WO2013134546A1 - Methods and materials for treating cancer - Google Patents

Abstract

This document provides methods and materials for treating cancers including renal cancer (e.g., renal cell carcinoma) as well as ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, and thyroid cancers and melanoma. For example, methods and material for using one or more inhibitors of an SCDl polypeptide to treat renal cell carcinoma (e.g., clear cell renal cell carcinoma (ccRCC)) or to increase the efficacy of a renal cell carcinoma treatment are provided. In addition, this document provides methods and materials for using elevated SCDl expression levels in diseased tissues as an indication that an SCDl inhibitor can be used as an appropriate therapeutic to ameliorate the disease.

Description

METHODS AND MATERIALS FOR TREATING CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Serial No.

61/607,961, filed on March 7, 2012, which is incorporated by reference in its entirety herein.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was made with government support under CA104505, CA136665, and CA104505-05 SI awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in treating cancer, for example, renal cell carcinoma, ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, thyroid cancers, and melanoma. For example, this document provides methods and material for using one or more inhibitors of a stearoyl-Coenzyme A desaturase 1 (SCDl) polypeptide to treat cancer.

2. Background Information

The incidence and deaths caused by renal cell carcinoma are increasing in the United States. Indeed, mortality from renal cell carcinoma has increased over 37% since 1950.

SUMMARY

This document provides methods and materials for treating cancer, for example, renal cell carcinoma, ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, thyroid cancers, and melanoma. For example, this document provides methods and material for using one or more inhibitors of an SCDl polypeptide to treat renal cell carcinoma (e.g., clear cell renal cell carcinoma (ccRCC)) or to increase the efficacy of a renal cell carcinoma treatment. As described herein, SCDl polypeptides are

overexpressed in certain cancer cells and are involved in the survival or proliferation of cancer cells. For example, reducing expression of renal cell carcinoma cells can result in reduced proliferation of renal cell carcinoma cells with minimal or no reduction in proliferation of normal kidney cells. In some cases, one or more inhibitors of an SCDl polypeptide can be used to reduce the number of cancer cells within a mammal (e.g., a human). In some cases, one or more inhibitors of an SCDl polypeptide can be used to increase the efficacy of a cancer treatment. For example, one or more inhibitors of an SCDl polypeptide can be used to increase the efficacy of a renal cell carcinoma treatment (e.g., treatment with Nexavar^®, Sutent^®, Torisel^®, Afmitor^®, and interleukin-2).

In general, one aspect of this document features a method for reducing the number of renal cell carcinoma cells within a mammal. The method comprises, or consists essentially of, administering, to the mammal, an inhibitor of an SCDl polypeptide under conditions wherein the number of viable renal cell carcinoma cells present within the mammal is reduced. The mammal can be a human. The administration can be an intratumoral, oral, intraperitoneal, intramuscular, or intravenous administration. The inhibitor can be A939572, MK-8245, CVT-11127, MF-152, or HYR-061. In another embodiment, one or more inhibitors of an SCDl polypeptide can be administered with one or more inhibitors of a mTor polypeptide. Non-limiting examples of such inhibitors include sirolimus (RAPAMUNE®), temsirolimus (CCI-779), everolimus (RADOOl), and ridaforolimus (AP-23573).

In another aspect, this document features a method for reducing the number of renal cell carcinoma cells within a mammal. The method comprises, or consists essentially of, administering, to the mammal, a composition under conditions wherein the number of viable renal cell carcinoma cells present within the mammal is reduced, wherein the composition comprises the ability to reduce SCDl mRNA expression or SCDl polypeptide expression. The mammal can be a human. The administration can be an intratumoral, oral, intraperitoneal, intramuscular, or intravenous administration. The composition can comprise a nucleic acid construct having the ability to express an shRNA directed against SCDl nucleic acid.

In another aspect, this document features a method for reducing the number of cancer cells overexpressing an SCDl polypeptide within a mammal. The method comprises, or consists essentially of, administering, to the mammal, an inhibitor of an SCDl polypeptide under conditions wherein the number of viable cancer cells overexpressing an SCDl polypeptide present within the mammal is reduced. Non- limiting examples of cancers include renal cell carcinoma, ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, thyroid cancers, and melanoma.

In another aspect, this document features a method for reducing the number of cancer cells overexpressing an SCDl polypeptide within a mammal. The method comprises, or consists essentially of, administering, to the mammal, a composition under conditions wherein the number of viable cancer cells overexpressing an SCDl polypeptide present within the mammal is reduced, wherein the composition comprises the ability to reduce SCDl mRNA expression or SCDl polypeptide expression. Non- limiting examples of cancers include renal cell carcinoma, ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, thyroid cancers, and melanoma.

In another aspect, this document features a method for identifying a mammal having cancer cells responsive to treatment with an inhibitor of an SCDl polypeptide. The method comprises, or consists essentially of, (a) detecting the presence of cancer cells expressing an elevated level of an SCDl mRNA or an SCDl polypeptide, and (b) classifying the mammal has having cancer cells responsive to treatment with the inhibitor of an SCDl polypeptide. The method can comprise measuring SCDl mRNA expression using real time PCR. The method can comprise measuring SCDl polypeptide expression using an immunohistochemical technique. The method can comprise measuring SCDl polypeptide expression using a Western blot analysis. Non- limiting examples of cancers include renal cell carcinoma, ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, thyroid cancers, and melanoma.

In a further aspect, this document features a method for reducing the number of cancer cells within a mammal. The method comprises, or consists essentially of, administering, to said mammal, an inhibitor of an SCDl polypeptide and an inhibitor of an mTor polypeptide under conditions wherein the number of viable cancer cells present within said mammal is reduced. In some cases, the inhibitor of an mTor polypeptide can be sirolimus (RAPAMUNE®), temsirolimus (CCI-779), everolimus (RAD001), or ridaforolimus (AP-23573). In certain cases, the mammal is a human. In some cases, the administration is an intratumoral, oral, intraperitoneal, intramuscular, or intravenous administration. In some cases, the inhibitor of an SCDl polypeptide is A939572, MK- 8245, CVT-11127, MF-152, or HYR-061. Non-limiting examples of cancer cells include one or more of ovarian cancer, breast cancer, prostate cancer, colon cancer, renal cancer, pancreatic cancer, bladder cancer, liver cancer, lung cancer, thyroid cancer, and melanoma.

Unless otherwise defined, 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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

Figure 1A is a graph plotting SCD1 mRNA levels in ccRCC tissue and matched normal tissue across stages I-IV. Figure IB contains photographs of representative ccRCC tissue and matched normal tissue stained for SCD1 polypeptide expression. Figure 1C is a graph plotting SCD1 mRNA levels in normal renal epithelial cell lines (347N, 355N, 359N, 360N, 365N, and 366N) versus ccRCC cell lines. Figure ID contains photographs of a Western blot analysis of SCD1 polypeptide expression by normal cell lines and ccRCC cell lines.

