Diet

Recently, the appreciation of the impact of diet on cancer has come to the fore, with a number of studies establishing unequivocal relationships between diet and cancer initiation and progression. Reflecting the accumulation of these data, the WHO has now stated that diet forms the second-most preventable cause of cancer (after smoking)⁽³⁾. This impact will rise further as a result of demographic factors, and quite possibly because of changing dietary habits worldwide, which will contribute further to the projected increase in cancer incidence in developing nations. High-profile malignancies such as breast, prostate, and colon cancer typify this scenario, in which the aetiology of the disease reflects the cumulative impact of dietary factors over an individual's lifetime⁽Reference Astorg^4–Reference Messina⁶⁾. The relationship between diet and disease is already exploited clinically, e.g. in the Selenium and Vitamin E Cancer Prevention Trial to assess the chemoprevention potential of vitamin E and Se in prostate cancer⁽Reference Djavan, Zlotta, Schulman, Teillac, Iversen, Boccon, Bartsch and Marberger^7–Reference Surh⁹⁾.

Despite the importance and potential clinical benefit of these relationships it remains unclear as to what is the critical time-frame when dietary factors may be protective against cancer development, e.g. during embryogenesis, childhood development or adult life. Resolving this issue is, understandably, highly challenging. Considerable resources were required to elucidate what is now established as a clear causal relationship between cigarette smoke and lung cancer. To address these issues the emerging field of nutrigenomics aims to dissect the impact of dietary factors on genomic regulation, and thereby physiology and pathophysiology, utilizing a range of post-genomic technologies⁽Reference Futreal, Kasprzyk, Birney, Mullikin, Wooster and Stratton¹⁰⁾.

The complex aetiology of cancer

The search for a genetic component(s) to many cancers in this post-genomic era has failed to yield significant results and only a few cancers appear to have a strong genetic component. For example, mutations in the BRCA1 and BRCA2 genes in breast cancer were identified in the 1990s and typically show strong penetrance with a strong familial-linked risk, but these mutations contribute to <5% of breast cancers⁽Reference Wooster, Bignell, Lancaster, Swift, Seal, Mangion, Collins, Gregory, Gumbs and Micklem^11, Reference Miki, Swensen and Shattuck-Eidens¹²⁾. A more recent study implementing genome-wide analyses has indicated five novel alleles that are common in the population and increase the risk of breast cancer, therefore suggesting a role for genetic background in the susceptibility to breast cancer⁽Reference Bergman, Karlsson, Berggren, Martinsson, Bjorck, Nilsson, Wahlstrom, Wallgren and Nordling¹³⁾. A contemporary view of cancer is that there are many low-penetrance genetic factors that combine with environmental insults over the lifetime of the individual to bring about cancer. It is thought that in most cells, two insults are required to lose control of the cell cycle control⁽Reference Hsu, Chao, Chang, Ho, Huang, Huang, Luh, Chen and Lin¹⁴⁾ and between six and ten mutational events to develop into a fully-mature cancer⁽Reference Rajagopalan and Lengauer¹⁵⁾. The recent announcement of plans to immunize girls between 12 and 13 years of age against the sexually-transmitted human papillomavirus applies this theory directly to prevent an essential environmental insult required before a vital step in the transformation of normal cells to cervical cancer can occur. This vaccination programme is estimated to prevent approximately 70% of cervical cancer cases in the UK⁽Reference Mao, Koutsky and Ault¹⁶⁾. It is this complex interaction between environmental and low-penetrance genetic factors that means that age is the single biggest risk factor for the development of cancer because, simply put, there has been more time for environmental insults to impact on precancerous cells.

The sporadic temporal acquisition of a cancer phenotype is also compatible with models of disruption of the self-renewal of epithelial tissues. It has become increasingly clear that breast, colon and prostate tissues, in common with other epithelial tissues and many other cell types in the adult human subject, are self-renewing and contain committed stem cell components⁽Reference Dontu, Al Hajj, Abdallah, Clarke and Wicha^17–Reference Huss, Gray, Werdin, Funkhouser and Smith²²⁾. These stem cells are slowly proliferating and are able to undergo asymmetric divisions to give rise both to other stem cells and to transiently-amplifying populations of progenitor cells. The latter in turn give rise to the differentiated cell types that typify the functions of these tissues and are subsequently lost through programmed cell death processes and replaced by newly-differentiated transiently-amplifying cells. The mechanisms that control the intricate balance of these processes of division, differentiation and programmed cell death are the subjects of major investigations. These studies have revealed common roles for Wnt and hedgehog signalling and the actions of other signal transduction processes that govern cell cycle progression, with gene targets such as the cyclin-dependent kinase inhibitor CDKN1A (p21^(waf1/cip1)) emerging as points of criticality upon which numerous signal pathways converge⁽Reference Reya and Clevers^18–Reference De Marzo, Nelson, Meeker and Coffey²¹⁾.

