WO2016094457A1

WO2016094457A1 - Methods for treating basal-like and claudin-low breast cancer and combination therapies thereof

Info

Publication number: WO2016094457A1
Application number: PCT/US2015/064573
Authority: WO
Inventors: Partha S. RAY
Original assignee: Ray Partha S
Priority date: 2014-12-08
Filing date: 2015-12-08
Publication date: 2016-06-16

Abstract

Provided herein are methods for treating breast cancer in a subject. In certain embodiments, the methods may include determining an expression level of FOXC1 in a population of tumor cells obtained from the subject and administering one or more proteasome inhibitors, and optionally, one or more Wnt inhibitors, to the subject to treat the breast cancer if the population of tumor cells expresses FOXC1. In certain embodiments, the breast cancer may be a basal-like breast cancer. In certain embodiments, the proteasome inhibitor may be bortezomib. Combination therapies for treating breast cancer comprising one or more Wnt inhibitors and one or more proteasome inhibitors are also provided herein.

Description

METHODS FOR TREATING BASAL-LIKE AND CLAUDIN-LOW BREAST CANCER

AND COMBINATION THERAPIES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the priority date of United States Provisional Application No. 62/089,214, filed December 8, 2014, and United States Provisional Application No. 62/090,323, filed December 10, 2014. The contents of each of these referenced applications are incorporated herein by reference in their entirety, as if fully set forth herein.

BACKGROUND

[0002] Cancer stem cells (CSCs) are considered to be an important contributing factor towards treatment failure, cancer recurrence, and mortality. CSCs are known to be more enriched in the basal-like and claudin-low subtypes of breast cancer. There is a need to develop new approaches to effectively target and treat basal-like and claudin- low subtypes of breast cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figure 1 illustrates the regulation of CSCs.

[0004] Figure 2 illustrates the parallel between mammary epithelium differentiation and breast cancer subtypes.

[0005] Figure 3 shows a proposed mechanism of FOXC1 regulation by Wnt/β catenin signaling.

[0006] Figure 4 shows FOXC1 as a downstream mediator of Wnt-driven regulation of CSCs in basal-like breast cancer.

[0007] Figure 5 shows an analysis of beta-catenin (CTNNB1 ) expression level and survival using a breast cancer transcriptomic database (Curtis et al., 2012).

[0008] Figure 6 shows the correlation between FOXC1 expression and Wnt activity upon biological and pharmacologic inhibition and stimulation with a natural ligand. Figure 6A shows FOXC1 expression upon biological inhibition with siRNA knockdown of LRP6, a canonical Wnt signaling cell surface receptor. Figure 6B shows the relative FOXC1 mRNA expression with the siRNA knockdown of LRP6. Figure 6C shows decreased FOXC1 expression upon pharmacological inhibition with iCRT-3. Figure 6D shows an increase in FOXC1 expression with addition of Wnt3a, a canonical Wnt signaling ligand.

[0009] Figure 7 shows the relative survival of representative breast cancer cell lines upon inhibition of Wnt/β catenin signaling with increasing doses of the inhibitor iCRT3. Figure 7A shows the relative survival of the MCF-7 (luminal) breast cancer cell line (see line with diamonds); the SKBR3 breast cancer cell line (overexpresses HER2) (see line with triangles); and the BT-549 breast cancer cell line (basal-like) (see line with squares). Figure 7B shows the relative survival of the BT-549 breast cancer cell line (high constitutive FOXC1 expression) (see line with squares); the HS578T breast cancer cell line (high constitutive FOXC1 expression) (see line with Xs); and the MDA- MB-231 breast cancer cell line (low constitutive FOXC1 expression) (see line with circles).

[0010] Figure 8 shows the relative survival of representative breast cancer cell lines upon proteasome inhibition with increasing doses of the inhibitor bortezomib. Figure 8A shows the relative survival of the MCF-7 (luminal) breast cancer cell line (see line with diamonds); the HS578T breast cancer cell line (basal-like) (see line with squares); and the SKBR3 breast cancer cell line (overexpresses HER2) (see line with triangles). Figure 8B shows the relative survival of the HS578T breast cancer cell line (basal-like) (bottom line); the BT-549 breast cancer cell line (basal-like) (see middle line); and the MDA-MB-231 breast cancer cell line (low constitutive FOXC1 expression) (see top line).

