WO2017058896A1 - Targets for treatment of hepatocellular carcinoma cancer and methods related thereto - Google Patents

Abstract

The present invention is based on the finding of a new tumor-suppressor role for HAF in immune cell function by preventing inappropriate HIF-1 activation in SART1^+/" male mice. The findings identify RANTES (Regulated on Activation, Normal T cell Expressed and Secreted; also named Chemokine (C-C motif) ligand 5 (CCL5)) as a therapeutic target for CCL5 mediated disease, such as cancer. As such, the present invention provides, in part, a method for treating CCL5 mediated disease by administering an agent that inhibits CCL5/CCR5 signal transduction.

Description

TARGETS FOR TREATMENT OF HEPATOCELLULAR CARCINOMA CANCER

AND METHODS RELATED THERETO

CROSS REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Serial No. 62/233,773, filed September 28, 2015, the entire contents of which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, name BURN1720_lWO_Sequence_Listing, was created on September 8, 2016, and is 2 kb. The file can be assessed using Microsoft Word on a computer that uses Windows OS.

STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made in part with government support under Grant Nos. R01CA181106, R01CA098920, R01CA164679 and P01CA177322, all of which were awarded by the National Institutes of Health. The United States government has certain rights in this invention.

BACKGROUND

FIELD OF THE INVENTION

[0004] The invention relates generally to treating disease, and more specifically to treatment and detection of CCL5 mediated disease.

BACKGROUND INFORMATION

[0005] Hypoxia is a state of reduced oxygen pressure below its physiological threshold, which impacts both normal and disease processes. Hypoxia characterizes virtually every site of inflammation, thus requiring infiltrating immune cells to undergo a metabolic switch toward anaerobic pathways to maintain energy requirements. The hypoxia-inducible factor (HIF) transcription factors are central regulators of hypoxic response. The HIFs are heterodimers comprising one of three major oxygen labile HIF-a subunits (HIF- la, HIF-2a, and HIF-3a), and a constitutive HIF-Ιβ subunit, which together form the HIF-1, HIF-2, and HIF-3 transcriptional complexes, respectively. HIF-1 plays an essential role in survival and function of immune cells by facilitating energy generation through anaerobic glycolysis. Under aerobic conditions, HIF-a is hydroxylated by oxygen-dependent prolyl hydroxylases, promoting ubiquitination by the von Hippel-Lindau protein (pVHL) E3 ligase complex resulting in HIF-a proteasomal degradation. Under hypoxic conditions, pVHL binding is abrogated and HIF-a is stabilized and heterodimerizes with HIF-Ιβ to transactivate a variety of hypoxia-responsive genes. HIF-Ια can also be induced under non-hypoxic conditions by proinflammatory cytokines, which allow initiation of an inflammatory response before tissues become hypoxic. In addition to pVHL, hypoxia-associated factor (HAF; encoded by SART1) is an isoform-specific E3 ubiquitin ligase that specifically degrades HIF-Ια (but not HIF-2a) in an oxygen-independent manner. Also known as SART1, HAF is expressed in both normal and malignant proliferating tissue and is important for spliceosome assembly and cell division.

[0006] Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver, and is the third leading cause of cancer deaths worldwide, with 750,000 new patients diagnosed each year. Despite advances in diagnosis and treatment of HCC, it remains a highly lethal disease due to recurrence of metastasis. HCC frequently develops in the context of chronic hepatitis characterized by liver inflammation and hepatocyte apoptosis. More than 90% of HCC cases are associated with chronic inflammation, which arise from Hepatitis B virus (HBV) or Hepatitis C virus (HCV) infection, and from alcoholic or nonalcoholic steatosis. The inflammatory response results in mobilization of immune cells, resulting in infiltration of inflamed tissue, which plays decisive roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion, and metastasis. A number of cytokines has been associated with HCC development including the interleukins (IL-la, IL-Ιβ, IL-2, IL-6, 1L-12) and non-interleukins such as tumor necrosis factor alpha (TNF-a) and Interferon gamma (IFN-y). Chemokines and their receptors such as the CXCL12-CXCR4 axis, CX3CL1- CX3CR1 axis, CCL5/CCL1-CCR3 and CCL20-CCR6 have been also been implicated in HCC. The complex interplay between hepatocytes and immune infiltrating cells in the presence of growth factors, cytokines and chemokines within the inflammatory tumor microenvironment is believed to drive HCC development and progression.

SUMMARY

[0007] The present invention is based on the finding of a new tumor-suppressor role for HAF in immune cell function by preventing inappropriate HIF-1 activation in SART1^+/" male mice. The findings identify RANTES (Regulated on Activation, Normal T cell Expressed and Secreted; also named Chemokine (C-C motif) ligand 5 (CCL5)) as a novel therapeutic target for cancer, such as nonalcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) as well as other liver pathologies which lead to HCC. As used herein, RANTES is used interchangeably with CCL5 and RANTES/CCL5 which is a protein, in humans, encoded by genomic reference sequence NG O 15990.1 in the NCBI genomic database available on the World Wide Web at ncbi.nlm.nih.gov/nuccore/NG_015990. l?from=5001&to=13883&report=genbank.

[0008] In embodiments, the invention provides a method of treating or preventing cancer in a subject. The method includes administering to the subject a therapeutically effective amount of an agent which inhibits CCL5/CCR5 signal transduction, thereby treating or preventing cancer in the subject. In one embodiment, the cancer is hepatocellular carcinoma.

[0009] In a related embodiment, the invention provides a method of treating or preventing a CCL5 mediated liver disease or disorder in a subject. The method includes administering to the subject a therapeutically effective amount of an agent which inhibits CCL5/CCR5 signal transduction, thereby treating or preventing the CCL5 mediated disease in the subject. In embodiments, the CCL5 mediated liver disease or disorder is inflammation, cancer, fatty liver disease, alcohol induced fatty liver disease, non-alcohol induced fatty liver disease, cirrhosis, steatosis, steatohepatitis, viral infection or fibrosis.

[00010] In another embodiment, the invention provides a method of diagnosing a subject as having, or at risk of having, hepatocellular carcinoma. The method includes obtaining a sample from the subject; detecting the presence or expression level of CCL5 in the sample; and diagnosing the subject as having, or at risk of having, hepatocellular carcinoma when the presence or expression level of CCL5 in the sample is elevated as compared to a corresponding normal sample. In embodiments, the method further includes administering to the subject a therapeutic regime, such as administering a chemotherapeutic agent or a therapeutically effective amount of an agent which inhibits CCL5/CCR5 signal transduction.

[0010] In yet another embodiment, the invention provides a method for determining susceptibility of a subject to a therapeutic regime to treat hepatocellular carcinoma, or monitoring progression of hepatocellular carcinoma in a subject. The method includes detecting the presence or expression level of CCL5 in a sample from the subject; and assessing the therapeutic regime or hepatocellular carcinoma progression based on the detection, thereby determining susceptibility of a subject to a therapeutic regime to treat hepatocellular carcinoma, or monitoring progression of hepatocellular carcinoma in a subject. [0011] In still another embodiment, the invention provides a transgenic mouse whose genome includes a heterozygous disruption of the squamous cell carcinoma antigen recognized by T-cells 1 (SART1) gene.

[0012] In another embodiment, the invention provides a method for identifying an agent for preventing or treating cancer utilizing the transgenic mouse of the disclosure. The method includes contacting the transgenic mouse of the disclosure with a test agent and monitoring tumor growth or liver neutrophilic infiltration in the mouse, wherein a reduction or inhibition of tumor growth or liver neutrophilic infiltration in the mouse is indicative of the test agent as an agent for preventing or treating cancer.

[0013] In another embodiment, the invention provides a kit which includes the transgenic mouse of the disclosure and one or more reagents for performing an assay, such as an assay to identifying an agent for preventing or treating cancer.

[0014] In yet another embodiment, the invention provides a method of screening for an agent to treat cancer, for example by inhibiting CCL5/CCR5 signal transduction. The method includes contacting a sample with a test agent; and detecting CCL5/CCR5 mediated signal transduction, wherein a reduction in CCL5/CCR5 mediated signal transduction as compared to a control sample is indicative of the test agent as being an agent to treat cancer. In another embodiment, the method includes contacting a sample with a test agent; and detecting binding of CCL5 to CCR5, wherein a reduction in binding as compared to a control sample is indicative of the test agent as being an agent to treat cancer by inhibiting CCL5/CCR5 signal transduction.

[0015] The invention also provides a method of identifying a CCR5 antagonist. The method includes contacting CCR5 with one or more test agents in the presence of CCL5, and identifying an agent that selectively inhibits CCR5 signal transduction, the test agent being characterized as a CCR5 antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A-1B are a series of schematic and graphical representations pertaining to generation of SART1+/- knockout heterozygous mice. Figure 1A is a schematic of a gene trap construct used to disrupt the HAF (SARTl) gene. Figure IB is a graphical representation of HAF levels in a panel of tissues from 2-month-old male SART1^+/" and WT mice. [0017] Figure 2 is a graphical representation relating to liver pathology in male SARTl^+/" and WT mice. The graph depicts liver steatosis grade and liver/body weight ratios of WT (n=6) and SART1^+/" (n=12) mice of age 10-12 months (mean 6 standard error).

[0018] Figures 3A-3F are a series of graphical and pictorial representations pertaining to hepatic steatosis in male SART1^+/" mice. Figure 3A includes images showing hematoxylin and eosin sections showing microvesicular hepatic steatosis and preneoplastic foci of cellular alteration in 6-month-old SART1^+/" livers. Figure 3B is a graph depicting quantitation along with a flowchart of age related progression of liver pathology in male SART1^+/" mice versus WT littermates [#mice]. Figure 3C is a western blot showing HAF expression in WT and SART1^+/" livers according to age with quantitation Figure 3D. Figure 3D is a graph depicting quantitation of Figure 3C. Figure 3E is a gene expression heatmap of a SART1^+/" liver tumor (T) normalized to a WT liver (N) showing regions enriched for genes involved in FAO and inflammatory response with enlarged heatmap showing FAO genes at RHS. Figure 3F depicts Taqman™ validation for a select number of FAO genes using 3 additional mice/group with western blotting validation. Abbreviations: Acaala, acetyl-coenzyme A acyltransferase 1A; Acoxl, acyl -coenzyme A oxidase 1.

[0019] Figures 4A-4D are a series of graphical representations depicting Seahorse (Seahorse Bioscience, North Billerica, MA) metabolic analysis of OCR. Figure 4A shows analysis of OCR of primary hepatocytes isolated from male SART1^+/" or WT mice (age, 4 months). Figure 4B shows quantitation of data (3 mice/group). Figure 4C shows analysis of OCR of Huh7 cells transfected with HAF siRNA. Figure 4D shows quantitation of data from three replicate wells of Figure 4C. Data are mean 6 standard deviation.

