USE OF CYP27A1 INHIBITORS, STATIN, OR LXR ANTAGONISTS ALONE OR IN COMBINATION WITH CONVENTIONAL THERAPY FOR THE TREATMENT OF

BREAST CANCER

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/896,815, filed October 29, 2013, which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under federal grant numbers DK048807 and K99CA172357, awarded by the National Institutes of Health, and W81XWH-10- 1-0034 and W81XWH-09-1-0613, awarded by the Department of Defense. The U.S.

Government has certain rights to this invention.

TECHNICAL FIELD

[0003] The present invention relates to methods of preventing and treating subjects suffering from estrogen dependent cancer or metastatic cancer, such as metastatic breast cancer, by administering a drug that lowers 27-hydroxy cholesterol (27HC) levels in the subject alone or in combination with a conventional cancer therapy.

BACKGROUND

[0004] Obesity and metabolic syndrome are established risk factors for estrogen receptor (ER) positive breast cancer in postmenopausal women. This has been attributed to increases in circulating insulin and insulin-like growth factors, local production of estrogens in adipose tissue, and the influence of adipokines and inflammatory cytokines on tumors and their microenvironment. Hypercholesterolemia, a comorbidity of obesity, is a risk factor for ER positive breast cancers and is associated with a decreased response of tumors to endocrine therapies.

[0005] A large minority of patients fails to respond to this therapy and the majority of patients eventually relapse into endocrine-therapy resistant disease. One mechanism behind the failure to respond and/or the development of resistance may be the formation of cholesterol metabolites. Metastasis is the primary cause of death in breast cancer patients and is the primary reason for breast cancer relapse. Tamoxifen and raloxifene have been approved as breast cancer chemopreventatives in women at elevated risk for breast cancer. However, given the significant side effect profile of these drugs, it is difficult to advocate for their use as chemopreventatives in women with average risk. It would be beneficial to have other preventative and treatment options.

SUMMARY

[0006] The present disclosure is directed to a method for preventing or treating a subject suffering from or at risk of suffering from an estrogen dependent cancer. The method comprises administering to the subject a drug, wherein the drug lowers 27-hydroxy cholesterol (27HC) levels in the subject. The drug may be administered alone or in combination with an endocrine therapy. The estrogen dependent cancer may be breast cancer, ovarian cancer or endometrial cancer. The subject may be suffering from estrogen receptor positive breast cancer. The drug may include a CYP27A1 inhibitor, a statin, a cholesterol uptake inhibitor, or a combination thereof. The CYP27A1 inhibitor may include GI268267X, GW273297X, cyclosporin A, an inhibitory RNA, or a retinoic acid receptor antagonist. The inhibitory RNA may target

CYP27A1. The statin may include Atorvastatin, Simvastatin, Lovastatin, Pitavastatin,

Fluvastatin, Mevastatin, Pravastatin, and Rosuvastatin. The drug may inhibit the conversion of cholesterol to 27HC. The cholesterol uptake inhibitor may include eztimibe. The drug may be administered in combination with an endocrine therapy. The endocrine therapy may include at least one of a selective estrogen receptor modulator (SERM), an aromatase inhibitor, a HER2 intervention drug, or combinations thereof. The SERM may include tamoxifen, raloxifene, toremifene, lasofoxifene, pipendoxifene, bazedoxifene, or ospemifene, the aromatase inhibitor comprises anastrozole, letrozole, exemestane, formestane, fadrozole, aminoglutethimide, or testolactone, and the HER2 intervention drug comprises Herceptin (trastuzumab), pertuzumab, or lapatinib. The drug may be administered as a neoadjuvant therapy or post-surgery. The subject may have high levels of cholesterol in the blood. The subject may have normal levels of cholesterol in the blood. A therapeutically effective amount of the drug may be administered.

[0007] The present disclosure is directed to a method of preventing or treating a subject suffering from or at risk of suffering from a metastatic cancer. The method comprises administering a drug that inhibits the activation of liver X receptor (LXR) in the subject. The drug may be administered alone or in combination with a conventional cancer therapy. The drug may inhibit activation of LXR by lowering the 27- hydroxy cholesterol (27HC) levels in the subject. The drug may include at least one of a CYP27A1 inhibitor, a statin, a LXR antagonist, or combinations thereof. The CYP27A1 inhibitor may include GI268267X, GW273297X, cyclosporin A, an inhibitory RNA, or a retinoic acid receptor antagonist, the statin comprises atorvastatin, and the LXR antagonist comprises GSK2033. The inhibitory RNA may target CYP27A1. The conventional cancer therapy may include at least one of endocrine therapy, surgery, radiation therapy, bone-directed therapy, chemotherapy, targeted therapy, or

combinations thereof. The conventional cancer therapy may include endocrine therapy. The endocrine therapy may include at least one of a selective estrogen receptor modulator (SERM), an aromatase inhibitor, a HER2 intervention drug, or combinations thereof. The SERM may include tamoxifen, raloxifene, toremifene, lasofoxifene, pipendoxifene, bazedoxifene, or ospemifene, the aromatase inhibitor comprises anastrozole, letrozole, exemestane, formestane, fadrozole, aminoglutethimide, or testolactone, and the HER2 intervention drug comprises Herceptin (trastuzumab), pertuzumab, or lapatinib. The metastatic cancer may include metastatic breast cancer, metastatic colon, metastatic melanoma, metastatic lung cancer, or metastatic pancreatic cancer. The metastatic cancer may include metastatic breast cancer. A

therapeutically effective amount of the drug may be administered.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 shows that elevated 27HC promoted ER-dependent breast tumor growth. (A) MCF7 xenografts. At day 40, 27HC treated mice were randomized onto continued 27HC, 27HC + ICI 182,780 (ICI), or vehicle treatment (27HC withdrawal) (mean +/- SEM, n = 9-10). (B) Tamoxifen resistant xenografts. Stars (*) indicate significant differences from 27HC treated tumors (mean +/- SEM, p<0.05, n = 5-9). (C/D) MMTV-PyMT mice were bred onto a

CYP7B1+/+ or -/- background. (C) Tumor latency. (D) Tumor growth. Significance between curves is indicated by a connecting black line and * (p<0.05, n = 10-28). (E) Tumor growth in MMTV-PyMT mice. 27HC and E2 curves are significantly different from Placebo and GW3965 curves (p<0.05, n = 110 total). (F) E0771 grafts. Stars indicate significant difference from placebo at that time point (mean +/- SEM, p<0.05, n = 7). [0009] Figure 2 shows that hypercholesterolemia promoted tumor growth in a CYP27A1 dependent manner and can be attenuated by CYP27A1 inhibitors or statins. (A,B) MMTV- PyMT mice were bred onto either a CYP27A1+/+ or -/- background and placed on a control diet (CD) or high cholesterol diet (HCD) at wean. (A) Tumor latency. (B) Tumor growth.

Significance between curves is indicated by a connecting black line and * (p<0.05, n = 9-25). (C and D) E0771 tumor growth in human APOE3 mice on a control diet (CD) or high fat diet (HFD). Stars (*) indicate statistically significant differences with the HFD + placebo group (mean +/- SEM, p<0.05, n = 6-12).

[0010] Figure 3 shows that elevated 27HC increased metastasis to the lung. (A)

Representative lung sections from MMTV-PYMT mice. (B) Quantification of lung metastases by determination of PyMT mRNA. Different letters denote statistical significance (mean +/- SEM, p<0.05, n= 4-13). (C) 27HC and GW3965 induce vimentin expression and change morphology of cells. Overlayed images of green (vimentin) and blue (dapi nuclear stain). All images were adjusted to 90% brightness and 100% contrast. Scale bar = ΙΟΟμιη (D) Lung colonization of pretreated cells (mean +/- SEM, p<0.05, n= 5).