Stem cells of any tissue also have a high proliferative capacity and are the ideal candidates for tumourigenesis because they are programmed for self-renewal. It is likely to take fewer disruptions to maintain this activation than switching it on de novo in a more differentiated cell. Furthermore, by self-renewing, stem cells are relatively long lived compared with other cells within tissues. Although it has become apparent that there are numerous mechanisms in place in stem cells to ensure genomic integrity, the longevity of these cells results in a greater likelihood of genetic, cytogenetic or epigenetic disruptions accumulating or being passed on to daughter progenitors⁽Reference Beachy, Karhadkar and Berman²³⁾.

Tissue self-renewal is controlled by intrinsic and extrinsic cues, including a range of intrinsic, e.g. niche signals, and extrinsic hormonal and dietary cues, which appear to regulate many of the processes associated with differentiation and programmed cell death⁽Reference Huang, Ma, Zhang, Qatanani, Cuvillier, Liu, Dong, Huang and Moore^24, Reference Ferbeyre²⁵⁾. The primary genomic sensor for many dietary and environmental (e.g. xenobiotic) factors is the nuclear receptor superfamily of ligand-activated transcription factors, which bind steroid hormones, vitamin micronutrients and macronutrients such as fatty acids, lipids and bile acids⁽Reference Reid, Hubner, Metivier, Brand, Denger, Manu, Beaudouin, Ellenberg and Gannon^26–Reference Nagy and Schwabe²⁹⁾.

Structure and function

A broad classification of the nuclear receptor superfamily can be outlined according to ligand affinities. The first group of receptors, exemplified by sex steroid and thyroid hormone receptors, binds ligands with high affinity. A number of nutrient-derived ligands are also bound with high affinity by specific receptors. For example, 1α,25-dihydroxycholecalciferol (1α,25(OH)₂D₃) and the retinoids (all-trans- and 9-cis-retinoic acid) are bound by the vitamin D receptor (VDR) and by the retinoic acid and retinoid X receptors respectively. The second group of receptors, e.g. the PPAR, liver X receptors and farnesoid X receptor, bind with broader affinity more-abundant lipophilic compounds such as macronutrients, PUFA and bile acids. Finally, a group of orphan receptors exists, which either has no functional ligand-binding domain or no ligands have been identified as yet. By contrast, phylogenetic classification has defined seven subfamilies, the VDR being in the group 1 subfamily, sharing homology with the liver X receptors and farnesoid X receptor and more distantly the PPAR⁽Reference Kotnis, Sarin and Mulherkar^36, Reference Hanahan and Weinberg³⁷⁾.

The nuclear receptors share a common architecture, which includes defined regions for DNA recognition, ligand binding and cofactor interactions. The DNA-binding domain recognizes specific response elements (RE), which were originally characterized in the enhancer–promoter regions of target genes. More recently, such functionally-responsive regions have been characterized in both intronic and 3′ regions and gene regulation is brought about through the coordinated actions in multiple responsive regions⁽Reference Reya and Clevers^18, Reference Collins, Berry, Hyde, Stower and Maitland³⁸⁾. Most receptors preferentially form homo- or heterodimeric complexes; retinoid X receptor is a central partner for VDR, PPAR, liver X receptors and farnesoid X receptor. Thus, simple RE are formed by two recognition motives and their relative distance and orientation contributes to receptor binding specificity, although more recently larger, composite and integrated elements have been identified, suggesting a more intricate control⁽Reference Beachy, Karhadkar and Berman^23, Reference Liu, Dontu and Wicha^39, Reference Sherley⁴⁰⁾.

Metabolism of cholecalciferol and major cholecalciferol functions

Systemic monitoring and regulation of serum Ca levels are fundamentally important processes because of the vital function that Ca plays in a wide range of cellular functions. The VDR plays a well-established endocrine role in the regulation of Ca homeostasis, in particular by regulating Ca absorption in the gut and regulating bone mineralization⁽Reference Tiosano, Weisman and Hochberg^41–Reference Dowdle, Schachter and Schenker⁴³⁾. In turn, 1α,25(OH)₂D₃ status is dependent on cutaneous synthesis initiated by solar radiation and also on dietary intake; a reduction in one or both sources leads to vitamin D insufficiency. Interestingly, the contribution from the UV-initiated cutaneous conversion of 7-dehydrocholesterol to vitamin D is the greater, contributing >90% towards 1α,25(OH)₂D₃ synthesis in a vitamin D-sufficient individual⁽Reference Norman⁴⁴⁾. The importance of the relationships between solar exposure and the ability to capture UV-mediated energy is underscored by the inverse correlation between human skin pigmentation and latitude; i.e. the individual capacity to generate 1α,25(OH)₂D₃ in response to solar UV exposure is intimately associated with forebear environmental adaptation⁽Reference Norman⁴⁴⁾. The correct and sufficient level of solar exposure and serum vitamin D are matters of considerable debate. Current recommendations for daily vitamin D intake are in the range of 10–20 μg/d. More recently, reassessment of the 1α,25(OH)₂D₃ impact on the prevention of osteoporosis has suggested that the correct level may be as high as 50–75 μg/d⁽Reference Metivier, Penot, Hubner, Reid, Brand, Kos and Gannon⁴⁵⁾, which may reflect more accurately ‘ancestral’ serum levels.

