiogenesis inhibitors such as D-Penicillamine, and thalidomide, are examples.
  • tumor capillaries When angiogenesis is blocked, tumor capillaries actually regress, and
  • Anti- angiogenic therapy could be used in conjunction with surgical removal of a primary tumor, and could be directed against occult or overt metastasis or might be used to shrink a localized tumor, facilitating definitive surgery, radiation or chemotherapy as curative treatments.
  • Targeting angiogenesis for cancer treatment has distinct advantages over other treatment modalities.
  • the potentially universal requirement for angiogenesis amongst tumors may surmount issues with genetic heterogeneity between diverse tumor subtypes.
  • a second advantage is that anti-angiogenic therapy is directed against normal endothelial cells that are genetically stable, so the development of drug resistance may be less pronounced than with antineoplastic agents.
  • the main disadvantage is lifelong therapy may be required. When anti-angiogenic therapy is discontinued, dormant but viable foci of tumors can re-establish malignant and metastatic properties.
  • many anti-angiogenic therapies developed involve VEGF antagonists, which can elicit compensatory increased production of either VEGF or non- VEGF pro-angiogenic agents in malignant tissue, thereby constituting resistance.
  • the present invention provides a method of effecting anti- angiogenic cancer therapy, which entails administering an amount of a pharmaceutical composition effective for inhibiting angiogenesis of the cancer.
  • the present invention also provides a pharmaceutical composition which contains a nano-carrier having conjugated thereto a vascular targeting group and at least one antagonist against miR-126.
  • the present invention provides a pharmaceutical mixture containing a pharmaceutical composition containing a nano-carrier having conjugated thereto a vascular targeting group and at least one antagonist against miR-126, and one or more small molecule VEGF antagonists.
  • the present invention also provides a method of inhibiting expression of one or more VEGF pathway genes in a mammal, particularly a human.
  • the present invention also provides a method for assessing the effects of global miR-126 deletion, miR-126 inhibition and use of m/R-726-targeted anti- miRs in mammalian, particularly human, cancer treatments and more particularly human breast cancer treatments.
  • a pharmaceutical composition which contains at least one antagonist against miR-126, and optionally with a vascular targeting group, and optionally with a nano-carrier, such as a carbon nanotube.
  • EgfU and miR 126 delta (“ ⁇ ") alleles were generated by flanking exons 5-7 of EgfU or a 289 bp segment of intron 7 containing miR-126 with LoxP sites, respectively, followed by in vivo deletion using Cre recombinase. Green arrowheads, remnant LoxP sites after Cre deletion. Blue arows, PCR primers used in E. Red line, Egfl7 epitope used for polyclonal antibody generation.
  • In situ hybridization for processed miR-126 (dark purple staining) demonstrates vascular expression in the trunk region of wild-type (wt) E14.5 mouse embryos (top panels) that is absent in miR-126*** embryos (bottom panels). Arrows in higher magnification images (taken from the boxed regions) highlight vascular miR-126 expression in the neural tube and carotid ariery in wild-type embryos, and arrowheads the absence thereof in miR-126 Nh embryos.
  • CA carotid artery
  • JV jugular vein
  • NT neural tube
  • OE esophagus
  • TR trachea
  • VA vertebral artery.
  • mice exhibit incompletely penetrant embryonic lethality, edema and vascular leakage.
  • FIG. 3 Angiogenesis phenotypes in miR-12 ⁇ ⁇ / ⁇ embryos.
  • A lsolectin B4 staining of P5 postnatal retinas. Retinal vascularization was normal in Egf/77 ⁇ / ⁇ mice but was severely delayed in miR-126 CJL mice as indicated by arrows. The dashed line indicates the edge of the optic cup. wt, wild type.
  • B Quantitation of retinal vascularization demonstrates a ⁇ 40% reduction of retinal vascular coverage in miR-126 ⁇ L mice.
  • C High-magnification images of retinal vascular sprouts.
  • Fig. 4 Regulation of p85 expression by miR-126.
  • A Quantitative PCR analysis confirms the almost complete absence of miR-126 expression in HUVEC transfected with a miR-126 hairpin inhibitor, as opposed to a scrambled control (scr).
  • B Impaired migration of HUVEC transfected with the miR-126 hairpin inhibitor, versus scr, in the in vitro scratch wound assay ( * P ⁇ 0.05 versus scrambled inhibitor-transfected).
  • C C
  • Mutation of the miR-126 binding site in the p85 ⁇ 3'UTR abrogates repression by miR-126, identifying p85 ⁇ as a direct target.
  • NS not significant.
  • F p85 is upregulated in primary brain endothelial cells isolated from miR-126 tJbk , but not Egfl ⁇ , mice as assessed by western blot with anti-pan p85 antibody; anti-actin antibody provided a loading control.
  • FIG. 5 Upregulation of p85 ⁇ expression in HUVEC transfected with a hairpin inhibitor targeting miR-126, versus scr.
  • H Adenoviral expression of p85 ⁇ in HUVEC is sufficient to inhibit VEGF-induced Akt phosphorylation.
  • Fig. 5. Generation of miR-126 and Egfl7 deletion alleles,
  • the miR-126 deletion allele (miR-126*) was generated by crossing miR- 126" OX/+ and HPRT-Cre mice to achieve in vivo germline deletion of miR- 126 by replacing the 289 bp intronic region containing miR-126 with a single LoxP site, (b) The Egfl7 targeting construct with exons 5-7 of Eg/77 flanked with LoxP sites was recombined into the endogenous Eg/77 locus, generating the EgflT" 0 * allele. Egfff 0 *** and CMV-Cre mice were crossed to achieve in vivo germline deletion of exons 5-7, generating the Egfl7 ⁇ allele.
  • Fig. 6. Confirmed targeting of Egfl7 and miR-126.
  • Fig. 7 Regulation of Spredi expression by miR-126.
  • (b) Spredi is a direct target of miR-126 as shown by miR-126 repression of luciferase activity of a renilla reporter construct containing the 3 ' UTR of Spredi. This activity was specific to the miR-126 binding site as shown by failure of miR-126 to repress luciferase activity of a renilla reporter construct containing the 3 1 UTR of Spredi with the miR-126 binding site mutated,
  • Spredi is upregulated in HUVEC cells transfected with a miR-126 hairpin inhibitor compared to the scrambled control inhibitor transfection.
  • Fig. 8 Modulation of VEGF signaling by miR-126.
  • a proposed schema is depicted in which miR-126 represses VEGF signaling by down regulation of p85 ⁇ and Spredi , such that miR-126 deletion leads to increased levels of these inhibitors and VEGF signaling antagonism.
  • Fig. 9 Immunofluorescence analysis of Egfl7 expression in normal adult lung.
  • Sections of lung from non-tumor bearing C57B1/6 adult mice were processed for Egfl7 immunofluorescence using an affinity purified rabbit anti-Eg/77 antisera developed in our laboratory (RED) or for CD31 (control pan-endothelial marker) (GREEN). Note that while CD31 delineates the entire lung vasculature, (both microvasculature and large vessels) (left panels), Egfl7 is expressed only in large vessels (middle panels)
  • Fig. 10 Immunofluorescence analysis of K-ras G12D ; p53 flox/flox tumors at 20 weeks after intranasal delivery of adenovirus Cre. Note the pan-endothelial expression of the control endothelial marker CD31 (left panels, GREEN) both within tumor nodules (nodules are within the dotted yellow lines) and the surrounding lung parenchyma. In contrast, Egfl7 expression (middle panels, RED) is strongly upregulated in the tumor nodules versus the normal surrounding lung parenchyma, as demonstrated by restriction of the merged signal (right panels, YELLOW) to the tumor nodules.
  • Fig. 11 Immunofluorescence analysis of K-ras G12D ; p53 flox/flox tumors at 20 weeks after intranasal delivery of adenovirus Cre. Note the pan-endothelial expression of the control endothelial marker CD31 (left panels, GREEN) both within
  • Egfl7 is expressed exclusively in the tumor vasculature of numerous transgenic tumor models. Complete colocalization of the Egfl7 and CD31 (pan- endothelial) signals are shown (yellow signal in merged panels).
  • the Egfl7 antibody is an affinity-purified polyclonal generated by our laboratory.
  • miR-126 is expressed exclusively in the tumor vasculature of the
  • transgenic tumor models Top, murine LSL Ras G12D lung tumors and normal mouse lung.
  • murine PyMT mammary tumor Fluorescent in situ hybridization was performed using a DIG-labeled LNA probe against miR-126 (Exiqon) combined with tyramide-based amplification of the fluorescence signal. The green miR-126 signal exhibits a vascular pattern that is absent with the scrambled control. Simultaneous nuclear DAPI staining (blue) is shown for the PyMT mammary tumor samples (bottom).
  • Quantitative real-time PCR confirms upregulation of miR-126 in microdissected K- ras G12D ; p53 flox/flox tumors versus normal lung.
  • Fig. 14 Egfl7 immunofluorescence in lymph nodes from tumor-bearing RIP1-Tag2 mice.
  • Fig. 15 Defective lymphatic-venous separation in miR-126* 1 * embryos.
  • E16.5 miR- (a,b) Histological analysis revealed blood filled jugular lymph sacs in miR-W ⁇ *** embryos but not wild-type with red blood cells extravasating into the surrounding tissue (b, #).
  • Double-positive lymphatic vessels for both the lymphatic marker LYVE-1 (green) and the endothelial marker CD31 (red) support the notion of a lymphatic-venous separation defect in miR-126 NL embryos (f) but not in wild-type (e).
  • BV blood vessel
  • CA carotid artery
  • CV cardinal vein
  • JLS jugular lymph sac
  • LY lymphatic vessel.
  • Fig. 16. Illustrates microRNA biogenesis and mode of action.
  • Fig. 17. Illustrates the structure and expression of the endothelial microRNA m/f?-726.
  • A illustrates the structure of unprocessed pre-miR-126 and the mature miR-126-3p strand
  • B illustrates identical embryonic endothelial expression of miR-126 and EgIfT,
  • left shows a whole mount in situ for miR-126 on E9.5 mouse embryo
  • (right) shows whole mount Egfl7 ISH on E9.5 embryo;
  • C shows miR-126 resides in Egfl7 intron 7 in mouse and human.
  • mice exhibited primarily adenocarcinoma (83%) with poorly circumscribed, multilobulated masses consisting of haphazard, disorganized, lobules and cribriform structures of neoplastic mammary tissue.
  • the miR-126 ko contained primarily adenoma (67%) with well-circumscribed, expansile, unencapsulated, multilobulated masses composed of distinct, discrete lobules of neoplastic mammary tissue, (bottom 400X): Note distinct, discrete lobules of neoplastic tissue and the thin, clear, non-reactive stroma of the mammary adenoma in miR-126 ko versus disorganized invasive lobules and cribriform structures of neoplastic tissue and the thicker, cellular, fibrotic pink stroma of the mammary adenocarcinomas in miR-126 v ⁇ .
  • VEGF vascular endothelial growth factor
  • nt nucleotide
  • siRNA silencing RNA, used interchangeably with RNAi
  • SWCNT single-walled carbon nanotubes
  • VTA vascular targeting agent
  • nano-carrier a nano-scale carrier, generally from 1-1 ,000nm of maximum dimension, for vascular targeting groups and antagonists against.
  • nano-carriers are nanoparticles of carbon, polymer or metal; carbon, polymer or metal nanotubes, where both single-walled and double-walled may be used.
  • miR-126 silencer siRNA or other antagonist that inhibits miR-126.
  • Anti-angiogenic therapy of cancer is an accepted treatment modality for numerous solid tumor types.
  • VEGF Vastin
  • the anti-Vascular Endothelial Growth Factor (VEGF) monoclonal antibody the vast majority of anti-angiogenic cancer treatments target VEGF, and are characterized by rather modest survival improvements ( ⁇ 2-4 months), inevitably progressive disease and high cost.
  • novel anti- angiogenic therapeutics would target non-VEGF and non-receptor tyrosine kinase based mechanisms to avoid redundancy with Avastin and small molecule VEGF antagonists, and to perhaps increase the likelihood of combinatorial efficacy with VEGF- based strategies.
  • miRNAs interact with target mRNAs at specific sites to induce cleavage of the message or to inhibit translation of miRNA, and are essential regulators of physiology and pathophysiology. About 2,000 human genes are thought to have miRNA target sites, however, the specific function of many mammalian, including human, miRNAs is unknown. The inadvertent dysregulation of intronic miRNAs has been predicted to be a general complication in the design and interpretation of mouse knockout studies. miR-126 (Mirn126- Mouse Genome Informatics) is an endothelial miRNA residing within intron 7 of Egfl7, resulting in pan-vascular developmental coexpression of miR-126 and Egfl7 and their abundant expression in cultured
  • Egfl7 is an endothelial secreted extracellular matrix protein, which, in zebrafish, regulates embryonic vascular tube assembly. In vitro, various functions have been ascribed to Egfl7, including the regulation of endothelial or vascular smooth muscle migration and adhesion.
  • Two different mouse knockout alleles of Egfl7 have been described: a gene-trap insertion into intron 2, and an IRES lacZ knock-in replacing exons 5-7, both upstream of miR-126 in intron 7. Both the Egfl7 gene-trap and lacZ knock-in are associated with edema, angiogenic deficits and -50% embryonic lethality.
  • Egfl7 has been previously believed to be a potent
  • the present invention thus identifies the endothelial micro(mi)RNA miR-126 as an advantageous anti-angiogenic cancer target.
  • miR-126 is the most abundant endothelial miRNA.
  • MicroRNAs are short 21-23 nucleotide RNAs that bind
  • the miR-126/Egfl7 locus is strongly upregulated in tumor endothelium versus only sporadic endothelial expression in normal adult tissues.
  • miR-126 ko phenotype indicates that miR-126 is a critical angiogenesis regulator
  • these studies indicate that miR-126 is a highly novel anti-angiogenic drug target which could function in a highly distinct manner from VEGF antagonists.
  • anti-s cholesterol-modified anti- sense RNAs targeting the microRNA.
  • these antagomirs appear to work most robustly for liver microRNAs, and many published cancer applications of antagomirs have had to rely upon direct intratumoral injection, which is not a relevant route for clinical translation.
  • the present invention provides a method of regulating angiogenesis and/or lymphangiogenesis by silencing miR-126.
  • the present invention also provides a nano-carrier having conjugated thereto both a vascular targeting group and/or at least one antagonist against miR-126.
  • the present invention also provides a method of effecting anti-angiogenic cancer therapy, which entails administering an amount of the above pharmaceutical
  • composition sufficient to inhibit or silence miR-126, to a patient in need thereof.
  • the present invention also provides a method of inhibiting expression of one or more VEGF pathway genes in a mammal, particularly a human.
  • the nano-carrier of the present invention may be a single-walled or double- walled carbon nanotube, a metal-based or polymer-based nanoparticle or nanotube.
  • the nano-carrier may be a single-walled carbon nanotube or may be a cyclodextrin polymer-based nanoparticle as described in Davis, Molecular Pharmaceutics, 2009, 6(3), pp.659-668.
  • the nanoparticles and methodologies of loading the same may be as described in US Publ. No. 2009-0087493-A1 , which is incorporated herein in the entirety.
  • the nanoparticle may be a carbon nanotube, boron nitride (BN), a grapheme sheet, a graphitic oxide, a graphitic-coated metal core, a cyclodextrin-based polymer or a nanocrystal containing metals, such as Au or metal alloys, such as FeCo.
  • the methodologies described in U.S. Publ. No. 2008-0253961 for loading nanoparticles for targeted cancer nanotherapeutics may be used. This patent ' publication is incorporated herein in the entirety.
  • any of the following may be loaded, and even two or more
  • chemotherapeutical drugs are simultaneously loaded, onto the nano-carrier.
  • chemotherapeutical drugs are simultaneously loaded, onto the nano-carrier.
  • targeting groups such as peptides, antibodies or nucleic acid aptamers; or miR-126 antagonists may be loaded onto the nano-carrier. Any known method may be used to load any of these cargo species onto the nano-carrier.
  • nanoparticles may be made using known methods.
  • Fe/Au nanoparticles may be made in accordance with U.S. 7,186,398, which is incorporated herein the entirety.
  • nucleic acid functionalized nanoparticles for therapeutic uses may be made in accordance with U.S. Patent Publication
  • tissue targeted nanoparticle compositions containing polymer conjugates and nucleic acid molecules that induce RNA interference may be made and used in accordance with U.S. 7,534,878, which patent is incorporated herein by reference in the entirety, however including at least one miR-126 antagonist in accordance with the present invention instead of the siRNA antagonists of VEGFs of U.S. 7,534,878.
  • the present invention provides a class of miR-126 antagonists using nano-carriers such as single walled carbon nanotubes (SWCNT).
  • nano-carriers such as single walled carbon nanotubes (SWCNT).
  • SWCNT single walled carbon nanotubes
  • carbon nanotubes are rolled-up seamless tubes of graphene with pure C-atoms
  • carbon nanotubes as described above are generally used, i.e., a width of about 1nrn and a length of about 50-200 nm, other aspect ratios, i.e., ratio of
  • the SWCNTs of the present invention generally have a surface area of at least about 500 m 2 /g, and preferably at least about 1 ,000 m 2 /g.
  • a loxP site (P1452) and a neomycin selection cassette plus a loxP site (P1451) were cloned into an A/7II site 5 1 of exon 5 and into an Nhe ⁇ site 3' of exon 7, respectively.
  • P1452 and P1451 were cloned into an Nhe ⁇ site 194 bp 5' of miR-126 and an ⁇ /s/1 site 22 bp 3'of miR-126, respectively (flanking 289 bp total) (for details, see Figs S1 and 52 in the supplementary material).
  • Delta ( ⁇ ) alleles were generated by crossing to CMV- or HPRT-Cre mice. Mutant mice were analyzed in a mixed 129sV/CI57B1/6 genetic background. All mice were treated according to the Stanford Institutional Animal Care and Use Committee and the Stanford Administrative Panel on Laboratory Animal Care . miRNA in situ hybridization
  • Antiserum was affinity purified against the C-terminal 112 amino acids of Egfl7 fused to the C-terminus of glutathione-S transferase (GST). PFA-fixed frozen uterus sections were stained with 0.1 ⁇ g of affinitypurified rabbit anti-Egfl7 antibody and imaged with a Zeiss Z1 Axioimager with Apotome.
  • miR-126 expression was analyzed using the Taqman MicroRNA Assay (Applied Biosystems) utilizing looped RT primers to detect processed miR-126, and expression was normalized to that of miR-16.
  • Egfl7 expression was determined using the SYBR Green Quantitect PCR Kit (Qiagen) and normalized to that of Gapdh.
  • Egfl7 primers 5'- TGCGACG GAC -ACAGAGCCTGCA-S 1 and 5 1 -CAAGTATCTCCCTGCCATCCCA-3 1 . Assays were performed in triplicate and results from at least three independent experiments are presented.
  • P5 eyes were dissected and fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C.
  • Retinas were isolated, blocked in PBS containing 1% BSA and 0.5% Triton X-100 overnight at 4°C, incubated overnight with 10 ⁇ g of FITC-conjugated isolectin B4 (Vector Labs) in 500 ⁇ l of the same solution, washed and then flat mounted.
  • PFA paraformaldehyde
  • Antibodies used were: rabbit anti-p85, rabbit anti-phospho-Akt (Akt1 -Mouse Genome Informatics) (Ser 473), rabbit anti-phospho-Erk (Mapki -Mouse Genome Informatics) (all from Cell Signaling), rabbit anti-Spred1 , rabbit anti-p85 ⁇ , rabbit anti- ⁇ - actin (all from Abeam) and rat anti-HA (Roche). Transfection of human umbilical vein endothelial cells (HUVEC) with miRNA inhibitor
  • Ant ⁇ -miR-126 hairpin inhibitors (Thermo Scientific Dharmacon) or negative control inhibitor were transfected into HUVEC at 100 nM using Dharmafecti . Cells were assayed for protein expression 48 hours after transfection.
  • HUVEC were serum starved overnight 24 hours after transfection of miRNA inhibitors, and scraped with a sterile P200 tip to generate a cell-free zone.
  • the cornal micropocket assay was performed as described (Kuo et al.,2001). miR-126 target luciferase reporter assay
  • the 3'UTR of Pik3r2 and Spredi were amplified and cloned downstream of a Renilla luciferase reporter gene.
  • the miR-126 binding sites were mutated from 5'- ACGGTAC-3 1 to 5'-GTAACGA-3' and from 5'-GGTACG-3' to 5'-AAGCAT-3' in the 3 1 UTR of Pik3r2 and Spredi, respectively.
  • the Lin41 (Trim71 - Mouse Genome Informatics) 3'UTR was used as a negative control.
  • 293T cells in 24-well plates were transfected with 3.35 ng/well of firefly luciferase, 0.667 ng/well of Renilla 3'UTR construct, and either 0, 10 or 100 ng/well of miR-126 expression vector. Empty vector was added to provide a total of 337 ng of DNA per transfection. Forty-eight hours after transfection, the Re ⁇ /7/a/firefly luciferase was measured using the Dual Reporter Fabricferase Kit (Prornega). Akt/Erk phosphorylation assay
  • Akt/Erk phosphorylation assays were performed as described (Gerber et al., 1998).
  • mice exhibited loss of Egfl7 by qPCR and by immunofluorescence with an affinity-purified rabbit anti-Egf17 antiserum (Fig. 1C 1 D). Furthermore,
  • Egfl7 ⁇ / ⁇ mice were phenotypically normal and born at the expected Mendelian ratios despite previous reports from genetrap and conventional knockout alleles (Schmidt et al, 2007) (Fig.2A).
  • miR-126 ⁇ / ⁇ mice recapitulated numerous previously described Egfl7 mutant phenotypes (Schmidt et al., 2007) including ⁇ 50% embryonic lethality (Fig. 2B), which appeared obligately associated with the development of prominent subcutaneous embryonic edema by E14.5 (Fig. 2C 1 D).
  • Fig. 3A-C Surviving miR-126 ⁇ / ⁇ neonates, which were obtained at ⁇ 50% of the expected frequency (Fig. 2B), exhibited delayed postnatal retinal angiogenesis (Fig. 3A-C). This was particularly notable in terms of compromised radial migration, a decreased area of retinal vascularization, and abnormally thickened endothelial sprouts (Fig. 3A-C), as previously described in Egfl7 gene-trap and knock-in mice (Schmidt et al., 2007). miR- 126 ⁇ / ⁇ mice further displayed delayed developmental cranial angiogenesis (Fig. 3D), again reminiscent of previously described Egfl7 mutant phenotypes (Schmidt et al., 2007).
  • RNA hairpin inhibitor induced a greater than 95% depletion of mature miR-126 in HUVEC (Fig. 4A). This was accompanied by significant decreases in migration in scratch assays, as well as impaired VEGF- dependent activation of the downstream kinase Akt (Fig. 4B, C). The basis for this impaired VEGF signaling in m/fi-726-deficient endothelium was examined at the level of miRNA target genes.
  • Edema was a prominent feature of miR-126 ⁇ / ⁇ embryos and was tightly correlated with the lethality observed in ⁇ 50% of embryos. This edema did not appear secondary to intrinsic cardiac defects (data not shown).
  • these miR-126 phenotypes occur cell-autonomously in endothelium as judged from the compartment-specific deletion phenotypes of miR-126 ⁇ / ⁇ ;T ⁇ e2-Cre embryos.
  • this cell-autonomous action allows miR-126 deficiency to derepress and overexpress the p85 ⁇ regulatory subunit of PI3K and Spredi , which represent negative regulators of PI3K and MAP kinase signaling, respectively (see Fig. 8).