[0011] Figure 9 shows mammosphere formation upon inhibition of Wnt/β catenin signaling by iCRT-3. Figure 9A shows mammosphere formation with addition of DMSO and Figure 9B shows mammosphere formation with the addition of iCRT-3.

[0012] Figure 10 is a bar graph showing the fold change of mRNA expression of the stem related genes BMI-1 , FOXC1 , KIT, NANOG, OCT4, and SOX2 upon inhibition of Wnt/β catenin signaling by addition of iCRT-3 on day 4 (left hand bar) and day 7 (right hand bar).

[0013] Figure 1 1 shows mammosphere formation upon simultaneous inhibition of Wnt/β catenin signaling by addition of iCRT-3 and inhibition of NF-κΒ signaling. Figure 1 1A shows mammosphere formation with addition of DMSO and Figure 1 1 B shows mammosphere formation with the addition of iCRT-3 and bortezomib.

[0014] Figure 12 shows the Human FOXC1 Amino Acid Sequence (SEQ ID NO: 1 ).

SUMMARY

[0015] Provided herein are methods for treating cancer in a subject. In certain embodiments, the methods may comprise determining an expression level of FOXC1 in a population of tumor cells obtained from the subject and administering one or more proteasome inhibitors, and optionally, one or more Wnt inhibitors, to the subject to treat the cancer if the population of tumor cells expresses FOXC1 . In certain embodiments, the proteasome inhibitor may be bortezomib. In certain embodiments, the one or more Wnt inhibitors may be iCRT3. In certain embodiments, the cancer may be a basal-like breast cancer. In certain embodiments, the basal-like breast cancer may be triple negative breast cancer. In certain embodiments, the basal-like breast cancer may be ER positive breast cancer. In certain embodiments, the basal-like breast cancer may be HER2 positive breast cancer. In certain embodiments, the cancer may be claudin-low cancer. In certain embodiments, determining the expression level of FOXC1 in the population of tumor cells may be performed via quantitative RT-PCR (qRT-PCR).

[0016] Also provided herein are combination therapies comprising one or more Wnt inhibitors and one or more proteasome inhibitors. In certain embodiments, the one or more proteasome inhibitors may be bortezomib. In certain embodiments, the one or more Wnt inhibitors may be iCRT3. In certain embodiments, the one or more Wnt inhibitors may be a therapeutically effective amount. In certain embodiments, the one or more proteasome inhibitors may be a therapeutically effective amount. [0017] Also provided herein are methods for treating cancer in a subject comprising administering one or more proteasome inhibitors, and optionally, one or more Wnt inhibitors, to the subject to treat the cancer. In certain embodiments, the one or more proteasome inhibitors may be bortezomib. In certain embodiments, the one or more Wnt inhibitors may be iCRT3. In certain embodiments, the cancer may be a basal-like breast cancer. In certain embodiments, the basal-like breast cancer may be triple negative breast cancer. In certain embodiments, the basal-like breast cancer may be ER positive breast cancer. In certain embodiments, the basal-like breast cancer may be HER2 positive breast cancer. In certain embodiments, the cancer may be claudin- low cancer.

DETAILED DESCRIPTION

[0018] The following description provides specific details for a thorough understanding of, and enabling description for, embodiments of the disclosure. However, one skilled in the art will understand that the disclosure may be practiced without these details. In other instances, well-known structures and functions have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments of the disclosure.

[0019] The Wnt/β catenin signaling pathway is well known as a regulator of embryonic development and stem cell biology, and is prominently active in basal-like and claudin-low breast cancers. It was previously reported that transcription factor FOXC1 plays a role in mediating aggressive cell traits in basal-like/claudin low breast cancer (see Ray et al., 2010). As shown in Example 1 below, FOXC1 is a target of Wnt/β catenin signaling and FOXC1 expression predicts response to Wnt inhibition. Further, Wnt mediated regulation of FOXC1 appears to be critical for cancer stem cells (CSCs) in basal-like/claudin low breast cancer.

