• Nuclear receptors form a large family of transcription factors that control many aspects of cellular metabolism including growth and differentiation, lipid and glucose homeostasis, and inflammation.
• Several highly successful drugs used for hormone therapy of cancer and the control of diabetes target nuclear receptors. These compounds serve as a model for the development of novel compounds designed to modulate nuclear receptor activity. The challenges are not insignificant, but given the involvement of nuclear receptor signaling in major diseases such as diabetes, cancer, and obesity, the commercial potential is considerable.
• Many of the major types of cancer including breast, prostate, and colon cancer are amenable to control or prevention through selective targeting of nuclear receptors. Clinical and pre-clinical data suggest that changing the activity of nuclear receptor controlled genes can control cancer growth and in all probability, act in a preventive fashion to inhibit transformation. These data highlight the enormous potential of a new strategy for the control and prevention of cancer

Introduction
The nuclear receptor (NR) family contains a large group of transcription factors, with 49 members presently identified in the human genome. The activity of most nuclear receptors is mediated by the binding of small lipophilic ligands such as steroid and thyroid hormones, bile acids, fatty acids, and retinoids. Nuclear receptors are active as either homo- or hetero-dimers and regulate transcription by binding to response elements in the regulatory regions of target genes. Over the past two decades significant advances have been made in the understanding of the regulation of gene expression by nuclear receptors. In many cases, the characterization of the receptor has preceded the study of its function and the identification of a ligand. In the past few years, the ligands for these orphan receptors have been identified and in many cases have turned out to be products of normal metabolism. These receptors regulate a diverse collection of genes that control cell differentiation and growth, lipid homeostasis, and insulin sensitivity.

Nuclear receptors are regulators of gene activity in the cells of the body, which makes them important drug targets for a number of different diseases. This group of proteins forms the second largest class of drug targets and the current market for nuclear receptor targeted drugs is estimated to be 10%-15% of the global pharmaceutical market of US$ 400 billion. Examples of drugs acting on nuclear receptors are estrogens for hormone replacement therapy, antiestrogens for treatment of cancer, and various steroids for treatment of inflammatory disorders. Most of the drugs on the market that act through the modulation of nuclear receptor activity were developed with an incomplete understanding of the receptor that they target. In consequence, they have side effects due to lack of receptor specificity or tissue selectivity. Increased understanding of the structure and function of nuclear receptors and their role in health and disease makes it possible to improve existing therapies and to treat new disorders with novel, more precise drugs that target nuclear receptors. This article will review some of the common forms of cancer and discuss the potential for intervention through modulation of NR activity.

Breast Cancer
Breast cancer is a leading cause of death for women worldwide. Despite recent improvements in mortality rates, predominantly due to earlier detection and better treatment options, novel and effective therapies remain elusive. The majority of breast cancers are sporadic, only 5-10% of all the women with disease inherited a predisposing mutation.

Estrogen Receptor. Estrogens are key regulator of growth, differentiation, and function in a large number of tissues. The biological effects of estrogens are mediated by two receptors, ERa and ERß. Although they are encoded by separate genes, the receptors have a high degree of sequence homology and show a similar binding affinity profile for an array of endogenous and synthetic estrogens. Despite these similarities each receptor has a distinct expression pattern in various tissues, suggesting that they play selective and specific biological functions. ERa is the predominant subtype, expressed in breast, uterus, cervix, and vagina. ERß expression is more limited and is predominantly localized to ovary, prostate, testis, spleen, lung, hypothalamus, and thymus.

In the absence of hormone, the ER is contained within a multi-protein inhibitory complex in the nuclei of target cells. Ligand binding induces a conformational change that promotes homodimerization and binding to DNA. The DNA-bound receptors activate transcription through the interaction with various activating proteins. Depending on the cell and promotor context, the ER exerts either a positive or negative effect on the expression of the downstream target gene. Understanding the control of positive or negative regulation is critical in the design of specific agonistic or antagonistic pharmaceuticals.

Newly diagnosed breast cancer is usually treated with a combination of surgery to remove the primary tumor and adjuvant chemotherapy to eliminate any tumor cells that might have spread to nearby lymph nodes. Developments in the pharmaceutical management of breast cancer include selective hormone-based treatments and next generation treatments such as cancer vaccines and antisense therapy. A large percentage of breast cancers are dependent upon the presence of estrogen for growth. The objective of selective estrogen receptor modulators (SERMs) is to inhibit tumor growth by interrupting the estrogen signaling process.