[0011] Figure 4 shows that 27HC was a SERM and LXR agonist. (A) Microarray analysis of MCF7 breast cancer cells indicates that 27HC induces a shared set of genes with E2 and pathways associated with LXR activation. (B) Heat map of genes with largest difference in regulation between 27HC and E2, with associated pathway analysis. (C) Cluster 2 genes from

(B) have significant overlap with LXR target genes as identified in dendritic cells.

[0012] Figure 5 shows that 27HC was a modulator of both ER and LXR target gene expression in cellular models of ERa+ breast cancer. (A) 27HC and synthetic LXR ligands T1317 and GW3965 induce ER target genes such as PS2 and LXR target genes such as ABCAl in MCF7, T47D, BT483 and BT474 cells. (B) Induction of ER target genes was ablated in the absence of ERa (siERa) or when ER activity was blocked with the antagonist ICI 182,780. On the other hand, induction of LXR target genes was enhanced in absence or antagonism of ERa.

(C) 27HC effect on LXR target genes was attenuated while its effect on ER target genes was enhanced in the absence of the LXRs (siLXRa/β) or when LXR activity was blocked with the antagonist GSK2033A. (D) 27HC induces MCF7 proliferation via the ER while its activation of LXRs decreases this effect. Different letters denote statistical significance (mean +/- SEM, p<0.05, n > 3). [0013] Figure 6 shows that 27HC and LXR modulation did not alter cellular proliferation in the ER-negative cell lines, SKBR3 and MDA-MB-231. (A) Activation of LXRs by 27HC or synthetic LXR ligands does not alter proliferation. Time course and bar-graph representation of day 7 is indicated. Dose of 27HC and synthetic LXR ligands GW3965 and T0901317 (T1317) is in Log(M). (B) Inhibition of CYP27A1 by the chemical inhibitors GW273297X (G297X) or GI268267X (G267X), or inhibition of LXRs by the antagonist GSK2033 or RNA interference does not alter proliferation. Bar-graph representation of day 7 is depicted. All data is presented as mean +/- SEM (n=3).

[0014] Figure 7 shows that MCF7 xenografts required estradiol or 27HC supplementation for take and growth. Supplemental data to Figure 1 indicating that when MCF7 cells are grafted into mice they do not grow unless mice are treated with either estradiol or 27HC. (mean +/- SEM, n = 5 for the no hormone group).

[0015] Figure 8 shows expression analysis of tumors from MMTV-PyMT mice treated with indicated ligands. (A) mRNA analysis of indicated genes by QPCR. Different letters denote statistical significance (mean +/- SEM, p<0.05, n = 6-18). (B) Micrographs depicting the proliferation marker Ki67. Scale bar = 200μιη (C) Micrographs depicting the endothelial (angiogenesis) marker CD31. Scale bar = 200μιη (D) Percentage of cells within tumor expressing the macrophage marker CD1 lb as determined by flow cytometry. Different letters denote statistical significance (mean +/- SEM, p<0.05, n = 4).

[0016] Figure 9 shows that CYP7B1 mRNA expression tracked with a better prognosis in patients with luminal A type breast cancer while CYP27A1 mRNA expression was not associated with relapse free survival in patients classified with any subtype of breast cancer. (A) Relapse free survival curve based on CYP7B 1 expression in patients classified with Luminal A type breast cancer (n = 1170). (B) Relapse free survival curves based on CYP27A1 expression in patients classified with any type of breast cancer or only those diagnosed with Luminal A type (n = 1372 or 482 respectively).

[0017] Figure 10 shows that CYP27A1 was highly expressed in macrophages within human benign and malignant mammary tissue. (A) Benign tissue depicting large duct with negatively staining epithelial lining and macrophages in lumen. (B) Benign tissue depicting weakly staining epithelium and strongly staining single stromal macrophage in center. (C) Negatively staining tumor nests with aggregate of strongly positive macrophages in stroma. (D) Negatively staining intraductal carcinoma with strongly positive macrophages in the lumen. A and C taken at lOOx, scale bar = 40μιη. B and D taken at 400x, scale bar = ΙΟμιη.

[0018] Figure 11 shows that conditioned media from macrophages stimulated MCF7 cell proliferation. Some of this activity is inhibited when macrophages are co-treated with the CYP27A1 inhibitor (A) GI268267X (G267X) or (B) GW273297X (G297X), or (C) using CYP27A1 null macrophages. Values at day 9 are indicated in bar graph format. (D) Conditioned media from macrophages induced the expression of ER target genes such as PS2. Different letters denote statistical significance (mean +/- SEM, p<0.05, n = 3-4). ICI = ICI 182,780.

[0019] Figure 12 shows that breast cancer cell intrinsic protein expression of CYP27A1 was associated with tumor grade. Supplemental data for Table 1. Representative breast cancer TMA cores demonstrate a wide range of CYP27A1 expression within cancer cells. (A) negative staining, score = 0. (B) weak staining, score = 1. (C) moderate staining, score = 2. (D) strong staining, score = 3. Scale bars = 80μιη. (E) Plot of probabilities indicating that an increase in CYP27A1 score from weak to overexpressed is associated with an increased probability of being grade 3 and a decreased probability of being a lower grade (1 or 2) (estimated odds ratio of 6.7 with interval of 1.7-26.6, p=0.007).

[0020] Figure 13 shows cholesterol and 27HC concentrations in MMTV-PyMT mice fed a high cholesterol diet (HCD). Supplemental data to Figure 2 A and B in text. (A) Plasma cholesterol levels (n = 7-9). (B) Intratumoral concentrations of 27HC. Values are ng of 27HC per g of wet tissue, (n = 3-6). ND = not detected. Statistics for (B) did not include CYP27A1-/- groups as no 27HC was detected. Values are mean +/- SEM and different letters denote statistical significance.

[0021] Figure 14 shows that CYP27A1 inhibitor GW273297X decreased plasma and intratumoral 27HC concentrations. Supporting data for Figure 2C. (A) A high fat diet (HFD) significantly increased total plasma cholesterol in APOE3 mice compared to a control diet (CD) (n = 3-9). (B) Daily treatment ΟΪΑΡΟΕ3 mice on a HFD with the CYP27A1 inhibitor

GW273297X (GW297X) decreases plasma 27HC concentrations (mean +/- SEM, n = 3-4). (C) Intratumoral concentration of 27HC decreased upon treatment with GW273297X in E0771 tumor bearing APOE3 mice. Values are ng of 27HC per g of wet tissue (mean +/- SEM, n = 4). Different letters denote statistical significance (p<0.05). [0022] Figure 15 shows that statin treatment effectively decreased total cholesterol and inhibited obesity driven tumor growth. Supporting data for Figure 2D. (A) apoE3 mice gain significant weight when fed a high fat diet (HFD) compared to a control diet (CD) for eight weeks (mean+/- SEM, n = 15/group). (B) Oral administration of atorvastatin (statin) from time of tumor graft decreases total plasma cholesterol to levels observed in CD fed mice. Different letters denote statistical significance (mean +/- SEM, n = 3-9). (C) There are linear relationships between obesity (% weight change) and final tumor volume, obesity and total plasma

cholesterol, and total plasma cholesterol and final tumor volume, p-values indicate difference probability that the slope≠ 0.