[0020] Figures 5A-5E are a series of representations illustrating that HAF loss is associated with increased HIF-la and RANTES production. Figure 5 A shows western blotting and quantitation of HIF-la levels in PBMCs and spleen-adherent and -nonadherent mononuclear cells from male SART1^+/" and WT mice (age, 6 months). Figure 5B is flow cytometry scatterplots showing LacZ-FITC intensity in peripheral blood cells from male SART1^+/" and WT mice (age, 4 months). Figure 5C depicts Lac Z intensities of immune cells from spleens of male SART1^+/" (Het) and wild-type (Wt) mice (age, 4 months; 4 mice/group). Each data point represents a single mouse with mean 6 standard deviation. Figure 5D depicts quantitation of secreted cytokines from KCs isolated from male SART1^+/" livers normalized to wild-type with arrays depicted inset (pooled from 4 mice/group). Figure 5E depicts quantitation of RANTES secretion by TFIP-1 cells transfected with siRNAs to HAF with western blotting validation on RHS. Data are mean 6 standard error.

[0021] Figures 6A-6C are a series of representations associated with biomarkers for assessing HCC progression and therapeutic efficacy. Figure 6A illustrates a timeline for liver dysfunction manifestation in SART1^+/" mice accompanied by observable elevation in biomarker levels compared to age matched wild-type mice. Figure 6B depicts levels of blood liver enzyme alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in SART1^+/" versus age-matched wild-type mice. Note that ALP levels are highest in younger mice. Figure 6C shows elevation of genes encoding glycolytic enzymes (Slc2Al, Hkl and Pkm) and CCL5 in livers of SART1^+/" mice of indicated ages normalized to levels in age-matched wild-type litter mates. Data were obtained from at least 3 mice per wild-type or SART1^+/" age group for each panel.

[0022] Figures 7A-7C are a series of representations pertaining to identification of a central role for RANTES/CCL5 in HCC in SART1^+/" mice. Figure 7A depicts measurement of cytokine/chemokine secretion from Kupffer cells isolated from livers of 6-month old SART1^+/" mice normalized to age matched wild-type mice. Note elevation of RANTES/CCL5 to >100-fold over control. Figure 7B shows elevation of CCL5 mRNA in livers of SART1^+/" mice of indicated ages normalized to levels in age-matched wild-type litter mates. Note: SART1^+/" mice develop visible HCC tumors at 10 months. Figure 7C shows percentage of neutrophils of total cell count determined by flow cytometry (Ly6G+) in livers of SART1^+/" and wild type mice of indicated ages.

DETAILED DESCRIPTION

[0023] The present invention is based on the discovery of a new tumor-suppressor role for HAF in immune cell function as well as identification of RANTES as a novel therapeutic target for NASH and NASH-driven HCC.

[0024] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0025] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.

[0027] SART1/HAF is a protein that acts as a molecular switch regulating the balance between the cellular levels of the hypoxia inducible proteins HIF-1 and HIF-2. The inventors have found that SART1/HAF knockout is embryonic lethal in mice. Unexpectedly it was found that SART1/HAF haploinsufficient mice with germ line deletion of 1 copy of the SART 1 gene develop hepatocellular carcinoma (HCC) after about 10 months. Gene expression and functional studies suggest that HCC development in these mice is promoted by RANTES/CCL5 and its receptor CCR5, which promote immune cell infiltration into the liver and chronic liver inflammation. This suggests that inhibition of RANTES/CCL5 or its receptor(s) might provide therapeutic benefit by shutting down the inflammatory response that drives HCC. Due to the accessibility of RANTES/CCL5 as a circulating ligand, and its receptor CCR5 located on the cell surface, these targets can be suitably inhibited to treat RANTES/CCL5 mediated diseases and disorders, such as cancer.

[0028] Accordingly, the invention provides a method of treating or preventing cancer in a subject. The method includes administering to the subject a therapeutically effective amount of an agent which inhibits CCL5/CCR5 signal transduction, thereby treating or preventing cancer in the subject.

[0029] The invention also provides a method of treating or preventing a CCL5 mediated liver disease or disorder in a subject. The method includes administering to the subject a therapeutically effective amount of an agent which inhibits CCL5/CCR5 signal transduction, thereby treating or preventing the CCL5 mediated disease in the subject.

[0030] As used herein, a CCL5 mediated liver disease or disorder may include inflammation, cancer, fatty liver disease, alcohol induced fatty liver disease, non-alcohol induced fatty liver disease, cirrhosis, steatosis, steatohepatitis, viral infection or fibrosis.

[0031] The term "cancer" as used herein, includes a variety of cancer types which are well known in the art, including but not limited to, dysplasias, hyperplasias, solid tumors and hematopoietic cancers. Many types of cancers are known, such as, but in no way limited to, the following organs or systems: brain, cardiac, lung, gastrointestinal, genitourinary tract, liver, bone, nervous system, gynecological, hematologic, skin, breast, and adrenal glands. Additional types of cancer cells include gliomas (Schwannoma, glioblastoma, astrocytoma), neuroblastoma, pheochromocytoma, paraganlioma, meningioma, adrenal cortical carcinoma, medulloblastoma, rhabdomyoscarcoma, kidney cancer, vascular cancer of various types, osteoblastic osteocarcinoma, prostate cancer, ovarian cancer, uterine leiomyomas, salivary gland cancer, choroid plexus carcinoma, mammary cancer, pancreatic cancer, colon cancer, and megakaryoblastic leukemia; and skin cancers including malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, sarcomas such as fibrosarcoma or hemangiosarcoma, and melanoma. In one embodiment, the cancer is hepatocellular carcinoma (HCC).

[0032] Clinical evidence has revealed that elevated levels of tissue or plasma CCL5 is a marker of an unfavorable outcome or metastatic disease in patients with a variety of cancer types which may be targeted by the present invention. RANTES is elevated in advanced breast carcinoma. Additionally, the expression of CCR5 and RANTES/CCL5 correlates with a metastatic phenotype of basal breast cancer, both in clinical samples and in cell lines, and treatment with CCR5 antagonists reduced the risk of lung metastasis in a mouse model of breast cancer. Serum RANTES/CCL5 concentration is also significantly elevated in ovarian cancer patients compared to benign ovarian cyst patients, and values correlated with the stage of disease and the extent of residual tumor mass. In patients with breast or cervical cancer, plasma RANTES/CCL5 levels are found to be higher with increasing stages. Furthermore, in these patients, marked increases in plasma RANTES/CCL5 level was found in patients with progressive malignancy but in none of those in clinical remission. Markedly elevated levels of plasma RANTES/CCL5 were also observed in patients with stage IV gastric cancer, and might be useful for identifying patients with metastatic disease, RANTES/CCL5 polymorphisms also conferred increased risk for development of pancreatic adenocarcinoma.

[0033] The axis of RANTES/CCL5 and its receptor (i.e., CCR5), alone, is central for HCC development, progression and metastasis. Also, the inhibition/neutralization of RANTES/CCL5 or its receptor(s) using, for example, targeted therapeutic antibodies has clinical utility for the treatment of all forms of HCC including that caused by chronic inflammation, such as Hepatitis B and Hepatitis C infection, alcohol induced fatty liver disease (AFLD), non-alcohol induced fatty liver-disease (NAFLD) and other metabolic overload causes; FLD, NAFLD and other liver conditions at high risk for progressing to cirrhosis, liver failure or HCC.

[0034] The SARTl/HAF heterozygous knockout mouse of the disclosure models the progression of human HCC from hepatic steatosis through cirrhosis without requiring additional manipulation with chemicals, diets or mutagens, which is currently the norm for producing mouse models of HCC. Additionally loss of 1 copy of the SART1 gene is sufficient to mediate a phenotype. The SART1/HAF haploinsufficient mice are therefore a more physiological model of human HCC and fatty liver disease. This model enabled identification of RANTES/CCL5, and its receptor CCR5 as playing a causal role in HCC development and progression. This model enables one to test the efficacy of agents specifically targeted to inhibit CCL5/CCR5 signal transduction, for example, specifically targeted antibodies blocking RANTES/CCL5/CCR5 activation for the treatment of HCC.

[0035] Accordingly, the invention provides a transgenic mouse whose genome includes a heterozygous disruption of the SARTl gene.

[0036] There are no effective therapies for HCC. HCC is the primary malignancy of the liver and the third leading cause of cancer deaths worldwide, with over 500,000 people affected. The incidence of HCC is highest in Asia and Africa, where the endemic high prevalence of hepatitis B and hepatitis C strongly predisposes to the development of chronic liver disease and subsequent development of HCC. HCC accounts for more than 12,000 deaths a year in the United States and is being diagnosed more frequently. It is more common in men than women and in African Americans than whites. Resection may benefit certain patients, albeit mostly transiently, although most patients are not candidates because of the advanced stage of their cancer at diagnosis. In these patients, local ablative therapies, including radiofrequency ablation, chemoembolization may extend life and provide palliation. HCC is minimally responsive to systemic chemotherapy. Among the agents tried, doxorubicin-based regimens appear to have the greatest efficacy. A variety of hormonal and biologic agents have been tried with minimal success, including tamoxifen, antiandrogens (eg, cyproterone, ketoconazole), interferon, interleukin 2 (IL-2), and octreotide. Sorafenib (Nexavar®) was approved in 2007 by the FDA for patients with unresectable HCC being able to extend the life of patients by 3 months, from 8 months to 11 months. [0037] As discussed in detail in Example 1, to investigate the physiological role of HAF, C57BL/6 mice were generated with germ line heterozygosity for HAF (SART1^+/"). These mice developed multiple large tumors (histologically confirmed as hepatocellular carcinoma, HCC) within their livers when they reached 10 months of age. This was accompanied by steatohepatitis- lipid deposition accompanied by extensive immune cell infiltration; hallmarks of fatty liver disease in humans. This is presently the only genetic model where haploinsufficiency promotes HCC development without requiring any further manipulation. The gene expression profiles of the livers of SART1^+/" mice were consistent with constitutive activation of HIF-1, including a 10-fold induction of the HIF-1 target gene: RANTES/CCL5. CCL5 is a chemoattractant for a variety of immune cells including neutrophils. Chronic liver inflammation mediated by liver-associated immune cells is a key component for HCC development in humans. Tumor development in the SART1^+/" mice was preceded at 6 months of age, by a >100- fold increase in CCL5 secretion by liver- associated Kupffer cells compared to age- matched wild-type littermates. This was accompanied by a >10-fold elevation in liver neutrophil infiltration. Hepatic steatosis and cellular alterations including pre-neoplastic lesions were already apparent in mice of this age although no neoplastic lesions were observed. Elevated liver neutrophil infiltration and CCL5 expression was observed in mice as early as 1 month of age, hence preceding hepatic steatosis and all other liver dysfunction and cellular alteration associated with HCC development. This suggests that increased RANTES/CCL5 expression in the livers of SART1+/- mice is a key factor promoting HCC initiation and progression.