  • PI3K and Spredi represent negative regulators of PI3K and MAP kinase signaling
  • miR-126 deletion phenotypes in mouse and knockdown in zebrafish were previously described with impaired angiogenesis and vascular integrity via dysregulation of SprecM and p85 ⁇ (Fish et al.. 2008; Wang et al., 2008). These phenotypes are both reinforced by similar findings in our experiments and are extended by our analysis of endothelial-specific deletion in miR-12 ( ? OX/ ⁇ ; Tie2-Cre embryos.
  • an added important feature of the present invention is the unexpected lack of abnormalities in Egf ⁇ ⁇ mice and the widespread phenocopying by miR-126 ⁇ / ⁇ mice of vascular deficits of previously described Egfl7 alleles, consisting of a gene-trap in intron 2 and a lacZ insertion into exons 5-7, both upstream of intron 7 that contains miR-126 (Schmidt et al., 2007).
  • mice allow selective in vivo analysis of Eg/77 without the confounding influence of miR-126.
  • Our data by no means exclude novel and essential Egr/77-specific functions, either alone or in conjunction with the paralog Egfl8, or as described in zebrafish knockdown, mouse overexpression and in vitro studies (Campagnolo et al., 2005; Lelievre et al., 2008; Soncin et al., 2003; Xu et al., 2008).
  • miR-126 as an essential regulator of angiogenesis directly suggests miR-126 regulation of tumor angiogenesis, with attendant therapeutic implications. Since miR-126 and Egfl7 are co-expressed from the same transcript and have identical expression patterns (Wang et al., 2008), the expression of Egfl7 can be used as a surrogate for miR-126.
  • Egfl7 is exclusively expressed in vasculature in adult lung (Fig. 9).
  • Egfl7 is restricted to the large vessels not the microvasculature (Fig. 9, arrows).
  • This system utilizes intranasal delivery of adenovirus Cre to induce extremely synchronous and penetrant primary lung adenocarcinomas that remarkably recapitulate human lung cancer in terms of nuclear atypia/dysplasia, stromal desmoplasia, invasion and metastasis (Jackson et al., 2005).
  • K-ras G12D after adenovims Cre treatment, K-ras G12D ; p53 flox/flox lung carcinomas exhibited dramatic Eg/77 expression in all the tumor microvasculature (i.e. capillaries) (Fig. 10).
  • the normal lung adjacent to K-ras G12D p ⁇ S" 0 ⁇ 0 " lung carcinomas expressed Egfl7 only in large vessels, with a notable lack of staining in the normal lung microvasculature/capillary beds (Fig. 10).
  • lymphangiogenesis and tumor lymphangiogenesis is suggested by the prominent expression of Egfl7 in lymph nodes.
  • Egfl7 immunofluorescence of lymph node samples from RIP1-Tag2 transgenic mice bearing pancreatic insulinomas revealed prominent lymphatic expression which was distinct from endothelial CD31 expression (Fig. 14).
  • These results suggest the potential utility of therapy directed against miR-126/Egfl7for lymphagiogenesis inhibition, such as during tumor lymphangiogenesis.
  • the miR-126 knockout embryos also exhibit vascular defects indicative of impaired developmental separation of lymphatics from blood vessels wherein the embryonic lymphatics exhibit an aberrant persistent connection to blood vessels resulting in abnormal filling with red blood cells (Fig. 15).
  • the present invention also provides nano-carrier-based compositions for regulating angiogenesis or lymphanogiogenesis in mammalian endothelial cells either in vivo or in vitro.
  • the nano-carier may be a nanoparlicle or a nanotube.
  • the nanoparticle or nanotube may be made of carbon, metal or polymer.
  • the nanotubes may be either single-walled or double-walled. See U.S. patent publ. nos. 2008-0253961 and 2009- 0087493- A1 , incorporated herein the entirety.
  • a composition in accordance with the present invention may contain single-walled carbon nanotubes having conjugated thereto both a vascular targeting agent (VTA) or moiety, and at least antagonist, such as siRNA against miR- 126.
  • VTA vascular targeting agent
  • the SWCNTs may be of any suitable size and aspect ratio, but are generally of a length of about 25-500 nm, preferably about 50-200 nm, and a width of about 0.5 to 5 nm, preferably about 0.5-2 nm.
  • the SWCNTs may be prepared using any known method. For example, the methods described in U.S. 6,183,714, which patent is hereby incorporated herein the entirety, may be used.
  • the miR-126 antagonists of the present invention are designed according to the principles set forth in Vermeulen et al, RNA, Vol. 13, No. 5, pp. 723- 730.
  • miR-126 inhibitor variants may be tested using the assay methodology set forth in Vermeulen et al, id.
  • Inhibitor length generally improves inhibitor potency.
  • the inhibitor function of 2'-O-methyl-modified oligonucleotides is strongly dependent upon length.
  • molecules having symmetrical flanking regions of > 10 nt inhibit target miR-126 to an appreciable extent, although the level of inhibition generally peaks in the range of 50-60 nt with symmetrical flanks in the range of about 10- 15 nt;
  • Flanking region sequence can affect overall inhibitor function; and 3) Incorporation of secondary structure improves inhibitor function, such as a hairpin structure. A similar effect may be obtained by the addition of double- stranded (ds) structures adjacent to the RC core.
  • ds double- stranded
  • enhancement secondary structure provides for the miRNA-RISC interactions with inhibitors.
  • Examples of a miR-126 inhibitor which may be used in the present invention are:
  • VTA hairpin RNA, small peptides obtained by phage display or monoclonal antibodies targeting other endothelial antigens, such as VEGFR2 or VE-Cadherin, etc.
  • the present nano-carrier-based compositions may be used to advantage in treating diseases or conditions in mammals, particularly humans, characterized by a hyper-angiogenetic state, such as macular degeneration or metastatic cancers, such as metastatic prostate cancer.
  • a hyper-angiogenetic state such as macular degeneration or metastatic cancers, such as metastatic prostate cancer.
  • Egfl7 which is co-regulated with miR-126, is strongly expressed in lymphatics, and since miR-126 knockout embryos exhibit some characteristics of defective lymphangiogenesis, the present invention may also be used to advantage in the regulation of lymphangiogenesis, and in the treatment of diseases characterized by a hyper- lympangiogenetic state.
  • the present nano-carrier-based compositions are administered, preferably intravenously, in an amount of about 1-100 mg/kg of body weight/dose.
  • Dosage is generally once per day, but multiple doses may be given per day as deemed appropriate by the treating physician.
  • the nano-carrier-based composition of the present invention may be used in conjunction (either concurrently or consecutively) with other therapies.
  • a VTA-based composition in the treatment of cancer, it is advantageous to use the present composition, a VTA-based composition, with other therapies that are particularly effective as tumor 'rim' or periphery therapies.
  • VTA-based therapies are sometimes most effective against tumor cores, hence, the usefulness of using 'rim 1 or peripheral tumor therapies therewith.
  • the construction and use of the nano-canier-based composition of the present invention may be as described in Feazell et al., J. Am. Chem. Soc. 2007, 129, 8438-8439. That is, the SWCNTs may be functionalized by non-covalent binding of phospholipid-tethered amines to the nanotube surface.
  • a polyethyleleglycol (PEG) chain between the amine and the anchoring phospolipid serves to solubilize the SWCNTs and extend functional groups away from the nanotube surface.
  • both 'core 1 and 'rim' treating agents nay used as a single compound. That is, it is specifically contemplated, for example, that both miR-126 antagonist and cis-platin or other chemotherapeutic agent, be loaded onto the same SWCNT in addition to the VTA, such as RGD. This may also be done using, for example, the procedure described in the above noted publication.
  • VTA such as RGD
  • miR-126 is strongly upregulated in leukemia, particularly acute myeloid leukemia (AML) subtypes M2 and M4. See, for example, Li et al, PNAS, October 7, 2008, vol. 105, no.40, 15535-15540.
  • AML acute myeloid leukemia
  • compositions disclosed herein in treating leukemia using the treatment protocols also described herein, as well as using the methodologies disclosed in U.S. 7,534,878, incorporated herein by reference in the entirety.
  • microRNA miR-126 represents the most abundant endothelial miRNA upon expression profiling, and is expressed in a pan-endothelial manner during
  • the present inventors have also prepared knockout (ko) mice lacking miR-126 which exhibit 50% embryonic lethality associated with edema, hemorrhage and angiogenic delay. In surviving miR-126 ko mice, adult angiogenesis is delayed, for instance, in corneal micropocket assays. miR-126 is present in intron 7 of a host gene, Egfl7. Thus, the miR-126 ko phenotype recapitulates previously described Egfl7 ko phenotypes, and previously described Egfl7 ko mice are now understood to have inadvertently disrupted miR-126 expression.
  • the present invention also explicitly contemplates analyzing, and quantifying, the import of miR-126 to tumor progression, particularly in the MMTV-PyMT transgenic breast cancer model.
  • the present invention also describes studies including extensive data including (a) generation of floxed miR-126 ko mice that do not perturb Egfl7 expression, (b) the developmental and adult angiogenesis phenotypes of miR-126 ko mice , (c) the prominent expression of the miR-126/Egfl7 locus in tumor vasculature, and (d) the significant inhibition of MMTV-PyMT mammary tumor progression upon miR- 126 deletion.
  • the present invention also provides parallel and complementary mouse genetic and pharmacological methodologies to evaluate miR-126 function.
  • a first methodology (1) entails genetic analysis of effects of global miR-126 deletion on MMTV- PyMT mammary tumor progression at the level of tumor angiogenesis, survival and metastasis.
  • a second methodology (2) entails genetic and pharmacological methods to evaluate the therapeutic potential of miR-126 inhibition against pre-established tumors, conditionally deleting miR-126 in pre-established tumors, and benchmarking this against pharmacological inhibition with miR- 126- targeted anti-miRs.
  • This second methodology also entails comparing and combining miR-126 inhibition by either genetic or
  • a third methodology (3) entails elucidating and evaluating mechanisms of miR-126 inhibition of mammary tumorigenesis by endothelial-specific miR-126 deletion by means of PDGFB-iCreER, by determining whether endothelial overexpression of miR-126 is sufficient to enhance, and applying mass spectroscopy to miR-126 target discovery to elucidate new modes of action.
  • tumor angiogenesis can be regulated by an endothelial miRNA, miR-126.
  • endothelial miRNA miR-126
  • Methodology 1 Requirement for miR-126 during mammary tumor progression
  • miR-126 is necessary for mammary tumor progression and genetic deletion of miR-126 impairs tumorigenesis in MMTV-PyMT mice.
  • Methodology 2 Evaluation of miR-126 inhibition as a therapy for treating breast cancer
  • Methodology 3 Mechanistic evaluation of miR-126 action during tumor progression Rationale: miR-126 is both necessary and sufficient to regulate tumor
  • angiogenesis by means of cell-autonomous action in endothelial cells
  • the general purpose of the present invention is the regulation of tumor progression and tumor angiogenesis by the endothelial microRNAs (miRNA), miR-126.
  • miRNA endothelial microRNAs
  • ncRNA small non-coding RNA
  • miRNAs microRNAs
  • siRNAs small-interfering RNAs
  • tasiRNAs trans-acting siRNAs
  • scnRNAs small-scan RNAs
  • rasiRNA repeat-associated siRNAs
  • Piwi-interacting RNAs piRNAs
  • ncRNAs long ncRNAs
  • the diverse ncRNA typically confer epigenetic regulation via translational repression.
  • the present invention focuses on the regulation of tumor progression and tumor angiogenesis by the microRNA (miRNA), miR-126.
  • miRNA microRNA
  • miRNA genes can be intronic or exonic to conventional protein-encoding mRNAs, and are transcribed by RNA polymerase Il yielding the primary miRNA (pri-miRNA) containing a stem-loop structure.
  • pri-miRNA undergoes sequential processing by the RNA endonucleases Dicer and Drosha, generating the mature 21 nt single stranded miRNA (Fig.16).
  • miRNAs down-regulate gene expression by annealing between the 5' ends of miRNA (nt 2-8, the "seed region") with the 3'UTR of target mRNAs in the RISC (the ribonucleoprotein "RNA-induced silencing complex") with direct repression of translation as well as mRNA degradation and de- adenylation, with the net effect of decreasing gene expression post- transcriptionally.
  • the base-pairing between miRNA and their mRNA targets is imprecise and degenerate, allowing a single miRNA to exert post-transcriptional regulation over the expression of hundreds of target mRNA.
  • miRNA functions in angioqenesis.
  • miRNA overexpression/gain-of-function to implicate miRNAs in diverse processes including metabolism, development and cancer.
  • the first strategy utilized for in vivo vertebrate loss-of-function analysis of miRNAs in vivo was systemic infusion of siRNA-cholesterol conjugate "antagomirs", or broad-spectrum ablation of all miRNAs in a particular tissue using Dicer ko.
  • HUVEC cultured endothelium
  • miRNA notably miR-126 but also miR-21 , -221 and -222 .
  • G inhibition of miRNA biogenesis in HUVEC by Dicer/Drosha siRNA reduced sprouting, tube formation or proliferation in vitro, and spheroid sprouting in vivo .
  • VEGF Vascular Endothelial Growth Factor
  • the present invention thus evaluates the endothelial microRNA, miR-126, as a novel anti-angiogenic target using complementary genetic and pharmacologic inhibition methods.
  • angiogenesis very little is known about the functions of individual miRNA during tumor angiogenesis. Initially, it was reported that the miR-17 ⁇ 92 cluster was expressed in tumor cells, and overexpression could repress tumor cell secretion of anti-angiogenic products such as TSP1 and CTGF, thereby increasing angiogenesis in adjacent tumor endothelial cells (EC). With respect to endothelial miRNAs, while miR-92a blockade by antagomirs augments neovasularization in hindlimb ischemia models, equivalent data for tumor angiogenesis has not been published . Although miR-17/20 antagomirs augment angiogenesis in matrigel plugs, no effect on tumor angiogenesis was noted.
  • miR-519c suppresses HIF-Ia expression in EC, and miR-519c antagomirs increase HIF-Ia and angiogenesis .
  • a brief 4-day antagomir treatment targeting miR-296 was reported to diminish tumor vasculature in subcutaneously xenografted U87 glioblastoma cells ; no data on tumor size, progression or survival were presented, however.
  • miR-17/20, miR-92a (i.e. miR-17 ⁇ 92 cluster miRNAs) or miR-519c suggests that these miRNAs are not optimal targets for pharmacologic inhibition in cancer, since in vivo antagomir inhibition of these increases angiogenesis.
  • miR-126 is advantageous for anti-angiogenic cancer therapy based upon miR-126 antagomir inhibition of angiogenesis post-hindlimb ischemia.Yet, surprisingly, prior to the present invention, the role of an individual miRNA in tumor angiogenesis was not rigorously explored using a genetic mouse knockout allele.
  • the miR-126/Epfl7 locus - a transcription unit encoding an endothelial protein-encoding gene with nested endothelial miRNA.
  • the present invention utilizes knockout mice deficient in miR-126, the most abundantly expressed endothelial miRNA. Until recently, no in vivo functional data for miR-126 had been described. In vivo endothelial expression of miR-126 was first described in a large-scale in situ screen of embryonic miRNA expression patterns. The present inventors were intimid both by the striking pan-vascular expression pattern of miR-126 as well as its chromosomal location. The miR-126 gene is present in intron 7 of the Eg/77 gene in both human and mouse (Fig.17).
  • Egfl7 is a secreted and matrix-associated protein with a signal peptide and 2 EGF repeats, and we and others have found it expressed in a pan-endothelial fashion in embryos (Fig. 17B) . Consequently, the identical vascular expression of miR-126 M was retrospectively not surprising since both Egfl7 and miR-126 likely arise from a common transcript, although an independent intronic promoter may co-exist .
  • the miR-126-Zp strand (5' UCGUACCGUGAGUAAUAAUGC 3') represents the predominant species in endothelial cells (EC), as the complementary m ⁇ ' R-126-5p strand is present at much lower levels and represents the " * " or degraded strand.
  • Egfl7 Various and conflicting in vitro functions have been ascribed to Egfl7 including regulation of EC or vascular smooth muscle migration and adhesion.
  • Egfl7 morpholino knockdown impaired vascular tube formation.
  • Two different mouse knockout alleles of Egfl7 were been described by Genentech: a gene trap insertion into intron 2, and an IRES lacZ knock-in allele replacing exons 5-7, both upstream of miR-126 in intron 7. Both Genentech Egfl7 alleles insert bulky cassettes with a 2xLTR-flanked PGK neo polyA module in the gene trap, and a PGK neo IRES b-Gal polyA module in the knock-in.
  • mice in which the miR-126 gene was flanked by loxP sequences we designate this as the "flox" allele, i.e. miR-126* 0X .
  • miR-126* 0X mice in which the miR-126 gene was flanked by loxP sequences.
  • miR-12& oxl * mice to an HPRT-Cre strain allowing deletion of the 289 nt floxed region in vivo, replacing miR- 126 with a single loxP site; we designate this as the delta, or " ⁇ " allele, i.e miR-126 A .
  • the cognate Egfl7 alleles were similarly referred to as Egfl7 flox and Egfl7 ⁇ .
  • mice were phenotypically normal and born at Mendelian ratios (Fig. 19A), while in contrast our miR-126 A/A ko mice fully recapitulated all the previously published Genentech Egfl7 mutant phenotypes including the highly characteristic 50% embryonic lethality and edema from vascular fragility. Further, miR- 126 ⁇ A mice and not Egfl7 A/A mice displayed atretic embryonic cranial angiogenesis previously described in Genentech Egfl7 ko mice 41 .
  • miR-126 knockdown in HUVEC decreases VEGF-dependent migration and inhibits VEGF-dependent activation of the downstream kinases Akt and Erk.
  • microRNAs associate with the 3'UTR of their target genes and repress their expression post-transcriptionally and thus miRNA knockout results in target gene upregulation.
  • miR-126 binds to the 3'UTR of signaling intermediates that normally repress VEGF receptor signaling, namely the PIK3R2 gene encoding the p85 ⁇ subunit of Pl 3-kinase (PI3K) and the intracellular ERK antagonist Spredi , with p85 ⁇ and Spredi overexpression upon knockout/knockdown of the predominant miR-126-3p strand in primary CD31+ miR-12 ⁇ ko EC or HUVEC, and p85 ⁇ overexpression being sufficient to impair VEGF-dependent Akt activation.
  • the coordinated upregulation of p85 ⁇ (Akt antagonist via interference with VEGFR RTK signaling) and Spredi (ERK antagonist) likely contribute to the compromised
  • miR-126 inhibition can sensitize tumor endothelium to pharmacologic VEGF inhibition in vivo for purposes of combinatorial anti-angiogenic therapy.
  • modulation of VEGF signaling is likely to be but a small subset of mechanisms by which miR-126 inhibition exerts anti-angiogenic effects, and any combinatorial anti-angiogenic effects could be based upon completely distinct mechanisms.
  • the miR-126 ko phenotype does not resemble published VEGF/VEGFR ko phenotypes.
  • the miR-126/Egfl7 locus is strongly expressed in tumor vasculature and neoangiogenic vessels in embryo and adult, versus sporadic.
  • Our data described below strongly support the hypothesis that miR-126 is expressed in tumor vasculature, and functionally required for tumor progression and tumor angiogenesis.
  • MMTV PyMT miR-126/Egfl7 transcription unit in 3 spontaneous transgenic mouse models of cancer: MMTV PyMT (mammary), LSL Ras GJ2D /intratracheal Ad Cre (lung) and RipTAg (pancreatic islet).
  • Egfl7 is a surrogate marker for miR-126 expression given their co- expression from a single transcript and identical developmental expression patterns.
  • Our affinity-purified Egfl7 antibody demonstrated clear localization of Egfl7 to CD31+ tumor EC and NOT the tumor parenchymal cells in all three transgenic cancer models.
  • direct in situ hybridization for miR-126 using a miR-126-specific LNA probe again showed miR-126 in the vasculature and not tumor parenchyma.
  • mice exhibit mammary hyperplasia by 2 months of age, multifocal, highly fibrotic, adenocarcinomas involving the entire mammary fat pad in virgin and breeder females with extremely high penetrance by 3 months, pulmonary metastases in essentially all tumor-bearing females by 4 months and lymph node metastases in ⁇ 50%.
  • This model resembles human breast cancer with progressive loss of estrogen and progesterone receptors and increasing overexpression of ErbB2 and cyclin D1.
  • MMTV-PyMT The onset of tumorigenesis with MMTV-PyMT (2-3 months) is much more rapid than with other primary breast cancer models (c.f. MMTV-Neu, ⁇ 1 year), and the MMTV-PyMT model has been used extensively to evaluate effects of angiogenesis regulators by cross to ko/transgenic strains.
  • miR-126* allele i.e. constitutive miR- 126 deletion
  • MMTV-PyMT females at 1/16 frequency followed by harvest of animals at 14 weeks of age.
  • miR-126 dJA MMTV-PyMT females (i.e miR-126ko) compared with miR-126 +l+ ; MMTV-PyMT control littermates (i.e miR-126v ⁇ ), that was evident simply upon gross dissection of the animals.
  • miR-126ko miR-126ko
  • miR-126v ⁇ miR-126v ⁇
  • miR-126 may also act cell-autonomously within tumor cells themselves.
  • the human breast cancer line MDA-MB-231 expresses miR-126, which was significantly decreased in lung- and bone-metastatic sublines, with miR-126 restoration suppressing tumor growth and lung metastasis.
  • miR-126 apparently undergoes significant downregulation in colon cancer cell lines versus normal colon, and miR-126 overexpression repressed in vitro growth of transfected colon cancer lines, although no in vivo analysis was performed and again spatial localization of miR-126 or Egfl7 in tumor samples was not performed.
  • miR-126 is overexpressed in leukemic blasts, consistent with frequent co-expression of molecules in endothelial and hematopoietic cells (c.f. VEGF receptors, Tie2) and common origins from
  • the present invention approaches miR-126 inhibition with an unbiased mindset, using a global/constuitive deletion of miR-126 (as in our miR-126* 1 *; MMTV-PyMT data) to establish both proof-of- principle for miR-126 essential action and a benchmark for comparison against systemically administered miR-126 therapeutics that act globally on miR-126 inhibition (c.f.
  • the present invention describes the functional relevance of miR-126 to tumor progression and as a therapeutic target, leveraging both mouse genetic tools in the form of rigorously characterized ko mice, and complementary and novel pharmacologic approaches in the form of antagomirs/antimirs.
  • the present inventors have also provided (a) generation of floxed miR-126 mice that do not perturb Egfl7 expression, (b) the developmental and adult angiogenesis phenotypes of miR-126 ko mice, (c) the prominent expression of the miR-126/Egfl7 locus in tumor vasculature, and (d) the significant inhibition of MMTV- PyMT mammary tumor progression upon global miR-126 deletion.
  • Methodology 1 is based upon our analysis of global miR-126 deletion (miR- 126 ⁇ ⁇ ) on MMTV-PyMT mammary tumor progression at the level of tumor
  • Methodology 2 affords pre-clinical translational evaluation of the therapeutic potential of miR-126 inhibition, using pre- established tumors and (1) conditionally deleting miR-126 via tamoxifen-sensitive CreER and (2) benchmarking this against a complementary strategy of pharmacologic systemic therapy with m/R-726-targeted antagomirs/antimiRs. Methodology 2 also assesses addition of miR-126 inhibition to VEGF inhibition for combinatorial anti- angiogenic therapy, and systematically compares miR-126 inhibition to VEGF antagonism.