The importance of the relationship between UV exposure and Ca homeostasis has been understood for >100 years and has driven the endocrine view of 1α,25(OH)₂D₃ signalling with spatially-distinct sites within the body of incremental vitamin D activation. Thus, vitamin D produced in the skin is converted in the liver to 25-hydroxycholecalciferol, (25OH-D), and circulating levels of this metabolite serve as a useful index of vitamin D status. A further hydroxylation occurs in the kidney at the C-1 position by 25-hydroxyvitamin D-1α-hydroxylase (encoded by CYP27b1) to produce the biologically-active hormone 1α,25(OH)₂D₃⁽Reference Norman⁴⁴⁾. A second mitochondrial cytochrome P450 enzyme, the 24-hydroxylase (encoded by CYP24) enzyme, can utilize both 25OH-D and 1α,25(OH)₂D₃ as substrates and is the first step in the inactivation pathway for these metabolites.

More recently, the expression of the 25OH-D activating enzyme, CYP27b1, has been identified in keratinocytes and a wide range of other cell types. In parallel, an autocrine–paracrine role for the local synthesis and signalling of 1α,25(OH)₂D₃ has been uncovered⁽Reference Zehnder, Bland, Williams, McNinch, Howie, Stewart and Hewison^46–Reference Townsend, Banwell, Guy, Colston, Mansi, Stewart, Campbell and Hewison⁵¹⁾. Thus, in multiple target tissues 25OH-D may enter into an intracellular VDR signalling axis that coordinates the local synthesis, metabolism and signal transduction of 1α,25(OH)₂D₃. The components of this axis have been shown to be regulated dynamically, as CYP27b1 is repressed by 1α,25(OH)₂D₃ and correspondingly CYP24 is positively regulated by 1α,25(OH)₂D₃. Thus, elevated levels of 1α,25(OH)₂D₃ appear to block its synthesis and induce its own inactivation⁽Reference Takeyama, Kitanaka, Sato, Kobori, Yanagisawa and Kato⁵²⁾ in a classical negative-feedback loop. The ability of the VDR to play roles in both transactivation and transrepression reflects emerging themes for other nuclear receptors, e.g. PPAR⁽Reference Rosenfeld, Lunyak and Glass^53, Reference Chen, Welsbie, Tran, Baek, Chen, Vessella, Rosenfeld and Sawyers⁵⁴⁾, and suggests a hitherto unsuspected flexibility of the VDR to associate with a diverse array of protein factors to adapt function⁽Reference Murayama, Kim, Yanagisawa, Takeyama and Kato^55, Reference Fujiki, Kim, Sasaki, Yoshimura, Kitagawa and Kato⁵⁶⁾. The biological importance of these autocrine actions have been the subject of intense investigation, and support the concept that the VDR has two, perhaps distinct, broad biological roles, i.e. the endocrine regulation of serum Ca and the autocrine–paracrine regulation of biological functions associated with the regulation of cell proliferation and differentiation and with the modulation of immune responses.

Apo and Holo nuclear receptor states

A current challenge in nuclear receptor biology, and especially pertinent for the VDR, is to define mechanisms that modulate and limit the transcriptional potential, and bring about promoter targeting specificity. Expression, localization and isoform composition of co-repressor complexes have emerged as important determinants of the spatio-temporal equilibrium point between the antagonistic actions of the apo and holo nuclear receptor complexes, and consequently target gene promoter responsiveness⁽Reference Gurevich, Flores and Aneskievich³⁴,Reference Baek, Ohgi, Rose, Koo, Glass and Rosenfeld⁵⁷–Reference Polly, Herdick, Moehren, Baniahmad, Heinzel and Carlberg⁶⁵⁾.

Efforts to understand nuclear receptor function have at their basis the antagonism between these apo and holo nuclear receptor complexes, a direct effect of which is the regulation of a diverse range of histone modifications. Histone modifications at the level of meta-chromatin architecture appear to form a stable and heritable ‘histone code’, such as in X chromosome inactivation (for review, see Turner⁽Reference Turner⁶⁶⁾). The extent to which similar processes operate to govern the activity of micro-chromatin contexts, such as gene promoter regions, is an area of debate⁽Reference Jenuwein and Allis^67, Reference Turner⁶⁸⁾. The apo and holo nuclear receptor complexes initiate specific and coordinated histone modifications⁽Reference Hartman, Yu, Alenghat, Ishizuka and Lazar^69, Reference Strahl, Briggs and Brame⁷⁰⁾ to govern transcriptional responsiveness of the promoter. There is good evidence that specific histone modifications also determine the assembly of transcription factors on the promoter and control individual promoter transcriptional responsiveness⁽Reference Shogren-Knaak, Ishii, Sun, Pazin, Davie and Peterson^71–Reference Varambally, Dhanasekaran and Zhou⁷³⁾. It is less clear to what extent nuclear receptors recognize basal histone modifications on target gene promoters; functional studies of the SANT motif contained in the co-repressor NCoR2/SMRT support this latter idea⁽Reference Yu, Li, Ishizuka, Guenther and Lazar⁷⁴⁾. This area is complex and rapidly evolving (for an excellent recent review, see Rosenfeld et al. ⁽Reference Rosenfeld, Lunyak and Glass⁵³⁾).