[0038] HAF is an E3 ligase for the hypoxia inducible factor, HIF-la, which has been implicated in the development of liver disease and HCC. To investigate the physiological role of HAF, mice with germline heterozygosity for HAF (SART1^+/") were generated. These mice developed multiple large tumors (histologically confirmed as hepatocellular carcinoma, HCC) within their livers when they reached 10 months of age. It was found that 60-80% of male SART1^+/" mice developed HCC at 12 months. This was accompanied by steatohepatitis - lipid deposition accompanied by extensive immune cell infiltration; hallmarks of fatty liver disease in humans. The gene expression profiles of the livers of SART1^+/" mice were consistent with constitutive activation of HIF-1, including induction of HIF target genes involved in glycolysis (Figure 6). In humans, hepatic HIF-1 a regulates the expression of glucose transporters as well as glycolytic enzymes, and is thought to contribute to the glycolytic phenotype of HCCs. Livers of SART1^+/" mice also show a 10- fold induction of the HIF-1 target gene RANTES/CCL5. CCL5 is a chemoattractant for a variety of immune cells including neutrophils. Tumor development in the SART1^+/" mice was preceded at 6 months of age, by a > 100-fold increase in CCL5 secretion by liver- associated Kupffer cells compared to age-matched wild-type littermates (Figure 7). This was accompanied by a >10 fold elevation in liver neutrophil infiltration. Hepatic steatosis and cellular alterations including pre-neoplastic lesions were already apparent in mice of this age although no neoplastic lesions were observed. Elevated liver neutrophil infiltration and CCL5 expression was observed in mice as early as 1 month of age, hence preceding hepatic steatosis and all other liver dysfunction and cellular alteration associated with HCC development. This suggests that RANTES/CCL5 plays a unique and central role in promoting HCC initiation and progression, and would thus be a suitable therapeutic target for the treatment of HCC.

[0039] In various embodiments, the present invention utilizes an agent that antagonizes or inhibits (i.e., blocks) CCL5/CCR5 signal transduction. One of skill in the art would appreciate that agents capable of inhibiting CCL5/CCR5 signal transduction can include a variety of different types of molecules. An agent or candidate agent useful in any method of the invention can be any type of molecule, for example, a polynucleotide, a peptide, a peptidomimetic, peptoids such as vinylogous peptoids, chemical compounds, such as organic molecules or small organic molecules, or the like.

[0040] In one embodiment, the agent or candidate agent may be a peptide, such as an antibody, or fragment thereof, that specifically binds CCL5 and/or CCR5 thereby blocking binding of CCL5 to CCR5. The term "polypeptide" is used in its broadest sense to refer to a polymer of subunit amino acids, amino acid analogs, or peptidomimetics, including proteins and peptoids. The polypeptides may be naturally occurring full length proteins or fragments thereof, processed forms of naturally occurring polypeptides (such as by enzymatic digestion), chemically synthesized polypeptides, or recombinantly expressed polypeptides. The polypeptides may comprise D- and/or L-amino acids, as well as any other synthetic amino acid subunit, and may contain any other type of suitable modification, including but not limited to peptidomimetic bonds and reduced peptide bonds.

[0041] The term "antibody," as used herein, refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as single chain antibodies and humanized antibodies. In various embodiments, the antibody may be a single chain antibody, a monoclonal antibody, a bi- specific antibody, a chimeric antibody, a synthetic antibody, a polyclonal antibody, a humanized antibody, a fully human antibody, and active fragments or homologs thereof.

[0042] An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules.

[0043] An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules.

[0044] The term "synthetic antibody" as used herein, is meant to include an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0045] The term "binding" refers to the adherence of molecules to one another, such as, but not limited to, enzymes to substrates, ligands to receptors, antibodies to antigens, DNA binding domains of proteins to DNA, and DNA or RNA strands to complementary strands. "Binding partner," as used herein, refers to a molecule capable of binding to another molecule.

[0046] As used herein, the term "biologically active fragments" or "bioactive fragment" of a polypeptide encompasses natural or synthetic portions of the full-length protein that are capable of specific binding to their natural ligand or of performing the function of the protein.

[0047] A "ligand" is a compound that specifically binds to a target receptor.

[0048] A "receptor" is a compound that specifically binds to a ligand. For example, receptor CCR5 specifically binds ligand CCL5.

[0049] A ligand or a receptor "specifically binds to" or "is specifically immunoreactive with" an agent when the ligand or receptor functions in a binding reaction which is determinative of the presence of the compound in a sample of heterogeneous compounds. Thus, under designated assay (e.g., immunoassay) conditions, the ligand or receptor binds preferentially to a particular compound and does not bind in a significant amount to other compounds present in the sample. For example, a polynucleotide specifically binds under hybridization conditions to a compound polynucleotide comprising a complementary sequence; an antibody specifically binds under immunoassay conditions to an antigen bearing an epitope against which the antibody was raised. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0050] In one embodiment, an agent or candidate agent is a polynucleotide, such as an antisense oligonucleotide or RNA molecule. In various embodiments, the agent or candidate agent may be a polynucleotide, such as an antisense oligonucleotide or RNA molecule, such as microRNA, dsRNA, siRNA, stRNA, and shRNA. In various aspects, the polynucleotide inhibits expression or activity of CCL5, CCR5 or both.

[0051] Polynucleotides of the present invention, such as antisense oligonucleotides and RNA molecules may be of any suitable length. For example, one of skill in the art would understand what length are suitable for antisense oligonucleotides or RNA molecule to be used to regulate gene expression. Such molecules are typically from about 5 to 100, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, or 10 to 20 nucleotides in length. For example the molecule may be about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45 or 50 nucleotides in length. Such polynucleotides may include from at least about 15 to more than about 120 nucleotides, including at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides or greater than 120 nucleotides.

[0052] The term "polynucleotide" or "nucleotide sequence" or "nucleic acid molecule" is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. As such, the terms include RNA and DNA, which can be a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. Furthermore, the terms as used herein include naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic polynucleotides, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR). It should be recognized that the different terms are used only for convenience of discussion so as to distinguish, for example, different components of a composition.

[0053] In general, the nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'- deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose. Depending on the use, however, a polynucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs. The covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond. However, depending on the purpose for which the polynucleotide is to be used, the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides.

[0054] A polynucleotide or oligonucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template.

[0055] In various embodiments antisense oligonucleotides or RNA molecules include oligonucleotides containing modifications. A variety of modification are known in the art and contemplated for use in the present invention. For example oligonucleotides containing modified backbones or non-natural internucleoside linkages are contemplated. As used herein, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0056] In various aspects modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3'-5' linkages, 2 -5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Certain oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0057] In various aspects modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. [0058] In various aspects, oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. In various aspects, oligonucleotides may include phosphorothioate backbones and oligonucleosides with heteroatom backbones. Modified oligonucleotides may also contain one or more substituted sugar moieties. In some embodiments oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are 0[(CH₂)_nO]_mCH₃, 0(CH.sub.₂)_nOCH₃, 0(CH₂)_n H₂, 0(CH₂)_nCH₃, 0(CH₂)_nO H₂ and 0(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, S0₂CH₃, ON0₂, N0₂, N3, H₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkyl amino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Another modification includes 2'-methoxyethoxy(2'OCH₂CH₂OCH₃, also known as 2'-0-(2-methoxyethyl) or 2'-MOE).

[0059] In related aspects, the present invention includes use of Locked Nucleic Acids (LNAs) to generate antisense nucleic acids having enhanced affinity and specificity for the target polynucleotide. LNAs are nucleic acid in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (— CH₂-)_n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.

[0060] Other modifications include 2'-methoxy(2'-0— CH₃), 2'-aminopropoxy(2'- OCH₂CH₂CH₂NH₂), 2'-allyl (2'-CH-CH-CH₂), 2'-0-allyl (2'-0-CH₂-CHCH₂), 2^*-fluoro (2'-F), 2' -amino, 2'-thio, 2'-Omethyl, 2'-methoxymethyl, 2' -propyl, and the like. The 2'- modification may be in the arabino (up) position or ribo (down) position. A preferred 2'- arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0061] Oligonucleotides may also include nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5- methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (lH-pyrimido[5,4-b][l,4]benzoxazi-n-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)- one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases are known in the art. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds described herein. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.

[0062] Another modification of the antisense oligonucleotides described herein involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The antisense oligonucleotides can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., dihexadecyl-rac- glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylaminocarbonyloxycholesterol moiety.

[0063] In embodiments, the invention provides a method for identifying an agent for preventing or treating cancer or other CCL5 mediated disease utilizing the SART1^+/" transgenic mouse of the disclosure. The method includes contacting the transgenic mouse of the disclosure with a test agent and monitoring tumor growth or liver neutrophilic infiltration in the mouse, wherein a reduction or inhibition of tumor growth or liver neutrophilic infiltration in the mouse is indicative of the test agent as an agent for preventing or treating cancer.

[0064] As used herein, "test agent" and "candidate agent" are used interchangeably and refer to agents that are known to, or are being investigated for their ability to inhibit CCL5/CCR5 signal transduction. [0065] The invention also provides a method of screening for an agent to treat cancer, for example by inhibiting CCL5/CCR5 signal transduction. The method includes contacting a sample with a test agent; and detecting CCL5/CCR5 mediated signal transduction, wherein a reduction in CCL5/CCR5 mediated signal transduction as compared to a control sample is indicative of the test agent as being an agent to treat cancer. In another embodiment, the method includes contacting a sample with a test agent; and detecting binding of CCL5 to CCR5, wherein a reduction in binding as compared to a control sample is indicative of the test agent as being an agent to treat cancer by inhibiting CCL5/CCR5 signal transduction.

[0066] The invention also provides a method of identifying a CCR5 antagonist. The method includes contacting CCR5 with one or more test agents in the presence of CCL5, and identifying an agent that selectively inhibits CCR5 signal transduction, the test agent being characterized as a CCR5 antagonist.

[0067] A screening assay used in a method of the invention for identifying a CCR5 antagonist can involve detecting a signal produced by binding of CCL5 to CCR5, i.e., CCL5/CCR5 mediated signal transduction. As used herein, the term "receptor signal" is intended to mean a readout, detectable by any analytical means, that is a qualitative or quantitative indication of activation of signal transduction through CCR5. Assays used to determine such qualitative or quantitative activation of signal transduction are referred to below as "signaling assays."

[0068] A signaling assay can be performed to determine whether a test agent is a CCR5 antagonist. A signaling assay can be performed to determine whether a test agent is a CCR5 antagonist. In such a signaling assay, CCR5 is contacted with one or more test agents under conditions wherein CCR5 produces a signal in response to an agonist, and an agent is identified that reduces production of the signal.

[0069] CCR5 is a G protein coupled receptor which functions as a chemokine receptor in the CC chemokine group. Signaling through G proteins can lead to increased or decreased production or liberation of second messengers, including, for example, arachidonic acid, acetylcholine, diacylglycerol, cGMP, cAMP, inositol phosphate, such as inositol-1,4,5- trisphosphate, and ions, including Ca⁺⁺ ions; altered cell membrane potential; GTP hydrolysis; influx or efflux of amino acids; increased or decreased phosphorylation of intracellular proteins; or activation of transcription. [0070] Various assays, including high throughput automated screening assays, to identify alterations in G-protein coupled signal transduction pathways are well known in the art. Various screening assay that measure Ca⁺⁺, cAMP, voltage changes and gene expression are reviewed, for example, in Gonzalez et al., Curr. Opin. in Biotech. 9:624-631 (1998); Jayawickreme et al., Curr. Opin. Biotech. 8:629-634 (1997); and Coward et al., Anal. Biochem. 270:2424-248 (1999). Yeast cell-based bioassays for high-throughput screening of drug targets for G-protein coupled receptors are described, for example, in Pausch, Trends in Biotech. 15:487-494 (1997). A variety of cell-based expression systems, including bacterial, yeast, baculovirus/insect systems and mammalian cells, useful for detecting G-protein coupled receptor agonists and antagonists are reviewed, for example, in Tate et al., Trends in Biotech. 14:426-430 (1996).