Figures 2A and 2D shows the knockdown of SCD1 in ccRCC as shown by decrease in both (A) mRNA and (D) protein expression using two separate lentiviral constructs shSCD780 and shSCD1200. Figures 2B and 2C show proliferation in (B) A498 and Cakil ccRCC cell lines and (C) NRE samples of NT versus shSCD lentiviral infected cells. Figure 2D contains photographs of an immunoblot for Poly-ADP ribose polymerase (PARP) cleavage and SCD1 expression in A498 and Cakil cell lines. Figure 3. Anti-pro liferative and apoptotic induction via loss of SCDl expression can be rescued with addition of oleic acid (OA-BSA). Figure 3A is a bar graph showing proliferation for SCDl and PARP cleavage in Cakil and A498 NT versus shSCD with or without OA-BSA supplementation. Figure 3B contains photographs of a Western blot analysis for SCDl and PARP cleavage in Cakil and A498 NT versus shSCD with or without OA-BSA supplementation. Figure 3C contains photographs of a phase-contrast microscopy representative ccRCC cell (Cakil) confluence at day 5 of proliferation assay with different treatment conditions.

Figure 4. Treatment of ccRCC cells with a small molecule SCDl inhibitor, A939572, inhibits cell growth and induces apoptosis. Figure 4A is a line graph showing cell proliferative response to dose out of A939572 in Cakil, A498, Caki2, and ACFiN ccRCC cell lines. Figure 4B is a bar graph displaying ccRCC proliferation rescue with OABSA in A939572 treated ccRCC cell lines. Figure 4C contains photographs of a Western blot analysis for PARP cleavage in A939572 treated vs. control, as well as OA- BSA rescue in ccRCC cell lines. Figure 4D contains representative phase contrast images of A939572 treated ccRCC cells (A498) +/-OA-BSA rescue at day 5.

Figure 5. Inhibition of SCDl activity in ccRCC induces cell death mediated by endoplasmic reticulum stress response. Figure 5A contains photographs of a Western blot analysis for expression of ER stress markers: BiP, CHOP, and spliced XBP1 in response to A939572 treatment or lentiviral silencing of SCDl in Cakil and A498. Figure 5B provides bar graphs showing QPCR analysis of ER stress gene expression in Cakil and A498 cells treated with A939572 or shSCD lentivirus +/-OA-BSA rescue. Figures 5C provides bar graphs showing relative luciferase activity of ER stress p5xATF6-GL3 (UPR) luciferase reporter transfected in Cakil and A498 cells treated with A939572 or shSCD lentivirus +/-OA-BSA supplementation.

Figure 6. Treatment of ccRCC cells with SCDl inhibitor in combination with the mTOR inhibitor Temsirolimus synergistically inhibits tumor cell growth in vivo. Figures 6A is a line graph illustrating in vivo tumor growth analysis and animal weight of A498 ccRCC subcutaneous xenografts in female athymic nude mice treated with A939572 and Temsirolimus alone or in combination versus placebo control (n=10 per group). Figure 6B contains photographs of IHC of tissue harvested from treatment groups stained for Ki67 and CC3 (quantitated by N-score), CD31 (quantitated by I-score), and phospho- mTOR (quantitated by H-score). Average group scores +/- the standard error are reported for each stain. Figure 6C contains photographs of Western blot and quantitation of CHOP expression in all four treatment groups. Figure 6D is an illustration of proposed SCD1 activity in ccRCC model: inhibition of SCD 1 blocks desaturation of SFA resulting in an accumulation of SFA species which trigger the ER stress response.

Figures 7A-D are line graphs comparing cell number to dose of A939572 or Gemicitabine in MiaPaca and pancreatic cancer cells. Figure 7E contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figures 8A-B are line graphs comparing cell number to dose of A939572 or

Sorafenib in SNU449 liver cancer cells. Figure 8C contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figures 9A-C are line graphs comparing cell number to dose of A939572 or Temodar in A375 AND Mela 11 melanoma cancer cells. Figure 9D contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figures 10A-B are line graphs comparing cell number to dose of A939572 or Capecitabine in CaCo2 and HT29 colon cancer cells. Figure IOC contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figures 11A-B are line graphs comparing cell number to dose of A939572 or cisplatin in T24 and HT1276 bladder cancer cells. Figure 11C contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figures 12A-B are line graphs comparing cell number to dose of A939572 or cisplatin in BCJ4T bladder cancer cells. Figure 12C contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figures 13A-B are line graphs comparing cell number to dose of A939572 or

Taxol in KTC3 and FF1 anaplastic thyroid cancer cells. Figure 13C contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figures 14A-B are line graphs comparing cell number to dose of A939572 or Taxol in A549 and CaLu-1 lung cancer cells. Figure 14C contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression. Figures 15A-B are line graphs comparing cell number to dose of A939572 or Taxol in OVCA420 and HOVTax2res ovarian cancer cells. Figure 15C contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figure 16A is a line graph comparing cell number to dose of A939572 in MCF-7 (ER+/PR+), MDA-231 (triple negative) and T47D (PR+) breast cancer cells. Figure 16B contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figure 17A is a line graph comparing cell number to dose of A939572 in DU-145 prostate cancer cells. Figure 17B contains photographs of Western Blot and quantitation of SCDl and beta-actin expression.

Figure 18A is a bar graph illustrating SCD1 protein expression in various cancer cell lines. Figure 18B contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figure 19A is a bar graph illustrating SCD1 protein expression in various cancer cell lines. Figure 19B contains photographs of Western Blot and quantitation of SCD1 and beta-actin expression.

Figures 20-22 provide structures for exemplary SCD1 inhibitors.

DETAILED DESCRIPTION

This document provides methods and materials for treating cancer, for example, for example, renal cell carcinoma, ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, thyroid cancers, and melanoma. In some embodiments, this document provides methods and material for using one or more inhibitors of an SCD1 polypeptide to treat cancer (e.g., clear cell renal cell carcinoma (ccRCC)) or to increase the efficacy of a cancer treatment.

As described herein, one or more (e.g., one, two, three, four, or more) inhibitors of an SCD1 polypeptide can be administered to a mammal (e.g., a human) having cancer (e.g., renal cancer) under conditions wherein the number of cancer cells within the mammal is reduced. In some embodiments, one or more (e.g., one, two, three, four, or more) inhibitors of an SCD1 polypeptide can be administered to a mammal (e.g., a human) having renal cancer (e.g., ccRCC) under conditions wherein the number of renal cancer cells within the mammal is reduced.