[0023] Figure 16 shows that a high fat diet failed to impact tumor growth in the MMTV- PyMT mouse model of mammary cancer, where circulating cholesterol levels were not altered by diet. (A) PyMT Mice fed a high fat diet (HFD) fed from wean are heavier compared to a control diet (CD) fed mice. Different letters denote statistical significance (mouse age 65-75 days, mean +/- SEM, n=7-8, p<0.05). (B) HFD fails to significantly alter total plasma cholesterol in this mouse strain (mean +/- SEM, n=4-8). (C) Tumor latency was determined by age at which a palpable tumor was detected. (D) Once a palpable tumor formed, its size was measured and plotted as time to reaching a size of 1000mm ³ . (n =14-15).

[0024] Figure 17 shows that 27HC and GW3965 modulated expression of genes associated with EMT. (A) 27HC and GW3965 modulate mRNA expression of genes associated with EMT. c: control, 2:27HC, G:GW3965. (B) 27HC and GW3965 induce Snaill expression. Overlayed images of green (Snaill) and blue (dapi nuclear stain). (C) 27HC and GW3965 induce FAPa (separase) expression. Overlayed images of green (FAPa) and blue (dapi nuclear stain). All images were adjusted to 90% brightness and 100% contrast. Scale bar = 200μιη.

[0025] Figure 18 shows the chemical structures of small molecule inhibitors of CYP27A1.

DETAILED DESCRIPTION

[0026] The present disclosure provides a method of preventing or treating a subject suffering from or at risk of suffering from estrogen dependent cancer. 27-hydroxycholesterol (27HC), an oxysterol produced in a stoichiometric manner from cholesterol, is an endogenous ligand for the ER and Liver X receptor (LXR). 27HC impacts the growth and metastasis of breast tumors by increasing ER dependent growth and LXR-dependent metastasis in animal models of breast cancer. The manipulation of the synthesis of 27HC may impact breast cancer pathophysiology. The effects of cholesterol on tumor pathology involved CYP27A1 dependent conversion to 27HC are shown herein to be attenuated by both CYP27A1 inhibitors and statins. The relevance of these studies to human disease is highlighted by the observation that CYP27A1 expression increased with grade in breast tumors and that elevated expression of this enzyme was observed within tumor associated macrophages. Described herein are methods of lowering circulating cholesterol or interfering with the conversion of cholesterol to 27HC to prevent and/or treat breast cancer using CYP27A1 inhibitors, statins, and/or cholesterol uptake inhibitors, alone or in combination with endocrine therapy. The disclosed methods may limit the presence of 27HC and likely increase the number of patients that respond to endocrine therapy and prolong relapse.

[0027] The present disclosure provides a method of preventing or treating a subject suffering from or at risk of suffering from metastatic cancer. Cholesterol via conversion to 27HC and subsequent activation of the LXRs promotes metastasis. The present disclosure provides methods of preventing and/or treating metastatic cancer by inhibiting the activation of LXR using CYP27A1 inhibitors, statins, cholesterol uptake inhibitors, LXR antagonists, or combinations thereof. The disclosed methods specifically target or prevent metastasis by limiting the presence of 27HC and/or limiting the activity of the LXR receptor and thus increase the time to relapse.

[0028] The present disclosure provides a method of using CYP27A inhibitors to reduce levels of 27HC in cells in vitro and in vivo as well as treat tumors. CYP27A inhibitors were not previously used to treat animals or humans because it was believed that such an inhibitor would be toxic. The CYP27A protein is involved in various processes and mutations in the CYP27A gene can cause hepatitis of infancy as well as cerebrotendinous xanthomatosis, a lipid storage disease.

1. Definitions

[0029] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0030] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0031] 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. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0032] The terms "27-hydroxycholesterol lowering drug", "27HC lowering drug", and "drug that lowers 27HC" as used interchangeably herein refer to a drug or compound that lowers the levels of 27HC in a subject.

[0033] The term "administration" or "administering," as used herein refers to providing, contacting, and/or delivery of the 27HC lowering drug by any appropriate route to achieve the desired effect. These agents may be administered to a subject in numerous ways including, but not limited to, orally, ocularly, nasally, intravenously, topically, as aerosols, suppository, etc. and may be used in combination.

[0034] "Aromatase inhibitor" as used herein refers to a compound that targets aromatase, which is an enzyme involved in the biosynthesis of estrogen. Aromatase inhibitors may block the production of estrogen or block the action of estrogen on receptors.

[0035] "Breast cancer" as used herein refers to a type of cancer that originates from and develops in the breast. "Metastatic breast cancer" refers to breast cancer that spreads outside the breast to the lymph nodes, bones, or other areas.

[0036] "Cancer" as used herein refers to the uncontrolled and unregulated growth of abnormal cells in the body. Cancerous cells are also called malignant cells. Cancer may invade nearby parts of the body and may also spread to more distant parts of the body through the lymphatic system or bloodstream. Cancers include Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor, Breast Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma of

Unknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer, Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family of Tumors (PNET), Extracranial Germ Cell Tumor, Intraocular Melanoma Eye Cancer, Gallbladder Cancer, Gastric Cancer (Stomach), Extragonadal Germ Cell Tumor, Gestational Trophoblastic Tumor, Head and Neck Cancer, Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cell cancer), Laryngeal Cancer, Acute Lymphoblastic Leukemia, Leukemia, Acute Myeloid, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Hairy Cell Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, AIDS-Related

Lymphoma, Central Nervous System (Primary) Lymphoma, Cutaneous T-Cell Lymphoma, Hodgkin's Disease Lymphoma, Non-Hodgkin's Disease Lymphoma, Malignant Mesothelioma, Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer with Occult Primary, Multiple Myeloma and Other Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplasia Syndrome, Myeloproliferative Disorders, Nasopharyngeal Cancer, euroblastoma, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Pancreatic Cancer, Exocrine, Pancreatic Cancer, Islet Cell Carcinoma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pituitary Cancer, Plasma Cell Neoplasm, Prostate Cancer, Rhabdomyosarcoma, Rectal Cancer, Renal Cell Cancer (cancer of the kidney), Transitional Cell Renal Pelvis and Ureter, Salivary Gland Cancer, Sezary Syndrome, Skin Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Testicular Cancer, Malignant Thymoma, Thyroid Cancer, Urethral Cancer, Uterine Cancer, Unusual Cancer of Childhood, Vaginal Cancer, Vulvar Cancer, and Wilms' Tumor.

[0037] "CYP27A1" as used herein refers to a gene encoding a cytochrome P450 oxidase, namely sterol 27-hydroxylase. The CYP27A1 enzyme is located in many different tissues where it is found within the mitochondria. It is most prominently involved in the biosynthesis of bile acids. The CYP27A1 enzyme participates in the degradation of cholesterol to bile acids in both the classic and acidic pathways. It is the initiating enzyme in the acidic pathway to bile acids, yielding oxysterols by introducing a hydroxyl group to the carbon at the 27 position in cholesterol. In the acidic pathway, it produces 27HC from cholesterol whereas in the classic or neutral pathway, it produces 3P-hydroxy-5-cholestenoic acid. [0038] "CYP27A1 inhibitor" as used herein refers to a drug or compound which interacts with a CYP27A1 enzyme and decreases CYP27A1 enzyme activity.

[0039] The term "effective dosage" as used herein means a dosage of a drug effective for periods of time necessary, to achieve the desired therapeutic result. An effective dosage may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the individual. This term as used herein may also refer to an amount effective at bringing about a desired in vivo effect in an animal, mammal, or human, such as reducing and/or inhibiting the function of CYP27A and the like. A therapeutically effective amount may be administered in one or more administrations (e.g. , the agent may be given as a preventative treatment or therapeutically at any stage of disease progression, before or after symptoms, and the like), applications or dosages and is not intended to be limited to a particular formulation, combination or administration route. It is within the scope of the present disclosure that the 27HC lowering drug may be administered at various times during the course of treatment of the subject. The times of administration and dosages used will depend on several factors, such as the goal of treatment (e.g., treating v. preventing), condition of the subject, etc. and can be readily determined by one skilled in the art.