[0071] Assays to detect and measure G-protein-coupled signal transduction can involve first contacting a sample containing CCR5, such as an isolated cell, membrane or artificial membrane, such as a liposome or micelle, with a detectable indicator. A detectable indicator can be any molecule that exhibits a detectable difference in a physical or chemical property in the presence of the substance being measured, such as a color change. Calcium indicators, pH indicators, and metal ion indicators, and assays for using these indicators to detect and measure selected signal transduction pathways are described, for example, in Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Sets 20-23 and 25 (1992-94). For example, calcium indicators and their use are well known in the art, and include agents like Fluo-3 AM, Fura-2, Indo-1, FURA RED, CALCIUM GREEN, CALCnJM ORANGE, CALCR7M CRFMSON, BTC, OREGON GREEN BAPTA, which are available from Molecular Probes, Inc., Eugene OR, and described, for example, in U.S. Patent Nos. 5,453,517, 5,501,980 and 4,849,362.

[0072] An assay to identify agents that function as CCR5 antagonists are generally performed under conditions in which contacting the receptor with a known receptor agonist would produce a receptor signal. An antagonist that prevents CCL5 from binding CCR5, or indirectly decreases the signaling activity of CCR5 can be identified. The test agent can be tested at a range of concentrations to establish the concentration where half-maximal signaling occurs; such a concentration is generally similar to the dissociation constant (Kd) for CCR5 binding.

[0073] A binding assay can be performed to identify agents that are CCR5 antagonists. In such an assay CCR5 is contacted with one or more test agents under conditions in which an agent that binds CCL5, an agent that binds CCR5 or an agent that reduces binding of CCL5 to CCR5 can be identified. Contemplated binding assays can involve detectably labeling a test agent, or competing an unlabeled test agent with a detectably labeled CCR5 agonist. A detectable label can be, for example, a radioisotope, fluorochrome, ferromagnetic substance, or luminescent substance. Exemplary radiolabels useful for labeling agents include ¹²⁵I, ¹⁴C and ³H. Methods of detectably labeling organic molecules, either by incorporating labeled amino acids into the agent during synthesis, or by derivatizing the agent after synthesis, are known in the art.

[0074] In order to determine whether a test agent decreases binding of detectably labeled CC15, the amount of binding of a given amount of the detectably labeled CC15 is determined in the absence of the test agent. Generally the amount of detectably labeled CC15 will be less than its K_d, for example, 1/10 of its K4. An exemplary assay for determining binding of detectably labeled CCL5 is the radioligand filter binding assay described in Li et al. Molecular Pharmacology 59:692-698 (2001)).

[0075] Either low- and high-throughput assays suitable for detecting selective binding interactions between a receptor and a ligand include, for example, fluorescence correlation spectroscopy (FCS) and scintillation proximity assays (SPA) reviewed in Major, J. Receptor and Signal Transduction Res. 15:595-607 (1995); and in Sterrer et al., J. Receptor and Signal Transduction Res. 17:511-520 (1997)). Binding assays can be performed in any suitable assay format including, for example, cell preparations such as whole cells or membranes that contain a CCR5, or substantially purified CCR5, either in solution or bound to a solid support.

[0076] The number of different test agents to test in the methods of the invention will depend on the application of the method. For example, one or a small number of test agents can be advantageous in manual screening procedures, or when it is desired to compare efficacy among several predicted ligands, agonists or antagonists. However, it will be appreciated that the larger the number of test agents, the greater the likelihood of identifying an agent having the desired activity in a screening assay. Additionally, large numbers of agents can be processed in high-throughput automated screening assays.

[0077] Assay methods for identifying agents that selectively bind to or inhibit signaling through a CCR5 generally involve comparison to a control. One type of a "control" is a preparation that is treated identically to the test preparation, except the control is not exposed to the test agent. Another type of "control" is a preparation that is similar to the test preparation, except that the control preparation does not express the receptor, or has been modified so as not to respond selectively to CCL5. In this situation, the response of the test preparation to a test agent is compared to the response (or lack of response) of the control preparation to the same agent under substantially the same reaction conditions.

[0078] An agent identified to be an agonist or antagonist of CCR5 can be tested for its ability to modulate one or more effects on the function of a cell or animal. For example, a CCR5 antagonist can be tested for an ability to reduce or inhibit tumor growth, reduce or inhibit liver neutrophilic infiltration, treat cancer, such as HCC, or treat a liver disease or disorder such as inflammation, cancer, fatty liver disease, alcohol induced fatty liver disease, non-alcohol induced fatty liver disease, cirrhosis, steatosis, steatohepatitis, viral infection, and fibrosis.

[0079] In another embodiment, the invention provides a method for diagnosing or prognosing cancer in a subject. As such, the invention provides a method of diagnosing a subject as having, or at risk of having, cancer, for example, hepatocellular carcinoma. The method includes obtaining a sample from the subject; detecting the presence or expression level of CCL5 in the sample; and diagnosing the subject as having, or at risk of having, hepatocellular carcinoma when the presence or expression level of CCL5 in the sample is elevated as compared to a corresponding normal sample. In embodiments, the method further includes administering to the subject a therapeutic regime, such as administering a chemotherapeutic agent or a therapeutically effective amount of an agent which inhibits CCL5/CCR5 signal transduction.

[0080] "Diagnosing" includes determining, monitoring, confirmation, subclassification, and prediction of the relevant disease, complication, or risk. "Determining" relates to becoming aware of a disease, complication, risk, and the like. "Monitoring" relates to keeping track of an already diagnosed disease, complication, or risk factor, e.g., to analyze the progression of the disease or the influence of a particular treatment on the progression of disease or complication. "Confirmation" relates to the strengthening or substantiating of a diagnosis already performed using other indicators or markers. "Classification" or "subclassification" relates to further defining a diagnosis according to different subclasses of the diagnosed disease, disorder, or condition, e.g., defining according to mild, moderate, or severe forms of the disease or risk. "Prediction" relates to prognosing a disease, disorder, condition, or complication before other symptoms or markers have become evident or have become significantly altered. [0081] The term "risk" relates to the possibility or probability of a particular event occurring either presently, or, at some point in the future. "Risk stratification" refers to an arraying of known clinical risk factors to allow physicians to classify patients into a low, moderate, high or highest risk of developing of a particular disease, disorder, or condition.

[0082] The term "subject" as used herein refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0083] The invention further provides a method for determining susceptibility of a subject to a therapeutic regime to treat cancer, i.e., hepatocellular carcinoma, or monitoring progression of cancer, i.e., hepatocellular carcinoma in a subject. The method includes detecting the presence or expression level of CCL5 in a sample from the subject; and assessing the therapeutic regime or hepatocellular carcinoma progression based on the detection, thereby determining susceptibility of a subject to a therapeutic regime to treat hepatocellular carcinoma, or monitoring progression of hepatocellular carcinoma in a subject.

[0084] In various embodiments, assessments be made over a particular time course in various intervals to assess a subject's progression and pathology. For example, analysis may be performed at regular intervals such as one day, two days, three days, one week, two weeks, one month, two months, three months, six months, or one year, in order to track level tumor progression or regression as a function of time. In the case of existing cancer patients, this provides a useful indication of the progression of the disease and assists medical practitioners in making appropriate therapeutic choices.

[0085] In any of the methods provided herein, additional analysis may also be performed to characterize disease to provide additional clinical assessment. For example, PCR techniques may be employed, such as multiplexing with primers specific for particular cancer markers to obtain information such as the type of tumor, metastatic state, and degree of malignancy. Additionally, cell size, DNA or RNA analysis, proteome analysis, or metabolome analysis may be performed as a means of assessing additional information regarding characterization of the patient's cancer. [0086] The additional analysis may provide data sufficient to make determinations of responsiveness of a subject to a particular therapeutic regime, or for determining the effectiveness of a candidate agent in the treatment of disease, i.e., cancer. Accordingly, the present invention provides a method of determining responsiveness of a subject to a particular therapeutic regime or determining the effectiveness of a candidate agent in the treatment of cancer. For example, once a drug treatment is administered to a patient, it is possible to determine the efficacy of the drug treatment using the methods of the invention. For example, a sample taken from the patient before the drug treatment, as well as one or more cellular samples taken from the patient concurrently with or subsequent to the drug treatment, may be processed using the methods of the invention. By comparing the results of the analysis of each processed sample, one may determine the efficacy of the drug treatment or the responsiveness of the patient to the agent. In this manner, early identification may be made of failed compounds or early validation may be made of promising compounds.

[0087] The terms "administration" or "administering" are defined to include an act of providing a compound and/or therapeutic agent, or agent of the invention to a subject in need of treatment. Administration may be via any appropriate route, depending on the type of therapeutic.

[0088] In one aspect, an agent that inhibits CCL5/CCR5 signal transduction may be coadministered with known chemotherapeutic agents, including but not limited to, Aclacinomycins, Actinomycins, Adriamycins, Ancitabines, Anthramycins, Azacitidines, Azaserines, 6-Azauridines, Bisantrenes, Bleomycins, Cactinomycins, Carmofurs, Carmustines, Carubicins, Carzinophilins, Chromomycins, Cisplatins, Cladribines, Cytarabines, Dactinomycins, Daunorubicins, Denopterins, 6-Diazo-5-Oxo-L-Norleucines, Doxifluridines, Doxorubicins, Edatrexates, Emitefurs, Enocitabines, Fepirubicins, Fludarabines, Fluorouracils, Gemcitabines, Idarubicins, Loxuridines, Menogarils, 6- Mercaptopurines, Methotrexates, Mithramycins, Mitomycins, Mycophenolic Acids, Nogalamycins, Olivomycines, Peplomycins, Pirarubicins, Piritrexims, Plicamycins, Porfiromycins, Pteropterins, Puromycins, Retinoic Acids, Streptonigrins, Streptozocins, Tagafurs, Tamoxifens, Thiamiprines, Thioguanines, Triamcinolones, Trimetrexates, Tubercidins, Vinblastines, Vincristines, Zinostatins, and Zorubicins.