An SCDl polypeptide can be a human SCDl polypeptide having the amino acid sequence set forth in GenBank^® Accession No. 000767 (GI No. 21431730) or a human SCDl polypeptide encoded by nucleic acid having the nucleic acid sequence set forth in GenBank^® Accession No. AF097514.1 (GI No. 4808600). Examples of inhibitors of an SCDl polypeptide include, without limitation, inhibitory anti-SCDl polypeptide antibodies, siRNA molecules, shRNA molecules, nucleic acid vectors designed to express siRNA or shRNA molecules, anti-sense molecules, and small molecule antagonists such as A939572 (Biofine International Inc., Urvashi et al, Mol. Cancer Res., 9: 1551 (2011); Bristol-Myers Squibb R&D, Roongta et al., Mol. Cancer Res., 9(11): 1551-61 (2011)), MK-8245 (Merck Research Laboratories, Oballa et al, J. Med. Chem., 54(14):5082-96 (2011)), CVT-11127, MF-152 (Merck, Li et al, Bioorganic & Medicinal Chemistry Letters, 19:5214 (2009)), LCF369, CVT-11,563, CVT-12,012, DSR-4029, and GSK993 (Uto et al, Eur. J. Med. Chem., 45:4788-4796 (2010)), MF-438 (Leger, S. et al, Bioorg Med Chem Lett. 20(2):499-502 (2010)), and HYR-061

(Medchem Express, Koltun et al., Bioorganic & Medicinal Chemistry Letters,

19(7):2048-2052 (2009), and Xin et al., Bioorganic & Medicinal Chemistry Letters, 18(15):4298-4302 (2008)). In some cases, an inhibitor of an SCDl polypeptide can be an inhibitor described elsewhere (Igal, Carcinogenesis, 31(9): 1509-1515 (2010); Oballa, J. Med. Chem., 54:5082-5096 (2011); Li et al., Bioorganic & Medicinal Chemistry Letters, 19:5214-5217 (2009); Uto et al, Eur. J. Med. Chem., 46: 1892-1896 (2011); Uto et al, Eur. J. Med. Chem., 45:4788-4796 (2010); Liu, G. Expert Opin Ter Pat, 19(9): 1169-91 (2009); Powell, D.A., Bioorg Med Chem Lett. 20(22):6366-9 (2010), Mason P, et al, PLoS ONE 7(3): 33823 (2012), and Roongta et al, Mol. Cancer Res., 9: 1551-1561 (2011)).

Cancers that may be treated by an inhibitor of an SCDl polypeptide,

compositions and methods described herein include, but are not limited to, the following:

Breast cancers, including, for example ER⁺ breast cancer, ER^" breast cancer, her2^" breast cancer, her2⁺ breast cancer, stromal tumors such as fibroadenomas, phyllodes tumors, and sarcomas, and epithelial tumors such as large duct papillomas; carcinomas of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma; and miscellaneous malignant neoplasms. Further examples of breast cancers can include luminal A, luminal B, basal A, basal B, and triple negative breast cancer, which is estrogen receptor negative (ER^~), progesterone receptor negative, and her2 negative (her2^~). In some embodiments, the breast cancer may have a high risk Oncotype score;

lung cancers, including, for example, bronchogenic carcinoma, e.g., squamous cell, undifferentiated small cell, undifferentiated large cell, and adenocarcinoma; alveolar and bronchiolar carcinoma; bronchial adenoma;

sarcoma; lymphoma; chondromatous hamartoma; and mesothelioma;

genitourinary tract cancers, including, for example, cancers of the kidney, e.g., adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, and leukemia; cancers of the bladder and urethra, e.g., squamous cell carcinoma, transitional cell carcinoma, and adenocarcinoma; cancers of the prostate, e.g., adenocarcinoma, and sarcoma; cancer of the testis, e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, and lipoma;

liver cancers, including, for example, hepatoma, e.g., hepatocellular carcinoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hepatocellular adenoma; and hemangioma;

gynecological cancers, including, for example, cancers of the uterus, e.g., endometrial carcinoma; cancers of the cervix, e.g., cervical carcinoma, and pre tumor cervical dysplasia; cancers of the ovaries, e.g., ovarian carcinoma, including serous cystadenocarcinoma, epithelial cancer, mucinous

cystadenocarcinoma, unclassified carcinoma, granulosa thecal cell tumors, Sertoli Leydig cell tumors, dysgerminoma, and malignant teratoma; cancers of the vulva, e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, and melanoma; cancers of the vagina, e.g., clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma, and embryonal rhabdomyosarcoma; and cancers of the fallopian tubes, e.g., carcinoma;

skin cancers, including, for example, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and

adrenal gland cancers, including, for example, neuroblastoma.

In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of an SCDl polypeptide can be used as described herein to treat cancer, including renal cancer, ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, and thyroid cancers as well as melanoma.

For example, a human having cancer can be administered one or more inhibitors of an SCDl polypeptide under conditions that result in reduced tumor size or stable disease. In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of an SCDl polypeptide can be used as described herein to increase the efficacy of a cancer treatment. In some embodiments (e.g., when compositions comprising one or more (e.g., one, two, three, four, or more) inhibitors of an SCDl polypeptide are administered in conjunction with another anticancer agent), one can create a synergistic effect among the agents administered and thereby improve the outcome for a patient. In some

embodiments, one or more (e.g., one, two, three, four, or more) inhibitors of an SCDl polypeptide (or a pharmaceutically acceptable salt form thereof) can be administered in combination with (i.e., before, during, or after) administration of a pain relief agent (e.g., a nonsteroidal anti-inflammatory drug such as celecoxib or rofecoxib), an antinausea agent, or an additional anticancer agent (e.g., paclitaxel, docetaxel, doxorubicin, daunorubicin, epirubicin, fluorouracil, melphalan, cis-platin, carboplatin,

cyclophosphamide, mitomycin, methotrexate, mitoxantrone, vinblastine, vincristine, ifosfamide, teniposide, etoposide, bleomycin, leucovorin, taxol, herceptin, avastin, cytarabine, dactinomycin, interferon alpha, streptozocin, prednisolone, irinotecan, sulindac, 5 -fluorouracil, capecitabine, oxaliplatin/5 FU, abiraterone, letrozole,

5aza/romidepsin, or procarbazine). In certain embodiments, the anticancer agent is paclitaxel or docetaxel. In other embodiments, the anticancer agent is cisplatin or irinotecan.