[0040] "Estrogen dependent cancer" or "estrogen receptor positive cancer" as used interchangeably herein refers to a tumor that contains estrogen receptor (ER) positive cells, i.e., cells that have estrogen receptors, that respond to the presence of estrogen with increased proliferation. Estrogen dependent cancers may include breast cancer, ovarian cancer, or endometrial cancer. "Estrogen receptor positive breast cancer" is a type of breast cancer that is sensitive to estrogen.

[0041] "Estrogen-receptor downregulators" as used herein refers to a drug or compound which binds and down-regulates the expression of an estrogen-receptor.

[0042] "Estrogen receptor negative breast cancer" or "Estrogen independent breast cancer" as used interchangeably herein refers to a tumor that does not contain estrogen receptor positive cells, i.e., cells that lack estrogen receptors, and does not depend on the presence of estrogen for ongoing proliferation.

[0043] "HEPv2 intervention drug" or "HER2 inhibitor" as used interchangeably herein refers to a compound that targets human Epidermal Growth Factor Receptor 2 (HER2). HER2 is a member of the epidermal growth factor receptor family and is involved in the development and progression of certain aggressive types of breast cancer, such as estrogen dependent breast cancer. A HER2 inhibitor may be a tyrosine kinase or a monoclonal antibody.

[0044] "LXR antagonist" as used herein refers to a drug of compound that binds to the LXR, which is a member of the nuclear receptor family of transcription factors. LXRs are regulators of cholesterol, fatty acid, and glucose homeostasis.

[0045] "Metastatic cancer" as used herein refers to a cancer that has spread from the part of the body where it started (the primary site) to other parts of the body.

[0046] "Selective estrogen receptor modulators" or "SERMs" as used interchangeably herein refers to a compound that interacts with an ER and whose relative agonist/antagonist activities are manifest in a cell selective manner. The prevention of estrogen binding to the estrogen receptor may lead to decreased proliferation of estrogen dependent cancer cells.

[0047] "Statin" as used herein refers to a class of drugs used to lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase. Statins are also known as HMG-CoA reductase inhibitors.

[0048] The term "subject", "patient" or "subject in the method" as used herein

interchangeably, means any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non- human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject or subject may be a human or a non- human. In some embodiments, the subject may be a human subject at risk for developing or already suffering from cancer.

[0049] "Treat", "treating" or "treatment" are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of the 27HC lowering drug to a subject that is not at the time of administration afflicted with the disease. "Preventing" also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease. "Treatment" and "therapeutically," refer to the act of treating, as "treating" is defined above.

2. Methods of treating estrogen dependent primary tumors

[0050] The present invention is directed to a method for preventing or treating a subject suffering from or at risk from suffering from an estrogen dependent cancer. The method includes administering to the subject a drug that lowers 27HC levels in the subject. The drug may be administered alone or in combination with an endocrine therapy. The estrogen dependent cancer may be any cancer that needs the hormone estrogen to grow. The estrogen dependent cancer may be a primary tumor, such as breast cancer, ovarian cancer or endometrial cancer. The estrogen dependent cancer may be estrogen receptor positive breast cancer.

3. Methods of treating or preventing of metastasis

[0051] The present invention is directed to a method of preventing or treating a subject suffering from or is at risk from suffering metastatic cancer. The method includes administering a drug that inhibits the activation of LXR in the subject. The drug may be administered alone or in combination with a conventional cancer therapy. A drug that inhibits the activation of LXR may include a 27HC lowering drug, such as a CYP27A1 inhibitor, a statin, or a cholesterol uptake inhibitor, a LXR antagonist, or combinations thereof. The metastatic cancer may be any metastatic cancer. The metastatic cancer may be metastatic breast cancer, metastatic colon cancer, metastatic melanoma, metastatic lung cancer or metastatic pancreatic cancer. The metastatic breast cancer may be estrogen receptor positive breast cancer or estrogen receptor negative breast cancer.

4. 27HC Lowering Drugs

[0052] Cholesterol, subsequent to its conversion to 27HC by the enzyme CYP27A1 , can drive the progression of breast cancer in mouse models. There is a robust stoichiometric relationship between circulating cholesterol and 27HC. A 27HC lowering drug lowers the levels of 27HC in a subject. The 27HC lowering drug may be any drug that inhibits the conversion of cholesterol to 27HC, such as a CYP27A1 inhibitor or a statin, or inhibits cholesterol uptake.

[0053] In some embodiments the 27HC lowering drug can be administered to a patient in an amount of about 10 mg/day to about 500 mg/day, about 10 mg/day to about 200 mg/day (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg/day), 100 mg/day to about 200 mg/day, or about 200 mg/day to about 500 mg/day (e.g., 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, or 700 mg/day), inclusive of any single or multi-dose daily administration regimen that falls within that total daily dose range. In some embodiments, the dose is from 150 - 325 mg/day. Additionally, one of ordinary skill in the art would also know how to adjust or modify variables such as dosage, dosage schedules, and routes of administration, as appropriate, for a given subject.

[0054] Dosage regimens may be adjusted to provide the optimum desired response (e.g. , a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0055] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of the 27HC lowering drug is a dose of between 0.1 and 200 mg/kg, for example between 0.1 and 10 mg/kg. The therapeutically or prophylactically effective amount of the 27HC lowering drug may be between 1 and 200 mg/kg, 10 and 200 mg/kg, 20 and 200 mg/kg, 50 and 200 mg/kg, 75 and 200 mg/kg, 100 and 200 mg/kg, 150 and 200 mg/kg, 50 and 100 mg/kg, 5 and 10 mg/kg, or 1 and 10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. Further, the 27HC lowering drug dose may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the 27HC lowering drug to elicit a desired response in the individual. The dose is also one in which toxic or detrimental effects, if any, of the 27HC lowering drug are outweighed by the therapeutically beneficial effects. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0056] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of the 27HC lowering drug is 0.1-20 mg/kg, more preferably 0.5-10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

a. CYP27A1 inhibitors

[0057] A CYP27A1 inhibitor may be used to treat the subject in the methods described above. The CYP27A1 inhibitor may be a small molecule, such as GI268267X (Lyons et al. (2001) Lipids 36:701-711; see Figure 18 for chemical structure), GW273297X (Lyons et al. (2001) Lipids 36:701-711; see Figure 18 for chemical structure), or cyclosporin A

((3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-Ethyl-33-[(lR, 2R,4E)-l-hydroxy-2-methyl-4- hexen- 1 -yl]-6,9, 18,24-tetraisobutyl-3 ,21 -diisopropyl- 1,4,7,10,12,15,19,25 ,28-nonamethyl- 1,4,7,10,13, 16,19,22,25,28,31-undecaazacyclotritriacontane-2,5,8,l 1,14,17,20,23,26,29,32- undecone). Cyclosporin A may down-regulate CYP27A1.

[0058] The CYP27A1 inhibitor may be an inhibitory RNA, such as a dsRNA, a siRNA, a piRNA, an antisense RNA, a RNAse external guide sequence, a miRNA, a ribozyme, or a shRNA comprising a sequence complementary to a portion of an RNA sequence encoding CYP27A, such mouse CYP27A (GenBank Accession No. NM_024264.4) or human CYP27A (GenBank Accession No. NM_000784.3).