  • Methodology 3 then affords the mechanism for miR-126 inhibition of mammary tumorigenesis, formally addressing a potential endothelial compartment of action by EC-specific versus tumor parenchymal miR-126 deletion, evaluating if endothelial overexpression of miR-126 is sufficient to augment the function of primary tumor endothelium in vitro, and applying novel mass spectroscopy approaches to miR- 126 target discovery to reveal unsuspected pathways of action.
  • tumor angiogenesis can be regulated by an endothelial miRNA, using complementary and mutually reinforcing genetic and pharmacologic approaches.
  • angiogenesis inhibition is now a commonly utilized
  • VEGF antagonists for cancers of the colon, lung, breast, kidney and liver, although despite modest survival advantages measured in months, substantial economic cost, and eventual cancer progression. Consequently, novel anti-angiogenic agents are clearly needed; these could be used in combination with or instead of VEGF inhibitors for increased efficacy.
  • This proposal seeks to fulfills this translational gap by evaluating the endothelial microRNA, miR-126, as a novel anti-angiogenic target using complementary genetic and pharmacologic inhibition methods.
  • miR-126 ko mice Our generation of miR-126 ko mice represents an unusual opportunity to provide rigorous genetic evidence for the involvement of a miRNA in tumor angiogenesis.
  • miR-126 is the first endothelial miRNA to be knocked out in mice, there are no previous studies of effects of genetic deletion of a particular miRNA, miR-126 or otherwise, on tumor angiogenesis in vivo.
  • Methodology 1 of this application evaluates the effects of genetic deletion of miR-126 on tumor angiogenesis and progression in the well-characterized MMTV-PyMT transgenic mammary tumor model which recapitulates numerous aspects of human breast cancer.
  • miR-17 ⁇ 92 cluster miRNAs miR-17, miR-20 and miR-92a, or miR-519c are not suitable targets for pharmacologic anti-angiogenic therapy, since antagomir blockade of these either increases angiogenesis in vivo or do not have effects on tumor angiogenesis.
  • miR-126 ⁇ *; MMTV-PyMT mice (Figs 8,9) as well as miR-126 antagomir inhibition of angiogenesis post-hindlimb ischemia both clearly indicate the anti-tumorigenic and anti-angiogenic potential of miR-126 inhibition.
  • Methodology 2 affords rigorous preclinical target validation for miR-126 inhibition by treating pre-established tumors, simulating the clinical setting. Notably, these treatment studies utilize complementary genetic and pharmacologic inhibition methods. Conditional deletion of miR-126 using our floxed allele in pre-established tumors serve as an extremely rigorous positive control benchmark for pharmacologic miR-126 inhibitors since it would be unlikely that a therapeutic agent could be superior to a genetic ko of the target.
  • Our miR-126 mouse is the only floxed allele available and thus represents the only possible method for temporally conditional adult ko.
  • antagomir For pharmacologic miR-126 inhibition, the current gold standard of cholesterol- conjugated antagomirs is compared to an innovative and promising second-generation "anti-mir" technology containing a 2' F/MOE backbone modification of the antisense RNA, that is not cholesterol modified, and appears superior to antagomirs ion vivo. These pharmacologic strategies are pursued using antagomirs and anti-mirs in quantities sufficient for in vivo experimentation, and therapeutic use.
  • preclinical validation includes the benchmarking of miR- 126 deletion or anti-miR treatment against VEGF inhibition to get some sense of relative efficacy.
  • Preclinical validation of VEGF inhibitors is well known as evidenced by publications in Nature Medicine and PNAS .
  • miR-126 inhibition might be additive or synergistic with VEGF blockade, either by sensitizing endothelium to VEGF inhibition or by promiscuous miRNA action on numerous pathways, VEGF and otherwise.
  • the present invention thus, has important implications for improved anti-angiogenic treatment strategies and the potential to alter clinical practice patterns.
  • miR-126 In situ hybridization for miR-126 reveals embryonic vascular expression of miR-126 and/or the surrogate marker Eg/77, while vascular deletion of miR-126 in miR- 12 ⁇ fi o ⁇ o ⁇ ; jj e 2-Cre embryos phenocopies the complete null miR-126 ⁇ * embryos (Figs. 19c, 19e,21).
  • miR-126 and Eg/77 are expressed exclusively in the vascular, not tumor parenchymal compartments, and miR- 12 ⁇ ; MMTV-PyMT tumors exhibit decreased tumor angiogenesis.
  • miR-126 may also have stimulatory or inhibitory cell-autonomous functions within tumor cells themselves although again our studies suggest a primarily tumor
  • Methodologies 1 and 2 utilize global deletion to establish proof-of-principle and to establish an benchmark for comparison of compartment-independent, systemically acting pharmacologic anti-mir strategies.
  • methodology 3 examines the hypothesis of primary endothelial action through in vivo endothelium-specific deletion and in vitro analysis of purified miR-126 ⁇ * tumor vasculature, with the alternative approach of deletion in the tumor parenchymal compartment by simply crossing to MMTV-Cre.
  • the present invention affords broad implications for diverse areas of biology, including the tumor biology, microRNA, vascular biology/angiogenesis and experimental therapeutics communities.
  • the present invention contemplates treatment of breast cancer.
  • phase III trials of conventional anti-angiogenic agents, such as Avastin have substantially delayed time to progression, but have not yet significantly extended survival, highlighting the need to identify additional anti- angiogenic strategies for this malignancy.
  • the concepts and therapeutics established with miR-126 in breast cancer, in accordance with the present invention are readily extendable to other solid tumors or even leukemia, in the preclinical and/or clinical settings, and have represent a highly novel miRNA-targeted therapeutic approach.
  • miR-126 is necessary for mammary tumor progression in MMTV- PyMT mice.
  • miR-126 is a positive angiogenesis regulator in ko mice, with developmental angiogenesis defects as well as impaired adult angiogenesis in the VEGF-dependent corneal micropocket assay, (b) the Egfl7/miR-126 transcription unit is strongly upregulated in tumor vasculature with strong expression of both Egfl7 and miR-126 in the MMTV PyMT, LSL Ras G12D and RipTAg tumor models, and (c) marked suppression of tumor progression and angiogenesis in m/7?- ⁇ 26 A/ ⁇ ;
  • mice This is consonant with other studies indicating Egfl7 upregulation in neoangiogenic beds (wound healing, corpus luteum, pregnant uterus and tumors).
  • angiogenesis in the MMTV-PyMT transgenic mammary tumor model is angiogenesis in the MMTV-PyMT transgenic mammary tumor model.
  • the use of global constitutive miR-126ko will cast of the widest initial net for miR-126 functions, capable of detecting effects on tumor incidence, progression, metastasis and angiogenesis, as well as unanticipated alternative roles in tumor parenchyma.
  • tumor incidence and tumor volume Differences in tumor incidence are sought in the cohorts with and without null miR-126 deletion.
  • null, heterozygous and wild-type miR-126 mice Prior to harvest, null, heterozygous and wild-type miR-126 mice are examined twice weekly for mammary tumor onset in a blinded fashion by palpation for nodules in all 10 mammary glands.
  • Kaplan-Meier curves using ages of individual mice at time of initial tumor detection to detect potential alterations in the onset of MMTV-PyMT tumor incidence associated with miR-126 deletion.
  • Primary mammary tumor volume measurements are calculated at week 14 as an oblong spheroid approximation using the formula L x W 2 x ⁇ /6 as a sum of the tumor burden in all mammary glands at sacrifice.
  • the Mann- Whitney U test as well as well as average and standard error is used to compare tumor burden. Histologic evaluation includes H&E staining of the mammary fat pads excised en bloc, whether grossly involved by tumor or not, using 3 individual sections separated by at least 100 microns. In some iterations, the inguinal fat pad is stained in whole mount with Carmine alum to reveal tumor. Blinded pathologic interpretation and classification as adenoma versus adenocarcinoma, and invasive and nuclear features will continue to be performed by Dr. Richard Luong in the Stanford Department of Veterinary Medicine. Standard proliferative and apoptotic indices are determined in the tumor parenchyma of ko, het and wild-type groups with anti- Ki67 and TUNEL.
  • ER ER
  • PR ER
  • Neu which undergo characteristic down-regulation
  • Neu up-regulation
  • miR-126-specific endpoints From total tumor tissue, Western blot and qPCR for the miR-126 target genes p85b and Spredi are performed, and miR-126 deletion confirmed by qPCR. Endothelium-specific analysis may be more revealing. Although we have not observed lung mets at 14 weeks, the lungs undergo similar histologic analysis. Endothelial staining is performed as described below.
  • Metastatic lung tumor development is documented as (a) absolute incidence and number of macroscopically evident lung nodules and (b) microscope caliper measurement of nodules in inflated lungs of 3 individual H&E sections separated by at least 100 microns.
  • the Mann-Whitney U test is used to compare metastatic burden amongst ko, het and wt. Further, regional lymph nodes are harvested and undergo similar gross/microscopic evaluation.
  • Endothelial cells comprising the invading angiogenic sprout exhibit significant molecular heterogeneity.
  • Receptors such as DII4 and VEGFR3 are uniquely expressed by endothelial "tip cells" at the leading edge, but not the trailing "stalk cells", with counterreceptors/ligands such as Notch 1 and Jagged are expressed in a paracrine manner by the adjacent stalk cells and surrounding non-endothelial tissues.
  • Neonatal m/R-t26 ⁇ / ⁇ mice exhibit extremely abnormal tip cells with thick endothelial sprouts and a highly characteristic multinucleate
  • tip/stalk markers in miR-126 ⁇ A tumor vasculature are analyzed by CD31 and nuclear DAPI staining (see above) in combination with: (1) Filopodial number and length assessed by isolectin B4 and phalloidin staining. (2) Anti-VEGFR3 (Santa Cruz) and anti-DII4 immunofluorescence on tip cells (R&D Systems). (3) Stalk cell markers (Notch 1 , Jagged-1 immunofluorescence) and (5) tip and stalk cell proliferation (BrdU) is quantitated.
  • the Zeiss LSM 510 and Leica TCS SP2 AOBS confocal microscopes are utilized for this analysis, specifically searching for multinucleate, broadened endothelial sprouts with atretic filopodia. If specifically associated with the null genotype these sprouts will be compared with retinas of non-tumor bearing miR-126 AIA mice. ⁇ Vascular integrity. A second developmental phenotype of miR-12 ⁇
  • mice is impaired vascular integrity, with disruption of the circumferential endothelial wall lining, loss of homotypic endothelial adhesion and resultant hemorrhage (10-20% penetrance) and/or edema (50% penetrance).
  • Evidence for similar findings in the miR-126 ko tumor vasculature is sought, by dextran-FITC (MW ⁇ 70 kDa) injection although our ability to detect such changes may be compromised by intrinsic leakiness of the tumor vasculature without miR-126 deletion.
  • EM is performed as a most specific measure to assess the possible presence of cytoplasmic loss, impairment of homotypic endothelial contacts or other ultrastructural defects specific to endothelial miR-126 deletion tumor vasculature. ⁇ Analysis of miR-126 deletion and miR-126 target gene/pathway
  • RNA from mammary primaries and lung mets are collected for confirmation of miR-126 deletion by qPCR with normalization to b- actin.
  • CD31 + tumor endothelium is isolated by liberase digestion and CD31 magnetic bead selection as in our published miR-126 ko work or CD31+ FACS sorting, followed by Western blotting to determine appropriate
  • upregulation of the miR-126 targets p85b and Spredi , in ko tumor EC as we previously described for miR-126 ⁇ * EC.
  • the potential stimulation of the Erk and Akt pathways by miR-126 overexpression is evaluated in cultured tumor endothelial cells by immunofluorescence with P-Erk or P- Akt antibodies (Cell Signaling) as described above or in VEGF- stimulated isolated CD31+ tumor endothelium by analogy to isolated miR-12 ⁇ endothelium.
  • R26-M2rtTA allows ubiquitous expression of the reverse tetracycline trans-activator/rtTA ("tet-on"/tet- inducible) from the ubiquitous ROSA R26 locus (JAX B6.Cg Gt(ROSA)26 Sortml (rtTA * M2) Jae/J #006965).
  • the TRE-Cre allele contains tet-operator sites driving Cre expression (JAX Tg(tetO-cre)1 Jaw/J #006224).
  • Treatment of miR-126 ftox/flox ; MMTV- PyMT; R26-M2rtTA; TRE-Cre mice with tetracycline in the drinking water (1.5 mg/ml tetracycline/5% sucrose) activates rtTA binding to the tet-operator sites and induces ubiquitous tet-regulated Cre expression (i.e. in both endothelial and mammary compartments), leading to temporally conditional, homozygous deletion of the floxed miR-126 alleles (miR-126F 0 ** 0 *).
  • miR-126 nox/flox ⁇ MMTV-PyMT mice are mated to miR-126 ⁇ oxMox ; R26- M2rtTA; TRE-Cre mice, yielding female miR-126 nox/flox ; MMTV-PyMT; R26-M2rtTA; TRE-Cre mice at 1/16 frequency.
  • females at 12 weeks exhibit a tumor burden in which approximately 50% are progressing to carcinoma, with tumor volume approximately 20% of that at 14 weeks.
  • pharmacologic inhibition of miR-126 is evaluated using the same experimental framework as the conditional genetic ko.
  • pharmacologic inhibition of miRNA in vivo has used "antagomirs" which are single-stranded RNA analogues that are complementary to the target miRNA, typically with 2'-0Me backbone modification throughout, phosphorothioate backbone with or without a cholesterol moiety at the 3'-end.
  • injection of antagomirs using regimens from 3 consecutive daily doses to 2x/week dosing elicits an impressive, durable (>21d) highly selective in vivo silencing of target miRNA in all tissues examined.
  • tumor angiogenesis only fragmentary data exists for inhibit tumor angiogenesis (miR-296), where no data on tumor size, progression or survival were presented.
  • antimirs are used for evaluation in the MMTV-PyMT model, allowing for the validation of promising new antimir oligonucleotide chemistry. antimir).
  • Our well validated miR-126-3p hairpin inhibitor is transfected in parallel; this decreases miR-126 by 90% as well as upregulates p85b but is not usable in vivo.
  • FACS or magnetic bead isolation of CD31+ tumor endothelium is performed as previously with Western to assess EC upregulation of the miR- 126 target p85b (antagomir, antimir) and qPCR (repression of mature m/R-726)(antagomir).
  • VEGF inhibition has been tempered by modest survival advantages and relentlessly progressive disease, highlighting the need for novel anti-angiogenic targets potentially useful in singly or in combination.
  • a priori the broad action of miRNAs on hundreds if not thousands of targets, with their concomitant fine-tuning of the proteome, suggests that targeting angiogenesis regulatory miRNAs, of which miR-126 is the founding member, could be useful in combination with VEGF inhibition.
  • miR-126 pleiotropically acting miR-126 influences tumor angiogenesis.
  • miR-126 genetic ko or pharmacologic inhibition will be combined with VEGF inhibition in the MMTV- PyMT model. Further, careful comparison of phenotypes of miR-126 gene deletion versus VEGF inhibition would yield mechanistic insight into rational combination in the future.
  • Our well-validated adenoviruses expressing soluble VEGFR1 or VEGFR2 ectodomains, which neutralize and sequester VEGF is used; single i.v.
  • animals receive either single i.v. injection of 10 9 pfu Ad FIkI-Fc (soluble VEGFR2 ectodomain) (group A1), or the control virus Ad Fc, (secreted murine lgG2a Fc antibody fragment) (group A2).
  • Ad FIkI-Fc soluble VEGFR2 ectodomain
  • Ad Fc secreted murine lgG2a Fc antibody fragment
  • Ad FIkI-Fc VEGF inhibition, group B1
  • Ad Fc control, group B2
  • Ad FIkI-Fc doses i.e. 10 8 pfu
  • Constitutive miR-126 ko is utilized for the VEGF inhibitor combinations - but a miR-126 "intervention model" is certainly possible with temporally conditional deletion at 12 weeks (i.e. pre-established tumor burden) if feasibility is established in methodology 2B (miR-126f ox/ ⁇ ox ; MMTV-PyMT; R26-M2rtTA; TRE-Cre).
  • ROSA CreERT2 could be used the miR-126 ko would require tamoxifen which could have confounding effects on MMTV-PyMT growth even though there is progressive (but not absolute) estrogen receptor loss.
  • miR-126 is both necessary and sufficient to regulate tumor angiogenesis via cell-autonomous action in endothelial cells and repression of downstream targets.
  • miR-126 appears expressed in tumor parenchyma with potential functional consequences, these being largely deduced from overexpression studies in cell lines.
  • endothelial versus mammary gland-specific miR-126 deletion will be performed in MMTV-PyMT mice (loss- of-function approach), and a converse gain-of-function approach will examine whether miR-126 overexpression is sufficient to enhance endothelial function upon restoration to miR-126 &/ * endothelium.
  • additional miR-126 target genes are sought using a mass spectroscopy-based proteomics approach to generate additional hypotheses for miR-126 action.
  • miR-126 ⁇ * vascular phenotypes are phenocopied by miR-126"° m ; Tie2-Cre mice. While Tie2-Cre has long been the standard for achieving EC deletion, many labs including our own have recently described that Tie2- Cre deletes in both the EC and pericyte lineages.
  • miR-126?*; MMTV-PyMT mice will be crossed to miR-126P ox//+ ; PDGFB- iCreER mice, yielding the desired female miR-12 €p ox/& ; MMTV-PyMT; PDGFB-iCreER (i.e. endothelial ko) mice and control littermate miR-126 +/+ ; MMTV-PyMT; PDGFB- iCreER. Maternal treatment with 1.0 mg tamoxifen i.p.
  • CD31+ EC will be isolated by FACS to confirm miR-126 deletion (qPCR) and p85b upregulation by Western blot.
  • miR-126 deletion does not impair MMTV-PyMT tumorigenesis, then miR-126 is deleted in the tumor parenchymal compartment using MMTV-Cre (JAX Tg(MMTV-cre)4Mam/J #003553) in place of PDGFB-iCreER.
  • miR-126 knockdown or knockout in cultured endothelium markedly attenuates VEGF stimulation of Akt, ERK and migration. Therefore the converse scenario of miR-126 overexpression could sensitize tumor vasculature to VEGF 1 allowing endothelium to respond to lower VEGF concentrations and to lower degrees of VEGFR kinase activity. Certainly, tumor vascular miR-126 overexpression could even facilitate clinical resistance to VEGF inhibitors via VEGF sensitization. Supporting this, Klf2a activation of miR-126 expression in zebrafish enhances VEGF signaling and miR- 126 overexpression in cultured EC enhances FGF signaling.
  • miR-126 iJti tumor endothelium is isolated as described, followed by overnight infection with increasing m.o.i. (0-100) of adenovirus miR-126 (already generated in our laboratory) as we have performed with adenovirus p85p 26 .
  • the miR- 126 ⁇ * tumor endothelium +/- Ad miR-126 infection at different m.o.i. is cultured in EGM- 2 with VEGF 1 starved overnight without VEGF and then stimulated with VEGF at 0, 0.1 , 0.3, 1 , 3 and 10 ng/ml.
  • Endpoints include migration, proliferation over 5 day culture as well as more acute measures (30 min) of VEGF/FGF-2-induced phosphorylation of Akt and Erk upon Western blot which we already have shown to be impaired upon miR-126 deletion.
  • overexpressing vs. wt endothelium is placed in EGM-2 starvation medium without VEGF and temporal kinetics of apoptosis evaluated.
  • Either enhanced VEGF signaling or proliferation upon miR-126 overexpression or decreased apoptosis following VEGF deprivation indicates that miR-126 is sufficient to increase angiogenic properties in tumor endothelium, complementing the necessity/loss-of-function studies in methodology 3A above.
  • miR-126 functions in both compartments, as globally inhibited by miR-126 antagomirs or antimirs.
  • an in vivo correlate may be contemplated to use our tet-inducible TRE-miR-126 transgenics with VE- Cadherin-tTA and MMTV-PyMT to test if miR-126 overexpression confers mammary tumor resistance to VEGF inhibition, i.e. with Ad Flk1-Fc/sVEGFR2.
  • the present invention broadly contemplates both the study and the treatment of mammalian cancer, particularly breast cancer.
  • mammalian cancer particularly breast cancer.
  • any known animal model of cancer may be used
  • generally transgenic mouse models of cancer are preferred.
  • any of the mouse models such as those described in U.S. 6,639,121 , which is incorporated herein in the entirety.
  • LSL K-Ras may be specifically mentioned.
  • the present invention may be used in the treatment of all cancers as miR-126 appears likely to be important for all vasculature, and thus tumor progression, in all parts of the mammalian body.
  • miR-126 broadly includes mice, cats, dogs, cows, sheep and horse, for example, but particularly humans.
  • the present invention provides an evaluation of tumor progression (or regression) and a treatment of tumors by pharmacologically inhibiting miR-126.
  • the design principles or guidelines for pharmacological inhibition of miR-126 are:
  • Newer Approach involves the use of "antimirs” which are single-stranded analogues that are complimentary to the target miRNA, with completely modified phosphorothioate backbones further modified with combinations of 2'-fluoro and 2'-O- methoxyethyl bases; and
  • Homo sapiens miR-126 stem loop sequence is well-known and may be used in a routine manner to obtain antagonists to miR-126 in the treatment of human cancers.
  • antimirs for miR-126 guidance may be obtained therefor from any of US 7,307,067 and SNs 11/141 ,407 and 11/273,640, which are each incorporated herein in the entirety.
  • guidance may also be obtained from US pat. publ. 2010/011356, which is incorporated herein in the entirety.
  • nucleic acid antagonists against miR-126 are nucleic acid antagonists against miR-126. Administration of nucleic acid antagonists against miR-126 to any mammal, particularly humans, in accordance with any of the incorporated documents above, as well as with US pat. publ. 2006/0058266, the latter of which is also incorporated herein by reference in the entirety.
  • nucleic acid antagonists against miR-126 contain the core sequence:
  • modified core sequences that contain at least 50% of the above sequence in the same sequential order, and preferably at least 75% of the above sequence in the same sequential order with the sequence starting at either end of the sequence or in the interior of the sequence.
  • scrambled versions of the above core sequence where no more than 50%, and preferably no more than 25%, of the core sequence above is scrambled, i.e., interchanged with other residues of the core sequence.
  • scrambled as used herein means that the total nucleic acid content of the scrambled sequence remains the same as the core sequence shown above, but that the exact sequence differs due to an interchange of the nucleic acid residues in the scrambled sequence as compared to the sequence shown above.
  • nucleic acid antagonists of the present invention may be from 10-50 nucleic acid units including a portion of the core sequence shown above or one of its variations as described above. However, it is preferred if the antagonist is from 20-30 nucleic acid residues in length with the above considerations for sequence and content in mind.
  • These nucleic acid antagonists are administered as a pharmaceutical composition with a suitable carrier described below.
  • the nucleic acid antagonists against miR-126 are administered in saline, dextrose-5%-saline and/or aqueous solution by intravenous-, subcutaneous- or intraperitoneal injection. These antagonists are administered from 1 to about 100 mg/kg of body weight, and from daily to once every four weeks.
  • the metric used to determine the success of the treatment may include prolongation of overall survival time or time to progression.
  • Time to progression may be measured by radiologic scans, such as MRI, CT, PET or x-rays; by physical palpitation of tumors and/or by histologic analyses.

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Abstract

A pharmaceutical composition, containing at least one antagonist or antimir against miR-126, and a suitable carrier. The composition is advantageously used in the treatment of tumors.