For example, a human having ccRCC can be administered one or more inhibitors of an SCD1 polypeptide under conditions that result in reduced tumor size or stable disease. In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of an SCD1 polypeptide can be used as described herein to increase the efficacy of a renal cell carcinoma treatment. Examples of such renal cell carcinoma treatments include, without limitation, treatment with Nexavar^®, Sutent^®, Torisel^®, Afinitor^®, or interleukin-2.

In some cases, one or more (e.g., one, two, three, four, or more) inhibitors of an SCD1 polypeptide can be used as described herein can be used in combination with one or more (e.g., one, two, three, four, or more) inhibitors of mammailian target of rapamycin (mTor) polypeptide. Non-limiting examples of mTor inhibitors include:

sirolimus (RAPAMUNE®), temsirolimus (CCI-779), everolimus (RAD001),

ridaforolimus (AP-23573).

Accordingly, provided herein is a method for reducing the number of cancer cells within a mammal, wherein the method comprises administering, to the mammal, an inhibitor of an SCD1 polypeptide and an inhibitor of an mTor polypeptide under conditions wherein the number of viable cancer cells present within said mammal is reduced. In some embodiments, the one or more mTor inhibitor can include a standard of care drug for a particular cancer cell type. For example, an SCD1 inhibitor can be administered with pacliltaxel and/or platin (cisplatin, carboplatin, or oxaliplatin) for the treatment of ovarian cancer. In some embodiments, the following standard of care drugs can be combined with an SCD1 inhibitor for the following cancers:

Lung - paclitaxel

Colon - capecitabine

Breast

Metastatic breast - capecitabine, paclitaxel, and/or gemcitabine

Hormonally responsive breast - aromatase inhibitors such as letrazole and/or antiestrogens such as tamoxifen

HER2 positive - Herceptin

Melanoma - temodar, and/or BRAF inhibitors Prostate - abiraterone

Bladder - gemcitabine and/or paclitaxel

Thyroid - paclitaxel and/or cisplatin

Pancreatic - gemcitabine

Liver - sorafanib

In some embodiments, the combination of one or more inhibitors of an SCD1 polypeptide and one or more inhibitors of mTor exhibit a synergistic response. In some embodiments, the one or more inhibitors of an SCD1 polypeptide can be administered before, during, or after administration of the one or more inhibitors of mTor.

An inhibitor of an SCD1 polypeptide can also be administered to a subject in combination with surgical methods to treat cancers, e.g., resection of tumors. The inhibitor can be administered to the individual prior to, during, or after the surgery. The inhibitor can be administered parenterally, intravenous or injected into the tumor or surrounding area after tumor removal.

Typically, one or more of the inhibitors of an SCD1 polypeptide provided herein can be formulated into a pharmaceutical composition that can be administered to a mammal (e.g., rat, dog, horse, cat, mouse, rabbit, pig, cow, monkey, or human). For example, A939572 or a pharmaceutically acceptable salt thereof can be in a

pharmaceutically acceptable carrier or diluent. A "pharmaceutically acceptable carrier" refers to any pharmaceutically acceptable solvent, suspending agent, or other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, without limitation, water, saline solutions, dimethyl sulfoxide, binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate), lubricants (e.g., starch, polyethylene glycol, or sodium acetate), disintegrates (e.g., starch or sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate).

The term "pharmaceutically acceptable salt" refers to the relatively non-toxic, inorganic and organic acid addition salts of a compound provided herein. These salts can be prepared in situ during the final isolation and purification of a compound provided herein, or by separately reacting the compound in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulphonate salts, and amino acid salts, and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19.)

In some embodiments, a compound provided herein may contain one or more acidic functional groups and, thus, is capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic inorganic and organic base addition salts of a compound provided herein. These salts can likewise be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al, supra).

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

General Materials and Methods

Cell Culture

ccRCC cell lines: RWV366T and KD265T (16) (both stage IV ccRCC patient tissue derived), A498, Cakil, Caki2, and ACHN (ATCC) and K347N, K355N, K359N, K360N, K365N, and K366N normal renal tissue derived mortal cells (NRE) were cultured in DMEM medium (Cellgro) containing 5%FBS (Hyclone) and lxpenicillin- streptomycin (Invitrogen) at 37°C in humidified conditions with 5%C0₂.

Proliferation, Treatment, and Rescue Assays

Cells were plated (0.5 or lxl0⁵/well) in 24-well plates for proliferation or treatment assays, in triplicate. Cells were trypsinized (0.25%) and counted using a Coulter Particle Counter at specified time intervals. For SCD1 rescue assays, oleic acid- albumin was added to media at 5 μΜ. Drug stocks were prepared in

DMSO. Monotherapeutic treatment identified drug dose-response. Combinatorial dosing ranged up to the IC50 for each inhibitor. Temsirolimus dosing was performed as described in the text. Soft agar cultures were prepared by diluting 2x growth medium 1 : 1 in 1.5% Seaplaque®GTG® agarose, with 500 cells/plate in 60mm culture dishes.

Colonies were stained with Giemsa (LabChem Inc.) and counted after 3 wks. Cell images were obtained with an 01ympusIX71 microscope at 20x magnification.

Lentivirus

MISSION shRNA pLKO. l constructs were used to make self-inactivating shRNA lentiviruses for human SCD1 (clones: NM_005063.3-1200slcl [shSCD1200],

NM_005063.3-780slcl [shSCD780]), and a non-target (NT) random scrambled sequence control (SHC002). Trans fection reagents Lipofectamine 2000 and ViraPower were used to generate lentiviruses using HEK293FT viral progenitor cells. ccRCC and NRE cells were incubated with lentivirus plus 5μg/mL polybrene for 24 hrs prior to clonal selection with Puromycin. Transfections and Luciferase Assays

For transient transfection, Cakil and A498 cells were transfected using

Lipofectamine 2000. Cells treated with DMSO vs. A939572 or infected using shSCD780 lentiviral constructs vs. NT control were harvested after 48hrs using Promega's Dual Luciferase assay kit per the manufacturer's protocol and luciferase activity was measured using a Veritas Luminometer; reported as relative luminescence. RNA Isolation and Quantitative PCR

An R Aqueous Midi Kit was utilized to extract and purify RNA from cell lines. Human tissue RNA was prepared using using TRIzol® per manufacturer's protocol followed by purification using the RNAqueous Midi Kit. The O.D. 260/280 ratio of the mRNA was at least 1.8 and the 18s/28s bands were verified on a 1% agarose gel. cDNA was prepared from purified RNA samples using High Capacity cDNA Reverse

Transcriptase Kit per manufacturer's instruction. TaqMan®Fast Universal PCR Master Mix and TaqMan®FAMTM dye-labeled probes including POLR2A (Hs00172187_ml) (normalization control), SCD1 (Hs01682761_ml), HSPA5 (Hs99999174_ml), CEBPp (CEBPB Hs00270923_sl), GADD45A (Hs00169255_ml), DDIT3 (Hs01090850_ml), and HERPUD1 (HsOl 124269 ml) were combined with prepared cDNA samples to analyze relative mRNA expression via qPCR. Fold change values were compared between normal and tumor, non-target scrambled lentiviral and target lentiviral infected, and DMSO vs. A939572 treated samples using the AACt method (Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc.