[0020] However, Wnt inhibitors as a single agent may not work well in treating basal-like/claudin low breast cancer. As such, treatment cocktails targeting multiple pathways governing CSCs need to be considered for rational design of effective targeted therapy against basal-like/claudin low breast cancer. As discussed herein, bortezomib, a proteasome inhibitor that inhibits nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κΒ), effectively overcomes cancer stem cell escape triggered by Wnt inhibitor therapy in Forkhead box C1 (FOXC1 ) positive basal- like/claudin-low breast cancer. This makes proteasome inhibitors such as bortezomib a candidate drug to treat all types of basal-like/claudin-low breast cancers. Bortezomib has previously been used in a clinical trial to treat triple-negative patients; however, this trial yielded inconclusive results because it was not possible to accurately select basal- like patients. Thus, the patients in the trial were composed of a mix of basal-like positive and basal-like negative patients. FOXC1 is a marker for basal-like breast cancer, which affects ~70% of triple negative breast cancer. Further, basal-like breast cancer includes more than just triple-negative breast cancer as it is present in up to 30% ER+ type cancers and in >15% HER2+ type cancers. Screening of patients using FOXC1 as a marker will help to more efficiently select patients with basal-like positive cancers for treatment with bortezomib.

[0021] Forkhead box transcription factors, including FOXC1 , also known as forkhead-like 7 (FKHL7)), are transcription factors characterized by a common 100- amino acid winged-helix DNA-binding domain termed the forkhead box domain, and play important roles in regulating the expression of genes involved in cell growth, survival, differentiation embryonic mesoderm development, migration, and longevity (Nishimura et al., 1998). The FOXC1/FKHL7 gene and protein sequences are known, and can be found in GenBank (Accession Nos. AR140209 (complete sequence), AR140210 (coding sequence) and AAE63616 (amino acid sequence). As a result of the studies described in International Patent Application No. PCT/US 10/44817, filed August 6, 2010, and entitled "Methods for Diagnosis, Prognosis, and Treatment of Primary and Metastatic Basal-Like Breast Cancer and Other Cancer Types," filed February 3, 2012, it was determined that FOXC1 expression in human breast cancer, both at the mRNA and at the protein level, occurs consistently and exclusively in basal-like breast cancers. It was shown that FOXC1 is elevated only in basal-like molecular subtypes of breast cancers, and has been demonstrated to be of high prognostic significance, as it is predictive of the high mortality and metastasis rate specifically associated with basal- like breast cancers. Furthermore, in a head-to-head comparison with other suggested biomarkers of basal-like breast cancer and as shown by statistically significant in both univariate as well as multivariate analyses, FOXC1 has emerged as the most indicative and the most characteristic biomarker of basal-like breast cancers, in its ability to diagnose, prognose, and treat basal-like breast cancers.

[0022] Provided herein are methods for treating a subject with breast cancer including first screening a population of breast cancer tumor cells of the subject to determine whether the breast cancer tumor cells express FOXC1 , and subsequently treating that subject having breast cancer tumor cells expressing FOXC1 with bortezomib. In certain embodiments, the breast cancer tumor cells express FOXC1 if the level of FOXC1 is higher than a predetermined cutoff level. In certain embodiments, if the level of FOXC1 is higher than a predetermined cutoff level, then the subject may be classified as having basal-like breast cancer. In certain embodiments, the predetermined cutoff level may be determined by a 90th percentile level of FOXC1 expression levels for a dataset of breast cancer tumors, the dataset comprising all breast cancer subtypes.