Description

TITLE OF THE INVENTION
METHOD OF REGULATING ANGIOGENESIS AND LYMPHANGIOGENESIS, AND A
PHARMACEUTICAL COMPOSITION FOR EFFECTING ANTI-ANGIOGENIC AND
ANTI-LYMPHANGIOGENIC CANCER THERAPY
The work leading up to the present invention was at least partly funded by the U.S. Government under grants NIH (1 R01 CA95654-01 , 1 R01 NS052830-01 and 1 R01 HL074267-01) and by the State of California under grant (TRDRP 18XT-0084). As such, the U.S. Government may have certain rights in this invention under 35 USC 203 et seq.
Field of the Invention
The present invention provides a method of regulating angiogenesis or neovascularization and lymphangiogenesis using an antagonist against miR-126, as well as a composition for effecting both anti-angiogenic and lymphangiogenic cancer therapy.
The present invention also provides a methods of analyzing the effects of global miR-126 deletion on mammary tumor progression, evaluating the efficacy of miR- 126- inhibition against pre-established tumors, and also benchmarking such inhibition against pharmacological inhibition with m/R-726-targeted anti-miRs.
Description of the Background
Much of current oncology research is directed at metastatic cancer.
Recent discoveries about tumor biology have uncovered vulnerable targets for cancer therapy, and several new classes of anti-cancer agents show promise in treating and preventing the fatal spread of cancer. A significant physiologic difference between normal tissue and cancer is blood supply. Some tumors can induce new growth of capillaries, termed angiogenesis, which enables expansion of the primary tumor by providing a nutrient blood supply to new tumor cells. An incomplete endothelial basement membrane in these capillaries also permits access for free tumor cells to the general endothelial basement membrane in these capillaries, and thereby access to general circulation. This is an enabling factor in metastasis.
Tumors produce some angiogenic or neovascular substances directly, or may induce the body to increase production of these factors that stimulate vascular endothelial cells. Angiogenic factors that have been identified include Vascular
Endothelial Growth Factor (VEGF), basic and acidic fibroblast growth factor, and TNF-α, for example. Of these, VEGF appears to be the most important as it induces vascular endothelial cell mitosis, and also increases vascular permeability, which is critical for angiogenesis.
Various classes of anti-angiogenic compounds have been discovered, and most act by blocking endothelial response. Protease inhibitors, such as cartilage-derived inhibitor (CDI), angiostatic steroids such as tetrahydrocortisol, fungus-derived
angiogenesis inhibitors such as D-Penicillamine, and thalidomide, are examples.
When angiogenesis is blocked, tumor capillaries actually regress, and
consequently, the tumor itself regresses due to oxygen and nutrient deprivation or "starvation." Anti- angiogenic therapy could be used in conjunction with surgical removal of a primary tumor, and could be directed against occult or overt metastasis or might be used to shrink a localized tumor, facilitating definitive surgery, radiation or chemotherapy as curative treatments.
Targeting angiogenesis for cancer treatment has distinct advantages over other treatment modalities. First, the potentially universal requirement for angiogenesis amongst tumors may surmount issues with genetic heterogeneity between diverse tumor subtypes. A second advantage is that anti-angiogenic therapy is directed against normal endothelial cells that are genetically stable, so the development of drug resistance may be less pronounced than with antineoplastic agents. The main disadvantage is lifelong therapy may be required. When anti-angiogenic therapy is discontinued, dormant but viable foci of tumors can re-establish malignant and metastatic properties. Further, many anti-angiogenic therapies developed involve VEGF antagonists, which can elicit compensatory increased production of either VEGF or non- VEGF pro-angiogenic agents in malignant tissue, thereby constituting resistance.
Thus, a need exists for anti-angiogenic or neovascular therapies for treating cancer that are both effective and selective, and which avoid or minimize the problems observed with existing therapies; and for methods for assessing the effects of global miR-126 deletion, miR-126 inhibition and use of m/R-726-targeted anti-miRs in mammalian, particularly human, tumor progression.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method of effecting anti- angiogenic cancer therapy, which entails administering an amount of a pharmaceutical composition effective for inhibiting angiogenesis of the cancer.
The present invention also provides a pharmaceutical composition which contains a nano-carrier having conjugated thereto a vascular targeting group and at least one antagonist against miR-126.
Further, the present invention provides a pharmaceutical mixture containing a pharmaceutical composition containing a nano-carrier having conjugated thereto a vascular targeting group and at least one antagonist against miR-126, and one or more small molecule VEGF antagonists. The present invention also provides a method of inhibiting expression of one or more VEGF pathway genes in a mammal, particularly a human.
The present invention, moreover, also provides a method for assessing the effects of global miR-126 deletion, miR-126 inhibition and use of m/R-726-targeted anti- miRs in mammalian, particularly human, cancer treatments and more particularly human breast cancer treatments.
The above objectives and more are provided by a pharmaceutical composition which contains at least one antagonist against miR-126, and optionally with a vascular targeting group, and optionally with a nano-carrier, such as a carbon nanotube.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1. Generation and validation of EgH7 and miR-126 deletion alleles. (A) EgfU and miR 126 delta ("Δ") alleles were generated by flanking exons 5-7 of EgfU or a 289 bp segment of intron 7 containing miR-126 with LoxP sites, respectively, followed by in vivo deletion using Cre recombinase. Green arrowheads, remnant LoxP sites after Cre deletion. Blue arows, PCR primers used in E. Red line, Egfl7 epitope used for polyclonal antibody generation. (B) In situ hybridization for processed miR-126 (dark purple staining) demonstrates vascular expression in the trunk region of wild-type (wt) E14.5 mouse embryos (top panels) that is absent in miR-126*** embryos (bottom panels). Arrows in higher magnification images (taken from the boxed regions) highlight vascular miR-126 expression in the neural tube and carotid ariery in wild-type embryos, and arrowheads the absence thereof in miR-126Nh embryos. CA, carotid artery; JV, jugular vein; NT, neural tube; OE, esophagus; TR, trachea; VA, vertebral artery. (C) Quantitative PCR (n=6) confirmed the absence of Egfff mRNA in Egfl^ embryos and the absence of mature (processed) miR-126 in miR-126^ά embryos. A looped RT primer specifically detecting the mature miR-126 processed end was utilized. Notably, Egfl^ embryos exhibited normal miR-126 processing and miR-126tJL embryos exhibited normal levels of Egfl7 mRNA, indicating that microdeletion did not disrupt physiological expression of the adjacent gene/miRNA in either case. *P<0.001 versus wild type. (D)
Immunofluorescence staining of uterus from a pregnant mouse with affinity-purified rabbit anti-Egfl7 antibody demonstrating loss of Egfl7 protein in adult Egfl7^L, but not adult miR-126NL, mice. (E) RT-PCR of full-length Egfl7 coding sequence from miR- 126^^ embryos indicates that microdeletion of miR-126 does not induce occult splicing of Egfl7 mRNA. A doublet is present in Eg/77Δ/Δ embryos representing out-of-frame splicing from exon 4 to exon 8 or 9.
Fig. 2. miR-126*1*, but not £gΛf7Δ/Δ, mice exhibit incompletely penetrant embryonic lethality, edema and vascular leakage. (A1B) Breeding tables from heterozygous intercrosses show that EgfU^^ mice are born at normal Mendelian ratios (^=0.741), whereas miR-126^& embryos exhibit ~50% embryonic/perinatal lethality (χ2<0.001). Numbers in parenthesis indicate embryos found with edema. Red numbers indicate deviation from Mendelian ratios. (C) Wild-type (wt) and Egfl7ά/ά embryos were phenotypically indistinguishable, whereas 50% of the miR-126LIL embryos displayed subcutaneous edema (*) at E14.5. (D) Hematoxylin and Eosin staining reveals the severity of the edema (*) in E14.5 embryos. (E) Varying degrees of subcutaneous hemorrhage are detected in ~20% of E15.5 m/7?-t26Δ/Λ embryos; a severely affected embryo is depicted. Histological analysis shows red blood cells extravasating from a representative ruptured, leaky vessel (arrow). (F) Tie2-Cre-mediated endothelial deletion of miR-126 phenocopies the miR-Wβ*1* edema (*) phenotype.
Fig. 3. Angiogenesis phenotypes in miR-12βΔ/Λ embryos. (A) lsolectin B4 staining of P5 postnatal retinas. Retinal vascularization was normal in Egf/77Δ/Λ mice but was severely delayed in miR-126CJL mice as indicated by arrows. The dashed line indicates the edge of the optic cup. wt, wild type. (B) Quantitation of retinal vascularization demonstrates a ~40% reduction of retinal vascular coverage in miR-126ωL mice. (C) High-magnification images of retinal vascular sprouts. Note the marked thickening (arrows) of vascular sprouts in miR-126Nt retinas as compared with wild-type or Egfl^ mice. (D) CD31 (Pecami) whole-mount staining of E12.5 heads. Note the delayed vascularization and reduced complexity (arrow) of the cranial vasculature in miR-12^ embryos. (E) Adult animals of the indicated genotypes (n=7) received corneal implants of slow-release hydron pellets containing VEGF. Neovascularization was quantified after 6 days by slit lamp examination. Arrows highlight the length of vessel growth. (F) Adult miR-126tlL mice exhibited ~50% impairment of corneal vascularization relative to Eg/77Δ/Λ or wild-type mice.
Fig. 4. Regulation of p85 expression by miR-126. (A) Quantitative PCR analysis confirms the almost complete absence of miR-126 expression in HUVEC transfected with a miR-126 hairpin inhibitor, as opposed to a scrambled control (scr). (B) Impaired migration of HUVEC transfected with the miR-126 hairpin inhibitor, versus scr, in the in vitro scratch wound assay (*P<0.05 versus scrambled inhibitor-transfected). (C)
Impaired VEGF-dependent Akt and Erk phosphorylation in HUVEC transfected with the miR-126 hairpin inhibitor, versus scr. (D) Target site alignment for miR-126 in the 31UTR of Pik3r2, which encodes p85β. (E) p85β (Pik3r2) is a direct target of miR-126 as shown by dosedependent repression by miR-126 of luciferase expression from the wild-type p85β 3'UTR, but not the control Lin41 31UTR1 reporters in 293T cells. Mutation of the miR-126 binding site in the p85β 3'UTR (p85β mut) abrogates repression by miR-126, identifying p85β as a direct target. *P<0.05 versus no miR-126 expression vector and tP<0.05 versus p85β mut and, Lin41 31UTR reporter construct, for a given dose of miR- 126 expression vector (0, 10 and 100 ng). NS, not significant. (F) p85 is upregulated in primary brain endothelial cells isolated from miR-126tJbk, but not Egfl^, mice as assessed by western blot with anti-pan p85 antibody; anti-actin antibody provided a loading control. (G) Upregulation of p85β expression in HUVEC transfected with a hairpin inhibitor targeting miR-126, versus scr. (H) Adenoviral expression of p85β in HUVEC is sufficient to inhibit VEGF-induced Akt phosphorylation. Fig. 5. Generation of miR-126 and Egfl7 deletion alleles, (a) The miR-126 targeting construct engineered to flank 289 bp of Egfl7 intron 7 containing miR-126 with LoxP sites was recombined into the endogenous locus to generate the floxed miR-126 allele (miR- 126"°*) in ES cells. The miR-126 deletion allele (miR-126*) was generated by crossing miR- 126"OX/+ and HPRT-Cre mice to achieve in vivo germline deletion of miR- 126 by replacing the 289 bp intronic region containing miR-126 with a single LoxP site, (b) The Egfl7 targeting construct with exons 5-7 of Eg/77 flanked with LoxP sites was recombined into the endogenous Eg/77 locus, generating the EgflT"0* allele. Egfff0*** and CMV-Cre mice were crossed to achieve in vivo germline deletion of exons 5-7, generating the Egfl7Δ allele. This generated a null allele in which exons 5-7 of Eg/77 were replaced by a single LoxP site as opposed to previously described Eg/77 mutant alleles in which a IRES-β-Gal PGK-neo cassette was knocked into exons 5-7 or in which a retroviral gene trap vector was inserted into intron 2 of Eg/77 (Schmidt et al. 2007). Alignment of Eg/77 message with EGFLT protein illustrates that the in vivo Cre mediated deletion removes the C-terminal half of the EMI domain and both EGF repeats of the encoded protein.
Fig. 6. Confirmed targeting of Egfl7 and miR-126. (a). Southern blot strategy to confirm proper targeting of Eg/77 and miR-126. Insertion of the Neomycin (Neo) selection cassette into the Nhel site in intron 7 of Eg/77 causes a 1.9 kb or 1.8 kb increase in an Ndel or an Nhel fragment respectively. Note that the change in the Nhel fragment is slightly smaller than that of the Ndel fragment because cloning the Neo cassette into the Nhei site in intron 1 of Eg/77 destroys the initial site but introduces a new Nhel site within the Neo cassette, (b) Southern blots of wild-type, Egfff0*** or miR- 126"01^+ ES cell genomic DNA following Ndel (51 strategy, left) or Nhel (31 strategy, right) digestion probed against regions 51 of the targeting construct's 5' homology arm (red line in a) or 3' of the 3' homology arm (blue line in a) generated bands that were 7.2 kb (wt allele) and 9.1 kb (Eg/77 and miR-126 floxed alleles) for the 5' probe as well as 4.6 kb (wt allele), 6.4 kb (Eg/77"OX allele), and.6.2 kb {miR- 126F0* allele) for the 3' probe, respectively. Fig. 7. Regulation of Spredi expression by miR-126. (a) Alignment of miR-126 to its binding site in the 31 UTR of Spredi. (b) Spredi is a direct target of miR-126 as shown by miR-126 repression of luciferase activity of a renilla reporter construct containing the 3 ' UTR of Spredi. This activity was specific to the miR-126 binding site as shown by failure of miR-126 to repress luciferase activity of a renilla reporter construct containing the 31 UTR of Spredi with the miR-126 binding site mutated, (c) Western blot analysis of Spredi expression. Spredi is upregulated in HUVEC cells transfected with a miR-126 hairpin inhibitor compared to the scrambled control inhibitor transfection.
Fig. 8. Modulation of VEGF signaling by miR-126. A proposed schema is depicted in which miR-126 represses VEGF signaling by down regulation of p85β and Spredi , such that miR-126 deletion leads to increased levels of these inhibitors and VEGF signaling antagonism.
Fig. 9. Immunofluorescence analysis of Egfl7 expression in normal adult lung.
Sections of lung from non-tumor bearing C57B1/6 adult mice were processed for Egfl7 immunofluorescence using an affinity purified rabbit anti-Eg/77 antisera developed in our laboratory (RED) or for CD31 (control pan-endothelial marker) (GREEN). Note that while CD31 delineates the entire lung vasculature, (both microvasculature and large vessels) (left panels), Egfl7 is expressed only in large vessels (middle panels)
(ARROWS). The merged YELLOW signal is only seen in large vessels. Bottom rows are high power images of the top row.
Fig. 10. Immunofluorescence analysis of K-rasG12D; p53flox/flox tumors at 20 weeks after intranasal delivery of adenovirus Cre. Note the pan-endothelial expression of the control endothelial marker CD31 (left panels, GREEN) both within tumor nodules (nodules are within the dotted yellow lines) and the surrounding lung parenchyma. In contrast, Egfl7 expression (middle panels, RED) is strongly upregulated in the tumor nodules versus the normal surrounding lung parenchyma, as demonstrated by restriction of the merged signal (right panels, YELLOW) to the tumor nodules. Fig. 11. Egfl7 is expressed exclusively in the tumor vasculature of numerous transgenic tumor models. Complete colocalization of the Egfl7 and CD31 (pan- endothelial) signals are shown (yellow signal in merged panels). The Egfl7 antibody is an affinity-purified polyclonal generated by our laboratory.
Fig. 12. miR-126 is expressed exclusively in the tumor vasculature of the
indicated transgenic tumor models. Top, murine LSL RasG12D lung tumors and normal mouse lung. Bottom, murine PyMT mammary tumor. Fluorescent in situ hybridization was performed using a DIG-labeled LNA probe against miR-126 (Exiqon) combined with tyramide-based amplification of the fluorescence signal. The green miR-126 signal exhibits a vascular pattern that is absent with the scrambled control. Simultaneous nuclear DAPI staining (blue) is shown for the PyMT mammary tumor samples (bottom).
Fig. 13. Quantitative real-time PCR confirms upregulation of miR-126 in microdissected K- rasG12D; p53flox/flox tumors versus normal lung.
Fig. 14. Egfl7 immunofluorescence in lymph nodes from tumor-bearing RIP1-Tag2 mice.
Fig. 15. Defective lymphatic-venous separation in miR-126*1* embryos. E16.5 miR- (a,b) Histological analysis revealed blood filled jugular lymph sacs in miR-Wβ*** embryos but not wild-type with red blood cells extravasating into the surrounding tissue (b, #). (c,d) Staining for the lymphatic marker LYVE-1 (black) marks blood-filled lymphatic vessels in the skin of E14.5 miR-126tJL embryos (d, arrow) which are not present in wild-type (c). Note the distinct separation of red blood cell-containing blood vessel (*) from lymphatics in wt skin (c). (e,f) Double-positive lymphatic vessels for both the lymphatic marker LYVE-1 (green) and the endothelial marker CD31 (red) support the notion of a lymphatic-venous separation defect in miR-126NL embryos (f) but not in wild-type (e). BV, blood vessel; CA, carotid artery; CV, cardinal vein; JLS, jugular lymph sac; LY, lymphatic vessel.
Fig. 16. Illustrates microRNA biogenesis and mode of action. Fig. 17. Illustrates the structure and expression of the endothelial microRNA m/f?-726. (A) illustrates the structure of unprocessed pre-miR-126 and the mature miR-126-3p strand, (B) illustrates identical embryonic endothelial expression of miR-126 and EgIfT, (left) shows a whole mount in situ for miR-126 on E9.5 mouse embryo, (right) shows whole mount Egfl7 ISH on E9.5 embryo; and (C) shows miR-126 resides in Egfl7 intron 7 in mouse and human.
Fig.18. Illustrates reduced tumor incidence in MMTV-PyMT and miR-126LIL mice (a,b) MMTV-PyMT; miR-126+/+ females (i.e. ko) and control littermate MMTV-PyMT; miR-126 +/+ females (i.e. w.t.) (n=5) were harvested at 14 weeks of age. Representative tumor spectrum from mammary fat pad dissection of a single mouse is depicted; (c,d,e) statistical analysis of MMTV-PyMT; m/'R-f 26Δ/A mice (ko) versus control littermate MMTV-PyMT; miR-126+l+ m]ce (w.t.) is depicted, (c) N=5 mice, (d, e) for miR-126NL mice n=18 tumors; miR-126*'+ n=33 tumors.
Fig.19 Histological analysis of MMTV-PyMT; miR-126Nh mammary tumors (A) (top, 20X) MMTV-PyMT; miR-1 '26** mice (i.e. ko) and control littermate MMTV-PyMT; miR- 126+l+ mice (i.e. w.t.) (n=5 mice, n=10 mammary glands/mouse) were harvested at 14 weeks. Representative histology (H&E) is depicted. The miR-126 w.t. mice exhibited primarily adenocarcinoma (83%) with poorly circumscribed, multilobulated masses consisting of haphazard, disorganized, lobules and cribriform structures of neoplastic mammary tissue. In contrast, the miR-126 ko contained primarily adenoma (67%) with well-circumscribed, expansile, unencapsulated, multilobulated masses composed of distinct, discrete lobules of neoplastic mammary tissue, (bottom 400X): Note distinct, discrete lobules of neoplastic tissue and the thin, clear, non-reactive stroma of the mammary adenoma in miR-126 ko versus disorganized invasive lobules and cribriform structures of neoplastic tissue and the thicker, cellular, fibrotic pink stroma of the mammary adenocarcinomas in miR-126 vΛ. (B) CD31 IHC. Tumors in miR-126 w.t. mice are well vascularized with abundant CD31 staining (brown), versus miR-126 ko mice with large regions of relatively poorly vascularized tumor. The tumor border is marked by the dashed line and "T" denotes the tumor mass. CD31 staining in the surrounding normal mammary gland serves as a positive control. Two independent tumors of each genotype are shown. 200X.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following term definitions are used throughout the present specification.
Abbreviational Definitions: mi = micro; miR= microRNA
ko = knockout
VEGF = vascular endothelial growth factor
si=silencing; I = interfering
nt=nucleotide
siRNA = silencing RNA, used interchangeably with RNAi
SWCNT = single-walled carbon nanotubes
RC = reverse complement
VTA = vascular targeting agent
nano-carrier = a nano-scale carrier, generally from 1-1 ,000nm of maximum dimension, for vascular targeting groups and antagonists against. Examples of nano-carriers are nanoparticles of carbon, polymer or metal; carbon, polymer or metal nanotubes, where both single-walled and double-walled may be used.
miR-126 silencer = siRNA or other antagonist that inhibits miR-126.
Anti-angiogenic therapy of cancer is an accepted treatment modality for numerous solid tumor types. As exemplified byAvastin, the anti-Vascular Endothelial Growth Factor (VEGF) monoclonal antibody, the vast majority of anti-angiogenic cancer treatments target VEGF, and are characterized by rather modest survival improvements (~2-4 months), inevitably progressive disease and high cost. Ideally, novel anti- angiogenic therapeutics would target non-VEGF and non-receptor tyrosine kinase based mechanisms to avoid redundancy with Avastin and small molecule VEGF antagonists, and to perhaps increase the likelihood of combinatorial efficacy with VEGF- based strategies.
MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or to inhibit translation of miRNA, and are essential regulators of physiology and pathophysiology. About 2,000 human genes are thought to have miRNA target sites, however, the specific function of many mammalian, including human, miRNAs is unknown. The inadvertent dysregulation of intronic miRNAs has been predicted to be a general complication in the design and interpretation of mouse knockout studies. miR-126 (Mirn126- Mouse Genome Informatics) is an endothelial miRNA residing within intron 7 of Egfl7, resulting in pan-vascular developmental coexpression of miR-126 and Egfl7 and their abundant expression in cultured
endothelium. Egfl7 is an endothelial secreted extracellular matrix protein, which, in zebrafish, regulates embryonic vascular tube assembly. In vitro, various functions have been ascribed to Egfl7, including the regulation of endothelial or vascular smooth muscle migration and adhesion. Two different mouse knockout alleles of Egfl7 have been described: a gene-trap insertion into intron 2, and an IRES lacZ knock-in replacing exons 5-7, both upstream of miR-126 in intron 7. Both the Egfl7 gene-trap and lacZ knock-in are associated with edema, angiogenic deficits and -50% embryonic lethality. We have explored the functions of both Egfl7 and its embedded miRNA, miR-126, using floxed alleles to selectively disrupt each gene without reciprocal perturbation.
Intronic microRNAs have been proposed to complicate the design and
interpretation of mouse knockout studies. The endothelial-expressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice (Schmidt et al., 2007). On the basis of these studies, Egfl7 has been previously believed to be a potent
angiogenesis regulator and an intriguing target for anti-angiogenic drug design. On the other hand, the presence of miR-126 within intron 7 of Egfl7, greatly complicates genetic analysis of this region. The present inventors pursued a detailed functional examination of this locus by generating two strains of knockout mice. First, we generated a floxed Egfl7Δ allele which disrupted Egfl7 without disrupting miR-126. Secondly, we generated a floxed miR-126Δ allele which disrupted miR-126 without disrupting Egfl7. Surprisingly, side-by-side analysis of these selectively floxed Egfl7Δ and miR-126Δ alleles revealed that Egfl7Δ/Δ mice were phenotypically normal, whereas miR-126Δ/Δ mice bearing a 289-nt
microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85β subunit of PI3 kinase and of Spredi , respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.