2008;3: 1101-8).

Gene Array Expression Analysis

Gene array expression analysis was performed using Affymetrix Human Genome U133 Plus 2.0 Array chip. The details of the data processing and methodology were previously described in (Tun HW, Marlow LA, von Roemeling CA, Cooper SJ, Kreinest P, Wu K, et al. Pathway signature and cellular differentiation in clear cell renal cell carcinoma. PLoS One. 2010;5:el0696). Gene expression data was deposited at Gene Expression Omnibus (Accession#GSE41485). Pathway analysis was performed using IP A (Ingenuity® Systems).

Western Blot Analysis

Protein extracts, electrophoresis, and membrane transfers were prepared as previously described (Copland JA, Marlow LA, Kurakata S, Fujiwara K, Wong AK, Kreinest PA, et al. Novel highaffinity PPARgamma agonist alone and in combination with paclitaxel inhibits human anaplastic thyroid carcinoma tumor growth via p21WAFl/CIPl . Oncogene. 2006;25:2304-17). Primary antibodies included SCD1, PARP, DDIT3, BiP, sXBPl, and β-actin. A Supersignal chemiluminescent kit was used to perform detection. IHC and ICC Analysis

Formalin fixed, paraffin-embedded tissue microarray (TMA) of patient ccRCC tumor and matched normal tissues and TMA of combinatorial in vivo mouse tumor tissue. The TMAs were mounted on slides from paraffin-embedded blocks according to IHC procedure and samples were blocked with Diluent that contained Background Reducing Components (Dakocytomation) for 30 min and then probed for SCD1, Ki67, Caspase-3, CD31, phospho-mTOR, DDIT3, and XBPl . ICC preparation and staining was performed as previously described (Cooper SJ, Von Roemeling CA, Kang KH, Marlow LA, Grebe SK, Menefee ME, et al. Reexpression of tumor suppressor, sFRPl, leads to antitumor synergy of combined HDAC and methyltransferase inhibitors in

Chemoresistant cancers. Mol Cancer Ther. 2012). Stain scoring was done using algorithms generated with Imagescope software created by a histologist. H-scores were calculated based upon signal intensity (0-3+) using the formula:

[(l+%xl)+(2+%x2)+(3+%x3)], intensity (I)-scores were calculated by dividing signal intensity by area, and nuclear (N)-scores were calculated by dividing % positive nuclei by total nuclei examined per area. Cases where insufficient tumor tissue presented were excluded from the study. 20x images were obtained using Scanscope XT and Imagescope software. RWV366T cell line validation was carried out as previously

described (Cooper SJ, Von Roemeling CA, Kang KH, Marlow LA, Grebe SK, Menefee ME, et al. Reexpression of tumor suppressor, sFRPl, leads to antitumor synergy of combined HDAC and methyltransferase inhibitors in chemoresistant cancers. Mol Cancer Ther. 2012).

In Vivo Analysis

A498 cells were subcutaneously implanted in athymic nu/nu mice at lxlO⁶ cells/mouse in 50%Matrigel. Tumors reached ~50 mm³ prior to treatment, which was carried out for 4 wks. A939572 was administered via oral feeding using strawberry flavored Kool-Aid® in sterilized H₂0 (0.2g/mL) vehicle at 30 mg/kg in a 50 μΐ dose twice daily/mouse. Temsirolimus was solubilized in 30% ethanol/saline and administered via intraperitoneal injection at 10 mg/kg in a 50 μΐ dose once every 72 hrs/mouse. Tumor volumes were calculated using the formula 0.5236(L*W*H) and body weight were measured every 3 days.

DNA isolation and STR Analysis

Genomic DNA was extracted from both RWV366T patient primary tissue and matching cell line using PurelinkTM Genomic DNA mini kit. Sixteen STR markers were PCR amplified using fluorescently labeled primers from ABI, and were analyzed using ABI 3130. Peak sizes were calculated versus a co-injected size standard using Gene Marker.

Statistical Analysis

Data values are presented as either percentage or fold change ±s.d. unless otherwise specified. Fold change values 1.5< are considered statistically significant. Treatment group comparisons were analyzed using two-tailed paired Student's t-test with p<0.05 being considered statistically significant. Statistically significant results are indicated by asterisk (*). Drug synergy statistics are indicated via combination index (CI) determined using CalcuSyn® as described in the text.

Example 1 - SCDl Polypeptide is Upregulated in ccRCC and is Involved

in Tumor Cell Survival

Normal kidney tissue and matched ccRCC tissue samples were obtained, and the levels of SCDl mRNA and SCDl polypeptide expression were determined. SCDl mRNA levels and SCDl polypeptide levels were elevated in matched ccRCC samples when compared to normal samples (Figures 1 A and IB).

The levels of SCDl mRNA and SCDl polypeptide expression also were determined in established ccRCC cell lines and normal renal epithelial cells. SCDl mRNA levels and SCDl polypeptide levels were elevated in established ccRCC cell lines when compared to normal renal epithelial cells (Figures 1C and ID). RWV366T is a newly established patient derived ccRCC cell line, whose patient and renal origins were validated by STR analysis and IHC for renal markers (data not shown).

To determine the involvement of SCD1 expression in renal cell carcinoma proliferation, two separate lentiviral constructs were designed to express shRNA molecules having the ability to reduce SCD1 expression. The first lentiviral construct was designed to express an shRNA designated SCD780. The sequence of SCD780 was as follows: 5'-CTACGGCTCTTTCTGATCATT-3' (SEQ ID NO: l). The second lentiviral construct was designed to express an shRNA designated SCD1200. The sequence of SCD1200 was as follows: 5 '-CGTCCTTATGACAAGAACATT-3 ' (SEQ ID NO:2). A non-target lentiviral construct was designed as a control.

Treatment of established ccRCC cell lines (Cakil and A498) with lentiviral constructs designed to express SCD780 or SCD1200 resulted in reduced SCD1 mRNA expression levels (Figure 2A) and reduced SCD1 polypeptide expression levels (Figure 2D). The reduction in SCD1 expression in ccRCC cells revealed the induction of apoptosis as demonstrated by poly ADP ribose polymerase (PARP) cleavage (Figure 2D). Treatment of normal renal epithelial cells (K359N and K360N) with lentiviral constructs designed to express SCD780 or SCD1200 resulted in reduced SCD1 mRNA expression levels (Figure 2A).