Although it is generally accepted that ERa and ERß control discrete cellular pathways, an additional layer of complexity in ER signaling became apparent with the discovery of synthetic compounds with mixed agonist/antagonist activity. SERMs show estrogenic effects in some tissues and anti-estrogenic effects in others. This is thought to result from the induction of distinct conformations in the two ERs, allowing the recruitment of different cofactors which, in turn, determine the genes activated by the ligands. Several SERMs are in clinical use for the treatment of cancer and osteoporosis. Raloxifene (Evista®, Eli Lilly), approved for the prevention and treatment of osteporosis, is in clinical trials for the treatment of breast cancer. Raloxifene exhibits agonistic activity in bone and cardiovascular tissue and antagonistic activity in breast. Tamoxifen (Nolvadex®, AstraZeneca), the first SERM introduced into clinical use, is approved for the first line treatment of breast cancer. Tamoxifen has antagonistic effects in breast and agonist effects in bone, uterine, and cardiovascular tissue. Obviously, an ideal pharmaceutical would retain the beneficial effects of estrogen in bone, brain, and cardiovascular tissues while inhibiting the mitogenic effects of estrogen in breast and uterus. Understanding the molecular mechanism behind activation of the ER will facilitate the development of such selective compounds.

Worldwide, over 400,000 women are diagnosed with breast cancer annually. Generating revenues of over $3 billion, the market is receptive for the introduction of new, targeted therapies. The unquestionable success of tamoxifen has established the efficacy of SERMs and highlighted the issues associated with tissue-specific agonist activity. Based on a growing knowledge of nuclear receptor signaling, the next generation of SERMs, should redefine the meaning of selective and be able to be prescribed for long-term prevention of breast cancer recurrence, significantly improving relapse rate and quality of life in breast cancer survivors.

Vitamin D Receptor (VDR) and Breast Cancer Prevention. The vitamin D receptor (VDR) binds 1a,25-dihydroxycholecalciferol D₃ (1,25-(OH)₂-D₃), the most active metabolite of vitamin D, and regulates transcription of vitamin D responsive genes, many of which are involved in calcium endocrinology or bone formation. VDR expression is relatively ubiquitous, and as well as in the expected vitamin D target organs (intestine, bone, kidney, and parathyroid glands), the VDR is found in tissues not involved in calcium homeostasis such as skin, muscle, pancreas, and reproductive organs. At the cellular level, vitamin D signaling affects proliferation, differentiation, and apoptosis of both normal and transformed cells.

First described in the 1920s, vitamin D can be obtained from the diet, although most foods have low concentrations of this essential vitamin. Human epidermal cells are able to synthesize vitamin D from 7-dehydrocholesterol, but this process requires ultraviolet radiation and especially in times of limited sun exposure can be highly variable. Despite the fortification of milk with vitamin D in the US and Canada, vitamin D deficiency is common, especially in the elderly or those who live in northern climates. Of particular significance to the role of vitamin D in breast cancer, both aging and estrogen deficiency are associated with low vitamin D concentrations. Aging reduces the production of vitamin D by the epidermis, and estrogen deficiency decreases both the metabolic activation of vitamin D and the expression of the VDR. Therefore postmenopausal women, the population at the greatest risk of developing breast cancer, are also increasingly likely to suffer from inadequate vitamin D levels.

A considerable amount of work has been devoted to understanding the effects of vitamin D on breast cancer cells and to applying this knowledge to design synthetic vitamin-D-based drugs for cancer therapy. The greatest challenge associated with the application of vitamin D analogs for cancer therapy is that the majority of these compounds induce hypercalcemia at a concentration required to suppress cancer cell proliferation. Nearly 400 structural analogs of vitamin D have been synthesized and evaluated for their efficacy and toxicity, but few have advanced past preliminary toxicity testing.

Another area of research into vitamin D and the VDR and their involvement in cancer focuses on the role of vitamin D signaling in the normal mammary gland and the impact of this pathway in breast cancer prevention. It has been proposed that an activated VDR induces genes that suppress proliferation and stimulate differentiation of cells in the normal mammary gland. Consequently, dysregulation of VDR-mediated gene expression would be expected to alter mammary gland development and possibly predispose cells to transformation, and optimization of VDR-signaling would be expected to protect against malignancy.