In the absence of ligand VDR–retinoid X receptor dimers exist in an ‘apo’ state, as part of large complexes (approximately 2·0 MDa)⁽Reference Yoon, Chan, Huang, Li, Fondell, Qin and Wong⁷⁵⁾, associated with co-repressors (e.g. NCoR2/SMRT) and bound to RE sequences. These complexes actively recruit a range of enzymes that post-translationally modify histone tails, e.g. histone deacetylases and methyltransferases, and thereby maintain a locally condensed chromatin structure around RE sequences⁽Reference Nagy and Schwabe²⁹⁾. Ligand binding induces a so-called holo state, facilitating the association of the VDR–retinoid X receptor dimer with co-activator complexes. A large number of interacting co-activator proteins, which can be divided into multiple families including the p160 family, the non-p160 members and members of the large ‘bridging’ DRIP/TRAP/ARC complex, have been described that link the receptor complex to the co-integrators CBP/p300 and basal transcriptional machinery⁽Reference Reid, Hubner, Metivier, Brand, Denger, Manu, Beaudouin, Ellenberg and Gannon^26, Reference Metivier, Penot, Hubner, Reid, Brand, Kos and Gannon^45, Reference Vaisanen, Dunlop, Sinkkonen, Frank and Carlberg^76, Reference Rachez, Gamble, Chang, Atkins, Lazar and Freedman⁷⁷⁾. These receptor–co-activator complexes coordinate the activation of an antagonistic battery of enzymes, such as histone acetyltransferases, and thereby induce the reorganization of local chromatin regions at the RE of the target gene promoter. The complex choreography of this event has recently emerged and involves cyclical rounds of promoter-specific complex assembly, gene transactivation, complex disassembly and proteosome-mediated receptor degradation⁽Reference Reid, Hubner, Metivier, Brand, Denger, Manu, Beaudouin, Ellenberg and Gannon^26, Reference Metivier, Penot, Hubner, Reid, Brand, Kos and Gannon^45, Reference Metivier, Reid and Gannon⁷⁸⁾.

The expression, localization and isoforms of co-repressor complexes have emerged as critical in determining the spatio-temporal equilibrium between the antagonistic actions of the apo and holo nuclear receptor complexes, and thus determine target gene promoter responsiveness in a range of physiological and pathological settings⁽Reference Hermanson, Jepsen and Rosenfeld^79–Reference Khanim, Gommersall and Wood⁸¹⁾.

Evidence of vitamin D receptor involvement in cancer

In 1981 1α,25(OH)₂D₃ was shown to inhibit human melanoma cell proliferation significantly in vitro at nanomolar concentrations⁽Reference Colston, Colston and Feldman⁸²⁾ and was subsequently found to induce differentiation in cultured mouse and human myeloid leukaemia cells⁽Reference Miyaura, Abe, Kuribayashi, Tanaka, Konno, Nishii and Suda^83, Reference Abe, Miyaura, Sakagami, Takeda, Konno, Yamazaki, Yoshiki and Suda⁸⁴⁾. Following these studies anti-proliferative effects have been demonstrated in a wide variety of cancer cell lines, including those from the prostate and breast⁽Reference Palmer, Sanchez-Carbayo, Ordonez-Moran, Larriba, Cordon-Cardo and Munoz^85–Reference Colston, Colston, Fieldsteel and Feldman⁹²⁾. Thus, common models of VDR responses include MCF-7 breast cancer cells, LNCaP prostate cancer cells and CaCo2 colon cancer cells.