A proliferation assay was performed to determine if reduced SCD1 expression preferentially reduced the ability of established ccRCC cell lines to proliferate as compared to normal kidney cells. Treatment of established ccRCC cell lines (Cakil and A498) with lentiviral constructs designed to express SCD780 or SCD1200 resulted in reduced proliferation as compared to the levels of proliferation observed with normal kidney cells (K359N and K360N) treated with the lentiviral constructs (Figures 2B and 2C).

These results demonstrate that inhibitors of SCD1 can be used to reduce the number of ccRCC cells present within a mammal, while having little or no effect on normal kidney cells. These results also demonstrate that loss of SCD1 in ccRCC cells can lead to apoptotic programmed cell death.

Example 2 - Oleic Acid Reverses Effects of Decreased SCD1 Expression in Tumor Cells As oleic acid (OA) is the principle product of SCDl mediated SFA dehydrogenation, a cell culture stable form of OA conjugated to albumin from bovine serum (OA-BSA) was utilized to perform rescue experimentation in order to confirm that decreased tumor cell growth and induction of cell death was due to lentiviral mediated suppression of SCD 1.

Media alone and BSA supplemented media served as control groups. Proliferation assay of NT control versus shSCD780 infected Cakil and A498 cells cultured in media with or without OABSA were counted after five days. Both Cakil and A498 shSCD780 cells exhibited significant decreases in growth when compared to controls; however the addition of OA-BSA rescued the proliferative capacity of these cells to near control rates (Figure 3A). Notably, addition of OABSA to Cakil NT cells marginally enhanced proliferation (Figure 3 A). SCDl knockdown by lentiviral infection was confirmed at the protein level (Figure 3B). In addition to growth rescue, supplementation with OA-BSA also decreased shSCD780 induced apoptosis as demonstrated by reduction in PARP cleavage shown by western blot (Figure 3B). Representative phase contrast images of ccRCC cells for each group are shown in Figure 3C.

Example 3 - Small Molecule Inhibition of SCDl Induces ccRCC Cell Death

A939572 was dosed out in four ccRCC cell lines- Cakil, A498, Caki2, and ACHN, and demonstrated a significant dose-dependent decrease in proliferation at day 5 (IC50s of 65 nM, 50 nM, 65 nM, and 6 nM, respectively) (Figure 4A). Molecular target specificity was confirmed by addition of OA-BSA to the growth inhibitory assay, with IC50 doses applied to all four cell lines versus DMSO+BSA control. Addition of OABSA prevented A939572 mediated growth inhibition which was comparable to control groups in all four cell lines (Figure 4B). In congruity with previous experimentation examining SCDl lentiviral knockdown models, A939572 induced apoptosis confirmed by PARP cleavage via western blot analysis in all four cell lines (Figure 4C). Addition of OA-BSA blocked apoptosis noted by lack of PARP cleavage (Figure 4C). Representative phase contrast cell images (Figure 4D) demonstrate marked reduction in confluence of A939572 treated ccRCC cells (day 5), which reflects decreased proliferation and induction of cell death as a result of treatment. OA-BSA supplemented cells display no visible alterations in phenotype. Thus, we have identified a specific small molecule SCD1 inhibitor that induces apoptotic cell death that can be rescued by oleic acid.

Example 4 - Treatment of ccRCC Cells with A939572 Induces Endoplasmic

Reticulum Stress

In order to determine the mechanism of decreased proliferation and induction of cell death associated with loss of SCD1 activity in ccRCC cells, gene array analysis was performed with Cakil, A498, Caki2, and ACFIN ccRCC cells treated for 24 hours with a 75 nM dose of A939572 compared to DMSO control. Gene expression data was analyzed using the Ingenuity® Systems (IP A) program and revealed increased expression of ER stress response genes associated with UPR.

Western blot of Cakil and A498 cells for protein expression of key ER stress markers including BiP (heat shock 70kDa protein, GRP78), CHOP (DNA damage inducible transcript 3, DDIT3), and spliced-XBPl (x-box binding protein 1, s-XBPl) revealed amplified expression in both drug treated (75nM) and shSCD780 lentiviral knockdown cells after 48 hours (Figure 5 A), confirming induction of ER stress upon loss of SCD1 activity or expression as implicated by the gene array analysis.

In order to validate the specificity of ER stress induction mediated by both A939572 and shSCD780, rescue assays were performed using OA-BSA in Cakil and A498 cells qPCR analysis of five ER stress genes identified in the gene array including BiP, CHOP, HERPUD1 (homocysteine-inducible, ER-stress inducible, ubiquitin-like-1), GADD45a (DNA damage inducible transcript 1, DDITl), and ΟΕΒΡβ (CCAAT/enhancer binding protein beta) were examined. In A939572 (SCDi) treated Cakil and A498 cells, all five ER stress related genes were expressed at significantly increased levels compared to DMSO+BSA control, and this elevated expression could be blocked with the addition of OA-BSA (Figure 5B). In shSCD780 lentiviral infected Cakil and A498 cells, all of the ER stress genes were significantly induced in the Cakil shSCD780 sample and 4 of the 5 were significantly induced in the A498 shSCD780 sample. Similar to the drug treated cells, OA-BSA successfully blocked shSCD780 induced expression of the ER stress genes (Figure 5B). Activating transcription factor 6 (ATF6) is a key bZIP transcription factor that mediates part of the UPR stress response. Upon stress induction ATF6 is proteolytically cleaved into the activated transcription factor allowing it to transcribe several downstream mediators in the ER stress response pathway including XBP1, BiP,

HSP90B1 (heat shock protein 90kDa beta), and CHOP (23). Cakil and A498 cells transfected with an ATF6 luciferase reporter (p5xATF6-GL3) were treated with a 75 nM dose of A939572 or were infected with shSCD780. Resulting luminescence was measured after 48 hours. Inhibiting SCD1 genetically or pharmacologically resulted in significant enhancement of luciferase activity as compared to DMSO and NT controls with Cakil A939572+BSA, Cakil shSCD780+BSA, A498 A939572+BSA, and A498 shSCD780+BSA cells expressing fold change inductions of 1.6, 1.7, 3.8, and 2.0 respectively (Figure 5C). The addition of OA-BSA significantly reduced reporter activation in response to A939572 and shSCD780, thereby confirming specificity of ATF6 stimulation by loss of SCD1 activity in ccRCC cells. Collectively, these data are indicative that SCD1 inhibition activates the UPR stress response. Tumor cells may therefore be prone to elevated levels of ER stress requiring the induction of protective factors such as SCD1 in order to preserve cell viability. Targeting ER protective constituents presents another potential route for therapeutic intervention not only in ccRCC, but likely in other cancers as well.