Animal models of breast cancer have shown that vitamin D analogs can reduce the incidence of mammary tumors. These analogs were administered as a dietary supplement to rats and shown to be free of side effects when administered chronically. In combination studies, vitamin D analogs enhanced the ability of tamoxifen to prevent tumors, suggesting that these compounds act through independent mechanisms. Cellular studies of vitamin D analogs have shown activity during both the initiation and promotion stages of cellular transformation, suggesting that vitamin D compounds can inhibit both early and late events in tumorigenesis. Finally, epidemiological studies on vitamin D status and breast cancer support a preventative role for this compound in cancer development and progression. The data show that total vitamin D intake is inversely associated with breast cancer risk. While the clinical and scientific data support a role for the VDR in breast cancer, the downstream targets that mediate these effects have yet to be identified. Definition of the affected genes will allow the clarification of the mechanism through which vitamin D and the VDR affect mammary carcinogenesis and will lay the groundwork for a new prevention strategy.

Endometrial Cancer

Cancer of the endometrium is the most common gynecologic malignancy and accounts for 6% of all cancers in women. A highly curable tumor, endometrial cancer is usually treated surgically by hysterectomy and bilateral salpingo-oophorectomy. For patients with more advanced disease, adjuvant radiotherapy is added to the treatment regimen. Current therapy is inadequate for patients with regional and distant metastases. This group of women occasionally respond to hormone therapy, but cures are rarely achieved.

An increased incidence of endometrial cancer has been found in association with prolonged, unopposed estrogen exposure. In contrast, combined estrogen and progesterone therapy prevents the increase in risk of endometrial cancer associated with supplemental estrogen use. An increased incidence of endometrial cancer has also been found in association with tamoxifen treatment of breast cancer, perhaps related to the estrogenic effect of tamoxifen on the endometrium. Because of this increase, patients on tamoxifen should have follow-up pelvic examinations and should be examined if there is any abnormal uterine bleeding.

The most common hormonal treatment for advanced endometrial cancer is progestational agents, which produce antitumor responses in 15% to 30% of patients. These responses are associated with significant improvement in survival and are correlated both with the presence and level of hormone receptors and the degree of tumor differentiation. In one study, progesterone receptor levels were identified as the single most important prognostic indicator of 3-year survival in early stage disease. Patients with high progesterone receptor levels had a 3-year disease-free survival of 93% compared with 36% for patients with poorly expressed receptors.

Progesterone Receptor

Progesterone is a critical steroid hormone that controls cell proliferation and differentiation in the female reproductive tract. Hormonal effects are mediated through interaction with one of two isoforms of the progesterone receptor (PR), PR-A and PR-B. The two isoforms are splice variants of a single gene, and their expression patterns vary according to the developmental or hormonal status of reproductive tissues. The primary tissue that depends on progesterone to limit cellular growth is the uterine epithelium, where progesterone is antagonistic to estrogen-mediated cell proliferation. Both isoforms of the PR are required for endometrial differentiation and down-regulation of one or both isoforms is a frequent occurrence in endometrial carcinogenesis. Progesterone treatment reverses premalignant endometrial hyperplasia and limits the spread of PR positive endometrial cancers, although the mechanisms by which it exerts its effects have not been well characterized. Many of the effects of progesterone are attributable to its ability to oppose the action of estrogen, inhibiting ER gene expression and enhancing degradation of ER proteins. Progesterone has also been shown to exert estrogen-independent effects on cell growth, mediated through PR. The growth inhibitory pathways of progesterone represent a potential target for novel antineoplastic drugs.

Peroxisome Proliferator Activated Receptors (PPARs)

Three subtypes of PPARs have been identified: PPARa , PPARg, and PPARd. First identified in 1990, when PPARa was shown to be the receptor of xenobiotics that induced peroxisome proliferation in rodent liver, the PPAR subfamily has been extensively studied and has been implicated in many cellular processes including lipid and glucose homeostasis, cellular proliferation and differentiation, and control of inflammation. The expression of PPAR genes is not ubiquitous throughout human tissues. PPARg is predominantly expressed in tissues involved in fatty acid catabolism such as liver, kidney, heart, and muscle. PPARd is preferentially expressed in brown and white adipose tissues and in the cells of the intestine where it controls cellular differentiation and lipid storage and modulates the activity of insulin. Both isoforms are expressed in the different cell types that combine to form the vascular wall and can be found in the lipid core of atherosclerotic plaques. The expression of PPAR? is more generalized, and this receptor has been found in many tissues including heart, brain, intestine, muscle, and adrenal glands. Although PPARd has been less widely studied than the other isoforms in this sub-family, it has been implicated in the control of lipid metabolism in the brain, high density lipoprotein (HDL) metabolism, epidermal cell proliferation, and fatty acid induced adipogenesis.