[0059] The CYP27A1 inhibitor may be a retinoic acid receptor (RAR) antagonist. The RAR antagonist may down-regulate CYP27A1. The RAR antagonist may include BMS 453 (4-[(lE)- 2-(5,6-Dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl] -benzoic acid), BMS 195614 (4- [[[5,6-Dihydro-5,5-dimethyl-8-(3-quinolinyl)-2-naphthalenyl] carbonyl]amino]benzoic acid), BMS 493 (4-[(lE)-2-[5,6-Dihydro-5,5-dimethyl-8-(2-phenylethynyl)-2- naphthalenyl]ethenyl]benzoic acid), CD 2665 (4-[6-[(2-Methoxyethoxy)methoxy]-7-t ricyclo[3.3.1.13,7]dec-l-yl-2-naphthalenyl)benzoic acid), ER 50891 (4-[5-[8-(l-Methylethyl)-4- phenyl-2-quinolinyl]-lH-pyrrolo-2-benzoic acid), LE 135 (4-(7,8,9,10-Tetrahydro-5,7,7,10,10- pentamethyl-5H-benzo[e]naphtho[2,3-b][l,4]diazepin-13-yl)ben zoic acid), and MM 11253 (6- [2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-l ,3-dithiolan-2-yl]-2-na

phthalenecarboxylic acid).

b. Statins

[0060] A statin may be used to treat the subject in the methods described above. Statins are a class of drugs used to lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase, which is involved in the production of cholesterol in the liver. A statin may include atorvastatin, simvastatin, lovastatin, pitavastatin, fluvastatin, mevastatin, pravastatin, and rosuvastatin

c. Cholesterol Uptake Inhibitors

[0061] Cholesterol uptake inhibitors (also known as cholesterol absorption inhibitors may be used to treat the subject in the methods described above. Cholesterol uptake inhibitors prevent the uptake of cholesterol from the small intestine into the circulatory system. A cholesterol uptake inhibitor may include eztimibe ((3i?,45)-l-(4-fluorophenyl)-3-[(35)-3-(4-fluorophenyl)-3- hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one).

d. LXR antagonists

[0062] A LXR antagonist may be used to treat the subject in the methods described above. A LXR antagonist may reduce cholesterol levels in serum and liver. The LXR antagonist may include GSK2033 (2,4,6-trimethyl-N-((3 ^* -(methylsulfonyl)biphenyl-4-yl)methyl)-N-((5- (trifluoromethyl)furan-2-yl)methyl)benzenesulfonamide).

5. Conventional Cancer Therapies

[0063] Conventional cancer therapies may include surgery, radiation therapy, bone-directed therapy, chemotherapy, hormone therapy, and targeted therapy. Examples of surgery include lumpectomy, quadrantectomy, mastectomy, such as simple mastectomy, skin-sparing

mastectomy, modified radical mastectomy, prophylactic mastectomy, and radical mastectomy, prophylactic ovary removal, cryotherapy, and lymph node surgery, such as axillary lymph node dissection and sentinel lymph node biopsy. Examples of radiation therapy include external beam radiation, such as accelerated breast irradiation and 3D-conformal radiotherapy, and

brachytherapy (internal radiation), such as interstitial brachytherapy, intracavitary brachytherapy, and intraoperative radiation. Examples of bone-directed therapy include bisphosphonates and denosumab. Examples of chemotherapy include anthracyclines, such as doxorubicin

(Adriamycin, Doxil), epirubicin (Ellence), and daunorubicin (Cerubidine, DaunoXome), capecitabine (Xeloda), carboplatin (Paraplatin), cisplatin, cyclophosphamide (Cytoxan), eribulin (Halaven), fluorouracil (also called 5-fluorouracil or 5-FU; Adrucil), gemcitabine (Gemzar), ixabepilone (Ixempra), methotrexate (Amethopterin, Mexate, Folex), mitoxantrone

(Novantrone), mutamycin (Mitomycin), taxanes, such as paclitaxel (Taxol, Abraxane), and docetaxel (Taxotere), thiotepa (Thioplex), vincristine (Oncovin, Vincasar PES, Vincrex), and vinorelbine (Navelbine). Examples of targeted therapy include trastuzumab (Herceptin), lapatinib (Tykerb), bevacizumab (Avastin), pertuzumab (Perjeta), and everolimus (Afinitor).

a. Endocrine Therapy (Hormone therapy)

[0064] Endocrine therapy, also known as hormonal therapy, hormone therapy, and hormone treatment, is a treatment that adds, blocks, or removes hormones. For example, hormones may be given to adjust low hormone levels. Synthetic hormones or other drugs may be given to block the body's natural hormones to slow or stop the growth of certain cancers (such as prostate and breast cancer). Endocrine therapy may also include surgery to remove the gland that makes a certain hormones.

[0065] Examples of hormone therapy include selective estrogen receptor modulators

(SERMs), such as tamoxifen, raloxifene, toremifene, lasofoxifene, pipendoxifene, bazedoxifene, and ospemifene, aromatase inhibitors, such anastrozole, letrozole, exemestane, formestane, fadrozole, aminoglutethimide, and testolactone, a HER2 intervention drug, such as a HER2 inhibitor, such as Herceptin (trastuzumab), pertuzumab, and lapatinib, and estrogen-receptor downregulators, such as fulvestrant (ICI 182,780).

6. Subject or Subject in the Method

[0066] The methods described above are directed to treating a subject with drug that lowers 27HC levels. The subject treated by the methods described above may be a subject or patient suffering from or at risk of suffering from an estrogen dependent cancer and/or metastatic cancer. The subject may be diagnosed or identified as having or at risk of having estrogen dependent cancer using known methods and assays, such as a biopsy. The subject may be treated with the 27HC lowering drug alone or in combination with a conventional cancer therapy, as described above. The subject may be treated with the 27HC lowering drug as a neoadjuvant therapy or post-surgery. The subject may have normal or high levels of cholesterol in the blood. The subject may be a hypercholesterolemic patient or normocholesterolemic patient. The subject may be identified or diagnosed as having normal or high levels of cholesterol by measuring total cholesterol, high density cholesterol, low density cholesterol, triglycerides, or combinations thereof.

[0067] The present invention has multiple aspects, illustrated by the following non-limiting examples.

7. Examples

[0068] The foregoing may be better understood by reference to the following examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention.

EXAMPLE 1

Methods and Materials

[0069] In vitro Assays. MCF7, T47D, BT483 and BT474 cells were maintained in

DMEM/F12, DMEM, RPMI 1640 or RPMI 1640, respectively, supplemented with 8% fetal bovine serum, non-essential amino acids and sodium pyruvate. For experiments, cells were plated in the same media lacking phenol red and supplemented with 8% charcoal-stripped fetal bovine serum. Expression profiling of 27HC in MCF7 cells, uploaded to GEO as GSE46924, was part of a larger study (GSE35428). All cells were treated with 27HC at ΙμΜ for 24h.

Microarray analysis was performed in R/Bioconductor using the Affy package. Raw data was background corrected with RMA, Log2 transformed, and summarized by median polish.

Differentially expressed genes were called using the LIMMA package and p-values were corrected for multiple testing with the Benjamini-Hochberg procedure. The expression heatrnap was generated in R and ontology enrichment was performed in HOMER. RNA isolation quantitative real time PCR (QPCR) and proliferation assays were performed as previously described (Nelson et al, (2011) Endocrinology 152:4691).

[0070] Statistics. Values were assessed for normality and where appropriate either Ln- transformed or a non-parametric test was selected. Figure IB, IF, Figure 2C, 2D: 2-way ANOVA followed by Bonferroni's post-hoc t-test. Figure 1C, ID, IE, Figure 2A, 2B, Figure 9, 16C, 16D: Kaplan-Meier curves compared to each other using log rank test. Figure 3B, 3D, Figures 13B, 14A, 14B, 15 A, 16A, and 16B: unpaired two tailed student's t-test compared between two groups. Figures 5, 6, 8, 11, 13A, 14C, 15B and 17: 1-way ANOVA followed by Student Newman-Keuls multiple comparison post-hoc test, or Kruskal-Wallis followed by Dunn's multiple comparison test. Figure 15: linear regression was performed and slope compared to null hypothesis of 0. Graphpad Prism was used for analysis unless otherwise stated. Statistical approaches for Table 1, Table 2 and Figure 12E have been described under CYP27A1 IHC Analysis.