[0089] The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE 1

HIF-1 a/RANTES-Driven Pathway to Hepatocellular Carcinoma Mediated by Germline

Haploinsufficiency of SART1/HAF in Mice

[0090] The hypoxia-inducible factor (HIF), HIF-1, is a central regulator of the response to low oxygen or inflammatory stress and plays an essential role in survival and function of immune cells. However, the mechanisms regulating non-hypoxic induction of HIF-1 remain unclear. Here, the impact of germline heterozygosity of a novel, oxygen-independent ubiquitin ligase for HIF-Ια: hypoxia-associated factor (HAF; encoded by SART1), is assessed. SARTT ^" mice were embryonic lethal, whereas male SART1^{+ 1 '} mice spontaneously recapitulated key features of nonalcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC), including steatosis, fibrosis, and inflammatory cytokine production. Male, but not female, SART1^{+ / "} mice showed significant up-regulation of HIF- la in circulating and liver-infiltrating immune cells, but not in hepatocytes, before development of malignancy. Additionally, Kupffer cells derived from male, but not female, SART1^{+ 1 '} mice produced increased levels of the HIF-1 -dependent chemokine, regulated on activation, normal T-cell expressed and secreted (RANTES), compared to wild type. This was associated with increased liver-neutrophilic infiltration, whereas infiltration of lymphocytes and macrophages were not significantly different. Neutralization of circulating RANTES decreased liver neutrophilic infiltration and attenuated HCC tumor initiation/growth in SART1^{+ 1 '} mice. Conclusion: This work establishes a new tumor- suppressor role for HAF in immune cell function by preventing inappropriate HIF-1 activation in male mice and identifies RANTES as a novel therapeutic target for NASH and NASH-driven HCC.

[0091] MATERIALS AND METHODS

[0092] GENERA TION OF MICE

[0093] SART1^{+ 1 '} mice were derived from the Texas Institute for Genomic Medicine gene trap C57BL/6 SARTl^{" "} ES cell line (Clone IST11321E11). Genotyping was performed using allele-specific polymerase chain reaction PCR (Supplemental Methods). All animal procedures were performed in accord with institutional animal care and use protocols. [0094] GENE EXPRESSION ANALYSES

[0095] Differential gene expression profiling of S ART In versus wild-type (WT) livers (three replicates each) were performed using the GeneChip Mouse Genome 430 2.0 Array™ and the 3 ' IVT PLUS™ reagent kit (P/N 902416; Affymetrix, Santa Clara, CA): Gene Expression Omnibus™ accession GSE71057. Data analysis was performed using Transcriptome Analysis Console™ 2.0, which generated the heatmaps, and Ingenuity Pathway Analysis™ (IP A; Qiagen Inc., Valencia, CA). Hit validation was performed by Taqman™ custom arrays (Life Technologies, Carlsbad, CA).

[0096] HUMAN LIVER SECTIONS

[0097] Human HCC (of unknown etiology) was purchased from US Biomax Inc (Rockville, MD). Additional sections were from formalin-fixed, paraffin-embedded nonalcoholic steatohepatitis (NASH)-associated HCC tumors collected during surgical resections and liver transplants (normal liver samples) at the Mayo Clinic between 1997 and 2013. Sections were examined by a pathologist, graded histologically, and classified by etiology. The study was approved by the Mayo Clinic Institutional Review Board.

[0098] MAGNETIC RESONANCE IMAGING AND REGULATED ON ACTIVATION, NORMAL T-CELL EXPRESSED AND SECRETED STUDY

[0099] Magnetic resonance imaging (MRI) was performed by operators blinded to study design using a Bruker 11.7T™ MRI system (Supporting Methods). After initial imaging, tumor-bearing mice were stratified into two groups and injected intraperitoneally with 400 μg of regulated on activation, normal T-cell expressed and secreted (RANTES)-neutralizing antibody (MAB478;R& D systems, Minneapolis MN) or saline twice per week for 5 weeks, after which mice were reimaged and tumor volumes were recalculated.

[00100] STA TISTICAL ANALYSES

[00101] These were performed using the Student t tests using GraphPad Prism™ (version 6; GraphPad Software Inc., La Jolla, CA) or Microsoft Excel™ software (Microsoft Corporation, Redmond, WA). P < 0.05 were considered significant with *P < 0.05 and **P < 0.01.

[00102] SUPPLEMENTAL METHODS

[00103] PCR PRIMERS FOR GENOTYPING

[00104] Wild-type gene: SARTl wt F/R (896 bp product in wild-type and heterozygous mice): GAAACGCGATGACGGCTACGAGG (SEQ ID NO: 1) /

CCTATGGGCAGACTGTGGGTTCC (SEQ ID NO:2)

[00105] LacZ disruption: V76 F/ SART1 wt R (het/ko yields product 578 bp - no product in wild-type mice):

CTTGCAAAATGGCGTTACTTAAGC (SEQ ID NO:3) /

CCTATGGGCAGACTGTGGGTTCC (SEQ ID NO:4)

[00106] CELL LINES AND SIRNA TRANSFEC TIONS

[00107] Huh7 human hepatoma cells and THP-1 human monocyte cells were purchased from Japanese Collection of Research Bioresources (JCRB, NIBIO Osaka, Japan) and American Type Culture Collection (ATCC, Manassas VA) respectively and verified by STR fingerprinting. Huh7 and THP-1 cells were transfected with siRNA using Lipofectamine™ 2000 (Invitrogen, Life Technologies, Grand Island NY) or Dharmafect™ 4 (GE Dharmacon Lafayette, CO) respectively according to the manufacturer's protocol. THP-1 cells were differentiated with 25ng/ml phorbol myristate acetate (PMA, Sigma - Aldrich, St. Louis MO) 24 hours prior to siRNA transfection. Both cell lines were transfected with siRNA 72 hours prior to assay. HAF (SARTI) siRNA was ON-Targetplus SMARTpool™ (Dharmacon L-017283, siHAF or siHAF l), and Hs_SARTl_3 (siHAF_2, Qiagen, Germantown MD). Control siRNA (siCon) was si GENOME™ Non-targeting siRNA#3 (Dharmacon). All siRNAs were transfected at a final concentration of 40nM.

[00108] WESTERN BLOTTING (WB) AND IMMUNOHISTOCHEMISTRY (IHC)

[00109] HIF-la/CD68 dual staining was performed using Chromoplexl Dual™ detection (DS 9477, Leica Biosystems, Buffalo Grove IL). IHC for HIF-la and HAF was performed as previously described. IHC antibodies for Ly6G, Cd68 and RANTES were from BD Pharmingen (#559286 BD Biosciences San Jose CA), Novus Biologicals (NB 100-2086 Littleton, CO) and Abeam (#ab9679, Cambridge, MA) respectively, whereas HIF-la and HIF-2a antibodies used in mouse tissue were from Novus (NBlOO-479, NBlOO-122). WB was performed using Acsll, Cyp2bl0, actin and HAF from Cell signaling Technology (4047S, Danvers MA), EMD Millipore (AB9916), Santa Cruz Biotechnology Inc. (1-19, Dallas, TX), or made-in house respectively.

[00110] GENE EXPRESSION ANALYSIS

[00111] Hit validation was performed by Taqman™ custom arrays (Life Technologies, Carlsbad CA) according to the manufacturer's protocol using the following predesigned primer/probe sets: B2m-Mm00437762_ml; Srebfl-Mm00550338_ml; Pparg- Mm01184322_ml; Mlxipl-Mm02342723_ml; Ppara-Mm00440939_ml; Acoxl- Mm01246831_ml; Acsll-Mm00484217_ml; Acaala-Mm00728460_sl; Pkm- Mm00834102_gH, Hkl-Mm00439344_ml; Slc2al-Mm00441480_ml; Ccl5- MmO 1302427_m 1 ; Sart 1 -Mm00600274_m 1.

[00112] All measurements were performed in duplicate per mouse with at least 2 mice/group (wild-type and SART1+/-) for each age group. Data were normalized using β2- microglobulin (B2M) as the internal reference by the ΔΔ-CT method (3) and GeneAmp 5700 SDS™ software (Life Technologies).

[00113] KUPFFER CELL (KC) ISOLA TIONAND CYTOKINE ANALYSIS

[00114] KCs were isolated using in vitro collagenase incubation following liver perfusion as previously described. After Percoll purification and RBC lysis, cells were washed twice in RPMI 1640™ media (Life Technologies) containing 10% FBS, and seeded at a density of 106 cells in 6-well plates. After washing to remove non-adherent cells, cells were left to recover for 24-36 hours, after which media was changed for an additional 6 hours. Media was then removed for cytokine analysis using the Mouse Cytokine Array™ C3 (Raybiotech Inc, Norcross GA) according to manufacturer's protocol. Films were scanned using the CanoScan 9000 scanner and quantitated by Raybiotech using in-house analysis software.

[00115] SEAHORSE METABOLIC ANALYSIS

[00116] Hepatocytes were isolated using proprietary Liver Perfusion and Liver Digest Medium™ (Life Technologies) according to the manufacturer's protocol. Purified hepatocytes were seeded at 8000 cells/well in XF96 cell culture microplates (Seahorse Bioscience, MA) pre-coated with rat tail Collagen I (12^g/cm2, Geltrex™), starved overnight, then run on the XFe96 extracellular flux analyzer the next day according to the manufacturer protocol. Huh7 cells (from JCRB, confirmed by STR fingerprinting), were reverse transfected with HAF or non-targeting siRNA (L-017283-00-0005, D-001210-05- 20; GE Dharmacon, Lafayette CO) using Dharmafect 4™ and seeded at 6500 cells/well in an XF96 cell culture microplate. OCR analysis was performed 72 hours after siRNA transfection.

[00117] ISOLATION OF MONONUCLEAR CELLS

[00118] PBMCs and spleen mononuclear cells were prepared by standard protocols (5). For induction of HIF-la, spleen cells were seeded at 4E6 cells/well 6-well plates and exposed to hypoxia (1% 0₂. 5% C0₂, InVivo2400, Baker Ruskinn Sanford ME) for 2 hours prior to lysis. PBMCs were lysed immediately after purification. [00119] FLOW CYTOMETRY

[00120] Cells were washed in FACS buffer (2% FBS in PBS), stained for 30 minutes on ice, then washed twice in FACS buffer, and analyzed on the FACSCanto™ (BD Biosciences, San Jose CA) flow cytometer using 488 nm and 633 mil excitation. Antibodies used were from Ebiosciences as below.

[00121] Mouse liver profiling: Ly6G-FITC, CDl lc-PE, CD3-APC, TCRgd-PE Cy7, CD45-Pacific Blue, CD 19 Alexa fluor700.

[00122] Spleen profiling: TCRp-PECy7, CD19-PE, F4/80-APC,Ly6G-APC Cy7, TCRy5- PE, CDl lc-APC, 7AAD-APCefiuor780.

[00123] LacZ activity was quantitated using the Fluoreporter LacZ™ kit (Life Technologies F-1930) according to manufacturer's protocol.

[00124] MRI PROCEDURES

[00125] During the MRI procedures, anesthesia was maintained using a 1-2% isoflurane- in-oxygen mixture. Body temperature was maintained using a thermally regulated water heating blanket. After standard MRI preparation (optimization of shimming, pulse power calibration, scout images to locate the tumor), a respiratory-gated, T2-weighted, rapid acquisition with relaxation enhancement (RARE) sequence was acquired with repetition time=2.2s, echotime=30ms, and 4 averages. A 25x35mm field of view with a 125x175 matrix was used with 24, contiguous 1.25mm-thick, transaxial slices that covered the entire liver tumor volume. Images were analyzed using Amira 5.5.0. Individual tumor nodules were discerned based on their separation in 3D space from neighboring tumor nodules. All tumor growth in the liver was manually segmented on each slice in which tumor growth was resolved using the Amira Segmentation Editor. The presence of hepatic tumor nodules was confirmed grossly at necropsy, which was performed 48 hours after imaging, and by histopathologic examination of H&E stained sections.