The present invention thus identifies the endothelial micro(mi)RNA miR-126 as an advantageous anti-angiogenic cancer target. miR-126 is the most abundant endothelial miRNA. MicroRNAs are short 21-23 nucleotide RNAs that bind
promiscuously to hundreds of RNA targets, degrading them or impeding their translation. As described above, although knockout mice for miR-126 have been developed which demonstrate severely impaired developmental angiogenesis, our work indicates that it is in fact miR-126 and not Egfl7 that is the critical angiogenic regulator accounting at least for the developmental angiogenesis phenotype.
The miR-126/Egfl7 locus is strongly upregulated in tumor endothelium versus only sporadic endothelial expression in normal adult tissues. Combined with the miR- 126 ko phenotype indicating that miR-126 is a critical angiogenesis regulator, these studies indicate that miR-126 is a highly novel anti-angiogenic drug target which could function in a highly distinct manner from VEGF antagonists. It is possible to inhibit microRNAs in vivo using so-called "antagomirs" which are cholesterol-modified anti- sense RNAs targeting the microRNA. Unforunately, these antagomirs appear to work most robustly for liver microRNAs, and many published cancer applications of antagomirs have had to rely upon direct intratumoral injection, which is not a relevant route for clinical translation.
The present invention provides a method of regulating angiogenesis and/or lymphangiogenesis by silencing miR-126.
The present invention also provides a nano-carrier having conjugated thereto both a vascular targeting group and/or at least one antagonist against miR-126.
The present invention also provides a method of effecting anti-angiogenic cancer therapy, which entails administering an amount of the above pharmaceutical
composition sufficient to inhibit or silence miR-126, to a patient in need thereof.
The present invention also provides a method of inhibiting expression of one or more VEGF pathway genes in a mammal, particularly a human.
Generally, the nano-carrier of the present invention may be a single-walled or double- walled carbon nanotube, a metal-based or polymer-based nanoparticle or nanotube. For example, the nano-carrier may be a single-walled carbon nanotube or may be a cyclodextrin polymer-based nanoparticle as described in Davis, Molecular Pharmaceutics, 2009, 6(3), pp.659-668.
Additionally, the nanoparticles and methodologies of loading the same may be as described in US Publ. No. 2009-0087493-A1 , which is incorporated herein in the entirety. For example, the nanoparticle may be a carbon nanotube, boron nitride (BN), a grapheme sheet, a graphitic oxide, a graphitic-coated metal core, a cyclodextrin-based polymer or a nanocrystal containing metals, such as Au or metal alloys, such as FeCo. Furthermore, the methodologies described in U.S. Publ. No. 2008-0253961 for loading nanoparticles for targeted cancer nanotherapeutics may be used. This patent' publication is incorporated herein in the entirety.
Moreover, any of the following may be loaded, and even two or more
simultaneously loaded, onto the nano-carrier. Notably, chemotherapeutical drugs;
targeting groups; such as peptides, antibodies or nucleic acid aptamers; or miR-126 antagonists may be loaded onto the nano-carrier. Any known method may be used to load any of these cargo species onto the nano-carrier.
Further, the various nanoparticles may be made using known methods. For example, Fe/Au nanoparticles may be made in accordance with U.S. 7,186,398, which is incorporated herein the entirety. Moreover, nucleic acid functionalized nanoparticles for therapeutic uses may be made in accordance with U.S. Patent Publication
20080306016, which is incorporated herein the entirety. Furthermore, tissue targeted nanoparticle compositions containing polymer conjugates and nucleic acid molecules that induce RNA interference (RNAi) may be made and used in accordance with U.S. 7,534,878, which patent is incorporated herein by reference in the entirety, however including at least one miR-126 antagonist in accordance with the present invention instead of the siRNA antagonists of VEGFs of U.S. 7,534,878.
As an example, the present invention provides a class of miR-126 antagonists using nano-carriers such as single walled carbon nanotubes (SWCNT). Generally, carbon nanotubes are rolled-up seamless tubes of graphene with pure C-atoms
(diameter !1 nm, length -50-200 nm), and have surface areas of ~1000m2/g for high- stoichiometry conjugation (>50-300 moles cargo/mole SWCNT) of diverse molecules including siRNA as well as targeting groups. Efficient in vivo delivery of SWCNT using an RGD peptide, which is commonly used as a vascular targeting group, has already been demonstrated. The present invention contemplates SWCNT simultaneously conjugated to both RGD for vascular delivery and to an siRNA against miR-126. This is injected i.v. at 20-100 mg/kg q 3 days into LLC tumor-bearing C57B1/6 mice, and tumor size, CD31+ tumor vascular density, and real-time PCR for miR-126 are used as endpoints.
While carbon nanotubes as described above are generally used, i.e., a width of about 1nrn and a length of about 50-200 nm, other aspect ratios, i.e., ratio of
length/width, may be used.
Further, the SWCNTs of the present invention generally have a surface area of at least about 500 m2/g, and preferably at least about 1 ,000 m2/g.
MATERIALS AND METHODS
Generation of Eg/77^ and miR-126"* mice.
For targeting Egfl7, a loxP site (P1452) and a neomycin selection cassette plus a loxP site (P1451) were cloned into an A/7II site 51 of exon 5 and into an Nhe\ site 3' of exon 7, respectively. For targeting the 73-bp miR-126 precursor, P1452 and P1451 were cloned into an Nhe\ site 194 bp 5' of miR-126 and an Λ/s/1 site 22 bp 3'of miR-126, respectively (flanking 289 bp total) (for details, see Figs S1 and 52 in the supplementary material). Delta (Δ) alleles were generated by crossing to CMV- or HPRT-Cre mice. Mutant mice were analyzed in a mixed 129sV/CI57B1/6 genetic background. All mice were treated according to the Stanford Institutional Animal Care and Use Committee and the Stanford Administrative Panel on Laboratory Animal Care . miRNA in situ hybridization
In situ hybridization was performed as described (Obernosterer et al., 2007). Mouse miR-126 locked nucleic acid (LNA) probes were from Exiqon.
Generation of rabbit anti-Egf!7 antibody and immunofluorescence staining Rabbits were immunized against the bacterially expressed C-terminal 112 amino acids of murine Egfl7 fused to the C-terminus of maltose binding protein (MBP).
Antiserum was affinity purified against the C-terminal 112 amino acids of Egfl7 fused to the C-terminus of glutathione-S transferase (GST). PFA-fixed frozen uterus sections were stained with 0.1 μg of affinitypurified rabbit anti-Egfl7 antibody and imaged with a Zeiss Z1 Axioimager with Apotome.
Quantitative real-time PCR
miR-126 expression was analyzed using the Taqman MicroRNA Assay (Applied Biosystems) utilizing looped RT primers to detect processed miR-126, and expression was normalized to that of miR-16. Egfl7 expression was determined using the SYBR Green Quantitect PCR Kit (Qiagen) and normalized to that of Gapdh. Egfl7 primers: 5'- TGCGACG GAC -ACAGAGCCTGCA-S1 and 51-CAAGTATCTCCCTGCCATCCCA-31. Assays were performed in triplicate and results from at least three independent experiments are presented.
Whole-mount retina staining
P5 eyes were dissected and fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C. Retinas were isolated, blocked in PBS containing 1% BSA and 0.5% Triton X-100 overnight at 4°C, incubated overnight with 10 μg of FITC-conjugated isolectin B4 (Vector Labs) in 500 μl of the same solution, washed and then flat mounted.
Western blot analysis
Antibodies used were: rabbit anti-p85, rabbit anti-phospho-Akt (Akt1 -Mouse Genome Informatics) (Ser 473), rabbit anti-phospho-Erk (Mapki -Mouse Genome Informatics) (all from Cell Signaling), rabbit anti-Spred1 , rabbit anti-p85β, rabbit anti-α- actin (all from Abeam) and rat anti-HA (Roche). Transfection of human umbilical vein endothelial cells (HUVEC) with miRNA inhibitor
Ant\-miR-126 hairpin inhibitors (Thermo Scientific Dharmacon) or negative control inhibitor were transfected into HUVEC at 100 nM using Dharmafecti . Cells were assayed for protein expression 48 hours after transfection.
Scratch wound assay
HUVEC were serum starved overnight 24 hours after transfection of miRNA inhibitors, and scraped with a sterile P200 tip to generate a cell-free zone. Cells were stimulated with human VEGF165 (R&D Systems) (10 ng/ml) for 24 hours. Migration was quantified by counting the number of cells per scratched area (π=6).
Corneal micropocket assay
The cornal micropocket assay was performed as described (Kuo et al.,2001). miR-126 target luciferase reporter assay
The 3'UTR of Pik3r2 and Spredi were amplified and cloned downstream of a Renilla luciferase reporter gene. The miR-126 binding sites were mutated from 5'- ACGGTAC-31 to 5'-GTAACGA-3' and from 5'-GGTACG-3' to 5'-AAGCAT-3' in the 31UTR of Pik3r2 and Spredi, respectively. The Lin41 (Trim71 - Mouse Genome Informatics) 3'UTR was used as a negative control. 293T cells in 24-well plates were transfected with 3.35 ng/well of firefly luciferase, 0.667 ng/well of Renilla 3'UTR construct, and either 0, 10 or 100 ng/well of miR-126 expression vector. Empty vector was added to provide a total of 337 ng of DNA per transfection. Forty-eight hours after transfection, the Reπ/7/a/firefly luciferase was measured using the Dual Reporter Lucrferase Kit (Prornega). Akt/Erk phosphorylation assay
Akt/Erk phosphorylation assays were performed as described (Gerber et al., 1998).
Statistical analysis
P-values were determined using a two-tailed Student's f-test assuming unequal variances.
RESULTS AND DISCUSSION
We explored the mouse Egfl7/miR-126 locus using selectively floxed Egfl7Δ and, rniR-126Δ alleles to replace either a 289 bp segment of intron 7 containing miR-126 or exons 5-7 of Eg/77 with a single loxP site, without disruption of the reciprocal gene or miRNA (Fig. 1A; see Figs. 5 and 6). miR-126Δ/Δ, but not Egfl7^, embryos exhibited loss of miR-126 expression as assessed by in situ hybridization or quantitative PCR (qPCR) using looped RT primers to detect processed miR-126 (Fig. 1B,C). Conversely, Egfl7Δ/Δ, but not miR-126Δ/Δ, mice exhibited loss of Egfl7 by qPCR and by immunofluorescence with an affinity-purified rabbit anti-Egf17 antiserum (Fig. 1C1D). Furthermore,
sequencing of the EgfU ORF amplified from miR-126Δ/Δ cDNA revealed a lack of occult Eglf7 splicing alterations resulting from the miR-126 microdeletion (Fig. 1E). These studies indicated the successful generation of two monospecific Δ alleles for miR-126 and Egfl7, respectively.
Surprisingly, Egfl7Δ/Δ mice were phenotypically normal and born at the expected Mendelian ratios despite previous reports from genetrap and conventional knockout alleles (Schmidt et al, 2007) (Fig.2A). By contrast, miR-126Δ/Δ mice recapitulated numerous previously described Egfl7 mutant phenotypes (Schmidt et al., 2007) including ~50% embryonic lethality (Fig. 2B), which appeared obligately associated with the development of prominent subcutaneous embryonic edema by E14.5 (Fig. 2C1D). At E15.5, multifocal, progressive hemorrhage of varying severity from ruptured blood vessels was observed in ~20'% of the miR-126Δ/Δ embryos, most prominently in the jugular and subcutaneous regions (Fig.2E), with resultant embryonic lethality becoming first apparent at E16.5. The embryonic edema was phenocopied by miR-126flox/Δ\ Tiu2- Cre embryos, consistent with a cell-autonomous mechanism in the endothelium (Fig. 2F).
Surviving miR-126Δ/Δ neonates, which were obtained at ~50% of the expected frequency (Fig. 2B), exhibited delayed postnatal retinal angiogenesis (Fig. 3A-C). This was particularly notable in terms of compromised radial migration, a decreased area of retinal vascularization, and abnormally thickened endothelial sprouts (Fig. 3A-C), as previously described in Egfl7 gene-trap and knock-in mice (Schmidt et al., 2007). miR- 126Δ/Δ mice further displayed delayed developmental cranial angiogenesis (Fig. 3D), again reminiscent of previously described Egfl7 mutant phenotypes (Schmidt et al., 2007). Consistent with a more substantial role for miR-126 in the regulation of adult angiogenic processes, surviving miR-126Δ/Δ mice demonstrated impaired angiogenesis in a VEGF-dependent corneal micropocket assay (Fig. 3E1F). None of the
aforementioned phenotypes was observe d. in Egfl7^a or wild-type mice (Fig. 3A-F), with the deficits in retinal, head and corneal vasculature all supporting the in vivo regulation of angiogenesis by miR- 126.
The mechanisms of miR-126 regulation of angiogenesis were further explored in cultured endothelial cells. Transfection of an RNA hairpin inhibitor induced a greater than 95% depletion of mature miR-126 in HUVEC (Fig. 4A). This was accompanied by significant decreases in migration in scratch assays, as well as impaired VEGF- dependent activation of the downstream kinase Akt (Fig. 4B, C). The basis for this impaired VEGF signaling in m/fi-726-deficient endothelium was examined at the level of miRNA target genes. miR-126 directly repressed expression of the Pik3r2-encoded p85β subunit of PI3 kinase (PI3K) in co-transfection assays, whereas p85β protein was increased in both primary m\R-126Δ/Δ endothelium and miR-126 knockdown HUVEC (Fig 4D-G). Either the knockdown of miR-126 or the overexpression of the target p85β in HUVEC was sufficient to impair VEGF-mediated activation of the PI3K downstream target Akt, paralleling inhibition of insulin receptor tyrosine kinase signaling by p85 overexpression (Barbour et al., 2005; Brachmann et al., 2005; Ueki et al., 2002) (Fig. 4C, H). miR-126 knockdown additionally impaired VEGF activation of Erk (Fig. 4C), further reiterating compromised signal transduction in angiogenesis by miR-126 knockdown in vivo. In this regard, the Erk pathway inhibitor Spredi (Taniguchi et a1., 2001) was directly repressed by miR-126 cotransfection and was upregulated in miR- 126 knockdown HUVEC (see Fig. 7).
Overall, the current studies describe essential in vivo regulation of angiogenesis by a miRNA as evidenced by delayed developmental vascularization in retina and brain, impaired adult VEGF-dependent corneal angiogenesis, and in vitro regulation of motility. Edema was a prominent feature of miR-126Δ/Δ embryos and was tightly correlated with the lethality observed in ~50% of embryos. This edema did not appear secondary to intrinsic cardiac defects (data not shown). The incompletely penetrant embryonic lethality and angiogenic delay of miR-126Δ/Δ mice contrast with the more classical embryonic lethal angiogenic phenotypes (Gale and Yancopoulos, 1999) and appear consonant with the comparatively subtle action of miRNAs in finetuning global gene expression profiles (Kloosterman and Plasterk, 2006; Zhao and Srivastava, 2007).
Consistent with its vascular expression pattern, these miR-126 phenotypes occur cell-autonomously in endothelium as judged from the compartment-specific deletion phenotypes of miR-126Δ/Δ;T\e2-Cre embryos. Mechanistically, this cell-autonomous action allows miR-126 deficiency to derepress and overexpress the p85β regulatory subunit of PI3K and Spredi , which represent negative regulators of PI3K and MAP kinase signaling, respectively (see Fig. 8). Although p85β and Spredi dysregulation clearly appears contributory to the miR-126 phenotype, the promiscuous action of miRNA suggests the likely action of numerous additional target genes. miR-126 deletion phenotypes in mouse and knockdown in zebrafish were previously described with impaired angiogenesis and vascular integrity via dysregulation of SprecM and p85β (Fish et al.. 2008; Wang et al., 2008). These phenotypes are both reinforced by similar findings in our experiments and are extended by our analysis of endothelial-specific deletion in miR-12(?OX/Δ; Tie2-Cre embryos. Furthermore, an added important feature of the present invention is the unexpected lack of abnormalities in Egf^Δ mice and the widespread phenocopying by miR-126Δ/Δ mice of vascular deficits of previously described Egfl7 alleles, consisting of a gene-trap in intron 2 and a lacZ insertion into exons 5-7, both upstream of intron 7 that contains miR-126 (Schmidt et al., 2007). These data suggest that miR-126 regulates the collective migration of
endothelium as has been proposed for Egfl7 (Schmidt et al., 2007). These miR-126Δ/Δ mice facilitate additional exploration of miR-126 function in settings of adult
angiogenesis, as well as of divergent miR-126 roles such as in metastasis suppression (Tavazoie et al., 2008). Conversely, the EgflΔ/Δ mice allow selective in vivo analysis of Eg/77 without the confounding influence of miR-126. Our data by no means exclude novel and essential Egr/77-specific functions, either alone or in conjunction with the paralog Egfl8, or as described in zebrafish knockdown, mouse overexpression and in vitro studies (Campagnolo et al., 2005; Lelievre et al., 2008; Soncin et al., 2003; Xu et al., 2008).
Our results comparing miR-126Δ/Δ and EgflΔ/Δ mice provide the most extensive documentation of inadvertent disruption of miRNA expression by deletion and gene trap knockout approaches to date.
Our identification of miR-126 as an essential regulator of angiogenesis directly suggests miR-126 regulation of tumor angiogenesis, with attendant therapeutic implications. Since miR-126 and Egfl7 are co-expressed from the same transcript and have identical expression patterns (Wang et al., 2008), the expression of Egfl7 can be used as a surrogate for miR-126. The tissue distribution of Egfl7, as described by others, indicates that lung is a predominant tissue expressing Egfl7 in the adult organism (Fitch et al., 2004). Expanding on this observation, we explored Egfl7 expression in the adult animal by immunofluorescence using an affinity-purified anti- Egfl7 polyclonal antibody raised by our group. These studies indicated that Egfl7 is exclusively expressed in vasculature in adult lung (Fig. 9). Interestingly, in lung, Egfl7 is restricted to the large vessels not the microvasculature (Fig. 9, arrows). We also examined the expression of Egfl7 in the K-rasG12D; p53flox/flox model of lung cancer. This system utilizes intranasal delivery of adenovirus Cre to induce extremely synchronous and penetrant primary lung adenocarcinomas that remarkably recapitulate human lung cancer in terms of nuclear atypia/dysplasia, stromal desmoplasia, invasion and metastasis (Jackson et al., 2005). Strikingly, after adenovims Cre treatment, K-rasG12D; p53flox/flox lung carcinomas exhibited dramatic Eg/77 expression in all the tumor microvasculature (i.e. capillaries) (Fig. 10). Again, similar to wild-type mice, the normal lung adjacent to K-rasG12D; pδS"0^0" lung carcinomas expressed Egfl7 only in large vessels, with a notable lack of staining in the normal lung microvasculature/capillary beds (Fig. 10).
We further surveyed expression of the miR-126/Egfl7 transcription unit using Egfl7 as a surrogate reporter in additional spontaneous transgenic mouse models of cancer: MMTV PyMT (mammary), LSL RasG12V/intratracheal Ad Cre (lung) and RipTAg (pancreatic islet). As with transgenic lung cancers, our affinity-purified Egfl7 antibody demonstrated clear localization of Egfl7 to the tumor endothelium and NOT the tumor parenchymal cells in all three transgenic cancer models. Indeed, all three tumor types demonstrated complete co-localization between Eg/77 and the endothelial marker CD31 without discernable parenchymal signal (Fig. 11).
Furthermore, to directly examine miR-126 expression we have recently successfully adopted a newly described protocol for amplified fluorescent miRNA in situ hybridization (46) to miR- 126 detection in these mouse tumor models. In both the MMTV PyMT and LSL RasG12V/intratracheal Ad Cre models, direct in situ hybridization for miR-126 using a /77/'R-726-specifrc LNA probe again demonstrated clear miR-126 localization to the vasculature and not the tumor parenchyma (Fig. I2). Additionally, increased miR-126 expression lung adenocarcinoma was confirmed, through performed real-time PCR for miR-126 on normal lung versus K-rasG12D; p53flox/flox tumor. This revealed a 3-fold upregulation in the tumor, likely corresponding to endothelial expression (Fig. 13).
An additional function of the miR-126/Egfl7 transcription unit in
lymphangiogenesis and tumor lymphangiogenesis is suggested by the prominent expression of Egfl7 in lymph nodes. Egfl7 immunofluorescence of lymph node samples from RIP1-Tag2 transgenic mice bearing pancreatic insulinomas revealed prominent lymphatic expression which was distinct from endothelial CD31 expression (Fig. 14). These results suggest the potential utility of therapy directed against miR-126/Egfl7for lymphagiogenesis inhibition, such as during tumor lymphangiogenesis. The miR-126 knockout embryos also exhibit vascular defects indicative of impaired developmental separation of lymphatics from blood vessels wherein the embryonic lymphatics exhibit an aberrant persistent connection to blood vessels resulting in abnormal filling with red blood cells (Fig. 15).
Nano-carrier-based Compositions
The present invention also provides nano-carrier-based compositions for regulating angiogenesis or lymphanogiogenesis in mammalian endothelial cells either in vivo or in vitro.
The nano-carier may be a nanoparlicle or a nanotube. The nanoparticle or nanotube may be made of carbon, metal or polymer. The nanotubes may be either single-walled or double-walled. See U.S. patent publ. nos. 2008-0253961 and 2009- 0087493- A1 , incorporated herein the entirety.
For example, a composition in accordance with the present invention may contain single-walled carbon nanotubes having conjugated thereto both a vascular targeting agent (VTA) or moiety, and at least antagonist, such as siRNA against miR- 126. The SWCNTs, for example, may be of any suitable size and aspect ratio, but are generally of a length of about 25-500 nm, preferably about 50-200 nm, and a width of about 0.5 to 5 nm, preferably about 0.5-2 nm.
Generally, the SWCNTs may be prepared using any known method. For example, the methods described in U.S. 6,183,714, which patent is hereby incorporated herein the entirety, may be used.
Generally, the miR-126 antagonists of the present invention are designed according to the principles set forth in Vermeulen et al, RNA, Vol. 13, No. 5, pp. 723- 730.
MicroRNA Design
Further, the functionality of various miR-126 inhibitor variants may be tested using the assay methodology set forth in Vermeulen et al, id.
Generally, the following guidelines are used when designing an appropriate miR- 126 inhibitor/antagonist for use in accordance with the present invention:
1) Inhibitor length generally improves inhibitor potency. For example, the inhibitor function of 2'-O-methyl-modified oligonucleotides is strongly dependent upon length. Generally, molecules having symmetrical flanking regions of > 10 nt inhibit target miR-126 to an appreciable extent, although the level of inhibition generally peaks in the range of 50-60 nt with symmetrical flanks in the range of about 10- 15 nt;
2) Flanking region sequence can affect overall inhibitor function; and 3) Incorporation of secondary structure improves inhibitor function, such as a hairpin structure. A similar effect may be obtained by the addition of double- stranded (ds) structures adjacent to the RC core.
To exemplify the use of the above guidelines, two important features associated with 2'-O-methyl-modified miRNA inhibitors in general may be noted. First, extending the sequences beyond the boundary of the RC core greatly increases overall potency and identifies a minimal length necessary for potent sequence-specific RNA inhibition. Moreover, substitution of reverse complement flanking sequences with arbitrary sequences results in only a small decrease in overall functionality. Second, secondary structure is important to inhibitor function.
The importance of secondary structure may, perhaps, best be explained by the enhancement secondary structure provides for the miRNA-RISC interactions with inhibitors.