EXAMPLE 2

[0071] The impact of 27HC on breast cancer biology was first assessed using a microarray analysis in MCF7 cells (Figure 4A). The data obtained from the 17 β estradiol (E2) arm of the study (GSE35428) was included for comparative purposes. Gene ontology analysis of the most robustly regulated genes (cutoff: p<0.01) revealed a role for 27HC in the regulation of cholesterol homeostasis, tamoxifen resistance and inflammatory IFN responses (Figure 4B). Notable also was the significant overlap between the genes identified and those shown to be regulated by the LXR agonist GW3965 in dendritic cells (GSE23073) (Figure 4C). Follow up studies revealed that 27HC regulated both ERa and LXR transcriptional activity in multiple ERa+ breast cancer cell lines (Figure 5 A). Further, it was observed that induction of the expression of the LXR responsive gene ABCA1 by 27HC, or synthetic LXR agonists, was increased upon inhibition of ERa by the antagonist ICI 182,780, or by ERa knockdown. It was also observed that inhibition of LXRa/β enhanced 27HC mediated induction of ER target gene expression (Figure 5B, 5C). Increased proliferation was also observed in both 27HC or E2 treated MCF7 cells; an activity that was ablated upon ERa knockdown or co-treatment with the ER antagonist ICI 182,780 (Figure 5D). Interestingly, the mitogenic activity of E2 was inhibited by cotreatment with a pure LXR agonist and conversely the ER-dependent effects of 27HC on proliferation were accentuated following siRNA mediated knockdown or antagonist mediated inhibition of LXRa and β activity (Figure 5D). The same treatments were without effect in the ERa negative SKBR3 and MDA-MB-231 cell lines. Similarly the proliferation of these ERa- negative cell lines was unaffected by inhibitors of 27HC synthesis (CYP27A1 inhibitors), LXR antagonists or by LXR knockdown (Figure 6). Taken together these data highlight the complex interactions between ER and LXR and implicate these receptors as mediators of 27HC action in established cellular models of breast cancer. EXAMPLE 3

Xenograft Experiments

[0072] MCF 7 Xenografts. Athymic nude mice were ovariectomized and either implanted with a timed-release E2 pellet (0.72 mg E2/60 days), placebo (no hormone) pellet, or treated with 27HC (40mg/kg/day). Seven days post-surgery, MCF7 tumors were orthotopically grafted into the axial mammary fat pad. When E2 tumors reached a size of 0.2cm ³ , this group was randomized into vehicle treated or ICI 182,780 treated (weekly injection of 5mg/mouse). After 40 days, the 27HC treated mice were randomized into continued 27HC, no hormone withdrawal or 27HC + ICI 182,780 groups.

[0073] Tamoxifen resistant MCF7 Xenograft. Tamoxifen resistant tumors were previously established by serial transfer in mice treated with tamoxifen (Conner et al. (2001) Cancer Res 61 :2917; Wardell et al., (2013) Clin Cancer Res 19:2420). Experimental mice were

ovariectomized and either implanted with a timed-release E2 pellet (0.72 mg E2/60 days), timed- release tamoxifen pellet (5 mg/60 days) or treated with either 27HC (40mg/kg/day) or vehicle (no hormone). Two days post-surgery, tumors (-0.8- lcm ³ ) grown in tamoxifen treated donor mice were removed, cut into 2mm ³ cubes and inserted orthotopically into the axial mammary fat pad of experimental mice.

[0074] E2, but not vehicle treatment, supported the growth of ER-positive MCF7 cell derived tumors propagated as xenografts (Figure 1 A, Figure 7). 27HC also supported the growth of these tumors and this activity was inhibited by cotreatment with ICI 182,780 or upon cessation of 27HC supplementation. The microarray analysis described above identified a potential association between 27HC exposure and the development of tamoxifen resistance. Thus the pharmacology of 27HC was evaluated in a clinically relevant in vivo model of tamoxifen resistance (TamR). It was observed that 27HC promoted tumor growth as well as, or better than, tamoxifen or E2 (Figure IB).

EXAMPLE 4

PyMT Tumor Studies

[0075] Genetic Elevation of 27HC : CYP7B 1 +/+ and CYP7B 1 -/- mice were maintained on a mixed 129/C57BL/6 background. CYP7B1-/- were bred with MMTV-PyMT+ mice on an FVB background. Resulting heterozygous (CYP7Bl+/-;PyMT+) progeny were crossed with either CYP7B1+/+ or CYP7B1-/- mice to create homozygous wildtype or knockout mice expressing PyMT. These mice were again crossed with their respective CYP7B1 line to create breeding PyMT+ males of the appropriate genotype. The males of this generation were bred with their respective CYP7B1 line to generate the PyMT+ females used in experiments. Thus,

CYP7B1+/+; PyMT+ and CYP7B1-/-; PyMT+ females were established. At ~5 weeks of age, mice were ovariectomized. Mice were monitored for first palpable tumors, the growth of which was then recorded through time.

[0076] Pharmacologic Elevation of 27HC. MMTV-PyMT+ mice on an FVB background were obtained from Jackson Labs and bred in house. At ~5 weeks of age mice were

ovariectomized. Mice were monitored for first palpable tumors. At detection, mice were given daily treatment with placebo, 27HC (40mg/kg) to elevate circulating 27HC, GW3965 (30mg/kg) or E2 (l(^g/kg). After 28 days of treatment, serum 27HC concentrations were increased by approximately 40 fold in 27HC treated mice. Resulting plasma concentrations after 28 days of treatment were approximately 4.5μΜ. This was similar to concentrations found in patients with mutations in the CYP7B1 gene, although higher levels are apparent in atherosclerotic lesions, (in the millimolar range). Plasma levels of 27HC were 40-50 folds higher in PyMT mice treated with 27HC compared to placebo. Mice were euthanized when their collective tumor burden reached 2cm ³ .

[0077] The impact of 27HC on tumor pathology in an immune competent MMTV-PyMT murine model of mammary cancer was also evaluated. These mice, which express the MMTV- PyMT transgene, develop spontaneous ERa-positive mammary adenocarcinomas that metastasize to the lung. For this study MMTV-PyMT mice were crossed onto a CYP7B1 ^+/+ or ^~ ' ^~ background. CYP7B1 is responsible for the catabolism of 27HC. In CYP7Br ^/" ;MMTV- PyMT+ mice, plasma and intra-tumoral 27HC concentrations were ~3 times higher than wildtype;MMTV-PyMT+ control mice. Tumor latency was dramatically reduced in the

CYP7Bl ^/_ mice (Figure 1C). Once palpable tumors formed they grew at a significantly increased rate in CYP7Bl ^/_ mice compared to CYP7B1 ^+/+ mice (Figure ID). Treatment of CYP7Bl ^/_ mice with ICI 182,780 resulted in tumor growth rates similar to that in wildtype mice confirming the role of ER in this process. In a separate study, at the time of tumor detection, otherwise wildtype MMTV-PyMT+ mice were treated daily with placebo, 27HC, E2 or the synthetic LXR agonist GW3965. As shown in Figure IE, treatment with E2 or 27HC significantly increased the growth of tumors compared to vehicle, while GW3965 slightly retarded tumor growth when compared to placebo, a result that mirrors the responses observed in vitro.