Example 5 - Combination of A939572 with Temsirolimus Synergistically Enhances

Tumor Cell Death

In order to target ccRCC using a multifaceted approach, synergy was examined through application of combinatorial treatment utilizing A939572 in congruence with a current FDA approved regimen for ccRCC treatment. These included the TKIs pazopanib and sunitinib, as well as the mTOR inhibitor temsirolimus.

After identifying appropriate cell proliferative dose responses for pazopanib and sunitinib in four ccRCC cell lines including A498, Cakil, Caki2, and ACFIN, both TKIs were dosed in combination with A939572 up to approximately the IC50 dose for each drug in the Cakil and the A498 cell lines. No synergy was noted in either Cakil or A498 cell proliferative responses with combinatorial treatment. Temsirolimus (Tern) when dosed out in the four ccRCC cell lines yielded a limited reduction in cell proliferation, and no dose response could be determined. Combinatorial treatments were therefore done using a fixed dose of Tern (O. lnM, InM, and lOnM) combined with a dose range of A939572 up to the IC50 in Cakil, A498, Caki2 and ACHN cells. Both drugs in combination yielded very strong synergy in all four cell lines as indicated by the combination index (CI) determined using CalcuSyn® based on the Chou-Talalay Method where CI values >1 represent an antagonistic effect and values <1 represent synergy, with lower values signifying enhanced synergy. Colony formation assay of A498 cells grown in soft agar treated with mono and combination doses of 5nM A939572 and 5nM Tern reflected synergistic effects observed in combination growth assays performed in 2-D culture and provided the rationale for in vivo analysis of combinatorial therapy.

Athymic nude (nu/nu) mice bearing A498 ccRCC xenografts were treated with A939572 and Tern individually or in combination over the course of four weeks, and tumor volume (mrm) was recorded (Figure 6A). A939572 and Tern monotherapy generated similar growth responses with approximately 20-30% reductions in tumor volume (vs. placebo control) being observed upon study completion, with values reaching statistical significance only within the last week of treatment. The combination group yielded over a 60% decrease in tumor volume (vs. placebo control) by study completion with significant reductions recorded after approximately 1 week of treatment. All of the animals maintained a healthy weight throughout the course of the treatment (Figure 6A), however those in both the A939572 and the Combo group exhibited increased blinking, and slight mucosal discharge from the eyes after the first week of treatment.

IHC analysis of tumors resected from each treatment group was analyzed for proliferation, angiogenesis, and cell death (Figure 6B). All treatment groups (A939572, Tern, and Combo) when compared to the placebo control exhibited decreased

proliferation as marked by reduction in percent positivity of nuclear Ki67 staining, with the combinatorial group demonstrating the most significant decline. Angiogenesis as examined by intensity of microvessel density demonstrated a slight decrease in both the Tern and the Combo groups; however the cumulative scores were not considered significant. Cell death as examined by cleaved caspase-3 (CC3) demonstrated significant increases in the Combo group when compared to all groups. A moderate increase in cell death was also seen in the A939572 and Tem groups compared to the placebo.

Phosphorylated mTOR was inspected as a marker for temsirolimus activity, and decreased expression was confirmed in both the Tem and the Combo groups as compared to the Placebo and A939572 groups. ER stress was examined via western blot of total protein extractions prepared from randomly selected tumor tissue samples representing each treatment group, and resulting quantitative expression was normalized to respective Pactin controls. Increased expression of CHOP was confirmed in all samples treated with A939572 (A939572 and Combo) (Figure 6C) confirming that inhibition of SCD1 in ccRCC contributes to ER stress in vivo. A proposed mechanism is summarized in Figure 6D. Interestingly, samples in the Tem group also exhibited induction of CHOP, although to a lesser extent when compared to A939572 and Combo groups. Temsirolimus has been previously reported to decrease SCD1 expression in breast cancer cells. Inhibition of mTOR in ccRCC could indirectly mediate ER stress through decrease of SCD1, thereby explaining our observations. No significant increase in CHOP expression was seen in any placebo samples, confirming specificity of ER stress induction as a result of drug treatment.

Example 6 - Inhibition of SCD1 Polypeptide in Various Cancer Cell Lines

A number of cancer cell lines were tested to determine whether SCD1 protein expression correlates with growth inhibition of an SCD1 inhibitor in human cancer cell lines.

Pancreatic Cancer

Cells (20,000/ml) were plated in 12 well cell culture plates, allowed to attach and treated with the indicated dose of SCD1 inhibitor (A939753) or standard of care

(gemcitabine). Cell number was counted using a Coulter Counter. As show in Figure 7, the data are expressed as percent of DMSO control. Each value represents triplicates. Western analysis for SCD1 protein expression was performed on each cell line with beta- actin as the loading control. The data indicated that MiaPaca cells express SCD1 and were growth inhibited in a dose dependent fashion while Pane cells expressed very low levels of SCDl and were growth inhibited at only high levels of SCDl inhibitor, A939572.

The following cell lines were studied using a similar method as that described above.

Liver Cancer

It was found that SNU449 liver cancer cells express SCDl protein and are growth inhibited in a dose dependent fashion by the SCDl inhibitor, A939572 (see Figure 8). An estimated IC₅₀ concentration occurred around 100 nM. Sorafenib is FDA approved for liver cancer treatment and is effective between 1 - 10 micromolar concentrations.

Melanoma

A375 melanoma cells express SCDl protein and were growth inhibited in a dose dependent fashion by the SCDl inhibitor, A939572 (see Figure 9). An estimated IC50 concentration occurred around 50 nM. Mela 11 melanoma cells do not express SCDl and were not growth inhibited. Standard of care, Temodar, dose responsively inhibits growth in A375 cells but not Mela 11.

Colon Cancer

Caco2 and HT29 colon cancer cells express SCDl protein and are growth inhibited in a dose dependent fashion by the SCDl inhibitor, A939572 (see Figure 10).

Bladder Cancer

T24 and HT1376 bladder cancer cells express SCDl protein and were growth inhibited in a dose dependent fashion by the SCDl inhibitor, A939572 (see Figure 11). The standard of care for bladder cancer, Cisplatin, has minimal growth inhibitory effects on these two cell lines.

BCJ4T bladder cancer cells do not express SCDl protein and were not growth inhibited by the SCDl inhibitor, A939572 (see Figure 12). The standard of care for bladder cancer, Cisplatin, has minimal growth inhibitory effects on these this cell line. Anaplastic Thyroid Cancer

KTC3 thyroid cancer cells express SCDl protein and were growth inhibited in a dose dependent fashion by the SCDl inhibitor, A939572 (see Figure 13). Taxol is growth inhibitory in KTC3 cells but has minimal growth inhibitory effects on FF1 cells.