[00126] RESULTS

[00127] SART1 DEFICIENCY IS EMBRYONIC 'ALLY LETHAL AT Ell.5

[00128] HAF heterozygous (het) mice (SART1^+/") were generated using C57BL/6 embryonic stem cells produced by the gene trap method (Figure 1 A). Tissue-wide profiling of male WT and SARTl^+/ mice confirmed expression of HAF in multiple organs, including skin, lung, heart, liver, kidney, spleen, and colon, with highest expression within the spleen (data not shown). HAF heterozygosity was associated with significant decreases of HAF in heart, liver, and kidney and caused more subtle decreases in other tissues in which HAF could be detected (Figure IB).

[00129] Of a total of 99 pups resulting from pairings with heterozygous parents, 70 were het, whereas 29 were WT, close to the expected ratio of 2: 1 : 1 for het; for WT, knockout was consistent with embryonic lethality. By genotyping embryos at various developmental stages, knockouts were only identified at El 1.5, and SARTl^7" embryos showed diminished size and vascularization compared to their het or WT littermates (data not shown). Thus, knockout of SART1 results in embryonic lethality at approximately El 1.5.

[00130] SART1 HAPLOINSUFFICIENCY PROMOTES HEPATIC STEATOSIS AND HCC

[00131] Despite the embryonic lethality of HAF knockout mice, mice heterozygous for HAF (SART1^+/") appeared to develop normally and displayed no anatomically observable defects. However, at >14 months, male SART1^+/" mice were significantly smaller and had ruffled fur compared to their WT counterparts (data not shown). Necropsy revealed multifocal large liver tumors (histologically confirmed as HCC) in -83% (5 of 6) of male SART1^+/" mice, which were absent from their WT littermates (data not shown), whereas both WT and SART1^+/" male mice exhibited severe hepatic steatosis (HS). Livers of the SART1^+/" mice displayed hallmarks of NASH-driven HCC, including HS (confirmed by Oil Red O staining), fibrosis (confirmed by Sirius Red staining), and foci of immune cell infiltration (data not shown). Visual analysis of additional male SART1+/- mice confirmed grossly visible tumors in an additional 90% (9 of 10) of mice. By contrast, livers of age- matched SART1^+/" females showed minimal steatosis, and no malignant tumors were detected either grossly or histologically (0 of 4, not shown). At age 10 months, HCC was detected in 42% (5 of 12) of mice, whereas preneoplastic foci of hepatocellular alterations and/or adenomas were present in 83% (10 of 12) of mice. By contrast, none were observed in WT littermates (data not shown). Consistent with enlarged livers and decreased overall body weight, average liver weights/body weight ratios in SART1^+/" mice were also significantly higher than their WT counterparts (Figure 2, Rapp-Hodgkin syndrome [RHS]). Liver tumors were detected by MRI in a further 57% (4 of 7) of male SART1^+/" mice ages 16-18 months (data not shown), but were not detected in male mice under 10 months (0 of 21) nor in female SART1^+/" mice ages 16-19 months (0 of 3). Rapid growth of the liver tumors was apparent when tumor growth was monitored in one 16- to 18-month-old mouse, where multiple large liver tumor nodules (data not shown) confirmed histologically as HCC were found. No other obvious abnormalities were observed in blood or any of the other major organs apart from age-related changes. Hence, germline haploinsufficiency of HAF promotes spontaneous HCC development only in male mice >age 10 months.

[00132] HS PRECEDES HCC FORMA TION IN LIVERS OF SART1 ^+/~ MICE

[00133] When examined histologically, livers of 1-month-old SART1^+/" mice were indistinguishable from those of their WT littermates (data not shown). However, at age 6 months, SART1^+/" livers showed moderate to marked microvesicular HS with increased severity in the centri-lobular/periacinar and midzonal regions and also showed multiple foci of altered hepatocytes with atypical nuclei (Figure 3A). These foci were composed of hypertrophic or cytomegalic hepatocytes and showed increased proliferation supported by the increased number of mitotic and binucleated hepato-cytes. Although these foci are considered preneoplastic lesions predisposing to tumor development, no neoplastic lesions were observed at this age. By contrast, livers of WT littermates showed only very mild steatosis and no preneoplastic lesions. Consistent with the age-related progression to HCC, elevation of the serum transaminases, alanine aminotransferase, aspartate aminotransferase, or alkaline phosphatase, in SART1^+/" mice versus WT become apparent from 6 months of age (data not shown). The age-associated liver phenotypes of SART1^+/" mice are summarized in Figure 3B. Intriguingly, although HAF levels in SART1+/- versus WT livers were not significantly different at age 1 month, HAF levels decreased with increasing age, which reflected development of liver pathology in SART1^+/" mice (Figures 3C-3D). Hence, although initially normal, livers of SART1^+/" mice first manifest foci of preneoplastic hepatocellular alteration, coinciding with development of HS, which then progress to HCC.

[00134] MICROARRAY ANALYSIS OF SART1^+/- MOUSE LIVERS INDICATE METABOLIC AND IMMUNE DYSFUNCTION

[00135] To evaluate global changes in gene expression associated with HCC in SART1^+/" mice, microarray analysis was performed comparing a SART1^+/" liver tumor (T) to an age- matched WT liver (N). Using a fold-change cutoff of 2.0-fold up- or down-regulated, and P < 0.05, 2,290 genes were differentially expressed in T versus N groups of 20, 185 total genes probed by the array (Figure 3E). Intriguingly, the top differentially expressed gene was alpha-fetoprotein (441 -fold induction vs. normal), which encodes a blood serum marker typically used for diagnosis of HCC in humans (data not shown). Using IP A, the top biological functions associated with these differentially expressed genes were those involved in the inflammatory response, cancer, and lipid metabolism (data not shown). Highly significant changes were also observed in genes associated with hepatocyte hyperplasia/proliferation, steatosis, and HCC. All these expression changes were consistent with the observed steatosis/ steatohepatitis/HCC phenotype in SART1^+/" livers.

[00136] SART1^+/- HEPATOCYTES HAVE DEFECTIVE FATTY ACID OXIDATION

[00137] To investigate the molecular pathogenesis of HS in SART1^+/" mice, expression of subsets of genes known to be involved in the various aspects of lipid metabolism were examined. According to IPA, 39 of 45 genes involved in fatty acid metabolism were significantly decreased (P = 6.86E-13; Figure 3E). This included acyl-CoA synthase long- chain family member 1 (Acsll), which catalyzes one of the first steps of fatty acid oxidation (FAO), and a large number of cytochrome P450 (CYP) genes, which have diverse roles in liver metabolism. By contrast, levels of the major transcription factors that promote lipogenesis and lipid uptake, sterol regulatory element-binding protein-lc (SREBP-lc, SREBF1), carbohydrate response element-binding protein (MLXIPL), and peroxisome proliferator-activated receptor gamma (PPAR-γ, PPARG), were not significantly altered in the data set, nor were their downstream gene expression profiles significantly altered. The trend of decreased expression of genes involved in FAO was validated using 3 additional SART1^+/" livers normalized to livers from 3 WT and was also confirmed at the protein level for Acsll and CYP2 0 (Figure 3F).

[00138] To determine whether FAO was indeed dysfunctional in SART1^+/" mice, hepatocytes from 4-month-old SART1^+/" mice and their WT littermates were subjected to metabolic analysis using the Seahorse XF Extracellular Flux™ analyzer (Seahorse Bioscience, North Billerica, MA). Using oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, it was found that SART1^+/" hepatocytes showed a lower basal level of respiration compared to WT (Figure 4A). When treated with the mitochondrial uncoupler, p-triflouromethoxyphenylhydrazone (FCCP), which induces maximal respiration, SART1^+/" hepatocytes showed an almost 4-fold decrease in the ability to utilize exogenous palmitate compared to WT hepatocytes (Figures 4A-4B). Similar results were obtained using Huh7 human hepatoma cells transiently transfected with control or HAF (SARTl) small interfering (si)RNA (Figures 4C-4D). Thus, the enhanced HS observed in the SART1^+/" livers is likely attributable to defective FAO.

[00139] SART1^+/- MICE SHOW MARKED UP-REGULA TION OF HIF-la IN IMMUNE CELLS [00140] In addition to metabolic dysfunction, SART1 ^" livers showed changes consistent with increased inflammatory cell activation and trafficking (Figure 3E). Indeed, SART1^+/" livers showed extensive immune cell infiltration, distributed randomly and sporadically, often in clusters, throughout liver parenchyma (data not shown). These included cells positive for F4/80 or Ly6G, suggesting that these clusters contained macrophages and neutrophils respectively (data not shown). Additionally, Kupffer cell (KC) hyperplasia (likely a result of phagocytosis of disrupted hepatocytes) was observed in 100% (6 of 6) of the SART1^+/" livers, but not in WT livers (data not shown). Significantly, liver-infiltrating immune cells were markedly HIF-la positive in SART1^+/", but not in WT, livers. Increases in HIF-la was manifest primarily in SART1^+/" immune cells, but not in SART1^+/" hepatocytes, suggesting that HIF-la up-regulation was not attributable to a general hypoxic environment within livers (data not shown). The changes observed in SART1^+/" immune cells may also reflect distribution of HAF expression, which was detected primarily in infiltrating immune cells in WT livers (data not shown). By contrast, HIF-2a was expressed in the immune infiltrating cells in livers of both WT and SART1^+/" mice (data not shown).

[00141] To establish whether HIF-la up-regulation played a causal role in HCC carcinogenesis in SARTl^+/~ mice, or occurred as a result of the HCC already present, the inventors isolated mononuclear cells from peripheral blood (PBMCs) and spleens of 6- month-old SARTl^+/~ and WT mice. HIF-la protein levels were significantly elevated in both PBMCs and splenocytes from SARTl^+/~ versus WT mice, but not in primary hepatocytes (Figure 5A). The inventors also noted that unlike PBMCs, splenocytes did not express detectable HIF-la unless exposed to hypoxia (only cells exposed to hypoxia are depicted), suggesting some intrinsic differences in HIF-la expression in these cell types. These findings were associated with a reduction in HAF protein levels expected of the SARTl^+/~ genotype (data not shown). By contrast, the inventors did not detect up-regulation of HIF-la in PBMCs or splenocytes isolated from 6-month-old female SARTl^+/~ mice and only detected subtle decreases in HAF levels (data not shown). Hence, the data suggest that HIF-la up-regulation in immune cells was intrinsic to male SARTl^+/~ mice and occurred independently of HCC.