[0023] Methods used to determine whether a population of tumor cells express FOXC1 may include any suitable method, including but not limited to, immunohistochemistry (or other immunoassay), PCR, RT-PCR, quantitative RT-PCR (qRT-PCR) (or any other PCR-based method), and/or the methods, assays and materials described in International Application Nos. PCT/US10/44817 entitled "Methods for Diagnosis, Prognosis, and Treatment of Primary and Metastatic Basal-Like Breast Cancer and Other Cancer Types;" and PCT/US12/23871 entitled "FOXC1 Antibodies and Methods of Their Use;" the subject matter of both of which are hereby incorporated by reference as if fully set forth herein.

[0024] In certain embodiments, detecting the presence or absence of FOXC1 may be accomplished by an in vitro immunoassay, such as immunocytochemistry (ICC), immunohistochemistry (IHC), Western blot or fluorescent in situ hybridization (FISH). Alternatively, an in vivo imaging modality may be used, such as magnetic resonance imaging (MRI), positron emission tomography (PET) or microPET, computed tomography (CT), PET/CT combination imager, cooled charged coupled device (CCD), camera optical imaging, optical imaging and single photon emission computed tomography (SPECT). When the presence or absence of FOXC1 is determined by an in vivo method, the FOXC1 antibody or functional fragment thereof should be conjugated to an intracellular delivery agent to facilitate deliver of the antibody or functional fragment thereof to the cytoplasm of target cells.

[0025] In certain embodiments, an anti-FOXC1 monoclonal antibody may be used to detect expression level of FOXC1 in a cell. As described in detail in PCT/US12/23871 , the antibody may specifically bind a target antigenic peptide sequence of human FOXC1 (Figure 12; SEQ ID NO: 1 ). In one aspect, the target antigenic peptide sequence is 5' -AHAEQYPGGMARAYGPYTPQPQPKD-3' (SEQ ID NO:2), which corresponds to amino acids 51 to 75 of SEQ ID NO:1 (see Figure 12). A cysteine residue may be added to the N-terminus (i.e., 5'-C- AHAEQYPGGMARAYGPYTPQPQPKD-3' (SEQ ID NO:3)) to assist in conjugation to the carrier protein as necessary.

[0026] In certain embodiments, if the tumor cells express FOXC1 , the subject may be administered one or more proteasome inhibitors, and optionally one or more Wnt inhibitors, to treat the cancer. In certain embodiments, the proteasome inhibitor may be bortezomib. In certain embodiments, the Wnt inhibitor may be beta-Catenin/Tcf Inhibitor III (i.e., iCRT3 or 2-[[[2-(4-ethylphenyl)-5-methyl-4-oxazolyl]methyl]thio]-N-(2- phenylethyl)acetamide). In certain embodiments, the cancer may be basal-like/claudin- low breast cancer. In certain embodiments, the cancer may be basal-like breast cancer. For example, the basal-like breast cancer may be a triple negative breast cancer, an ER positive breast cancer, or a HER2 positive breast cancer. In certain embodiments, the cancer may be claudin-low breast cancer.

[0027] Also provided herein are methods for treating cancer in a subject. In certain embodiments, the methods for treating a cancer in a subject include determining the expression level of FOXC1 in a population of tumor cells obtained from the subject; and administering one or more proteasome inhibitors, and optionally one or more Wnt inhibitors, to the subject to treat the cancer if the population of tumor cells expresses FOXC1 . In certain embodiments, the proteasome inhibitor may be bortezomib. In certain embodiments, the cancer may be basal-like/claudin-low breast cancer. In certain embodiments, the cancer may be basal-like breast cancer. For example, the basal-like breast cancer may be triple negative breast cancer, ER positive breast cancer, or HER2 positive breast cancer. In certain embodiments, the cancer may be claudin-low breast cancer.

[0028] Also provided herein are combination therapies comprising one or more Wnt inhibitors and one or more proteasome inhibitors. In certain embodiments, the proteasome inhibitor may be bortezomib. In certain embodiments, the one or more Wnt inhibitors may be iCRT3. In certain embodiments, the combination therapy may be used to treat a FOXC1 positive cancer. In certain embodiments, the FOXC1 positive cancer may be a basal-like breast cancer. For example, the basal-like breast cancer may be a triple negative breast cancer, an ER positive breast cancer, or a HER2 positive breast cancer. In certain embodiments, the cancer may be claudin-low breast cancer.