The endogenous ligands for PPARs are fatty acids and fatty acid derived compounds, and these receptors play an important role in the maintenance of lipid homeostasis. PPARg was originally described as a regulator of adipogenesis, but subsequent data have shown this receptor to influence diverse processes such as insulin sensitization, cell differentiation, and atherosclerosis.

PPARg Agonists in Chemoprevention. Ligand activation of PPARg has been shown to cause growth arrest in several tumor cell lines in vitro, and mutations in the PPARg gene have been detected in a proportion of sporadic colorectal carcinomas and prostatic adenocarcinomas. Each mutation produced an inactive receptor that is unable to bind either natural or synthetic ligands. These observations suggest that in addition to its reported role in the maintenance of lipid and glucose homeostasis, PPARg may function as a tumor suppressor gene. PPARg is an attractive target for cancer therapy because while only a subset of patients would be expected to respond to PPARg activation, those without PPARg-inactivating mutations, PPARg ligands are used extensively for the treatment of type-2 diabetes and have very few side effects. The evidence for the effects of PPARg ligands on tumor growth are based on pre-clinical and early clinical observations and need to be confirmed by larger clinical trials designed to define the subset of colorectal and prostate cancer patients who may benefit from such treatment.

Other experiments suggest that PPARg activation has chemopreventive potential. This receptor is expressed in human breast cancer cell lines and breast adenocarcinomas, and in some cases the PPAR ligands, thiazolidines (TZDs), have been shown to inhibit growth and induce differentiation, presumably due to the activation of PPARg regulated genes. The relevance of PPARg activation for chemoprevention has been demonstrated in rodent mammary cancer models, where PPARg ligands reduced carcinogen-induced tumor development. A portion of this chemopreventive effect may be due to a reduction in the expression of aromatase, the rate limiting enzyme in estrogen biosynthesis. Given the success of tamoxifen for the chemoprevention of breast cancer, the selectivity and efficacy of PPARg ligands deserves further investigation.

Colorectal Cancer
Cancer of the colon and rectum are often considered together, and the term colorectal cancer (CRC) is used when the diagnosis, treatment, or epidemiological data refer to both types of cancer concurrently. CRC is the third most common malignancy worldwide and the second leading cause of cancer death, regardless of gender, in the United States. The risk of developing CRC increases with age, beginning to increase after age 40 and rising sharply between ages 50 and 55. The risk doubles with each subsequent decade of life, continuing to rise exponentially. CRC is highly treatable and often curable when localized disease is detected early. For localized disease, surgery is the primary form of treatment, and produces a cure in about 50% of patients. Recurrence following surgery is a major problem and frequently results in advanced and untreatable disease. The prognosis for colon cancer patients is associated with the degree of penetration of the tumor through the bowel wall and the presence or absence of lymph node involvement. Despite advances in surgical techniques and adjuvant therapy protocols, there has only been a slight improvement in survival for patients who present with advanced disease. The need for preventative approaches, screening techniques, and more effective therapies is obvious. As the population ages, the incidence of CRC can be expected to rise, and the availability of better patient management tools will become critical.

Vitamin D Receptor and Colorectal Cancer. A contributing factor to the deleterious effects of a high fat diet is an associated increase in the excretion of fecal bile acids, the most toxic of which is the secondary bile acid lithocholic acid (LCA). LCA is poorly reabsorbed into the enterohepatic circulation, and in consequence, most LCA produced passes into the colon. At high concentrations LCA induces DNA strand breaks, inhibits DNA repair enzymes, and can promote colon cancer in animal models.