Examples of a miR-126 inhibitor which may be used in the present invention are:
(51
AGAAGAGAGAAAUCUCUUCUCGCAUUACUCACGGUACGAUCUUCUCUUUCGAGA GAAGA 31)
Any suitable VTA may be used, such as hairpin RNA, small peptides obtained by phage display or monoclonal antibodies targeting other endothelial antigens, such as VEGFR2 or VE-Cadherin, etc.
Therapies Using the Nano-carrier-based Compositions
Generally, the present nano-carrier-based compositions may be used to advantage in treating diseases or conditions in mammals, particularly humans, characterized by a hyper-angiogenetic state, such as macular degeneration or metastatic cancers, such as metastatic prostate cancer. Further, since Egfl7, which is co-regulated with miR-126, is strongly expressed in lymphatics, and since miR-126 knockout embryos exhibit some characteristics of defective lymphangiogenesis, the present invention may also be used to advantage in the regulation of lymphangiogenesis, and in the treatment of diseases characterized by a hyper- lympangiogenetic state.
Generally, the present nano-carrier-based compositions are administered, preferably intravenously, in an amount of about 1-100 mg/kg of body weight/dose.
Dosage is generally once per day, but multiple doses may be given per day as deemed appropriate by the treating physician.
Furthermore, the nano-carrier-based composition of the present invention may be used in conjunction (either concurrently or consecutively) with other therapies. For example, in the treatment of cancer, it is advantageous to use the present composition, a VTA-based composition, with other therapies that are particularly effective as tumor 'rim' or periphery therapies. VTA-based therapies are sometimes most effective against tumor cores, hence, the usefulness of using 'rim1 or peripheral tumor therapies therewith.
For example, the construction and use of the nano-canier-based composition of the present invention may be as described in Feazell et al., J. Am. Chem. Soc. 2007, 129, 8438-8439. That is, the SWCNTs may be functionalized by non-covalent binding of phospholipid-tethered amines to the nanotube surface. A polyethyleleglycol (PEG) chain between the amine and the anchoring phospolipid serves to solubilize the SWCNTs and extend functional groups away from the nanotube surface.
Further, in using conjunctive 'rim' therapies in the treatment of cancer, both 'core1 and 'rim' treating agents nay used as a single compound. That is, it is specifically contemplated, for example, that both miR-126 antagonist and cis-platin or other chemotherapeutic agent, be loaded onto the same SWCNT in addition to the VTA, such as RGD. This may also be done using, for example, the procedure described in the above noted publication. For the use of cis-platin as a cancer therapy, see U.S.
4,177,263, which is incorporated herein in the entirety. For the attachment of cis-platin to nanoparticles, see Int. J. Nanomedicine, 2007 December; (4): 667 -674. However, any other chemotherapeutic compound may be used, such as methotrexate or vincristine, for example.
Use of mJR-126 in Treating Leukemia
It is known that miR-126 is strongly upregulated in leukemia, particularly acute myeloid leukemia (AML) subtypes M2 and M4. See, for example, Li et al, PNAS, October 7, 2008, vol. 105, no.40, 15535-15540.
Thus, the present invention specifically contemplates using the compositions disclosed herein in treating leukemia using the treatment protocols also described herein, as well as using the methodologies disclosed in U.S. 7,534,878, incorporated herein by reference in the entirety.
FURTHER USES OF THE PRESENT INVENTION IN ANALYSIS OF TUMOR PROGRESSION AND INHIBITION THEREOF
The microRNA miR-126 represents the most abundant endothelial miRNA upon expression profiling, and is expressed in a pan-endothelial manner during
embryogenesis and at sites of active angiogenesis, including tumors, in the adult. The present inventors have also prepared knockout (ko) mice lacking miR-126 which exhibit 50% embryonic lethality associated with edema, hemorrhage and angiogenic delay. In surviving miR-126 ko mice, adult angiogenesis is delayed, for instance, in corneal micropocket assays. miR-126 is present in intron 7 of a host gene, Egfl7. Thus, the miR-126 ko phenotype recapitulates previously described Egfl7 ko phenotypes, and previously described Egfl7 ko mice are now understood to have inadvertently disrupted miR-126 expression.
The present invention also explicitly contemplates analyzing, and quantifying, the import of miR-126 to tumor progression, particularly in the MMTV-PyMT transgenic breast cancer model. The present invention also describes studies including extensive data including (a) generation of floxed miR-126 ko mice that do not perturb Egfl7 expression, (b) the developmental and adult angiogenesis phenotypes of miR-126 ko mice , (c) the prominent expression of the miR-126/Egfl7 locus in tumor vasculature, and (d) the significant inhibition of MMTV-PyMT mammary tumor progression upon miR- 126 deletion.
The present invention also provides parallel and complementary mouse genetic and pharmacological methodologies to evaluate miR-126 function. Specifically, a first methodology (1) entails genetic analysis of effects of global miR-126 deletion on MMTV- PyMT mammary tumor progression at the level of tumor angiogenesis, survival and metastasis. A second methodology (2) entails genetic and pharmacological methods to evaluate the therapeutic potential of miR-126 inhibition against pre-established tumors, conditionally deleting miR-126 in pre-established tumors, and benchmarking this against pharmacological inhibition with miR- 126- targeted anti-miRs. This second methodology also entails comparing and combining miR-126 inhibition by either genetic or
pharmacological methods with VEGF inhibition towards development of combinatorial anti-angiogenic therapy. A third methodology (3) entails elucidating and evaluating mechanisms of miR-126 inhibition of mammary tumorigenesis by endothelial-specific miR-126 deletion by means of PDGFB-iCreER, by determining whether endothelial overexpression of miR-126 is sufficient to enhance, and applying mass spectroscopy to miR-126 target discovery to elucidate new modes of action.
The above methodologies are based upon the discovery, provided by the present invention, that tumor angiogenesis can be regulated by an endothelial miRNA, miR-126. The above methodologies may be summarized as follows:
Methodology 1: Requirement for miR-126 during mammary tumor progression
Rationale: miR-126 is necessary for mammary tumor progression and genetic deletion of miR-126 impairs tumorigenesis in MMTV-PyMT mice.
A. Evaluate effects of miR-126 deletion on mammary tumor progression in the MMTV-PyMT model;
B. Evaluate impact of miR-126 deletion upon survival and metastasis in the MMTV-PyMT model; and
C. Evaluate tumor angiogenesis in miR-126 Δ/Δ; MMTV-PyMT mice.
Methodology 2: Evaluation of miR-126 inhibition as a therapy for treating breast cancer
Rationale: Treatment of pre-established tumors by either conditional genetic deletion of miR-126 or by miR-126 anti-miR administration impairs mammary tumorigenesis
A. Effect temporally conditional deletion in miR-126 flox/flox; MMTV-PyMT; R26- M2rtTA; TRE-Cre mice;
B. Effect pharmacological antagomir and antimir inhibition of miR-126 in the MMTV-PyMT model; and
C. Compare and combine miR-126 inhibition with VEGF inhibition
Methodology 3: Mechanistic evaluation of miR-126 action during tumor progression Rationale: miR-126 is both necessary and sufficient to regulate tumor
angiogenesis by means of cell-autonomous action in endothelial cells and
repression of downstream targets
A. Evaluate endothelial cell autonomy in miR-126 ^* MMTV-PyMT; PDGFB- iCreER mice;
B. Evaluate miR-126 sufficiency in primary miR-126 Δ/Δ tumor endothelium; and
C. Utilize proteomic methods to miR-126 targets using pSILAC
The three methodologies described above will now be described in greater detail below in order to further exemplify the present invention.
(a). Significance
The general purpose of the present invention is the regulation of tumor progression and tumor angiogenesis by the endothelial microRNAs (miRNA), miR-126.
!. MicroRNA structure, biogenesis and function. The biological functions
of RNA have been recently greatly expanded by the description of
myriad types of small non-coding RNA (ncRNA). These ncRNA are typically 19-31 nucleotides in length, including microRNAs (miRNAs), small-interfering RNAs (siRNAs), trans-acting siRNAs (tasiRNAs),
small-scan RNAs (scnRNAs), repeat-associated siRNAs (rasiRNA),
Piwi-interacting RNAs (piRNAs), and long ncRNAs such as Xist and
HOTAIR. The diverse ncRNA typically confer epigenetic regulation via translational repression. The present invention focuses on the regulation of tumor progression and tumor angiogenesis by the microRNA (miRNA), miR-126. Following the initial discovery of miRNA in C. elegans, hundreds of miRNA genes have been described in essentially all metazoans. miRNA genes can be intronic or exonic to conventional protein-encoding mRNAs, and are transcribed by RNA polymerase Il yielding the primary miRNA (pri-miRNA) containing a stem-loop structure. This pri-miRNA undergoes sequential processing by the RNA endonucleases Dicer and Drosha, generating the mature 21 nt single stranded miRNA (Fig.16). miRNAs down-regulate gene expression by annealing between the 5' ends of miRNA (nt 2-8, the "seed region") with the 3'UTR of target mRNAs in the RISC (the ribonucleoprotein "RNA-induced silencing complex") with direct repression of translation as well as mRNA degradation and de- adenylation, with the net effect of decreasing gene expression post- transcriptionally. The base-pairing between miRNA and their mRNA targets is imprecise and degenerate, allowing a single miRNA to exert post-transcriptional regulation over the expression of hundreds of target mRNA. It has been thus postulated that a significant portion of the transcriptome is under miRNA regulation and that miRNA "sculpt" or "fine-tune" global protein expression patterns to critical threshold levels during homeostasis or in response to stimuli. miRNA functions in angioqenesis. In vivo analysis in vertebrates initially utilized miRNA overexpression/gain-of-function to implicate miRNAs in diverse processes including metabolism, development and cancer. The first strategy utilized for in vivo vertebrate loss-of-function analysis of miRNAs in vivo was systemic infusion of siRNA-cholesterol conjugate "antagomirs", or broad-spectrum ablation of all miRNAs in a particular tissue using Dicer ko. Individual murine miRNA knockout mice were first reported in 2007 in lymphocytes and myocardium with miR-1-2 or miR-208 ko resulting in either embryonic lethality or cardiac hypertrophic, conduction or heart structural defects in adults. Until recently .there had been no in vivo data for individual endothelial miRNAs, for either gain- or loss-of-f unction. Ablation of the processing enzymes Dicer and Drosha completely inhibits all miRNA biogenesis which is not surprisingly embryonic lethal at E7.5, precluding analysis of effects on vasculogenesis or angiogenesis. A hypomorphic Dicer allele exhibited later lethality at E12.5-E14.5 with impaired yolk sac angiogenesis. With regards to individual miRNA, cultured endothelium (i.e. HUVEC), highly express miRNA, notably miR-126 but also miR-21 , -221 and -222 . Global inhibition of miRNA biogenesis in HUVEC by Dicer/Drosha siRNA reduced sprouting, tube formation or proliferation in vitro, and spheroid sprouting in vivo .
Since our report in Development in 2008, describing the first endothelial miRNA knockout mice for miR-126, no additional endothelial miRNA knockout mice have been reported. In fact, the few other instances where in vivo loss-of-f unction analysis has been subsequently performed have used antagomir-based inhibition to demonstrate that miR-92a or miR-126 augment and inhibit revascularization, respectively, in hindlimb ischemia models or that miR-17/20 suppresses in matrigel plug angiogenesis.
3^ Tumor angiogenesis and miRNA. Folkman hypothesized in 1971 that tumor growth is dependent on angiogenesis and that targeting a tumor's blood supply inhibits neoplastic growth and progression through deprivation of oxygen and nutrients. The discovery of Vascular Endothelial Growth Factor (VEGF) as a key regulator of angiogenesis has ultimately led to U.S. FDA approval of numerous VEGF antagonists for cancers of the colon, lung, breast, kidney and liver. However, (a) current anti-angiogenic therapies only provide survival advantages measured in months, (b) this is accomplished at great economic cost, and (c) progressive cancer inevitably supervenes. Thus, there is a pressing need for novel anti-angiogenic agents, for use in combination with or instead of VEGF inhibitors for increased efficacy. The present invention thus evaluates the endothelial microRNA, miR-126, as a novel anti-angiogenic target using complementary genetic and pharmacologic inhibition methods.
Compared with miRNA functions in developmental and post-ischemic
angiogenesis, very little is known about the functions of individual miRNA during tumor angiogenesis. Initially, it was reported that the miR-17~92 cluster was expressed in tumor cells, and overexpression could repress tumor cell secretion of anti-angiogenic products such as TSP1 and CTGF, thereby increasing angiogenesis in adjacent tumor endothelial cells (EC). With respect to endothelial miRNAs, while miR-92a blockade by antagomirs augments neovasularization in hindlimb ischemia models, equivalent data for tumor angiogenesis has not been published . Although miR-17/20 antagomirs augment angiogenesis in matrigel plugs, no effect on tumor angiogenesis was noted. miR-519c suppresses HIF-Ia expression in EC, and miR-519c antagomirs increase HIF-Ia and angiogenesis . A brief 4-day antagomir treatment targeting miR-296 was reported to diminish tumor vasculature in subcutaneously xenografted U87 glioblastoma cells ; no data on tumor size, progression or survival were presented, however.
Clearly, the published data with miR-17/20, miR-92a (i.e. miR-17~92 cluster miRNAs) or miR-519c suggests that these miRNAs are not optimal targets for pharmacologic inhibition in cancer, since in vivo antagomir inhibition of these increases angiogenesis. miR-126 is advantageous for anti-angiogenic cancer therapy based upon miR-126 antagomir inhibition of angiogenesis post-hindlimb ischemia.Yet, surprisingly, prior to the present invention, the role of an individual miRNA in tumor angiogenesis was not rigorously explored using a genetic mouse knockout allele.
4^ The miR-126/Epfl7 locus - a transcription unit encoding an endothelial protein-encoding gene with nested endothelial miRNA. The present invention utilizes knockout mice deficient in miR-126, the most abundantly expressed endothelial miRNA. Until recently, no in vivo functional data for miR-126 had been described. In vivo endothelial expression of miR-126 was first described in a large-scale in situ screen of embryonic miRNA expression patterns. The present inventors were intrigued both by the striking pan-vascular expression pattern of miR-126 as well as its chromosomal location. The miR-126 gene is present in intron 7 of the Eg/77 gene in both human and mouse (Fig.17). Egfl7 is a secreted and matrix-associated protein with a signal peptide and 2 EGF repeats, and we and others have found it expressed in a pan-endothelial fashion in embryos (Fig. 17B) . Consequently, the identical vascular expression of miR-126 M was retrospectively not surprising since both Egfl7 and miR-126 likely arise from a common transcript, although an independent intronic promoter may co-exist . The miR-126-Zp strand (5' UCGUACCGUGAGUAAUAAUGC 3') represents the predominant species in endothelial cells (EC), as the complementary mι'R-126-5p strand is present at much lower levels and represents the "*" or degraded strand.
Various and conflicting in vitro functions have been ascribed to Egfl7 including regulation of EC or vascular smooth muscle migration and adhesion. In zebrafish, Egfl7 morpholino knockdown impaired vascular tube formation. Two different mouse knockout alleles of Egfl7 were been described by Genentech: a gene trap insertion into intron 2, and an IRES lacZ knock-in allele replacing exons 5-7, both upstream of miR-126 in intron 7. Both Genentech Egfl7 alleles insert bulky cassettes with a 2xLTR-flanked PGK neo polyA module in the gene trap, and a PGK neo IRES b-Gal polyA module in the knock-in. As Genentech published in Development in 2007, both gene trap and lacZ knock-in Egfl7 mice exhibited edema, angiogenic delay in embryonic brain and heart, a highly characteristic postnatal retinal angiogenesis phenotype with thickened sprouts, and intriguing ~ 50% embryonic lethality.
We created knockout mice for both Egfl7 and miR-126. Because of the intimate relationship between these two genes, we designed floxed targeting constructs carefully designed to ablate Egfl7 function without affecting miR-126 and vice versa. Our Egfl7 ko successfully replaced Egfl7 exons 5-7 with a single loxP site, and our miR-126 ko produced a 289 nt microdeletion replacing miR-126 with a single loxP site within Egfl7 intron 7 without disrupting the surrounding Egfl7 locus, in contrast to the bulky cassettes that were inserted by the Genentech group into Egfl7 upstream of miR-126. This yielded mice in which the miR-126 gene was flanked by loxP sequences, we designate this as the "flox" allele, i.e. miR-126*0X. Subsequently, we crossed these miR-12&oxl* mice to an HPRT-Cre strain allowing deletion of the 289 nt floxed region in vivo, replacing miR- 126 with a single loxP site; we designate this as the delta, or "Δ" allele, i.e miR-126A. The cognate Egfl7 alleles were similarly referred to as Egfl7floxand Egfl7Δ .
The present inventors published an analysis of these mice in Development in 2008. Surprisingly, Egfl7A/A mice were phenotypically normal and born at Mendelian ratios (Fig. 19A), while in contrast our miR-126A/A ko mice fully recapitulated all the previously published Genentech Egfl7 mutant phenotypes including the highly characteristic 50% embryonic lethality and edema from vascular fragility. Further, miR- 126^A mice and not Egfl7A/A mice displayed atretic embryonic cranial angiogenesis previously described in Genentech Egfl7 ko mice 41. In surviving miR-126A/A neonates, (50% of the expected frequency), but not Egfl7έk/A mice, postnatal retinal angiogenesis was substantially delayed with compromised radial migration and decreased area of retinal vascularization which was particularly notable for characteristically thickened endothelial sprouts all of which had been previously described in Egfl7 gene trap and knock-in mice. Consistent with a larger role for miR-126 in regulation of adult angiogenic processes such as cancer, surviving m\'R-126tJA but not EgfI7A/A mice demonstrated impaired angiogenesis in a VEGF corneal micropocket assay. A cell- autonomous mechanism of miR-126 action in the vasculature was supported by phenocopy of the miR-126A/A edema in miR-126fl0X//A ; Tie2-Cre embryos).
Similar phenotypes from miR-126 deletion were also deduced by the Olson group, which independently knocked out miR-126 but did not have the benefit of a parallel Egfl7 ko and could not draw as strong conclusions regarding potential inadvertent Egfl7 perturbation in their mice. We have unequivocally demonstrated with in vivo data that (1) the published Egfl7 angiogenic phenotypes were in fact attributable to the microRNA miR-126 and that (2) miR-126 is a novel positively-acting angiogenic regulator. The inadvertent dysregulation/deletion of intronic miRNA by gene trap and conventional knockin/ko strategies had been a theoretical concern; however our study was the first actual documentation of this complication, indicating that consideration of intronic miRNA should be a general concern in mouse gene targeting strategies.
We have also performed extensive in vitro mechanistic evaluation of miR-126 in cultured endothelial cells (EC). miR-126 knockdown in HUVEC decreases VEGF- dependent migration and inhibits VEGF-dependent activation of the downstream kinases Akt and Erk. microRNAs associate with the 3'UTR of their target genes and repress their expression post-transcriptionally and thus miRNA knockout results in target gene upregulation. It has been found that miR-126 binds to the 3'UTR of signaling intermediates that normally repress VEGF receptor signaling, namely the PIK3R2 gene encoding the p85β subunit of Pl 3-kinase (PI3K) and the intracellular ERK antagonist Spredi , with p85β and Spredi overexpression upon knockout/knockdown of the predominant miR-126-3p strand in primary CD31+ miR-12^ ko EC or HUVEC, and p85β overexpression being sufficient to impair VEGF-dependent Akt activation. The coordinated upregulation of p85β (Akt antagonist via interference with VEGFR RTK signaling) and Spredi (ERK antagonist) likely contribute to the compromised
angiogenesis signal transduction and anti-angiogenic phenotypes of miR-126 knockout animals. Thus, the important possibility that miR-126 inhibition can sensitize tumor endothelium to pharmacologic VEGF inhibition in vivo for purposes of combinatorial anti-angiogenic therapy is examined in methodology 2. At the same time, given the given the promiscuous action of miRNA, modulation of VEGF signaling is likely to be but a small subset of mechanisms by which miR-126 inhibition exerts anti-angiogenic effects, and any combinatorial anti-angiogenic effects could be based upon completely distinct mechanisms. Indeed, the miR-126 ko phenotype does not resemble published VEGF/VEGFR ko phenotypes.
5,. The mι'R-126/Egfl7 locus in tumor progression and angioαenesis. The miR-126/Egfl7 locus is strongly expressed in tumor vasculature and neoangiogenic vessels in embryo and adult, versus sporadic. Our data described below strongly support the hypothesis that miR-126 is expressed in tumor vasculature, and functionally required for tumor progression and tumor angiogenesis. Initially we surveyed expression of the miR-126/Egfl7 transcription unit in 3 spontaneous transgenic mouse models of cancer: MMTV PyMT (mammary), LSL RasGJ2D/intratracheal Ad Cre (lung) and RipTAg (pancreatic islet). Egfl7 is a surrogate marker for miR-126 expression given their co- expression from a single transcript and identical developmental expression patterns. Our affinity-purified Egfl7 antibody demonstrated clear localization of Egfl7 to CD31+ tumor EC and NOT the tumor parenchymal cells in all three transgenic cancer models. Furthermore, we used a newly described protocol for amplified fluorescent miRNA in situ hybridization for miR-126 detection in these mouse tumor models. In both the MMTV PyMT and LSL RasG12V lung Ad Cre models, direct in situ hybridization for miR-126 using a miR-126-specific LNA probe again showed miR-126 in the vasculature and not tumor parenchyma.
We have focused on the well-described MMTV-PyMT mammary transgenic tumor model allowing extremely synchronous and penetrant mammary carcinogenesis with a single allele cross. Female MMTV-PyMT mice exhibit mammary hyperplasia by 2 months of age, multifocal, highly fibrotic, adenocarcinomas involving the entire mammary fat pad in virgin and breeder females with extremely high penetrance by 3 months, pulmonary metastases in essentially all tumor-bearing females by 4 months and lymph node metastases in ~ 50%. This model resembles human breast cancer with progressive loss of estrogen and progesterone receptors and increasing overexpression of ErbB2 and cyclin D1. The onset of tumorigenesis with MMTV-PyMT (2-3 months) is much more rapid than with other primary breast cancer models (c.f. MMTV-Neu, ~ 1 year), and the MMTV-PyMT model has been used extensively to evaluate effects of angiogenesis regulators by cross to ko/transgenic strains. For initial proof of principle, we crossed the miR-126* allele (i.e. constitutive miR- 126 deletion) into the MMTV-PyMT background to generate miR-126^A; MMTV-PyMT females at 1/16 frequency, followed by harvest of animals at 14 weeks of age. Notably, we observed a prominent suppression of tumor size in miR-126dJA; MMTV-PyMT females (i.e miR-126ko) compared with miR-126+l+; MMTV-PyMT control littermates (i.e miR-126vΛ), that was evident simply upon gross dissection of the animals. There are 5 mammary fat pads on each side, (10 per female), and there was a trend towards decreased number of mammary glands involved by tumor per animal (6.5 +/+ vs. 3.6 Δ/Δ, p=0.125). More significantly, there was also a striking inhibition of tumor size upon miR-126 deletion with a 68% decrease in tumor mass per measurable tumor (p=0.011) and 80% decrease in tumor size per mammary gland (p = 0.004). Histologic analysis at week 14 revealed that the miR-126 wt tumors were predominantly adenocarcinoma (83%) versus predominantly adenoma (67%) in miR-126^A. Additionally, miR-IΣ^ tumors exhibited strongly decreased CD31+ microvessel density as opposed to miR- 126 wt, consistent with a potential anti-angiogenic mechanism. Overall, these data strongly indicate that miR-126 regulates tumor progression and angiogenesis in the MMTV-PyMT model, providing feasibility for the tumor progression, therapeutic and mechanistic studies in accordance with the present invention. See Fig. 18 (A-E).