[0029] Optimal dosages of the combination therapy (i.e., the one or more Wnt inhibitors and one or more proteasome inhibitors) to be administered may be

determined by those skilled in the art, and will vary with the particular Wnt inhibitor and/or proteasome inhibitor used, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular subject being treated, include, without limitation, subject age, weight, gender, diet, time of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Administration of the pharmaceutical composition may be effected continuously or intermittently. In any treatment regimen, the pharmaceutical composition may be administered to a subject either singly or in a cocktail containing one or more Wnt inhibitors and one or more proteasome inhibitors, other therapeutic agents, compositions, or the like, including, but not limited to, tolerance-inducing agents, potentiators and side-effect relieving agents. All of these agents are administered in generally-accepted efficacious dose ranges such as those disclosed in the Physician's Desk Reference, 41 st Ed., Publisher Edward R. Barnhart, N.J. (1987), which is herein incorporated by reference as if fully set forth herein. [0030] "Treating" or "treatment" of a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof. Treatment may also mean a prophylactic or preventative treatment of a condition.

[0031] In certain aspects of the embodiments described above, the use of a proteasome inhibitor (e.g., bortezomib) and optionally, one or more Wnt inhibitors, for treating a FOXC1 positive cancer is provided herein. In certain embodiments, the FOXC1 positive cancer may be a basal-like breast cancer. For example, the basal-like breast cancer may be a triple negative breast cancer, an ER positive breast cancer, or a HER2 positive breast cancer. In certain embodiments, the cancer may be claudin-low breast cancer. All of the embodiments described herein apply to such uses.

[0032] As used herein, a "subject" refers to a mammal, such as a human. In some embodiments, the subject is a patient.

[0033] In certain embodiments, the one or more proteasome inhibitors, and optionally, one or more Wnt inhibitors, may be administered in combination with a therapeutic agent, radiotherapy, surgery, or any combination thereof.

[0034] As used herein, a "therapeutically effective amount," "therapeutically effective concentration" or "therapeutically effective dose" is an amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.

[0035] This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the one or more Wnt inhibitors and one or more proteasome inhibitors or pharmaceutical compositions thereof (including activity, pharmacokinetics, pharmacodynamics, and bioavailability thereof), the physiological condition of the subject treated (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. Further, an effective or therapeutically effective amount may vary depending on whether the one or more Wnt inhibitors and one or more proteasome inhibitors disclosed herein or the pharmaceutical composition thereof is administered alone or in combination with other drug(s), other therapy/therapies or other therapeutic method(s) or modality/modalities. One skilled in the clinical and pharmacological arts will be able to determine an effective amount or therapeutically effective amount through routine experimentation, namely by monitoring a cell's or subject's response to administration of the one or more Wnt inhibitors and one or more proteasome inhibitors or the pharmaceutical composition thereof and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21 st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins,

Philadelphia, PA, 2005, which is hereby incorporated by reference as if fully set forth herein for additional guidance for determining a therapeutically effective amount.

[0036] In some embodiments, the one or more proteasome inhibitors, and optionally, one or more Wnt inhibitors, used in the methods and therapies herein may be administered as a combination of one or more therapeutic agents for the treatment of cancer. "A combination" or "in combination with," as used herein, means in the course of treating the same cancer in the same subject using two or more agents, drugs, treatment regimens, treatment modalities or a combination thereof, in any order. This includes simultaneous administration, as well as in a temporally spaced order of up to several days apart. Such combination treatment may also include more than a single administration of any one or more of the agents, drugs, treatment regimens or treatment modalities. Further, the administration of the two or more agents, drugs, treatment regimens, treatment modalities or a combination thereof may be by the same or different routes of administration.

[0037] The treatment as described herein may be administered by any suitable route of administration, alone or as part of a pharmaceutical composition. A route of administration may refer to any administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, transdermal (e.g., topical cream or ointment, patch), or vaginal. "Transdermal" administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. "Parenteral" refers to a route of administration that is generally associated with injection, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.