In order to identify critical target genes that mediate these actions, comprehensive genome-wide in silico and transcriptomic screens have analysed the anti-proliferative VDR transcriptome and revealed broad consensus on certain targets, but has also highlighted variability⁽Reference Palmer, Sanchez-Carbayo, Ordonez-Moran, Larriba, Cordon-Cardo and Munoz⁸⁵,Reference Eelen, Verlinden, Van Camp, Mathieu, Carmeliet, Bouillon and Verstuyf⁹³–Reference Wang, Tavera-Mendoza and Laperriere⁹⁵⁾. This heterogeneity may in part reflect experimental conditions, cell line differences and genuine tissue-specific differences in cofactor expression that alter the magnitude and extent of VDR transcriptional actions. The common anti-proliferative VDR functions are associated with arrest at G₀/G₁ of the cell cycle, coupled with up-regulation of a number of cell cycle inhibitors including p21^(waf1/cip1) and p27^(kip1). Promoter characterization studies have demonstrated a series of vitamin D-responsive elements in the promoter–enhancer region of CDKN1A, a primary 1α,25(OH)₂D₃-responding gene⁽Reference Liu, Lee, Cohen, Bommakanti and Freedman^96, Reference Saramaki, Banwell, Campbell and Carlberg⁹⁷⁾. By contrast, p27^(kip1) protein levels appear to be regulated by a range of post-transcriptional mechanisms, such as enhanced mRNA translation, and attenuating degradative mechanisms, often in a cell-type-specific manner⁽Reference Li, Li, Zhao, Zhang, Nicosia and Bai^98–Reference Hengst and Reed¹⁰⁰⁾. The up-regulation of p21^(waf1/cip1) and p27^(kip1) principally mediate G₁ cell cycle arrest, but 1α,25(OH)₂D₃ has been shown to mediate a G₂/M cell cycle arrest in a number of cancer cell lines via direct induction of GADD45α⁽Reference Akutsu, Lin, Bastien, Bestawros, Enepekides, Black and White^94, Reference Jiang, Li, Fornace, Nicosia and Bai^101, Reference Khanim, Gommersall and Wood¹⁰²⁾. Again, this regulation appears to combine direct gene transcription and a range of post-transcriptional mechanisms. These studies highlight the difficulty of establishing strict transcriptional effects of the VDR, as a range of post-transcriptional effects act in concert to regulate target protein levels. Another VDR effect is associated with elevated expression of a number of brush-border-associated enzymes such as alkaline phosphatase, as well as intermediate filaments, vinculin, ZO-1, ZO-2, desmosomes and E-cadherin, which collectively enhance adhesion and suppress migration⁽Reference Palmer, Gonzalez-Sancho and Espada¹⁰³⁾.

Another VDR action, notably in MCF-7 breast cancer cells, is a profound and rapid induction of apoptosis, irrespective of p53 content, which may reflect the VDR role in the involution of the post-lactating mammary gland. The direct transcriptional targets that regulate these actions remain elusive, although there is growing evidence of an involvement of the BAX family of proteins⁽Reference Blutt, McDonnell, Polek and Weigel^104, Reference Mathiasen, Lademann and Jaattela¹⁰⁵⁾. Induction of programmed cell death following 1α,25(OH)₂D₃ treatment is also associated with increased generation of reactive oxygen species. 1α,25(OH)₂D₃ treatment up regulates VDUP1 encoding vitamin D up-regulated protein 1, which binds to the disulfide-reducing protein thioredoxin and inhibits its ability to neutralize reactive oxygen species, thereby potentiating stress-induced apoptosis⁽Reference Song, Cho, Jeon, Han, Hur, Kim and Choi^106, Reference Han, Jeon and Ju¹⁰⁷⁾. In other cells the apoptotic response is delayed and not so pronounced, probably reflecting less-direct effects. Taken together, these data suggest that the extent and timing of apoptotic events depend on the integration of VDR signalling with other cell signalling systems.

Epidemiological evidence

Epidemiological studies by Garland and associates have demonstrated that intensity of local sunlight is inversely correlated with risk of certain cancers including breast, prostatic and colo-rectal carcinoma⁽Reference Garland and Garland^108–Reference John, Schwartz, Koo, Van Den and Ingles¹¹³⁾. Supportively, levels of 25OH-D, the major circulating metabolite of vitamin D, are significantly lower in patients with breast cancer than in age-matched controls⁽Reference Lowe, Guy, Mansi, Peckitt, Bliss, Wilson and Colston¹¹⁴⁾. Furthermore, there are reduced CYP27b1 mRNA and protein levels in breast cancer cell lines and primary tumours⁽Reference Townsend, Banwell, Guy, Colston, Mansi, Stewart, Campbell and Hewison¹¹⁵⁾. Comparative genome hybridization studies have found that CYP24 is amplified in human breast cancer and CYP24 is associated with altered patterns of 1α,25(OH)₂D₃ metabolism⁽Reference Townsend, Banwell, Guy, Colston, Mansi, Stewart, Campbell and Hewison^51, Reference Albertson, Ylstra, Segraves, Collins, Dairkee, Kowbel, Kuo, Gray and Pinkel¹¹⁶⁾. Thus, over-expression of 24-hydroxylase may further abrogate growth control mediated by 1α,25(OH)₂D₃, via target cell inactivation of the hormone. It has therefore been proposed that breast cancer is associated with low circulating concentrations of 25OH-D, arising as a result of reduced exposure to sunlight, altered dietary patterns and impaired generation of 1α,25(OH)₂D₃ within breast tissue⁽Reference Townsend, Banwell, Guy, Colston, Mansi, Stewart, Campbell and Hewison⁵¹,Reference Feskanich, Ma, Fuchs, Kirkner, Hankinson, Hollis and Giovannucci¹¹⁷–Reference Garland, Shekelle, Barrett-Connor, Criqui, Rossof and Paul¹²¹⁾.