Lung Cancer

A549 nonsmall cell lung cancer cells express SCDl protein and were growth inhibited in a dose dependent fashion by the SCDl inhibitor, A939572 while Calu-1 lung cancer cells do not express SCDl and are not growth inhibited by A939572 (see Figure 14). Taxol is growth inhibitory in A549 but was not tested in Calu-1 cells.

Ovarian Cancer

OVCA420 and HOV TAX2 ovarian cancer cells express SCDl protein and were growth inhibited in a dose dependent fashion by the SCDl inhibitor, A939572 while Calu-1 lung cancer cells do not express SCDl and were not growth inhibited by A939572 (see Figure 15). Taxol is growth inhibitory in HOV Tax2 cells but was not tested in OVCA420 cells.

Breast Cancer

MCF-7 (ER+/PR+), MDA-231 (triple negative) and T47D (PR+) breast cancer cells express SCDl protein and were growth inhibited in a dose dependent fashion by the SCDl inhibitor, A939572 (see Figure 16).

Prostate Cancer

DU-145 and LNCAP prostate cancer cells express SCDl protein and were growth inhibited in a dose dependent fashion by the SCDl inhibitor, A939572 (see Figure 17).

Quantitation (relative) of SCDl protein expression in different cancer cell lines

As shown in Figure 18, Western analysis was performed on SCDl and beta actin. Quantitation was performed by first normalizing to each respective beta-actin followed by normalization to A498 control. SNU449 cells appeared to have the highest SCDl protein expression while BCJ4, Melal l and PANC had the lowest protein levels. Protein levels appear to correlate with growth inhibition of the SCDl inhibitor.

As shown in Figure 19, Western analysis was also performed on SCDl and beta actin. Quantitation was performed by first normalizing to each respective beta-actin followed by normalization to A498 control. LN Cap cells appeared to have the highest SCDl protein expression while Calul, FFl and KTC3 cells had the lowest protein levels. Protein levels appear to correlate with growth inhibition of the SCDl inhibitor.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for reducing the number of cancer cells within a mammal, wherein said method comprises administering, to said mammal, an inhibitor of an SCDl polypeptide and an inhibitor of an mTor polypeptide under conditions wherein the number of viable cancer cells present within said mammal is reduced.

2. The method of claim 1, wherein said inhibitor of an mTor polypeptide is sirolimus (RAPAMUNE®), temsirolimus (CCI-779), everolimus (RADOOl), or ridaforolimus (AP- 23573).

3. The method of claim 1, wherein said mammal is a human.

4. The method of claim 1, wherein said administration is an intratumoral, oral, intraperitoneal, intramuscular, or intravenous administration.

5. The method of claim 1, wherein said inhibitor of an SCDl polypeptide is

A939572, MK-8245, CVT-11127, MF-152, or HYR-061.

6. The method of claim 1, wherein said cancer cells comprise one or more of ovarian cancer, breast cancer, prostate cancer, colon cancer, renal cancer, pancreatic cancer, bladder cancer, liver cancer, lung cancer, thyroid cancer, and melanoma.

7. A method for reducing the number of renal cell carcinoma cells within a mammal, wherein said method comprises administering, to said mammal, an inhibitor of an SCDl polypeptide under conditions wherein the number of viable renal cell carcinoma cells present within said mammal is reduced.

8. The method of claim 7, wherein said mammal is a human.

9. The method of claim 7, wherein said administration is an intratumoral, oral, intraperitoneal, intramuscular, or intravenous administration.

10. The method of claim 7, wherein said inhibitor is A939572, MK-8245, CVT- 11127, MF-152, or HYR-061.

11. The method of claim 7, wherein said mammal is further administered an inhibitor of a mTor polypeptide.

12. The method of claim 11, wherein said inhibitor is sirolimus (RAPAMUNE®), temsirolimus (CCI-779), everolimus (RAD001), or ridaforolimus (AP-23573).

13. A method for reducing the number of renal cell carcinoma cells within a mammal, wherein said method comprises administering, to said mammal, a composition under conditions wherein the number of viable renal cell carcinoma cells present within said mammal is reduced, wherein said composition comprises the ability to reduce SCDl mR A expression or SCDl polypeptide expression.

14. The method of claim 13, wherein said mammal is a human.

15. The method of claim 13, wherein said administration is an intratumoral, oral, intraperitoneal, intramuscular, or intravenous administration.

16. The method of claim 13, wherein said composition comprises a nucleic acid construct having the ability to express an shRNA directed against SCDl nucleic acid.

17. A method for reducing the number of cancer cells overexpressing an SCDl polypeptide within a mammal, wherein said method comprises administering, to said mammal, an inhibitor of an SCDl polypeptide under conditions wherein the number of viable cancer cells overexpressing an SCDl polypeptide present within said mammal is reduced.

18. The method of claim 17, wherein the cancer is selected from the group consisting of: renal cell carcinoma, ovarian cancer, breast cancer, prostate cancer, colon cancer, pancreatic cancer, bladder cancer, liver cancer, lung cancer, thyroid cancer, and melanoma.

19. The method of claim 18, wherein the cancer is renal cell carcinoma.

20. A method for reducing the number of cancer cells overexpressing an SCDl polypeptide within a mammal, wherein said method comprises administering, to said mammal, a composition under conditions wherein the number of viable cancer cells overexpressing an SCDl polypeptide present within said mammal is reduced, wherein said composition comprises the ability to reduce SCDl mR A expression or SCDl polypeptide expression.

21. The method of claim 20, wherein the cancer is selected from the group consisting of: renal cell carcinoma, ovarian cancer, breast cancer, prostate cancer, colon cancer, pancreatic cancer, bladder cancer, liver cancer, lung cancer, thyroid cancer, and melanoma.

22. The method of claim 21, wherein the cancer is renal cell carcinoma.

23. A method for identifying a mammal having cancer cells responsive to treatment with an inhibitor of an SCDl polypeptide, wherein said method comprises:

(a) detecting the presence of cancer cells expressing an elevated level of an SCDl mRNA or an SCDl polypeptide, and

(b) classifying said mammal has having cancer cells responsive to treatment with said inhibitor of an SCDl polypeptide.

24. The method of claim 23, wherein said method comprises measuring SCDl mRNA expression using real time PCR.

25. The method of claim 23, wherein said method comprises measuring SCDl polypeptide expression using an immunohistochemical technique.

26. The method of claim 23, wherein said method comprises measuring SCDl polypeptide expression using a Western blot analysis.