[0038] Examples of therapeutic agents that may be administered as a treatment include, but are not limited to, chemotherapeutic agents, therapeutic antibodies and fragments thereof, toxins, radioisotopes, enzymes (e.g., enzymes to cleave prodrugs to a cytotoxic agent at the site of the tumor), nucleases, hormones, immunomodulators, antisense oligonucleotides, nucleic acid molecules (e.g., mRNA molecules, cDNA molecules or RNAi molecules such as siRNA or shRNA), chelators, boron compounds, photoactive agents and dyes. The therapeutic agent may also include a metal, metal alloy, intermetallic or core-shell nanoparticle bound to a chelator that acts as a radiosensitizer to render the targeted cells more sensitive to radiation therapy as compared to healthy cells.

[0039] Chemotherapeutic agents that may be used in accordance with the embodiments described herein are often cytotoxic or cytostatic in nature and may include, but are not limited to, alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors hormone therapy, targeted therapeutics and immunotherapeutics. In some embodiments the chemotherapeutic agents that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, 13-cis-Retinoic Acid, 2-Chlorodeoxyadenosine, 5- Azacitidine, 5-Fluorouracil, 6-Mercaptopurine, 6-Thioguanine, actinomycin-D, adriamycin, aldesleukin, alemtuzumab, alitretinoin, all-transretinoic acid, alpha interferon, altretamine, amethopterin, amifostine, anagrelide, anastrozole, arabinosylcytosine, arsenic trioxide, amsacrine, aminocamptothecin, aminoglutethimide, asparaginase, azacytidine, bacillus calmette-guerin (BCG), bendamustine, bevacizumab, bexarotene, bicalutamide, bleomycin, busulfan, calcium leucovorin, citrovorum factor, capecitabine, canertinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cortisone, cyclophosphamide, cytarabine, darbepoetin alfa, dasatinib, daunomycin, decitabine, denileukin diftitox, dexamethasone, dexasone, dexrazoxane, dactinomycin, daunorubicin, decarbazine, docetaxel, doxorubicin, doxifluridine, eniluracil, epirubicin, epoetin alfa, erlotinib, everolimus, exemestane, estramustine, etoposide, filgrastim, fluoxymesterone, fulvestrant, flavopiridol, floxuridine, fludarabine, fluorouracil, flutamide, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin, granulocyte - colony stimulating factor, granulocyte macrophage-colony stimulating factor, hexamethylmelamine, hydrocortisone hydroxyurea, ibritumomab, interferon alpha, interleukin - 2, interleukin-1 1 , isotretinoin, ixabepilone, idarubicin, imatinib mesylate, ifosfamide, irinotecan, lapatinib, lenalidomide, letrozole, leucovorin, leuprolide, liposomal Ara-C, lomustine, mechlorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nelarabine, nilutamide, octreotide, oprelvekin, oxaliplatin, paclitaxel, palbociclib, pamidronate, pemetrexed, panitumumab, PEG Interferon, pegaspargase, pegfilgrastim, PEG-L-asparaginase, pentostatin, plicamycin, prednisolone, prednisone, procarbazine, raloxifene, rituximab, romiplostim, ralitrexed, sapacitabine, sargramostim, satraplatin, sorafenib, sunitinib, semustine, streptozocin, tamoxifen, tegafur, tegafur-uracil, temsirolimus, temozolamide, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, trimitrexate, alrubicin, vincristine, vinblastine, vindestine, vinorelbine, vorinostat, or zoledronic acid.

[0040] Therapeutic antibodies and functional fragments thereof, that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, alemtuzumab, bevacizumab, cetuximab, edrecolomab, gemtuzumab, ibritumomab tiuxetan, panitumumab, rituximab, tositumomab, and trastuzumab and other antibodies associated with breast cancer.