Parallel epidemiological studies have also linked the incidence of prostate cancer to vitamin D insufficiency as a result of either diet or environment. In 1990 Schwartz and colleagues suggested a role for vitamin D in decreasing the risk for prostate cancer based on the observation that mortality rates in the USA are inversely related to incident solar radiation⁽Reference Schwartz and Hulka¹¹²⁾. Recently, a study of men in the San Francisco Bay area has reported a reduced risk of advanced prostate cancer associated with high sun exposure, and similar relationships have been established in UK populations⁽Reference Luscombe, French, Liu, Saxby, Jones, Fryer and Strange^110, Reference Luscombe, French, Liu, Saxby, Jones, Fryer and Strange¹²²⁾. As with breast cancer, the proposed mechanism for the protective effects of sunlight on prostate risk involves the local generation of 1α,25(OH)₂D₃ from circulating 25OH-D in prostate epithelial cells. Cancerous prostate cells express reduced 1α-hydroxylase activity. Prediagnostic serum levels of 25OH-D have been assessed in several prospective studies, with some reporting increased risk among men with low circulating levels of the vitamin D metabolite and a suggestion of an inverse relationship with advanced disease⁽Reference John, Schwartz, Koo, Van Den and Ingles¹¹³,Reference Ahonen, Tenkanen, Teppo, Hakama and Tuohimaa¹¹⁸–Reference Chen, Wang, Whitlatch, Flanagan and Holick¹²⁰⁾.

As with breast and prostate cancer, some epidemiological studies have noted that colon cancer risk and mortality increase with increasing latitude; for example, adjusted death rates from colon cancer in Caucasian males in the USA are nearly three times higher in north eastern states than in sunnier more southerly states⁽Reference Slattery, Neuhausen, Hoffman, Caan, Curtin, Ma and Samowitz¹²³⁾.

In vivo studies

The vitamin D receptor in DNA damage and repair

The role of vitamin D in the skin is also suggestive of its chemopreventive effects. UV light from sun exposure has several effects in the skin; UVA light induces DNA damage through increasing the level of reactive oxygen species, but importantly UVB light also catalyses the conversion of 7-dehydroxycholesterol to 25OH-D and induces the expression of VDR.

Several lines of evidence suggest that vitamin D may be protective of solar-induced DNA damage. The anti-proliferative p21^(waf1/cip1) and GADD45α genes are direct targets of both VDR and the tumour suppressor p53. In fact, at least two VDR and p53 RE that lie within the promoter and enhancer regions of p21^(waf1/cip1) are so closely localized that functional interaction between promoter-bound VDR and p53 may be possible⁽Reference Saramaki, Banwell, Campbell and Carlberg⁹⁷⁾. Cooperation between the VDR and p53 may therefore be vital in mediating cell cycle arrest and the repair of DNA within cells with solar and other types of DNA damage.

In addition, antimicrobial and anti-inflammatory genes are another subset of VDR targets that are induced by UV radiation. Suppression of the adaptive inflammatory response is thought to be protective for several reasons; inflamed tissues contain more reactive oxygen species that can damage DNA and prevent proper function of DNA repair machinery, also the induction of cytokines and growth factors associated with inflammation act to increase the proliferative potential of the cells. NF-κB is a key mediator of inflammation and the VDR attenuates this process by negatively regulating NF-κB signalling⁽Reference Szeto, Sun, Kong, Duan, Liao, Madara and Li¹³⁴⁾. This control by VDR is underscored by studies showing that vdr−/− mice are more sensitive to chemicals that induce inflammation than their wild-types counterparts⁽Reference Froicu and Cantorna¹³⁵⁾. The normally protective effect of inflammation that occurs under other conditions is lost through this VDR-mediated suppression but is compensated by the induction of a cohort of antimicrobial and antifungal genes via the innate immune response⁽Reference Gombart, Borregaard and Koeffler^136–Reference Mallbris, Wiegleb, Sundblad, Granath and Stahle¹³⁸⁾. The induction of antimicrobials not only prevents infection in damaged tissue but can be cytotoxic for cells with increased levels of anion phospholipids within their membranes, a common feature of transformed cells⁽Reference Zasloff¹³⁹⁾; experimental results are, however, conflicting. Antimicrobials such as DCDMNQ show potent anti-proliferative effects in prostate cancer cells lines such as PC-3 and Du-145⁽Reference Copeland, Das, Bakare, Enwerem, Berhe, Hillaire, White, Beyene, Kassim and Kanaan¹⁴⁰⁾ and derivatives of 1,2,4-trizole are cytotoxic against some colon and breast cancer cell lines⁽Reference Sztanke, Tuzimski, Rzymowska, Pasternak and Kandefer-Szerszen¹⁴¹⁾. However, the direct VDR target LL-37, also a potent antimicrobial, appears to promote cellular proliferation in HaCaT cells⁽Reference Heilborn, Nilsson, Jimenez, Sandstedt, Borregaard, Tham, Sorensen, Weber and Stahle¹⁴²⁾.