[00142] To determine whether HAF expression in immune cells was cell-type specific, the inventors measured activity of the LacZ reporter transgene used to disrupt the SART1 gene. The inventors detected LacZ positivity in a wide variety of cell populations, including spleen- and bone-marrow-derived cells and peripheral blood leukocytes (Fig 5B). To identify cell types that expressed LacZ (and therefore endogenous HAF), the inventors stained splenocytes with surface markers CD 19 (B cells), TCRb (T cells), Ly6G (neutrophils), and F4/80 (macrophages) before the Lac Z activity assay. Here, the inventors observed significant LacZ/FITC (fluorescein isothiocyanate) positivity in all cell types, with highest expression in B and T cells and macrophages, whereas no significant differences between immune cell populations from SART1^+/" and WT mice were observed (Figure 5C). Thus, HAF is highly expressed in all immune cells, and loss of HAF in male SART1^+/" mice is associated with HIF-la up-regulation in all immune cell types.

[00143] HCC IS PRECEDED BY INCREASED RANTES SECRETION AND ELEVATED NEUTROPHILIC INFILTRATION

[00144] To determine whether immune cells played a causal role in development of HCC, the inventors isolated KCs from livers of 5-month-old SART1^+/" mice and compared their secreted cytokine expression profiles to that of WT littermates. In general, SART1^+/" KCs had higher levels of secreted cytokines than WT KCs (Figure 5D). Intriguingly, levels of the chemokine, RANTES, was increased by > 100-fold in SART1^+/" KCs compared to WT KCs (Figure 5D). RANTES plays a major role in recruiting leukocytes to sites of injury and is transcriptionally regulated by HIF-Ια by multiple functional HREs within its promoter. Knockdown of HAF using siRNA in THP-1 cells also resulted in significant up-regulation of RANTES in both normoxia and hypoxia (Figure 5E). RANTES was significantly up- regulated in livers of SART1^+/" mice over WT mice from 6 months of age, although increased transcription was already detected at 1 month of age (data not shown). Consistent with HIF-la up-regulation, the inventors also detected an age-dependent, albeit delayed, increase (compared to RANTES) in transcription of HIF-1 target genes facilitated glucose transporter 1 (Slc2al), hexokinase 1 (Hkl), and pyruvate kinase (muscle) 1 (Pkml) in SART1^+/" livers, indicating a general shift toward glycolytic metabolism. By contrast, the inventors did not observe any differences in secretion of RANTES or other cytokines by KCs from similar-aged female SART1^+/" livers compared to WT (data not shown).

[00145] To determine whether immune cell subpopulations present within SART1^+/" livers were altered before HCC initiation, the inventors profiled livers of 66-month-old mice. Intriguingly, the inventors found a 6-fold increase in Ly6G⁺ neutrophils in SART1^+/" livers relative to WT, also observed by immunohistochemistry (IHC), whereas levels of CDHcl macrophages, CD4, CD8, gamma delta, and natural killer (NK) T cells were not significantly altered (data not shown). The inventors also observed a 3 -fold increase in Ly6G⁺ neutrophils within livers of 1-month-old SART1^+/" mice, which was not observed in spleens, suggesting that increased neutrophilic infiltration was an early event independent of HS and was not attributable to systemic elevation of the neutrophil population (data not shown). Thus, male, but not female, SART1^+/" KCs secrete elevated levels of cytokines/chemokines, including a > 100-fold increase in RANTES. This was associated with increased neutrophilic infiltration in male SART1^+/" livers before development of steatosis or HCC.

[00146] RANTES NEUTRALIZATION INHIBITS HCC INITIATION/ GROWTH AND DECREASES NEUTROPHIL INFILTRATION

[00147] To determine the impact of RANTES secretion on liver neutrophilic infiltration, and HCC initiation/progression, the inventors used a neutralizing antibody to RANTES and monitored treatment efficacy by MRI. Imaging of SART1^+/" mice ages 14-18 months revealed that only 4 of 15 mice bore tumors with a total volume of >100 mm³ clearly detectable by MRI (data not shown). Mice were treated with RANTES-neutralizing antibody (or saline) for 5 weeks and reimaged. Upon reimaging, the inventors observed the appearance of new nodules in the control group, but not in the RANTES-treated group, suggesting that RANTES treatment prevented liver tumor initiation/ promotion (data not shown). Growth of individual nodules was also markedly reduced in RANTES-treated mice (RANTES 6.709 ± 3.872 fold [n = 5] vs. control 13.20 ± 6.546 fold [n = 5]). The inventors also observed a significant decrease in Ly6G⁺-infiltrating neutrophils in both tumor and adjacent normal tissue of mice treated with RANTES compared to those treated with saline alone (data not shown). Hence, RANTES neutralization inhibits both initiation/promotion and growth of liver tumors and attenuates neutrophilic infiltration in livers of SART1^+/" mice.

[00148] HUMAN HCC SHOW UP-REGULATION OF HIF-loJRANTES AND HAF DO WN-REGULA TION

[00149] Similar to SART1^+/" livers (data not shown), HIF- la-positive immune infiltrating cells in the context of largely HIF-la-negative hepatocytes was also observed in human HCC, including NASH-derived HCC (data not shown). RANTES expression in both hepatocytes and immune infiltrating cells was also up-regulated in NASH-derived HCC compared to normal liver (data not shown). By contrast, HAF staining was detected primarily in infiltrating cells in normal human liver, but was largely negative in human NASH-derived HCC (data not shown), thus supporting a role for HAF as a tumor suppressor for HCC.

[00150] DISCUSSION

[00151] HCC is the most common primary malignancy of the liver, with more than 750,000 new patients diagnosed globally each year. HCC frequently develops in the context of chronic hepatitis, which may arise from infection with hepatitis B or hepatitis C virus (HCV), and from alcoholic or nonalcoholic fatty liver disease (NAFLD). The current obesity epidemic has been associated with an increasing prevalence of NAFLD and its inflammatory component, NASH, which leads to HCC. The complex interplay between damaged hepatocytes and immune infiltrating cells within an inflammatory hepatic microenvironment is believed to drive HCC initiation and progression. Hence, HCC pathogenesis has been described by the "two-hit" hypothesis whereby the first hit— steatosis/ viral infection— sensitizes the liver to a variety of second hits, such as oxidative stress and abnormal cytokine production, which lead to necroinflammation and fibrosis.

[00152] Here, the inventors show that germline haploinsufficiency of SART1 induces HCC in the context of NASH only in male mice. This is the only genetic model where haploinsufficiency promotes HCC without requiring any further manipulation. Livers of SART1^+/" mice showed decreased FAO, providing a likely mechanism for HS (or first hit), and also showed constitutive stabilization of HIF-la, specifically in immune cells, elevated cytokine secretion by KCs, and increased neutrophilic infiltration before HCC development (together constituting the second hit). Activation of these immune cells likely promotes oxidative damage and inflammation, driving progression to plethora of inflammatory cytokines up-regulated in HCC. Although infiltrating immune cells are known human HCC have confounded the identification of to be involved in HCC initiation and progression, the individual key cytokines to target for therapy. The robust timeline for HCC development in the male SART1^+/" mice enabled identification of RANTES as an early, non-redundant event driving HCC initiation and progression, likely by promoting neutrophil infiltration. Intriguingly, increased circulating RANTES has been associated with obesity in humans and is up-regulated in patients with NAFLD/ NASH, suggesting that RANTES might be involved in the progression to NASH-driven HCC. The evaluation of the relevance of HIF- 1/RANTES to NASH-driven HCC is currently being investigated in a larger patient set. Intriguingly, RANTES elevation has also been observed in patients with alcoholic and viral hepatitis, whereas RANTES polymorphisms correlate to susceptibility to HCC, suggesting that RANTES may be a valid therapeutic target for HCC regardless of etiology.

[00153] An intriguing observation in the studies was that HCC did not occur in female SART1^+/" mice. This male predominance also occurs in human HCC and in chemically induced HCC in mice (with the hepatic procarcinogen, N-nitrosodiethylamine [DEN]). The male bias for DEN-induced HCC has been attributed to the suppressive effect of estrogen on interleukin (IL)-6 production, a driving factor for DEN-induced HCC. Whether the male bias observed in the model is related to IL-6 production is unclear, but it does appear to be associated with an intrinsic resistance of both WT and SART1^+/" female mice to HS, an initiating factor for HCC in male SART1^+/" mice. Female SART1^+/" and WT mice, even at >16 months of age, develop only mild HS, if at all, and do not become obese like their male counterparts (unpublished observations). The age-dependent decrease in HAF levels in male SART1^+/" mice, which was accompanied by immune-cell-specific up-regulation of HIF-la, was not observed in female mice, suggesting that the predisposition to steatosis in males may drive further decreases in HAF, hence exacerbating development of liver pathology (Figs. 3C and 5 A). Additional factors driving the male bias may be the sex-dependent production of RANTES (Figure 5D), which has been previously reported in obese mice. Nevertheless, steatosis per se is insufficient to induce HCC because germline VHL+/ mice, or mice bearing hepatocyte-specific VHL deletion, develop severe HS, but not HCC. Because VHL is unable to degrade HIF-la under hypoxic conditions, it is possible that HAF, as an oxygen-independent ubiquitin ligase, may play a more central role in attenuating inappropriate HIF-la up-regulation under hypoxic, inflammatory conditions. Indeed, the high expression levels of endogenous HAF within mouse spleen and in immune infiltrating cells (Figs. IB) supports an immune-specific physiological role of HAF as a suppressor of HIF-la. In view of the systemic activation of immune mediators in male SART1^+/" mice, the inventors also observed increased lymphohistiocytic infiltration in lungs, gastrointestinal tract, and lymph nodes of SART1^+/" mice compared to WT (data not shown). Hence, it is likely that other organ-specific stresses may lead to inflammation- induced cancer in these organs, and this is currently under investigation. In addition to its role as a ubiquitin ligase for HIF-la, HAF has also been known to play a role in pre- messenger RNA splicing and inhibit HCV replication in hepatoma cells by changes in splicing. Hence, splicing changes in the SART1^+/" mouse are also currently being explored. In conclusion, this study identifies a novel tumor-suppressor function of HAF by maintaining regular hepatic metabolism, and in preventing inappropriate immune cell activation, possibly by suppressing HIF-Ια. Additionally, the inventors described a novel mouse model of NASH-derived HCC, which closely recapitulates the human disease. These findings highlight a central role of the HIF-l/RANTES axis in HCC initiation and progression, thus identifying a novel target for therapy.

EXAMPLE 2

Treatment Studies

[00154] Having discovered this critical role for CCL5/RANTES in HCC pathogenesis, the following studies will be performed to validate the targeting of the RANTES/CCL5/CCR5 axis as a therapeutic target for HCC.

[00155] PREVENTION STUDIES

[00156] Proof-of concept studies will be performed using commercially available CCR5 antagonists including maraviroc, clinically approved for the treatment of patients with HIV infection. The efficacy of maraviroc will be monitored in inhibiting tumor initiation in the HCC GEM mouse model the inventors have developed. In mice this will be monitored by measuring levels of blood liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALK), and non-invasive measurement of tumor size using ultrasound.