EXAMPLE 5

E0771 Tumor Studies

[0078] The tumor promoting effects of 27HC were also confirmed in ERa positive E0771 derived tumors propagated syngenically (Figure IF). C57BL/6 mice were purchased from Charles River Laboratories Inc. and ovariectomized. E0771 cells were cultured in RPMI plus non-essential amino acids, sodium pyruvate and 8% FBS. 10-days post-ovariectomy, E0771 were orthotopically grafted into the axial mammary fat pad. Treatments were administered daily by subcutaneous injection. Doses of indicated ligand were the same as for the PyMT tumor growth studies. Tumor growth was increased by either E2 or 27HC supplementation; an activity that was blocked with ICI 182,780. Given the anti-estrogenic activity of LXR agonists noted in vitro, GW3965 significantly decreased tumor growth.

EXAMPLE 6

MMTV-PyMT Tumor Gene Expression

[0079] Assessment of mRNA expression in 27HC treated tumors in the MMTV-PyMT model confirmed elevated expression of both ER and LXR target genes (Figure 8). For the MMTV- PyMT models, mice were euthanized when their collective tumor size reached 2cm ^3' Mice were randomized into their lungs being extracted and frozen, or inflated with 10% formalin. RNA was extracted from frozen lungs and probed for PyMT transcript by QPCR. Formalin inflated lungs were fixed overnight and then preserved in 70% ethanol. They were then dehydrated, paraffin embedded, sectioned and stained with hematoxylin and eosin. Expression of markers for proliferation, macrophage infiltration, angiogenesis and invasion were increased in 27HC treated mice. Taken together these studies confirm the ER and LXR agonist activity of 27HC and implicate ER as the mediator of the effects of this oxysterol on primary tumor growth. EXAMPLE 7

Relapse Free Survival Analysis for CYP27A1 and CYP7B1 mRNA

[0080] An integrative database comprising 4022 patients from 22 publicly available datasets was assembled for querying. The raw data was downloaded from GEO, normalized with RMA and batch corrected using the COMBAT algorithm within R. Clinical data was also aggregated from GEO and duplicate patient samples were filtered out. Each tumor was then classified into tumor subtypes using the PAM50, MODI, and MOD2 gene modules in Genefu (Haibe-Kains et al. (2012) R package version 1.8.0; Parker et al. (2009) J Clin Oncol 27:1160). Gene expression was split by the median into Low and High classifications. Reported p-values were calculated using the log-rank method. Datasets used: GSE11121, GSE12093, GSE12276, GSE1456, GSE16391, GSE16446, GSE19615, GSE22035, GSE22093, GSE23720, GSE26639, GSE3494, GSE5327, GSE6532, GSE7390, GSE9195, GSE17705, GSE2034, GSE20685, GSE21653, GSE4922, GSE6532.

[0081] Human Tissues. Tissue microarrays were used that included duplicate 1 mm cores of formalin fixed, paraffin embedded primary human breast carcinomas from two independent cohorts of patients, as previously described: a group of 59 interpretable tumors from Vienna, Austria (Heller et al. (2007) Breast Cancer Res Treat 103:283) and a group of 112 interpretable tumors from Buffalo, NY (Sood et al. (2007) Hum Pathol 38: 1628). For all tumors, grade and ER/PR/HER2 biomarker data were available. The molecular subclasses were defined as follows: luminal A = positive for ER and progesterone receptor (PR), negative for HER2; luminal B = positive for ER and negative for PR and/or positive for HER2; H2 = negative for ER and PR, positive for HER2; triple negative (TN) = negative for ER, PR and HER2.

[0082] Analysis of CYP7B 1 mRNA expression (rate limiting step in 27HC catabolism) across human breast cancer datasets revealed that its elevated expression was associated with a good outcome in luminal A types (P=0.0469) (Figure 9). Expression of CYP27A1 (rate limiting step in 27HC synthesis) mRNA did not correlate with outcome prompting a closer examination of the importance of enzyme expression. CYP27A1 was highly expressed in macrophages. The studies revealed that regardless of where macrophages reside in human breast tissue (benign vs. malignant or intraductal vs. stromal), they consistently stain very strongly for CYP27A1 (Figure 10). This was in contrast to benign epithelial cells where minimal expression of this enzyme was detected. The well-established correlation between macrophage infiltration and breast cancer outcome suggests that macrophage produced 27HC may be able to support the growth of ER- positive breast tumors.

[0083] It was investigated as to whether conditioned media from bone-derived macrophages could support the growth of ER-positive breast cancer cells and if media quality was influenced by CYP27A1 activity. Using this approach it was determined that (a) macrophage conditioned media stimulated the MCF-7 (a breast cancer cell line) cell growth and that this was inhibited by ICI 182,780 (Fulvestrant), and (b) the basal effect of conditioned media from CYP27Al ^/_ macrophages on MCF-7 cells was increased by 27HC supplementation (Figure 11). The ability of conditioned media to support the growth of MCF-7 cells was compromised by two different CYP27A1 inhibitors (GI268267X and GW273297X), but could be restored by adding 27HC. Cumulatively, these data suggested that local production of 27HC by tumor-associated macrophages is likely to have a significant impact on tumor pathology. Unexpectedly, it was determined that the CYP27A1 protein was expressed, to varying degrees, in cancer cells themselves (human biopsies, Figure 12). In one cohort of breast tumors, overexpression of this enzyme increased the likelihood of also having a higher tumor grade (estimated odds ratio of 6. 7, CI 1.7-27, p=0. 0007; (Table 1, Figure 12). Furthermore, high CYP27A1 was found to be associated with ER negative as well as PR negative tumors (Table 1). As shown in Table 1, CYP27A1 expression was associated with higher grade, ER-negative and PR negative tumors. Frequencies are shown with absolute tallies in brackets. P-values were calculated by exact test demonstrating association (grade and HER2) or ordinal logistic regression to determine estimated odds ratio (ER, PR status).

Table 1

(OR=6.7, P=0.0007) (OR=0.19) (OR=0.21)

[0084] CYP27A1 was strongly expressed in the triple-negative molecular subtype of breast carcinomas (Table 2). CYP27A1 expression in Cohort 1 was associated with molecular class. Frequencies are shown with absolute tallies in brackets. P-value = 0.08 as determined by an Exact test.

Table 2

[0085] This observation was confirmed in a second independent cohort of primary breast carcinomas (Table 3). CYP27A1 expression in Cohort 2 was associated with molecular class. Frequencies are shown with absolute tallies in brackets. P-value = 0.10 as determined by an Exact Test.

Table 3

[0086] Collectively these data confirm the expression of CYP27A1 in breast tumor cells and suggest that in addition to ER-positive cancers, 27HC may also impact the pathology of those tumors that do not express ER. EXAMPLE 8

High Cholesterol Diet Studies

[0087] MMTV-PyMT+ mice were bred with either wildtype or CYP27A1 -/- mice on a C57BL/6 background using a similar strategy to the CYP7B1 mice described above. Thus, CYP27A1+/+; PyMT+ and CYP27A1-/-; PyMT+ females were established. At wean (21 days) mice were placed on either a control diet (TestDiet 5001) or a high cholesterol diet (2% cholesterol, 0.5% sodium cholate), ad libitum. The addition of cholate abrogates the bile acid synthesis deficit and thus poor cholesterol absorption in CYP27A1-/- mice). At ~5 weeks of age mice were ovariectomized. Mice were monitored for first palpable tumors, the growth of which was then recorded through time.