In contrast to the positive correlation between high fat diets and colon cancer, dietary intake of vitamin D is related to a reduced incidence of colorectal cancer. In experimental models, vitamin D supplementation has been demonstrated to inhibit LCA induced carcinogenesis. One mechanism for the elimination of LCA is through catabolism by the enterohepatic enzyme CYP3A, a target gene of vitamin D. Studies have shown that the vitamin D receptor (VDR) can function as an LCA sensor in vivo and that binding by LCA results in increased expression of CYP3A. By binding to VDR, both LCA and vitamin D may activate a pathway that increases CYP3A expression and leads to detoxification of LCA. This model explains how the enteric system could protect itself from the harmful effects of LCA and why vitamin D is protective against colon cancer in normal conditions. Epidemiological data support this relationship, showing that those areas with the highest death rates from colon cancer also have high prevalence of clinical conditions of vitamin D deficiency such as rickets. This association between the VDR and colon cancer suggests a potential for dietary contribution to cancer prevention, balancing fat intake and vitamin supplementation to reduce cancer risk.

Prostate Cancer
Prostate cancer afflicts one man in nine over the age of 65 and is the most frequently diagnosed cancer in men in the United States. Worldwide incidence varies, and Asian countries in particular have a much lower incidence of prostate cancer than is found in the US. The incidence of prostate cancer increases with age, and advanced age is one of the most significant risk factors for developing prostate cancer. Autopsy studies have shown that 30% of men in their 50s, 40% of men in their 60s, and virtually all men in their 9th decade have evidence of prostate cancer. Significantly, most of these cancers are small and of low histological grade, and most men with prostate cancer do not live long enough to develop clinically significant disease. Early detection through serum testing for prostate specific antigen and improved surgical and radiotherapy procedures have reduced the mortality rate of this disease, but no effective cure exists for men with advanced cancer. In the US, prostate cancer is the second leading cause of death due to cancer in men, accounting for 11% of male cancer-related deaths.

Androgen Receptor. Androgens mediate a wide range of physiological responses and are especially important in male sexual development and maturation, the maintenance of spermatogenesis, and male gonadotropin regulation. These effects are mediated through the androgen receptor (AR) and are modulated by coregulatory proteins. While androgens are essential for normal development and functioning of the prostate, many forms of prostate cancer are also sensitive to androgen activity. The predominant form of treatment for advanced prostate cancer is surgical and/or pharmacological androgen ablation, often combined with nonsteroidal anti-androgens to block residual adrenal androgen activity. Although most patients respond to androgen ablation, tumors ultimately become resistant to therapy and progress in an androgen independent fashion. The control of AR activity is a developing field, and as additional data accumulates the key activating pathways and interesting targets for pharmaceutical intervention will become evident.

Flavonoids and AR Activity. The polyphenolic flavonoid silymarin (SM) has been shown in several models of human cancer to effectively inhibit cell growth with little evidence of toxicity. Extracted from the milk thistle, SM has been used clinically in Europe and Asia for the treatment of alcoholic liver disease for many years. Silibinin, the major active component of SM has been shown in laboratory studies to cause cell cycle arrest in a prostate cancer cell lines. This cytostatic activity has been attributed to the down-regulation of several androgen-regulated genes primarily through inhibition of the transactivation activity of the AR. Mechanistically, SM appears to inhibit AR activity by blocking nuclear localization of the ligand bound receptor. These observations could lead to development of novel approaches to blocking AR mediated cell growth that are independent of the presence of activating ligand; through reduction of nuclear concentrations of AR, the activation of AR mediated genes will be inhibited.

Conclusions
Nuclear receptors are an attractive and relatively unexploited target for drug development. Functioning as transcription factors and thereby controlling cellular processes at the level of gene expression, modulation of NR activity produces selective alterations in downstream gene expression. These characteristics combined with their involvement in significant diseases, make NRs a key target for the development of disease-specific therapy.

Several members of the NR family control the expression of genes that promote cellular growth and differentiation and therefore are targets for cancer prevention and therapy. The success of this approach has been clearly demonstrated by marketed drugs such as tamoxifen and raloxifene, which were developed without a full understanding of their mechanism of action. The rapidly growing understanding about the cellular mechanisms controlled by NRs will facilitate the design of increasingly specific NR modulators. The ultimate goal is to produce compounds that can be administered to healthy individuals as chemopreventives, reducing the need for cancer therapeutics and significantly improving the outlook of those patients at risk of developing malignancy.