Lastly, it is also possible that miR-126 may also act cell-autonomously within tumor cells themselves. The human breast cancer line MDA-MB-231 expresses miR-126, which was significantly decreased in lung- and bone-metastatic sublines, with miR-126 restoration suppressing tumor growth and lung metastasis. miR-126 apparently undergoes significant downregulation in colon cancer cell lines versus normal colon, and miR-126 overexpression repressed in vitro growth of transfected colon cancer lines, although no in vivo analysis was performed and again spatial localization of miR-126 or Egfl7 in tumor samples was not performed. Finally, miR-126 is overexpressed in leukemic blasts, consistent with frequent co-expression of molecules in endothelial and hematopoietic cells (c.f. VEGF receptors, Tie2) and common origins from
hemangioblasts. It has been reported by others that miR-126 overexpression
enhanced proliferation of leukemic blasts versus growth suppressive effects in breast and colon cancer cells. As described above, in contrast to other reports we have not been able to obtain any evidence for tumor parenchymal miR-126 expression, since our extensive studies in multiple transgenic tumor models uniformly localize miR-126 and Egfl7 expression to the vascular compartment. Despite this, the present invention approaches miR-126 inhibition with an unbiased mindset, using a global/constuitive deletion of miR-126 (as in our miR-126*1*; MMTV-PyMT data) to establish both proof-of- principle for miR-126 essential action and a benchmark for comparison against systemically administered miR-126 therapeutics that act globally on miR-126 inhibition (c.f. antagomirs and anti-mirs), but also evaluates compartment-specific deletion in endothelium versus tumor parenchyma to elaborate mechanism. Regardless of the site of miR-126 expression within tumors, our data indicate that complete genetic deletion of miR-126 strongly impairs tumor angiogenesis and progression in the MMTV-PyMT breast cancer model (see Fig.19 A,B).
(x Summary. The present invention describes the functional relevance of miR-126 to tumor progression and as a therapeutic target, leveraging both mouse genetic tools in the form of rigorously characterized ko mice, and complementary and novel pharmacologic approaches in the form of antagomirs/antimirs. The present inventors have also provided (a) generation of floxed miR-126 mice that do not perturb Egfl7 expression, (b) the developmental and adult angiogenesis phenotypes of miR-126 ko mice, (c) the prominent expression of the miR-126/Egfl7 locus in tumor vasculature, and (d) the significant inhibition of MMTV- PyMT mammary tumor progression upon global miR-126 deletion.
Methodology 1 is based upon our analysis of global miR-126 deletion (miR- 126^κ) on MMTV-PyMT mammary tumor progression at the level of tumor
angiogenesis, survival and metastasis, establishing proof-of-principle for an essential role for miR-126 in mammary tumorigenesis. Methodology 2 affords pre-clinical translational evaluation of the therapeutic potential of miR-126 inhibition, using pre- established tumors and (1) conditionally deleting miR-126 via tamoxifen-sensitive CreER and (2) benchmarking this against a complementary strategy of pharmacologic systemic therapy with m/R-726-targeted antagomirs/antimiRs. Methodology 2 also assesses addition of miR-126 inhibition to VEGF inhibition for combinatorial anti- angiogenic therapy, and systematically compares miR-126 inhibition to VEGF antagonism. Methodology 3 then affords the mechanism for miR-126 inhibition of mammary tumorigenesis, formally addressing a potential endothelial compartment of action by EC-specific versus tumor parenchymal miR-126 deletion, evaluating if endothelial overexpression of miR-126 is sufficient to augment the function of primary tumor endothelium in vitro, and applying novel mass spectroscopy approaches to miR- 126 target discovery to reveal unsuspected pathways of action.
Overall, these studies provide strong evidence that tumor angiogenesis can be regulated by an endothelial miRNA, using complementary and mutually reinforcing genetic and pharmacologic approaches.
(b) Innovative Pharmaceutical Aspects
As described above, angiogenesis inhibition is now a commonly utilized
component of the therapeutic armamentarium for cancer, with FDA approval of numerous VEGF antagonists for cancers of the colon, lung, breast, kidney and liver, although despite modest survival advantages measured in months, substantial economic cost, and eventual cancer progression. Consequently, novel anti-angiogenic agents are clearly needed; these could be used in combination with or instead of VEGF inhibitors for increased efficacy. This proposal seeks to fulfills this translational gap by evaluating the endothelial microRNA, miR-126, as a novel anti-angiogenic target using complementary genetic and pharmacologic inhibition methods.
Our generation of miR-126 ko mice represents an unusual opportunity to provide rigorous genetic evidence for the involvement of a miRNA in tumor angiogenesis. As miR-126 is the first endothelial miRNA to be knocked out in mice, there are no previous studies of effects of genetic deletion of a particular miRNA, miR-126 or otherwise, on tumor angiogenesis in vivo. Thus, Methodology 1 of this application evaluates the effects of genetic deletion of miR-126 on tumor angiogenesis and progression in the well-characterized MMTV-PyMT transgenic mammary tumor model which recapitulates numerous aspects of human breast cancer.
Currently there are no FDA-approved agents targeting miRNA for either anti- angiogenic therapy or any other medical disorder, for that matter. As summarized above, the miR-17~92 cluster miRNAs miR-17, miR-20 and miR-92a, or miR-519c are not suitable targets for pharmacologic anti-angiogenic therapy, since antagomir blockade of these either increases angiogenesis in vivo or do not have effects on tumor angiogenesis. In contrast, our preliminary data in miR-126^*; MMTV-PyMT mice (Figs 8,9) as well as miR-126 antagomir inhibition of angiogenesis post-hindlimb ischemia both clearly indicate the anti-tumorigenic and anti-angiogenic potential of miR-126 inhibition. To date, extremely little functional evidence links a specific miRNA to the promotion of tumor angiogenesis, with the strongest example being an brief 4-day study in which miR-296 antagomir treatment reduced angiogenesis in s.c. xenografted U87 glioblastoma tumors without tumor size or survival data.
Methodology 2 affords rigorous preclinical target validation for miR-126 inhibition by treating pre-established tumors, simulating the clinical setting. Notably, these treatment studies utilize complementary genetic and pharmacologic inhibition methods. Conditional deletion of miR-126 using our floxed allele in pre-established tumors serve as an extremely rigorous positive control benchmark for pharmacologic miR-126 inhibitors since it would be unlikely that a therapeutic agent could be superior to a genetic ko of the target. Our miR-126 mouse is the only floxed allele available and thus represents the only possible method for temporally conditional adult ko.
For pharmacologic miR-126 inhibition, the current gold standard of cholesterol- conjugated antagomirs is compared to an innovative and promising second-generation "anti-mir" technology containing a 2' F/MOE backbone modification of the antisense RNA, that is not cholesterol modified, and appears superior to antagomirs ion vivo. These pharmacologic strategies are pursued using antagomirs and anti-mirs in quantities sufficient for in vivo experimentation, and therapeutic use.
An important aspect of the preclinical validation includes the benchmarking of miR- 126 deletion or anti-miR treatment against VEGF inhibition to get some sense of relative efficacy. Preclinical validation of VEGF inhibitors is well known as evidenced by publications in Nature Medicine and PNAS . Further, we also evaluate the important possibility that miR-126 inhibition might be additive or synergistic with VEGF blockade, either by sensitizing endothelium to VEGF inhibition or by promiscuous miRNA action on numerous pathways, VEGF and otherwise. The present invention, thus, has important implications for improved anti-angiogenic treatment strategies and the potential to alter clinical practice patterns.
Our investigations into miR-126 revised prior preconceived notions whereby comparison of miR-126^* versus Egfff^ mutant phenotypes revealed that previously described developmental angiogenic phenotypes of the Egfl7 locus were in fact attributable to the embedded miRNA miR-126. Accordingly, methodology 3 of the present invention also explores potentially conflicting models of the site of miR-126 action. On one hand, miR-126 clearly appears to function cell-autonomously in endothelium. In situ hybridization for miR-126 reveals embryonic vascular expression of miR-126 and/or the surrogate marker Eg/77, while vascular deletion of miR-126 in miR- 12βfioχΛoχ ; jje2-Cre embryos phenocopies the complete null miR-126^* embryos (Figs. 19c, 19e,21). In all transgenic tumors models we examined, miR-126 and Eg/77 are expressed exclusively in the vascular, not tumor parenchymal compartments, and miR- 12^; MMTV-PyMT tumors exhibit decreased tumor angiogenesis. On the other hand, miR-126 may also have stimulatory or inhibitory cell-autonomous functions within tumor cells themselves although again our studies suggest a primarily tumor
endothelial expression for both miR-126 and co-expresed Egfl7 in the models we have examined. Methodologies 1 and 2 utilize global deletion to establish proof-of-principle and to establish an benchmark for comparison of compartment-independent, systemically acting pharmacologic anti-mir strategies. Subsequently, methodology 3 examines the hypothesis of primary endothelial action through in vivo endothelium-specific deletion and in vitro analysis of purified miR-126^* tumor vasculature, with the alternative approach of deletion in the tumor parenchymal compartment by simply crossing to MMTV-Cre.
Overall, the present invention affords broad implications for diverse areas of biology, including the tumor biology, microRNA, vascular biology/angiogenesis and experimental therapeutics communities. The present invention contemplates treatment of breast cancer. In comparison, phase III trials of conventional anti-angiogenic agents, such as Avastin, have substantially delayed time to progression, but have not yet significantly extended survival, highlighting the need to identify additional anti- angiogenic strategies for this malignancy. The concepts and therapeutics established with miR-126 in breast cancer, in accordance with the present invention, are readily extendable to other solid tumors or even leukemia, in the preclinical and/or clinical settings, and have represent a highly novel miRNA-targeted therapeutic approach.
(c) The Three Methodologies and Preferred Embodiments For Each
Methodology 1. Requirement for miR-126 during mammary tumor progression
Hypothesis: miR-126 is necessary for mammary tumor progression in MMTV- PyMT mice.
Rationale: We have established (a) miR-126 is a positive angiogenesis regulator in ko mice, with developmental angiogenesis defects as well as impaired adult angiogenesis in the VEGF-dependent corneal micropocket assay, (b) the Egfl7/miR-126 transcription unit is strongly upregulated in tumor vasculature with strong expression of both Egfl7 and miR-126 in the MMTV PyMT, LSL RasG12D and RipTAg tumor models, and (c) marked suppression of tumor progression and angiogenesis in m/7?-^26A/Λ;
MMTV-PyMT mice. This is consonant with other studies indicating Egfl7 upregulation in neoangiogenic beds (wound healing, corpus luteum, pregnant uterus and tumors).
Here, we extend our encouraging miR-126^A; MMTV-PyMT data to assess effects of constitutive global miR-126 deletion on progression, survival, metastasis and
angiogenesis in the MMTV-PyMT transgenic mammary tumor model. The use of global constitutive miR-126ko will cast of the widest initial net for miR-126 functions, capable of detecting effects on tumor incidence, progression, metastasis and angiogenesis, as well as unanticipated alternative roles in tumor parenchyma.
A. Effects of miR-126 deletion on mammary tumor progression in the
MMTV-PyMT model
1÷ Breeding schema and fixed endpoint analysis. As a stringent test of mι'R-126 function during tumor angiogenesis, we have bred our well- described null miR-126* allele into the MMTV-PyMT background.
Accordingly, miR-126*1*; MMTV-PyMT mice are crossed to miR-126^*; MMTV-PyMT , and female virgin miR-126^*; MMTV-PyMT mice (null; 1/16 incidence), as well as control littermate miR-126sl*; MMTV-PyMT (het, 1/8), and miR-126*'*; MMTV-PyMT (wild-type, 1/16) are used (n=20 each). These cohorts are sacrificed at week 14 for the analyses below; in our experience wild-type MMTV-PyMT animals have a high proportion of multifocal adenocarcinoma without significant morbidity at this stage; we have already accumulated n=5 as in Figs 18 and 19.
2. Tumor incidence and tumor volume. Differences in tumor incidence are sought in the cohorts with and without null miR-126 deletion. Prior to harvest, null, heterozygous and wild-type miR-126 mice are examined twice weekly for mammary tumor onset in a blinded fashion by palpation for nodules in all 10 mammary glands. We generate Kaplan-Meier curves using ages of individual mice at time of initial tumor detection to detect potential alterations in the onset of MMTV-PyMT tumor incidence associated with miR-126 deletion. Primary mammary tumor volume measurements are calculated at week 14 as an oblong spheroid approximation using the formula L x W2 x π/6 as a sum of the tumor burden in all mammary glands at sacrifice. The Mann- Whitney U test as well as well as average and standard error is used to compare tumor burden. Histologic evaluation includes H&E staining of the mammary fat pads excised en bloc, whether grossly involved by tumor or not, using 3 individual sections separated by at least 100 microns. In some iterations, the inguinal fat pad is stained in whole mount with Carmine alum to reveal tumor. Blinded pathologic interpretation and classification as adenoma versus adenocarcinoma, and invasive and nuclear features will continue to be performed by Dr. Richard Luong in the Stanford Department of Veterinary Medicine. Standard proliferative and apoptotic indices are determined in the tumor parenchyma of ko, het and wild-type groups with anti- Ki67 and TUNEL.
Additional markers for tumor progression that are evaluated include ER, PR and Neu, which undergo characteristic down-regulation (ER/PR) and up-regulation (Neu) in MMTV-PyMT carcinomas. miR-126-specific endpoints. From total tumor tissue, Western blot and qPCR for the miR-126 target genes p85b and Spredi are performed, and miR-126 deletion confirmed by qPCR. Endothelium-specific analysis may be more revealing. Although we have not observed lung mets at 14 weeks, the lungs undergo similar histologic analysis. Endothelial staining is performed as described below.
B. Impact of miR-126 deletion upon survival and metastasis in the
MMTV-PyMT model.
1. Survival analyses. Identical cohorts of virgin female miR-126^*;
MMTV-PyMT (null), miR-126**; MMTV-PyMT (het), and miR-126+l+; MMTV-PyMT (wild-type) (n=20) are observed daily for morbidity or premorbid symptoms requiring sacrifice. Additionally, mice with combined tumor burden of > 3000 mm3 require euthanasia as per Stanford regulations and are sacrificed for the histologic analyses described below. Kaplan-Meier curves are generated for the survival data over a 12-month observation period. With n=20 per group this affords a statistical power of 80% to detect a survival difference with p<0.05 with a hazard ratio of 2.8. Histologic analysis of tumor grade, proliferation and apoptosis is performed as above.
2. Metastatic lung tumor development is documented as (a) absolute incidence and number of macroscopically evident lung nodules and (b) microscope caliper measurement of nodules in inflated lungs of 3 individual H&E sections separated by at least 100 microns. The Mann-Whitney U test is used to compare metastatic burden amongst ko, het and wt. Further, regional lymph nodes are harvested and undergo similar gross/microscopic evaluation.
C. Tumor angiogenesis in miR-126&/A; MMTV-PyMT mice.
The developmental angiogenic phenotypes of miR-126 ko, combined with prominent miR-126 expression in tumor vasculature and our miR-126^*; MMTV-PyMT data, suggest that miR-126 regulates tumor angiogenesis.
1÷ Endothelium and pericytes. The effects of null, heterozygous and wild- type miR-126 status on MMTV-PyMT angiogenesis are systematically analyzed in both mammary primaries and lung metastases by CD31 (endothelial) and PDGFRb/NG2 (pericyte) immunofluorescence on cryosections with determination of microvessel density and endothelial proliferative indices are determined as described above and in our publications.
Z Tip cells. Endothelial cells comprising the invading angiogenic sprout exhibit significant molecular heterogeneity. Receptors such as DII4 and VEGFR3 are uniquely expressed by endothelial "tip cells" at the leading edge, but not the trailing "stalk cells", with counterreceptors/ligands such as Notch 1 and Jagged are expressed in a paracrine manner by the adjacent stalk cells and surrounding non-endothelial tissues. Neonatal m/R-t26Δ/Δ mice exhibit extremely abnormal tip cells with thick endothelial sprouts and a highly characteristic multinucleate
morphology. Possible alterations in tip/stalk markers in miR-126^A tumor vasculature are analyzed by CD31 and nuclear DAPI staining (see above) in combination with: (1) Filopodial number and length assessed by isolectin B4 and phalloidin staining. (2) Anti-VEGFR3 (Santa Cruz) and anti-DII4 immunofluorescence on tip cells (R&D Systems). (3) Stalk cell markers (Notch 1 , Jagged-1 immunofluorescence) and (5) tip and stalk cell proliferation (BrdU) is quantitated. The Zeiss LSM 510 and Leica TCS SP2 AOBS confocal microscopes, such as those in the Stanford Cell Imaging Core, are utilized for this analysis, specifically searching for multinucleate, broadened endothelial sprouts with atretic filopodia. If specifically associated with the null genotype these sprouts will be compared with retinas of non-tumor bearing miR-126AIA mice. ^ Vascular integrity. A second developmental phenotype of miR-12^
mice is impaired vascular integrity, with disruption of the circumferential endothelial wall lining, loss of homotypic endothelial adhesion and resultant hemorrhage (10-20% penetrance) and/or edema (50% penetrance). Evidence for similar findings in the miR-126 ko tumor vasculature is sought, by dextran-FITC (MW ~70 kDa) injection although our ability to detect such changes may be compromised by intrinsic leakiness of the tumor vasculature without miR-126 deletion. EM is performed as a most specific measure to assess the possible presence of cytoplasmic loss, impairment of homotypic endothelial contacts or other ultrastructural defects specific to endothelial miR-126 deletion tumor vasculature. ÷ Analysis of miR-126 deletion and miR-126 target gene/pathway
analysis. RNA from mammary primaries and lung mets are collected for confirmation of miR-126 deletion by qPCR with normalization to b- actin. Although not feasible for all cases, in isolated tumors, CD31 + tumor endothelium is isolated by liberase digestion and CD31 magnetic bead selection as in our published miR-126 ko work or CD31+ FACS sorting, followed by Western blotting to determine appropriate
upregulation of the miR-126 targets p85b and Spredi , in ko tumor EC as we previously described for miR-126^* EC. The potential stimulation of the Erk and Akt pathways by miR-126 overexpression is evaluated in cultured tumor endothelial cells by immunofluorescence with P-Erk or P- Akt antibodies (Cell Signaling) as described above or in VEGF- stimulated isolated CD31+ tumor endothelium by analogy to isolated miR-12^ endothelium.
Methodology 2. Evaluation of miR-126 inhibition as a therapeutic approach for breast cancer
Hypothesis: Treatment of pre-established tumors by either conditional genetic deletion of miR-126 or by pharmacologic administration of miR-126 anti-mirs will impair mammary tumorigenesis.
Rationale: There are currently no FDA-approved therapeutic agents targeting miRNAs for any indication, including cancer or tumor angiogenesis. This methodology validates miR-126 as a therapeutic target for breast cancer using treatment of pre- established mammary tumors, simulating the clinical setting. Complementary
approaches of conditional genetic deletion and pharmacologic inhibition are evaluated in pre-established tumors whereby the genetic approach of inducible deletion of the miR-12&°* allele serves as a reference standard for the pharmacologic approach of systemic treatment with miR- 126 targeting cholesterol-conjugated antagomirs, as well as a novel anti-mir technology with non-cholesterol conjugated siRNA with a 2'F/methoxyester backbone modification. Importantly, miR-126 inhibition by either genetic or pharmacologic approaches is compared with and combined with the "gold standard" of VEGF inhibition to benchmark the efficacy of miR-126 antagonism, as well as to explore miR-126 inhibition in the context of combinatorial anti-angiogenic therapy.
A. Temporally conditional deletion in miR-12^ox/nox; MMTV-PyMT;
R26-M2rtTA; TRE-Cre mice.
Our data and the studies in methodology 1 utilize constitutive miR-126 deletion in miR-126^*; MMTV-PyMT mice. To allow temporally inducible deletion, the R26- M2rtTA; TRE-Cre system is used with the miR-126?°* allele. Here, R26-M2rtTA allows ubiquitous expression of the reverse tetracycline trans-activator/rtTA ("tet-on"/tet- inducible) from the ubiquitous ROSA R26 locus (JAX B6.Cg Gt(ROSA)26 Sortml (rtTA*M2) Jae/J #006965). The TRE-Cre allele contains tet-operator sites driving Cre expression (JAX Tg(tetO-cre)1 Jaw/J #006224). Treatment of miR-126ftox/flox; MMTV- PyMT; R26-M2rtTA; TRE-Cre mice with tetracycline in the drinking water (1.5 mg/ml tetracycline/5% sucrose) activates rtTA binding to the tet-operator sites and induces ubiquitous tet-regulated Cre expression (i.e. in both endothelial and mammary compartments), leading to temporally conditional, homozygous deletion of the floxed miR-126 alleles (miR-126F0**0*). We have already demonstrated the ability to
conditionally delete the miR-126"°* allele via cross to Tie2-Cre (Fig. 5e).
Here, miR-126nox/flox\ MMTV-PyMT mice are mated to miR-126ϋoxMox ; R26- M2rtTA; TRE-Cre mice, yielding female miR-126nox/flox; MMTV-PyMT; R26-M2rtTA; TRE-Cre mice at 1/16 frequency. In our own experience as well as in published descriptions of this model, females at 12 weeks exhibit a tumor burden in which approximately 50% are progressing to carcinoma, with tumor volume approximately 20% of that at 14 weeks. Thus, 12 week old female miR-126nox/flox; MMTV-PyMT; R26- M2rtTA; TRE-Cre mice are exposed to tetracycline (n=10) or sucrose alone (n=10) for 7 days to generate miR-126 ko or wt mice, respectively. The conditional miR-126 ko or wt mice (i.e. +/- tetracycline) are allowed to progress for an additional 4 weeks (i.e. until 16 weeks of age), at which the animals are sacrificed for endpoint analysis (tumor size, grade, histology, angiogenesis) as in methodology 1A.
Decreases in these endpoints upon conditional miR-126 ko strongly support the therapeutic potential of miR-126 inhibition for pre-established tumors using a genetic ko "gold standard" approach.
B. Pharmacologic antagomir and antimir inhibition of miR-126 in the MMTV-PyMT model.
In parallel, pharmacologic inhibition of miR-126 is evaluated using the same experimental framework as the conditional genetic ko. Traditionally, pharmacologic inhibition of miRNA in vivo has used "antagomirs" which are single-stranded RNA analogues that are complementary to the target miRNA, typically with 2'-0Me backbone modification throughout, phosphorothioate backbone with or without a cholesterol moiety at the 3'-end. l.v. injection of antagomirs using regimens from 3 consecutive daily doses to 2x/week dosing elicits an impressive, durable (>21d) highly selective in vivo silencing of target miRNA in all tissues examined. Silencing of miR-16, miR-122, miR-192 and miR-194 has been obtained in vivo, with phenotypic correction of hyperlipidemia with miR-122 antagomirs via hepatic cholesterol metabolism 136263. The first use of antagomirs in cancer was reported in 2010, where miR-10b antagomirs inhibited EMT and pulmonary metastasis from 4T1 human breast cancer cells implanted orthotopically in mammary fat pad. For angiogenesis, antagomirs against have been successfully used to stimulate (miR-92a) or inhibit (miR-126) post-ischemic
angiogenesis; only fragmentary data exists for inhibit tumor angiogenesis (miR-296), where no data on tumor size, progression or survival were presented.
.1 Synthesis of miR-126 antagomirs and antimirs : Antagomirs and
antimirs are used for evaluation in the MMTV-PyMT model, allowing for the validation of promising new antimir oligonucleotide chemistry. antimir). Our well validated miR-126-3p hairpin inhibitor is transfected in parallel; this decreases miR-126 by 90% as well as upregulates p85b but is not usable in vivo.