[0088] A diet high in fat and cholesterol decreases latency and increased tumor growth and metastasis in MMTV-PyMT mice. Tumor grafts grew faster when propagated in the

hyperlipidemic Apoe ^~ mouse model. However, since a high fat/high cholesterol diet was used in both of these studies the specific contribution of cholesterol (or its metabolites) on tumor biology could not be assessed. Tumor pathology was evaluated in the MMTV-PyMT model as a function of a high cholesterol diet (HCD). HCD results in significantly elevated cholesterol and 27HC levels, reflecting levels observed in human patients without any known genetic predisposition to elevated cholesterol. MMTV-PyMT mice fed a HCD from wean developed palpable tumors earlier than mice on a control diet (Figure 2A). Furthermore, once tumors were detected, they grew at a faster rate in mice fed a HCD compared to mice on a control diet (Figure 2B). The intratumoral concentrations of 27HC in high cholesterol diet fed mice were elevated (Figure 13). However, in CYP27Al ^"/_ ;MMTV-PyMT+ mice, in which 27HC was undetectable, HCD did not significantly alter tumor latency or growth velocity (Figure 2A,B). The impaired growth of mammary tumors in CYP27A1 ^"7" mice could be restored to that observed in CYP27A1 intact mice by daily administration of 27HC from the point of detection of a palpable tumor. Taken together these data indicate that 27HC and not cholesterol per se, was pathologic in breast tumors. EXAMPLE 9

High fat diet studies in human apoE3 mice

[0089] Human apoE3 transgenic replacement mice were generated previously (39). The control (CD) and high fat diets (HFD) were purchased from TestDiet (5TJS and 5TJN

respectively). To evaluate the effect of a high fat diet male apoE3 mice were placed on a CD or HFD for 8 weeks prior to blood draw and/or tumor graft. For tumor studies, females were ovariectomized between 6-8 weeks of age and placed on their respective diets for 8 weeks. At this point treatment with indicated ligands was initiated and E0771 cells orthotopically grafted into the mammary fat pad. The CYP27A1 inhibitor GW273297X was administered daily (lOOmg/kg) subcutaneously. Atorvastatin was administered by daily oral gavage (40mg/kg).

[0090] Unlike humans, mice do not normally become hypercholesterolemic when fed only a high fat diet (HFD). Therefore, the APOE3 targeted replacement mouse model was used, in which the mouse Apoe gene has been replaced with the human APOE3 allele. A HFD significantly increased both total cholesterol and 27HC in these mice. The elevated 27HC can be decreased by treatment with a CYP27A1 inhibitor (GW273297X) (Figure 14). E0771 tumors grew at an increased rate in HFD APOE3 mice compared to mice on a control diet; a response that was attenuated by treatment with GW273297X (Figure 2C). As plasma 27HC

concentrations are correlated with circulating cholesterol, it was tested whether inhibition of de novo cholesterol synthesis would impact tumor growth. Oral atorvastatin treatment reduced circulating cholesterol and attenuated the exaggerated tumor growth associated with a HFD (Figure 2D, 15). MMTV-PyMT mice on a HFD do not develop hypercholesterolemia and tumor growth in these animals was indistinguishable from those on a control diet (Figure 16). Thus, in mice that model human cholesterol biology, HFD was associated with a negative effect on tumor pathology; an activity that was attenuated by inhibition of cholesterol or 27HC biosynthesis.

EXAMPLE 10

[0091] CYP27A1 IHC Analysis. TMA sections were deparaffinized, treated with sub-boiling antigen retrieval buffer (citrate, pH 6) for 20 minutes, and then reacted with an anti-CYP27Al rabbit monoclonal antibody (abl26785 from Abeam) at 1 :500 for 2 hours. The detection reaction utilized the rabbit Envision kit from Dako. Diaminobenzidine (DAB) was used as chromogen, with hematoxylin as counterstain. The immunohistochemistry (IHC) experiments were performed on an automated immunostainer (Intellipath from Biocare). Paraffin-embedded cell blocks of HEPG2 cells and normal human liver tissue served as external positive controls. Positive cells showed granular cytoplasmic reactivity. Positive cells showed granular cytoplasmic reactivity. A blocking peptide (ab 139504) was able to ablate the signal. All analysis including cell type identification and staining intensity was performed by a board certified pathologist. Macrophages were identified by morphology. Staining intensity in tumor cells was scored as 0 (absent), 0.5 (borderline), 1 (weak), 2 (moderate) or 3 (strong). For statistical analysis, the tumors were categorized as weak (0, 0.5, 1), and overexpressed (2,3). Ordinal logistic regression was used for binary outcomes and proportional odds regression was used for Grade. Score was modeled as a binary predictor with levels weak and overexpressed. For outcomes with low cell counts an exact test of association was used. Analyses were conducted in SAS version 9.3 (SAS Institute, Cary, NC) and the R environment for statistical computing (RCoreTeam (2012) A language and environment for statistical computing. R

Foundation for Statistical Computing).

[0092] Lung Colonization. Near-infrared fluorescent protein (IRFP) expressing plasmid (Addgene 31857) was subcloned into pENTR2B (Invitrogen) to generate the Gateway®- compatible entry vector, pENTR2B-iRFP construct. To generate a lentivirus expressing IRFP, pENTR2B-IRFP was recombined with the destination vector, pLenti-CMV/TO Neo DEST (Addgene 17292) using LR reaction. Stable IRFP expressing Metl cells were established by flow cytometry. Metl cells were stably expressing IRFP were pretreated in culture with vehicle, 27HC or GW3965 for 72hrs. They were then injected (iv) into syngeneic recipient mice. 28 days later, mice were sacrificed, lungs were canulated, re-inflated with PBS and imaged with an IVIS- Kenetic machine (Caliper Life Sciences).

[0093] Whether this oxysterol impacted metastasis in the PyMT model was addressed. As shown in Figure 3A, a large difference in the number of metastatic foci was observed in sections of lungs from mice treated with 27HC. The effect of 27HC on metastasis was confirmed by measuring lung PyMT mRNA expression. A dramatic decrease in metastasis was observed in the CYP27Al ^/_ mice with a similarly impressive increase observed in CYP7bl ^~/_ mice. A similar relationship between elevated 27HC and metastasis was observed in mice that were treated daily with 27HC (Figure 3B). In contrast to the effects on the growth of primary tumors (above), the actions of 27HC on metastasis do not appear to involve ER as E2 was without effect. Conversely, whereas LXR activation attenuates E2 mediated breast cancer cell proliferation in vitro and in the tumor models described above, it was significant that the LXR agonist GW3965 increased lung metastasis, albeit less efficiently than 27HC.

[0094] 27HC and LXR agonists induce the expression of several genes implicated in epithelial-mesenchymal-transition (EMT) (Figure 17). Furthermore, the treatment of breast cancer cell lines in vitro with 27HC, or a synthetic LXR agonist, results in them adopting a spindle like morphology, the degree of which tracks with increased expression of vimentin, Snaill and FAPa, established markers of EMT (Figure 3C, Figure 17). To test the functional consequence of these observations, the metastatic potential of ER-negative Metl cells pretreated in vitro with 27HC was assess. 27HC increased their ability to metastasize to the lung (Figure 3D). Therefore, it is likely that 27HC, acting through LXR, increased lung metastasis secondary to effects on EMT.

EXAMPLE 11

[0095] Treatment of mice with 27-hydroxycholesterol increased the metastasis of colon cancer. See Table 4. Mice were treated with placebo or 27HC for 10 days prior to cell inoculation. Mouse colon cancer MC38 cells were injected IV. After three weeks, organs were harvested and examined for visible metastatic nodules on the lungs. A Fisher's Exact test was used to determine that the proportion of mice with metastatic nodules was significantly greater in 27HC treated mice compared to placebo (p=0.029).

Table 4

[0096] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0097] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.