[0041] Toxins that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0042] Radioisotopes that may be used as therapeutic agents in accordance with the embodiments of the disclosure include, but are not limited to, ³²P, ⁸⁹Sr, ⁹⁰Y, ^99mTc, 99i Mo, ³ 1, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re, ²¹³Bi, ²²³Ra and ²²⁵Ac.

[0043] The treatment and administration steps described herein may include any suitable treatment used in accordance with standard practice for treatment of breast cancer. The treatment is not limited to any particular treatment. One skilled in the art will appreciate that any United States Food and Drug Administration (FDA) approved therapeutic treatment or off-label treatment may be used in accordance with the methods provided herein.

[0044] In some embodiments, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response,

immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0045] The following examples are intended to illustrate various embodiments of the invention. As such, the specific embodiments discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.

EXAMPLES

Example 1 : Proteasome Inhibitor Bortezomib Inhibits NF-κΒ and Effectively Overcomes Cancer Stem Cell Escape Triggered by Wnt Inhibitor Therapy in FOXC1 Positive Basal-Like/Claudin-Low Breast Cancer. [0046] The Wnt/β catenin signaling pathway is active in basal-like/claudin-low breast cancers (Scheel et al., 2001 ). It was previously shown that FOXC1 plays a critical role in mediating aggressive cell traits in such cancers (see Figure 1 and 2; Ray et al, 2010). As provided herein, the link between Wnt signaling and FOXC1 and its potential in regulating cancer stem cell (CSC) biology in basal-like/claudin-low breast cancer was investigated.

[0047] The proposed mechanism of FOXC1 regulation by Wnt/β catenin signaling is shown in Figure 3. Figure 4 shows that FOXC1 acts as a downstream mediator of Wnt-driven regulation of CSCs in basal-like/claudin-low breast cancer.

[0048] Beta-catenin (CTNNB1 ) expression level and survival was analyzed using a breast cancer transcriptomic database (see Figure 5; Curtis et al., 2012). Additionally, it was observed that exposure of the MDA-MB-231 basal-like/claudin-low cell line (low constitutive FOXC1 expressor) to Wnt3a (a canonical Wnt signaling ligand), resulted in increased expression of FOXC1 (see Figure 6D). Reciprocally, overexpression of FOXC1 in MCF10A human mammary epithelial cells led to a pronounced increase in Wnt signaling activity, strongly suggestive of a direct or indirect positive feedback loop between Wnt signaling and FOXC1. More importantly, BT549 and HS578T basal- like/claudin-low cells (high constitutive FOXC1 expressors) proved to be more sensitive to treatment with the Wnt inhibitor iCRT3 as evidenced by decreased cell viability when compared to MCF7 (luminal) or SKBR3 (HER2) breast cancer cell lines (see Figures 7A and 7B). Furthermore the decrease in cell viability appeared to be proportionate to the level of FOXC1 expression (see Figures 7A and 7B).

[0049] Upon pharmacological inhibition with iCRT3 and biological inhibition with siRNA knockdown of LRP6, a canonical Wnt signaling cell surface receptor, a decrease in FOXC1 expression level was observed in a dose and time dependent manner (see Figures 6A and 6B). This effect was particularly pronounced in mammosphere cultures enriched for BT549 cancer stem-like cells. Inhibition of Wnt signaling reduced mammosphere formation efficiency of BT549 cells, suggesting that Wnt inhibition targets cancer stem cells (CSCs) in the basal-like/claudin-low breast cancer subtype (see Figures 9A and 9B). More importantly, however, after an initial 4 day incubation period, some cells were observed to persist and later display renewed enhancement of mammosphere formation ability. Profiling of such cells interestingly revealed depletion of FOXC1 positive cells, but persistence of cells displaying pronounced up regulation of stereotypical embryonic stem cell transcription factors OCT4, SOX2 and NANOG, strongly suggestive of a potential primitive stem cell/quiescent cell state escape mechanism (see Figure 10). qRT-PCR based pathway activation analysis revealed marked activation of NF-κΒ signaling in the residual cells that withstood Wnt inhibition.