Combined, these epidemiological, in vivo and cell line studies have supported the clinical evaluation of vitamin D compounds in a range of cancer settings. Recent high-dose and combination clinical trials targeting the VDR in prostate cancer have proved encouraging and continue to support therapeutic exploitation of this receptor⁽Reference Beer^143–Reference Beer, Myrthue and Eilers¹⁴⁶⁾. The proposed chemoprotective role of the VDR in the skin in terms of its interactions with p53, the suppression of inflammation and promotion of innate immune responses underscores the importance of vitamin D compounds in the prevention of cancer as well as providing a novel therapeutic target.

Genetic resistance

The gene encoding the VDR protein is known to display polymorphic variation. Thus, polymorphisms in the 3′ and 5′ regions of the gene have been described and variously associated with risk of breast, prostate and colon cancer, although the functional consequences remain to be established clearly⁽Reference Ruggiero, Pacini, Aterini, Fallai, Ruggiero and Pacini^149–Reference Dunning, McBride and Gregory¹⁵⁵⁾. For example, a start codon polymorphism in exon II at the 5′ end of the gene, determined using the fok-I restriction enzyme, results in a truncated protein⁽Reference Gsur, Madersbacher, Haidinger, Schatzl, Marberger, Vutuc and Micksche^156, Reference Morrison, Qi, Tokita, Kelly, Crofts, Nguyen, Sambrook and Eisman¹⁵⁷⁾. At the 3′ end of the gene three polymorphisms have been identified that do not lead to any change in either the transcribed mRNA or the translated protein. The first two sequences generate BsmI and ApaI restriction sites and are intronic, lying between exons 8 and 9. The third polymorphism, which generates a TaqI restriction site, lies in exon 9 and leads to a silent codon change (from ATT to ATC), either of which insert an isoleucine residue at position 352. These three polymorphisms are linked to a further gene variation, a variable-length adenosine sequence within the 3′ untranslated region. The poly(A) sequence varies in length and can be segregated into two groups: long sequences of eighteen to twenty-four adenosines; short sequences⁽Reference John, Schwartz, Koo, Van Den and Ingles¹¹³,Reference Guy, Lowe, Bretherton-Watt, Mansi and Colston¹⁵⁸–Reference Ma, Stampfer, Gann, Hough, Giovannucci, Kelsey, Hennekens and Hunter¹⁶⁰⁾. The length of the poly(A) tail can determine mRNA stability⁽Reference Gorlach, Burd and Dreyfuss^161–Reference Kuraishi, Sun, Aoki, Imakawa and Sakai¹⁶³⁾, so the polymorphisms resulting in long poly(A) tails may increase the local levels of the VDR protein.

Multiple studies have addressed the association between VDR genotype and cancer risk and progression. In breast cancer the ApaI polymorphism shows an association with breast cancer risk, as indeed have the BsmI and the long-sequence poly(A) variant. Similarly, the ApaI polymorphism is associated with metastases to bone⁽Reference Schondorf, Eisberg, Wassmer, Warm, Becker, Rein and Gohring^164, Reference Lundin, Soderkvist, Eriksson, Bergman-Jungestrom and Wingren¹⁶⁵⁾. The functional consequences of the BsmI, ApaI and TaqI polymorphisms are unclear but because of genetic linkage may act as a marker for the poly(A) sequence within the 3′ untranslated region, which in turn determine transcript stability. Interestingly, combined polymorphisms and serum 25OH-D levels have been shown to further compound breast cancer risk and disease severity⁽Reference Guy, Lowe, Bretherton-Watt, Mansi, Peckitt, Bliss, Wilson, Thomas and Colston¹⁶⁶⁾.

Earlier studies have suggested that polymorphisms in the VDR gene might also be associated with risk of prostate cancer. Ntais and co-workers have performed a meta-analysis of fourteen published studies with four common gene polymorphisms (Taq1, poly(A) repeat, Bsm1 and Fok1) in individuals of European, Asian and African descent. They have concluded that these polymorphisms are unlikely to be major determinants of susceptibility to prostate cancer on a wide population basis⁽Reference Ntais, Polycarpou and Ioannidis¹⁶⁷⁾. Equally, studies in colon cancer have yet to reveal conclusive relationships and may be dependent on the ethnicity of the population studied.