Z Antapomir and antimir treatment and endpoint analysis. Paralleling our conditional genetic ko (miR-126F0X/fl0X; MMTV-PyMT; R26-M2rtTA; TRE-Cre, Aim 2A above), 12 week MMTV-PyMT females (i.e. miR-126 wt) will receive antagom/f?-726-3p (n=6) or the generic control antagomir (n=6) at 12.5, 25 and 50 mg/kg, twice weekly, by i.v. tail vein injection. All groups will be harvested at 16 weeks (i.e. after 4 weeks of treatment). Endpoint analysis of tumor size, histology and vascular content will be performed identically to methodology 1 and 2A. FACS or magnetic bead isolation of CD31+ tumor endothelium is performed as previously with Western to assess EC upregulation of the miR- 126 target p85b (antagomir, antimir) and qPCR (repression of mature m/R-726)(antagomir).
C. Comparison and combination of miR-126 deletion with VEGF inhibition
The clinical successes of VEGF inhibition have been tempered by modest survival advantages and relentlessly progressive disease, highlighting the need for novel anti-angiogenic targets potentially useful in singly or in combination. A priori, the broad action of miRNAs on hundreds if not thousands of targets, with their concomitant fine-tuning of the proteome, suggests that targeting angiogenesis regulatory miRNAs, of which miR-126 is the founding member, could be useful in combination with VEGF inhibition. Additionally, the impairment of VEGFR/Akt/Erk signaling upon in vitro miR- 126 knockdown suggests that miR-126 inhibition could sensitize tumor endothelium to pharmacologic VEGF inhibition in vivo for purposes of combinatorial anti-angiogenic therapy, with this likely being but a small subset of mechanisms by which the
pleiotropically acting miR-126 influences tumor angiogenesis. Thus, miR-126 genetic ko or pharmacologic inhibition will be combined with VEGF inhibition in the MMTV- PyMT model. Further, careful comparison of phenotypes of miR-126 gene deletion versus VEGF inhibition would yield mechanistic insight into rational combination in the future.
1. In vitro modeling. Initially, cultured wild-type versus m/7?-726Δ/Δ; primary endothelium from MMTV PyMT tumors will be subjected to culture with or without decreasing concentrations of VEGF (10, 3, 1 , 0.3, 0.1 ng/ml) in EGM-2 medium without growth factor supplementation. Proliferation is determined by MTT assay over a 5 day period to assess if miR-126 deletion is synergistic with decreasing VEGF concentrations in vitro. Conversely, enhanced apoptotic response (accelerated kinetics, increased magnitude) to abrupt VEGF starvation (medium change from 10 ng/ml to 0 ng/ml VEGF) in miR-126^* vs. wt EC will be assessed by TUNEL/cleaved caspase-3 Western.
2. In vivo modeling. VEGF inhibition is combined with either constitutive deletion of miR-126 in miR-126^; MMTV-PyMT mice (ko - Cohort "A", n=20) or control littermate miR-126+/+; MMTV-PyMT mice (wt - Cohort "B", n=20 ); see methodology 1 for breeding schema. Our well-validated adenoviruses expressing soluble VEGFR1 or VEGFR2 ectodomains, which neutralize and sequester VEGF is used; single i.v.
injection of these Ad results in high-efficiency liver transduction, and hepatic secretion of soluble VEGFR ectodomains into the blood for ~4 weeks and >98% decrease of angiogenesis in cornea, corpus luteum, and potent repression of tumor
growth/angiogenesis in numerous models.
At 12 weeks of age, tumor-bearing animals from Cohort A (miR-126 ko) are randomly assigned to two groups of n=10 each as determined by caliper measurements of external tumor burden. At randomization, animals receive either single i.v. injection of 109 pfu Ad FIkI-Fc (soluble VEGFR2 ectodomain) (group A1), or the control virus Ad Fc, (secreted murine lgG2a Fc antibody fragment) (group A2). We have previously used such a tumor burden-based randomization protocol to successfully demonstrate a significant treatment effect of Ad FIkI-Fc in the TRAMP transgenic mouse model of prostate cancer, in which the prostate-specific probasin promoter drives expression of T antigen. Tumor-bearing littermate controls from Cohort B (wild-type miR-126) are similarly randomized by tumor burden to two groups (n=10) receiving Ad FIkI-Fc (VEGF inhibition, group B1) or Ad Fc (control, group B2). Together, this 2x2 design with Cohorts A and B generate MMTV-PyMT experimental groups with miR-126 deletion, VEGF inhibition, both miR-126 deletion and VEGF inhibition, or neither.
Equivalent experimental design are used to combine miR-126 antagomir or antimirs (whichever proves optimal in methodology 2B) with VEGF inhibition/Ad FIkI-Fc.
3. Endpoint analysis. Tumor burden in groups A1/A2 and B1/B2 are monitored for 4 weeks (i.e. from weeks 12-16) after adenoviral treatment with Ad FIkI-Fc (VEGF inhibition) of Ad Fc (control) with generation of individual and aggregate tumor growth curves for each group. The blinded histologic analysis of this experiment follows the form of the previous submethod A. Analysis of tumor angiogenesis includes
determination of microvessel density, endothelial tip cells, vascular integrity, and miR- 126-specific endpoints of loss of miR-126 RNA and upregulation of miR-126 targets (p85b, Spredi). Emphasis is paid to the differences in tumor progression and
angiogenesis phenotypes of miR-126 deletion vs. VEGF inhibition and the combination As an alternative to adenoviral inhibition of VEGF using soluble receptor ectodomains as we have published in numerous journals (c.f. PNAS, Circulation, Nature Medicine) we have also extensively used pharmacologic VEGF inhibitors such as VEGF Trap or DC101 which available in our lab. However, this would lose the convenience and efficacy of single injection adenovirus, combined with durable PK from continuous adenoviral transgene secretion. Our experience with the PyMT model suggests that the ~4 week duration of adenoviral expression matches well with an increase in tumor burden from 12 to 16 weeks, which allows determination of therapeutic effects. Other transgenic models may also be used. S.c. or orthotopic fat pad tumor models are possible after appropriate backcrossing but less physiologic. Sub-maximal Ad FIkI-Fc doses (i.e. 108 pfu) may be used to study miR-126 deletion in combination with partial VEGF inhibition. Constitutive miR-126 ko is utilized for the VEGF inhibitor combinations - but a miR-126 "intervention model" is certainly possible with temporally conditional deletion at 12 weeks (i.e. pre-established tumor burden) if feasibility is established in methodology 2B (miR-126fox/βox; MMTV-PyMT; R26-M2rtTA; TRE-Cre). As an alterative to this aforementioned R26-M2rtTA; TRE-Cre system, ROSA CreERT2 could be used the miR-126 ko would require tamoxifen which could have confounding effects on MMTV-PyMT growth even though there is progressive (but not absolute) estrogen receptor loss.
Methodology 3. Mechanistic investigation of miR-126 action during tumor progression
Hypothesis: miR-126 is both necessary and sufficient to regulate tumor angiogenesis via cell-autonomous action in endothelial cells and repression of downstream targets.
Rationale: The exclusively vascular expression of miR-126 in embryos and tumors, the angiogenesis phenotypes of miR-126 ko mice, the effects of miR-126 knockout/knockdown on endothelial RTK signaling and migration, and the reduced microvessel density in miR-126^^; MMTV-PyMT mice are all consistent with a
endothelial cell-autonomous mechanism. On the other hand, miR-126 appears expressed in tumor parenchyma with potential functional consequences, these being largely deduced from overexpression studies in cell lines. Here, endothelial versus mammary gland-specific miR-126 deletion will be performed in MMTV-PyMT mice (loss- of-function approach), and a converse gain-of-function approach will examine whether miR-126 overexpression is sufficient to enhance endothelial function upon restoration to miR-126&/* endothelium. Finally, additional miR-126 target genes are sought using a mass spectroscopy-based proteomics approach to generate additional hypotheses for miR-126 action.
A. Endothelial cell-autonomy in miR-126nox/ti\ MMTV-PyMT; PDGFB- iCreER mice. (1). Effects of mJR-126 deletion in endothelium and other compartments on MMTV-PyMT tumor anqiogenesis. Endothelial deletion is performed using the PDGFB- iCreER line (gift of Marcus Fruttiger). miR-126^* vascular phenotypes are phenocopied by miR-126"°m; Tie2-Cre mice. While Tie2-Cre has long been the standard for achieving EC deletion, many labs including our own have recently described that Tie2- Cre deletes in both the EC and pericyte lineages. Thus, we and others are now utilizing PDGFB-iCreER in which CreER is driven by the endothelial-expressed PDGFB promoter, allowing taxmoxifen-regulated deletion of floxed target genes. We have extensive experience with this line which is in our colony, and have confirmed
tamoxifen-dependent deletion in endothelium not pericytes using a floxed YFP reporter, and have shown that a GPCR null allele in our lab can be phenocopied by PDGFB- iCreER mediated deletion of the equivalent floxed allele.
Here, miR-126?*; MMTV-PyMT mice will be crossed to miR-126Pox//+; PDGFB- iCreER mice, yielding the desired female miR-12€pox/&; MMTV-PyMT; PDGFB-iCreER (i.e. endothelial ko) mice and control littermate miR-126+/+; MMTV-PyMT; PDGFB- iCreER. Maternal treatment with 1.0 mg tamoxifen i.p. at E8.5 allows full embryonic miR-126 deletion in endothelial ko but not wild-type mice; this small dose is well documented not to incur embryonic lethality and in our own GPCR studies affords extremely efficient endothelial (>90%) but not pericyte deletion. This embryonic EC miR-126 ko likely produces partial embryonic lethality as seen with miR-126ά/&L and miR- 126nox/h; Tie2-Cre embryos thus confirming deletion efficacy; miR-126 qPCR on FACS isolated EC will be performed in parallel. Cohorts of surviving female miR-126fs/*\ MMTV-PyMT; PDGFB-iCreER mice (endothelial ko) (n=6) and control wt littermates (n=6) will be sacrificed at 14 weeks for analysis of tumor progression and tumor angiogenesis identically to methodology 1 as with miR-126έi/h\ MMTV-PyMT mice. The only tamoxifen exposure is during embryogenesis and both endothelial ko and wt embryos are equally exposed; thus there are not confounding effects on adult mammary tumorigenesis. Differences in tumor size, grade, ER/PR/Neu expression, metastases, or endothelial microvessel density, proliferation, vascular integrity or tip cell markers would strongly support miR-126 endothelial cell-autonomous function during MMTV- PyMT tumor progression. CD31+ EC will be isolated by FACS to confirm miR-126 deletion (qPCR) and p85b upregulation by Western blot.
If endothelial miR-126 deletion does not impair MMTV-PyMT tumorigenesis, then miR-126 is deleted in the tumor parenchymal compartment using MMTV-Cre (JAX Tg(MMTV-cre)4Mam/J #003553) in place of PDGFB-iCreER.
B. Evaluation of miR-126 sufficiency in primary miR-126&/& tumor endothelium.
In parallel to our endothelial-specific loss-of-f unction studies, a reciprocal gain-of- function approach examines if miR-126 overexpression is sufficient to enhance tumor endothelial function. miR-126 knockdown or knockout in cultured endothelium markedly attenuates VEGF stimulation of Akt, ERK and migration. Therefore the converse scenario of miR-126 overexpression could sensitize tumor vasculature to VEGF1 allowing endothelium to respond to lower VEGF concentrations and to lower degrees of VEGFR kinase activity. Certainly, tumor vascular miR-126 overexpression could even facilitate clinical resistance to VEGF inhibitors via VEGF sensitization. Supporting this, Klf2a activation of miR-126 expression in zebrafish enhances VEGF signaling and miR- 126 overexpression in cultured EC enhances FGF signaling.
Here, miR-126iJti tumor endothelium is isolated as described, followed by overnight infection with increasing m.o.i. (0-100) of adenovirus miR-126 (already generated in our laboratory) as we have performed with adenovirus p85p26. The miR- 126^* tumor endothelium +/- Ad miR-126 infection at different m.o.i. is cultured in EGM- 2 with VEGF1 starved overnight without VEGF and then stimulated with VEGF at 0, 0.1 , 0.3, 1 , 3 and 10 ng/ml. Endpoints include migration, proliferation over 5 day culture as well as more acute measures (30 min) of VEGF/FGF-2-induced phosphorylation of Akt and Erk upon Western blot which we already have shown to be impaired upon miR-126 deletion. Conversely, overexpressing vs. wt endothelium is placed in EGM-2 starvation medium without VEGF and temporal kinetics of apoptosis evaluated. Either enhanced VEGF signaling or proliferation upon miR-126 overexpression or decreased apoptosis following VEGF deprivation indicates that miR-126 is sufficient to increase angiogenic properties in tumor endothelium, complementing the necessity/loss-of-function studies in methodology 3A above.
For miR-126 endothelial function, the present inventors have extensive experience with PDGFB-iCreER, which for us yields extremely penetrant endothelial- specific deletion. Certainly, Tie2-Cre is an option that we already have used to phenocopy the complete miR-126^* null allele. While deleting in both endothelium and a subset of pericytes, Tie2-Cre still allows rigorous demonstration of miR-126 function in the vascular compartment as opposed to the tumor parenchymal compartment. VE- Cadherin-CreERT2 may also be used. If the miR-126^*; MMTV-PyMT tumor
progression phenotype is not phenocopied by endothelial deletion, we will vigorously pursue deletion in the tumor parenchyma with MMTV-Cre. It is also conceivable that miR-126 functions in both compartments, as globally inhibited by miR-126 antagomirs or antimirs. In studying if miR-126 overexpression is sufficient to induce VEGF hyperresponsiveness and resistance to VEGF starvation in vitro, an in vivo correlate may be contemplated to use our tet-inducible TRE-miR-126 transgenics with VE- Cadherin-tTA and MMTV-PyMT to test if miR-126 overexpression confers mammary tumor resistance to VEGF inhibition, i.e. with Ad Flk1-Fc/sVEGFR2.
Thus, the present invention broadly contemplates both the study and the treatment of mammalian cancer, particularly breast cancer. In studying mammary tumors and the progression thereof, any known animal model of cancer may be used However, generally transgenic mouse models of cancer are preferred. For example, in addition to the mouse models mentioned above, any of the mouse models, such as those described in U.S. 6,639,121 , which is incorporated herein in the entirety. Also, LSL K-Ras may be specifically mentioned.
Further, the present invention may be used in the treatment of all cancers as miR-126 appears likely to be important for all vasculature, and thus tumor progression, in all parts of the mammalian body. It is understood that as used herein, the term "mammal" broadly includes mice, cats, dogs, cows, sheep and horse, for example, but particularly humans.
Generally, the present invention provides an evaluation of tumor progression (or regression) and a treatment of tumors by pharmacologically inhibiting miR-126. The design principles or guidelines for pharmacological inhibition of miR-126 are:
1) Traditional Approach: involves the use of "antagomirs" which are single- Stranded RNA analogues that are complimentary to the target miRNA, typically with 2'- OMe backbone modification throughout, phosphorothioate backbone with or without a cholesterol moiety at the 3'-end;
2) Newer Approach: involves the use of "antimirs" which are single-stranded analogues that are complimentary to the target miRNA, with completely modified phosphorothioate backbones further modified with combinations of 2'-fluoro and 2'-O- methoxyethyl bases; and
3) Other Permutations: involve "hairpin inhibitors" with double-stranded regions At the 5'- and/or 3'- ends, or incorporation into nano-carriers, such as carbon
nanotubes.
Homo sapiens miR-126 stem loop sequence is well-known and may be used in a routine manner to obtain antagonists to miR-126 in the treatment of human cancers.
Furthermore, in designing antimirs for miR-126, guidance may be obtained therefor from any of US 7,307,067 and SNs 11/141 ,407 and 11/273,640, which are each incorporated herein in the entirety. For antagonists, guidance may also be obtained from US pat. publ. 2010/011356, which is incorporated herein in the entirety. Of particular note for purposes of the present invention are nucleic acid antagonists against miR-126. Administration of nucleic acid antagonists against miR-126 to any mammal, particularly humans, in accordance with any of the incorporated documents above, as well as with US pat. publ. 2006/0058266, the latter of which is also incorporated herein by reference in the entirety.
Generally, the nucleic acid antagonists against miR-126 contain the core sequence:
5'-GCATTATTACTCACGGTACGA-S'
However, it is also acceptable to include modified core sequences that contain at least 50% of the above sequence in the same sequential order, and preferably at least 75% of the above sequence in the same sequential order with the sequence starting at either end of the sequence or in the interior of the sequence. Additionally, it is also acceptable to use scrambled versions of the above core sequence, where no more than 50%, and preferably no more than 25%, of the core sequence above is scrambled, i.e., interchanged with other residues of the core sequence. The term
"scrambled" as used herein means that the total nucleic acid content of the scrambled sequence remains the same as the core sequence shown above, but that the exact sequence differs due to an interchange of the nucleic acid residues in the scrambled sequence as compared to the sequence shown above.
Additionally, the nucleic acid antagonists of the present invention may be from 10-50 nucleic acid units including a portion of the core sequence shown above or one of its variations as described above. However, it is preferred if the antagonist is from 20-30 nucleic acid residues in length with the above considerations for sequence and content in mind. These nucleic acid antagonists are administered as a pharmaceutical composition with a suitable carrier described below. Generally, the nucleic acid antagonists against miR-126 are administered in saline, dextrose-5%-saline and/or aqueous solution by intravenous-, subcutaneous- or intraperitoneal injection. These antagonists are administered from 1 to about 100 mg/kg of body weight, and from daily to once every four weeks.
The metric used to determine the success of the treatment may include prolongation of overall survival time or time to progression. Time to progression may be measured by radiologic scans, such as MRI, CT, PET or x-rays; by physical palpitation of tumors and/or by histologic analyses.
Having described the present invention, it will be clear that changes and modifications may be made to the above-described embodiments without departing from the spirit and scope of the same.

Claims

WHAT IS CLAIMED IS:
1. A pharmaceutical composition, comprising at least one nucleic acid antagonist against miR-126, and a pharmaceutically-acceptable carrier.
2. The pharmaceutical composition of claim 1 , wherein the at least one nucleic acid antagonist against miR-126 contains from 10 to 50 nucleic acid units and comprises a sequence that contains all or a portion of the sequence (1):
5'-GC ATTATTACTCACGGTACGA-3' .
3. The pharmaceutical composition of claim 2, wherein the at least one nucleic acid antagonist comprises at least 50% of the identical sequence of sequence (1).
4. The pharmaceutical composition of claim 3, wherein the at least one nucleic acid antagonist comprises at least 75% of the identical sequence of sequence (1).
5. The pharmaceutical composition of claim 1 , which further comprises a vascular targeting moiety, which is the peptide, RGD.
6. The pharmaceutical composition of claim 5, which further comprises a single-walled carbon nanotube having a cargo density of at least about 50 moles of vascular targeting groups and antagonist against miR-126 per mole of single-walled carbon nanotubes.
7. The pharmaceutical composition of claim 6, wherein the cargo density is about 50- 300 moles per mole of single-walled carbon nanotubes.
8. The pharmaceutical composition of claim 5, wherein the vascular targeting moiety is conjugated to a nano- carrier.
9. The pharmaceutical composition of claim 8, which further comprises a moiety which targets tumor vasculature, which moiety is a chemotherapeutic compound.
10. The pharmaceutical composition of claim 1 , which is a dry composition.
11. The pharmaceutical composition of claim 1 , which is a liquid composition suitable for injection.
12. The pharmaceutical composition of claim 1 , wherein the nano-carrier comprises a polymer or copolymer.
13. The pharmaceutical composition of claim 1 , wherein the nano-carrier comprises boron nitride.
14. The pharmaceutical composition of claim 1 , wherein the nano-carrier comprises a graphitic surface.
15. The pharmaceutical composition of claim 12, wherein the nano-carrier comprises a polymer, which is a cyclodextrin-based polymer.
16. The pharmaceutical composition of claim 1 , wherein the nano-carrier comprises a graphene sheet.
17. The pharmaceutical composition of claim 1 , wherein the nano-carrier comprises a nanocrystal of graphitic-coated metal core.
18. The pharmaceutical composition of claim 1 , wherein the nano-carier comprises a nanocrystal which comprises FeCo or Au.
19. The pharmaceutical composition of claim 1 , wherein the miR-126 antagonist is a 21- O-methyl modified siRNA.
20. The pharmaceutical composition of claim 19, wherein the 2'-O-methyl modified siRNA has at least one flanking region with a total length of at least 10nt.
21. A pharmaceutical mixture, comprising the composition of claim 1 , and a small molecule VEGF antagonist.
22. A method for effecting anti-angiogenic cancer therapy, which comprises the step of administering an amount of the composition of claim 1 , to a patient having cancer sufficient to inhibit angiogenesis of said cancer.
23. The method of claim 22, wherein said composition is administered intravenously to said patient.
24. The method of claim 22, wherein said composition is administered in an amount of at least about 10 mg/kg of body weight.
25. The method of claim 24, wherein said composition is administered in an amount of at about 20-100 mg/kg of body weight.
26. The method of claim 25, wherein said composition is administered once daily for at least three days.
27. The method of claim 22, which further comprises administering cis-platin to the patient before, during or after the administration of the composition.
28. The method of claim 22, wherein said cancer is breast cancer.
29. The method of claim 28, wherein said breast cancer is metastatic
30. A method of manipulating angiogenesis in mammalian endothelial cells, which comprises introducing an miR-126 silencer into said cells.
31. The method of claim 30, wherein the mammalian cells are in vivo cells.
32. The method of claim 30, wherein the mammalian cells are in vitro cells.
33. The method of claim 30, wherein the silencer is an RNA hairpin inhibitor or other nucleic acid-based inhibitor of miR-126.
34. A method of regulating lymphanogiogenesis in mammalian lymphatic endothelial cells, which comprises delivery of a miR-126 silencer in the form an RNA hairpin inhibitor or other nucleic acid-based inhibitor of miR-126.
35. The method of claim 34, wherein the mammalian cells are in vitro cells.
36. The method of claim 34, wherein the mammalian cells are in vivo cells.
37. The method of claim 34, wherein the silencer is an RNA hairpin inhibitor or other nucleic acid-based inhibitor or miR-126.
38. A method of inhibiting expression of one or more VEGF pathway genes in a mammal, which comprises the step of administering to a mammal having cancer an amount of the composition of claim 1 , effective to inhibit angiogenesis of said cancer.
39. The method of claim 38, wherein the mammal is a human.
40. A method of treating cancer in a mammal having the same, which comprises the step of administering to a mammal having cancer an amount of the composition of claim 1 , effective to inhibit said leukemia.
41. The method of claim 40, wherein said mammal is a human.
42. The method of claim 40, wherein the cancer is leukemia.
43. The method of claim 40, wherein the cancer is breast cancer.
44. A method of treating breast cancer in a mammal, which comprises the step of administering one or more antagomirs and/or antimirs of miR-126 to the mammal.
45. The method of claim 44, wherein the mammal is a human.
46. A method of evaluating tumor progression in a mammal, which comprises the steps of:
a) silencing all or a portion of epithelial miR-126 of said mammal, and
b) detecting an effect of said silencing on said tumor progression.
47. The method of claim 46, wherein said tumor is a mammary tumor.
PCT/US2010/002045 2009-07-21 2010-07-21 Method of regulating angiogenesis and lymphangiogenesis, and a pharmaceutical composition for effecting anti-angiogenic and anti-lymphangiogenic cancer therapy WO2011011061A2 (en)

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