[0050] Simultaneous pharmacologic inhibition with Wnt inhibitor iCRT3 and the proteasome inhibitor bortezomib, which is known to inhibit NF-κΒ signaling, effectively targeted BT549 cancer stem cells in mammosphere culture and prevented the persistence/emergence of any residual cells (see Figures 1 1A and 1 1 B). The combination treatment of iCRT3 with bortezomib was so effective that no mammosphere cells were viable at the end of the combined treatment (i.e., iCRT3 and bortezomib) and therefore there were no mammosphere cells to be profiled using qRT- PCR (see Figures 1 1A and 1 1 B). Taken together, these findings suggest that combination therapy approaches are likely required to effectively target breast cancer stem cells.

[0051] In certain embodiments, the tumor cells of a patient with cancer may be screened for FOXC1 expression to select patients with basal-like cancers. In certain embodiments, the patients may be treated with a combination therapy of a Wnt inhibitor and a proteasome inhibitor.

REFERENCES

The references, patents and published patent applications listed below, and all references cited in the specification above are hereby incorporated by reference in their entirety, as if fully set forth herein.

1 . Scheel, C, et al. (201 1 ) Paracrine and Autocrine Signals Induce and Maintain Mesenchymal and Stem Cell States in the Breast. Cell 145, 926-940.

2. Ray, P. S., et al. (2010) FOXC1 Is a Potential Prognostic Biomarker with

Functional Significance in Basal-like Breast Cancer. Cancer Research 70,

3870-3876.

3. Curtis, C, et al. (2012) The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486, 346-352.

4. International Application No. PCT/US10/44817, entitled "Methods for Diagnosis, Prognosis, and Treatment of Primary and Metastatic Basal-Like Breast Cancer and Other Cancer Types," filed August 6, 2010.

5. International Application No. PCT/US 12/23871 , entitled "FOXC1 Antibodies and Methods of Their Use," filed February 3, 2012.

Claims

CLAIMS What is claimed is:

1 . A method for treating cancer in a subject comprising: determining an expression level of FOXC1 in a population of tumor cells obtained from the subject; and administering one or more proteasome inhibitors, and optionally, one or more Wnt inhibitors, to the subject to treat the cancer if the population of tumor cells expresses FOXC1.

2. The method of claim 1 , wherein the proteasome inhibitor is bortezomib.

3. The method of claim 1 , wherein the Wnt inhibitor is iCRT3.

4. The method of claim 1 , wherein the cancer is a basal-like breast cancer.

5. The method of claim 4, wherein the basal-like breast cancer is triple negative breast cancer.

6. The method of claim 4, wherein the basal-like breast cancer is ER positive breast cancer.

7. The method of claim 4, wherein the basal-like breast cancer is HER2 positive breast cancer.

8. The method of claim 1 , wherein the cancer is claudin-low cancer.

9. The method of claim 1 , wherein determining the expression level of FOXC1 in the population of tumor cells is performed via quantitative RT-PCR (qRT-PCR).

10. A combination therapy comprising one or more Wnt inhibitors and one or more proteasome inhibitors.

1 1 . The combination therapy of claim 10, wherein the one or more proteasome inhibitors is bortezomib.

12. The combination therapy of claim 10, wherein the one or more Wnt inhibitors is a therapeutically effective amount.

13. The combination therapy of claim 10, wherein the one or more proteasome inhibitors is a therapeutically effective amount.

14. The method of claim 10, wherein the Wnt inhibitor is iCRT3.

15. A method for treating basal-like cancer in a subject comprising administering one or more proteasome inhibitors, and optionally, one or more Wnt inhibitors, to the subject to treat the basal-like cancer.

16. The method of claim 15, wherein the proteasome inhibitor is bortezomib.

17. The method of claim 15, wherein the basal-like breast cancer is triple negative breast cancer.

18. The method of claim 15, wherein the basal-like breast cancer is ER positive breast cancer.

19. The method of claim 15, wherein the basal-like breast cancer is HER2 positive breast cancer.

20. The method of claim 15, wherein the Wnt inhibitor is iCRT3.