[00157] TREA TMENT OF ESTABLISHED HCC

[00158] Proof-of concept studies will be performed using commercially available neutralizing anti-RANTES/CCL5 or CCR5 antibodies for the treatment of established HCC in the HCC mouse model described herein, and other HCC models described below. Novel, neutralizing antibodies for RANTES/CCL5/CCR5 preferably using epitopes of high homology between the human and mouse proteins and amenable for humanization will be generated for future clinical use. When these are available, proof-of-concept studies will be performed for the treatment of established HCC in the mouse models described below. The ability of antibodies to ameliorate the symptoms associated with each of the models described will be tested using non-invasive methods such as liver ultrasound and/or MRI, and non-terminal measurement of blood liver enzymes AST, ALT and ALK. Terminal histological analysis for liver tissue will be performed at the end of the studies to identify effects of treatment on tumor size, and other liver dysfunction such as steatosis, steatohepatitis and fibrosis.

[00159] TREA TMENT OF HEP A TIC DYSFUNCTION AND INFLAMMA TION [00160] The studies described herein will be expanded to include other inflammatory or liver metabolism-related dysfunction such as alcoholic and non-alcoholic hepatic steatosis, steatohepatitis and cirrhosis, common manifestations of alcoholic, or non-alcoholic fatty liver disease in humans.

[00161] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A method of treating or preventing cancer in a subject comprising administering to the subject a therapeutically effective amount of an agent which inhibits CCL5/CCR5 signal transduction, thereby treating or preventing cancer in the subject.

2. The method of claim 1, wherein the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer.

3. The method of claim 1, wherein the cancer is hepatocellular carcinoma.

4. The method of claim 1, wherein the agent inhibits CCL5 binding to CCR5.

5. The method of claim 1, wherein the agent is a CCR5 antagonist.

6. The method of claim 1, wherein the agent inhibits or reduces CCL5 expression or activity.

7. The method of claim 1, wherein the agent is a small molecule, a peptide, a nucleic acid, or a combination thereof.

8. The method of claim 7, wherein the agent is a nucleic acid selected from the group consisting of siRNA, shRNA, miRNA, locked nucleic acid (LNA), antisense oligonucleotide, chemically modified oligonucleotide, and combinations thereof.

9. The method of claim 1, wherein the agent is an antibody, or fragment thereof.

10. The method of claim 9, wherein the antibody, or fragment thereof, specifically bind to CCL5 and blocks binding of CCL5 to CCR5.

11. The method of claim 9, wherein the antibody, or fragment thereof, specifically bind to CCR5 and blocks binding of CCL5 to CCR5.

12. The method of claim 9, wherein the antibody is selected from the group consisting of a single chain antibody, a monoclonal antibody, a bi-specific antibody, a chimeric antibody, a synthetic antibody, a polyclonal antibody, a humanized antibody, a fully human antibody, and active fragments or homologs thereof.

13. The method of claim 7, wherein the agent is conjugated to another molecule or structure.

14. The method of claim 13, wherein the molecule or structure is selected from the group consisting of an antibody, a protein, a pro-drug, a drug, a toxin, a protein toxin, a liposome, a radioactive isotope, and an enzyme.

15. The method of claim 1, wherein the agent is soluble CCR5.

16. The method of claim 1, further comprising administering to the subject a chemotherapeutic agent.

17. The method of claim 1, further comprising detecting expression or activity of CCL5.

18. A method of diagnosing a subject as having, or at risk of having, hepatocellular carcinoma, comprising:

a) obtaining a sample from the subject;

b) detecting the presence or expression level of CCL5 in the sample; and

c) diagnosing the subject as having, or at risk of having, hepatocellular carcinoma when the presence or expression level of CCL5 in the sample is elevated as compared to a corresponding normal sample.

19. The method of claim 18, further comprising administering to the subject a chemotherapeutic agent or a therapeutically effective amount of an agent which inhibits CCL5/CCR5 signal transduction.

20. A method for determining susceptibility of a subject to a therapeutic regime to treat hepatocellular carcinoma, or monitoring progression of hepatocellular carcinoma in a subject comprising:

a) detecting the presence or expression level of CCL5 in a sample from the subject; and

b) assessing the therapeutic regime or hepatocellular carcinoma progression based on (a),

thereby determining susceptibility of a subject to a therapeutic regime to treat hepatocellular carcinoma, or monitoring progression of hepatocellular carcinoma in a subject.

21. The method of claim 20, wherein the therapeutic regime comprises administration of a chemotherapeutic agent or an agent which inhibits CCL5/CCR5 signal transduction.

22. The method of claim 20, further comprising characterizing the subject as not susceptible to the therapeutic regime when the presence or expression level of CCL5 in the sample is elevated as compared to a corresponding normal sample or a reference sample from the subject.

23. A transgenic mouse whose genome comprises a heterozygous disruption of the squamous cell carcinoma antigen recognized by T-cells 1 (SART1) gene, wherein the mouse exhibits decreased expression of SART 1(800) and SART 1(259).

24. The transgenic mouse of claim 23, wherein the mouse exhibits liver neutrophilic infiltration and develops hepatocellular carcinoma upon growth.

25. A method for identifying an agent for preventing or treating cancer, comprising contacting the transgenic mouse of any of claims 23-24 with a test agent and monitoring tumor growth or liver neutrophilic infiltration in the mouse, wherein a reduction or inhibition of tumor growth or liver neutrophilic infiltration in the mouse is indicative of the test agent as an agent for preventing or treating cancer.

26. The method of claim 25, wherein the rate of tumor growth is reduced as compared to the rate of tumor growth in a corresponding transgenic mouse not contacted with the test agent.

27. The method of claim 25, wherein tumor size is reduced as compared to tumor size in the mouse prior to contacting with the test agent.

28. The method of claim 25, wherein the rate of liver neutrophilic infiltration is reduced as compared to the rate of liver neutrophilic infiltration in a corresponding transgenic mouse not contacted with the test agent.

29. The method of claim 25, wherein the rate of liver neutrophilic infiltration is reduced as compared to the rate of liver neutrophilic infiltration as compared to the rate of liver neutrophilic infiltration in the mouse prior to contacting with the test agent.

30. A kit comprising the transgenic mouse of any of claims claim 23-24 and one or more reagents for performing an assay.

31. A method of treating or preventing a CCL5 mediated liver disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of an agent which inhibits CCL5/CCR5 signal transduction, thereby treating or preventing the CCL5 mediated disease in the subject.

32. The method of claim 31, wherein the CCL5 mediated liver disease or disorder is inflammation, cancer, fatty liver disease, alcohol induced fatty liver disease, non-alcohol induced fatty liver disease, cirrhosis, steatosis, steatohepatitis, viral infection, and fibrosis.

33. The method of claim 31, wherein the cancer is hepatocellular carcinoma.

34. The method of claim 31, wherein the agent inhibits CCL5 binding to CCR5.

35. The method of claim 31, wherein the agent is a CCR5 antagonist.

36. The method of claim 31, wherein the agent inhibits or reduces CCL5 expression or activity.

37. The method of claim 31, wherein the agent is a small molecule, a peptide, a nucleic acid, or a combination thereof.

38. The method of claim 37, wherein the agent is a nucleic acid selected from the group consisting of siRNA, shRNA, miRNA, locked nucleic acid (LNA), antisense oligonucleotide, chemically modified oligonucleotide, and combinations thereof.

39. The method of claim 31, wherein the agent is an antibody, or fragment thereof.

40. The method of claim 39, wherein the antibody, or fragment thereof, specifically bind to CCL5 and blocks binding of CCL5 to CCR5.

41. The method of claim 39, wherein the antibody, or fragment thereof, specifically bind to CCR5 and blocks binding of CCL5 to CCR5.

42. The method of claim 39, wherein the antibody is selected from the group consisting of a single chain antibody, a monoclonal antibody, a bi-specific antibody, a chimeric antibody, a synthetic antibody, a polyclonal antibody, a humanized antibody, a fully human antibody, and active fragments or homologs thereof.

43. The method of claim 37, wherein the agent is conjugated to another molecule or structure.

44. The method of claim 43, wherein the molecule or structure is selected from the group consisting of an antibody, a protein, a pro-drug, a drug, a toxin, a protein toxin, a liposome, a radioactive isotope, and an enzyme.

45. The method of claim 31, wherein the agent is soluble CCR5.

46. The method of claim 31, further comprising administering to the subject a chemotherapeutic agent.

47. The method of claim 31, further comprising detecting expression or activity of CCL5.

48. A method of screening for an agent to treat cancer by inhibiting CCL5/CCR5 signal transduction, comprising:

a) contacting a sample with a test agent; and

b) detecting CCL5/CCR5 mediated signal transduction in the sample, wherein a reduction in CCL5/CCR5 mediated signal transduction as compared to a control sample is indicative of the test agent as being an agent to treat cancer.

49. The method of claim 48, wherein the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer.

50. The method of claim 48, wherein the cancer is hepatocellular carcinoma.

51. The method of claim 48, wherein the agent inhibits CCL5 binding to CCR5.

52. The method of claim 48, wherein the agent is a CCR5 antagonist.

53. The method of claim 48, wherein the agent inhibits or reduces CCL5 expression or activity.

54. The method of claim 48, wherein the agent is a small molecule, a peptide, a nucleic acid, or a combination thereof.

55. The method of claim 54, wherein the agent is a nucleic acid selected from the group consisting of siRNA, shRNA, miRNA, locked nucleic acid (LNA), antisense oligonucleotide, chemically modified oligonucleotide, and combinations thereof.

56. The method of claim 48, wherein the agent is an antibody, or fragment thereof.

57. The method of claim 56, wherein the antibody, or fragment thereof, specifically bind to CCL5 and blocks binding of CCL5 to CCR5.

58. The method of claim 56, wherein the antibody, or fragment thereof, specifically bind to CCR5 and blocks binding of CCL5 to CCR5.

59. The method of claim 56, wherein the antibody is selected from the group consisting of a single chain antibody, a monoclonal antibody, a bi-specific antibody, a chimeric antibody, a synthetic antibody, a polyclonal antibody, a humanized antibody, a fully human antibody, and active fragments or homologs thereof.

60. The method of claim 54, wherein the agent is conjugated to another molecule or structure.

61. The method of claim 60, wherein the molecule or structure is selected from the group consisting of an antibody, a protein, a pro-drug, a drug, a toxin, a protein toxin, a liposome, a radioactive isotope, and an enzyme.

62. The method of claim 48, further comprising detecting expression or activity of CCL5.

63. The method of claim 48, wherein the contacting is performed in-vitro.

64. The method of claim 48, wherein detecting comprises detecting binding of the test agent to CCL5, CCR5, or combination thereof.

65. The method of claim 48, wherein detecting comprises detecting binding of CCL5 to CCR5.

66. A method of screening for an agent to treat cancer by inhibiting CCL5/CCR5 signal transduction, comprising:

a) contacting a sample with a test agent; and

b) detecting binding of CCL5 to CCR5 in the sample, wherein a reduction in binding as compared to a control sample is indicative of the test agent as being an agent to treat cancer by inhibiting CCL5/CCR5 signal transduction.

67. The method of claim 66, wherein the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer.

68. The method of claim 66, wherein the cancer is hepatocellular carcinoma.

69. The method of claim 66, wherein the contacting is performed in-vitro.