Epigenetic resistance

To date no cytogenetic abnormalities of the VDR have been reported. Thus, exploration of epigenetic mechanisms that disrupt VDR signalling is being undertaken by the authors and by other groups. The lack of an anti-proliferative response is reflected by a suppression of the transcriptional responsiveness of anti-proliferative target genes such as p21 ^(waf/cipi1), p27 ^(kip1), GADD45α and BRCA1 ⁽Reference Campbell, Elstner, Holden, Uskokovic and Koeffler^87, Reference Khanim, Gommersall and Wood^102, Reference Rashid, Moore, Walker, Driver, Engel, Edwards, Brown, Uskokovic and Campbell^168, Reference Campbell, Gombart, Kwok, Park and Koeffler¹⁶⁹⁾. Paradoxically, VDR transactivation is sustained or even enhanced, as measured by induction of the highly 1α,25(OH)₂D₃-inducible CYP24 gene⁽Reference Miller, Stapleton, Hedlund and Moffat^170, Reference Rashid, Mountford, Gombart and Campbell¹⁷¹⁾. Together these data suggest that the VDR transcriptome is skewed in cancer cells to disfavour anti-proliferative target genes, and that lack of functional VDR alone cannot explain resistance. It has been proposed that apparent 1α,25(OH)₂D₃ insensitivity is the result of epigenetic events that skew the promoter responsiveness to suppress responsiveness of specific target gene promoters⁽Reference Banwell, O'Neill, Uskokovic and Campbell^172, Reference Banwell, Singh, Stewart, Uskokovic and Campbell¹⁷³⁾.

In support, frequently elevated co-repressor mRNA expression has been found, most commonly involving NCoR2/SMRT, in malignant prostate primary cultures and cell lines, with reduced 1α,25(OH)₂D₃ anti-proliferative response⁽Reference Khanim, Gommersall and Wood^81, Reference Campbell, Elstner, Holden, Uskokovic and Koeffler^87, Reference Campbell, Gombart, Kwok, Park and Koeffler^169, Reference Abedin, Banwell, Colston, Carlberg and Campbell¹⁷⁴⁾. These data indicate that the VDR: co-repressor maybe critical in determining 1α,25(OH)₂D₃ responsiveness in cancer cells. It has been reasoned that this molecular lesion could be targeted by co-treatment of ligand (1α,25(OH)₂D₃) plus the histone deacetylase inhibitors such as trichostatin A. These approaches restore the 1α,25(OH)₂D₃ response of the androgen-independent PC-3 cells to levels indistinguishable from those of control normal prostate epithelial cells. This reversal of 1α,25(OH)₂D₃ insensitivity is associated with re-expression of gene targets associated with the control of proliferation and induction of apoptosis, notably GADD45α. A small interfering RNA approach towards NCoR2/SMRT has demonstrated the important role this co-repressor plays in regulating this response, with its repression resulting in profound enhancement of the induction of GADD45α in response to 1α,25(OH)₂D₃. These data support a central role for elevated NCoR2/SMRT levels to suppress the induction of key target genes, resulting in loss of sensitivity to the anti-proliferative action of 1α,25(OH)₂D₃⁽Reference Khanim, Gommersall and Wood^81, Reference Campbell, Elstner, Holden, Uskokovic and Koeffler^87, Reference Campbell, Gombart, Kwok, Park and Koeffler¹⁶⁹⁾.

In parallel studies a similar spectrum of reduced 1α,25(OH)₂D₃ responsiveness between non-malignant breast epithelial cells and breast cancer cell lines has been demonstrated⁽Reference Banwell, O'Neill, Uskokovic and Campbell^172, Reference Banwell, MacCartney and Guy¹⁷⁵⁾. Again, this reduction is not determined entirely by a linear relationship between the levels of 1α,25(OH)₂D₃ and VDR mRNA expression. Rather, elevated co-repressor mRNA levels, notably NCoR1, in oestrogen receptor α-negative breast cancer cell lines and primary cultures are associated with 1α,25(OH)₂D₃ insensitivity. Again targeting this molecular lesion through co-treatments of 1α,25(OH)₂D₃ with histone deacetylases inhibitors coordinately regulates VDR targets such as p21 ^(waf/cipi1) and GADD45α and restores anti-proliferative responsiveness⁽Reference Banwell, O'Neill, Uskokovic and Campbell^172, Reference Banwell, MacCartney and Guy¹⁷⁵⁾.

Together these data support the concept that altered patterns of co-repressors inappropriately sustains histone deacetylation around the vitamin D-responsive element of target gene promoter–enhancer regions, and shifts the dynamic equilibrium between apo and holo receptor conformations to favour transcriptional repression of key target genes such as p21 ^(waf1/cip1) or GADD45α. Thus, VDR gene targets are less responsive in 1α,25(OH)₂D₃-insensitive cancer cells compared with non-malignant counterparts. Furthermore, targeting this molecular lesion with co-treatments of cholecalciferol compounds plus histone deacetylases inhibitors generates a temporal window in which the equilibrium point between apo and holo complexes is shifted to favour a more transcriptionally permissive environment.

These findings complement a number of parallel studies undertaken by other groups, which have established cooperation between 1α,25(OH)₂D₃ and butyrate compounds, such as sodium butyrate⁽Reference Costa and Feldman^176–Reference Tanaka, Bush, Klauck and Higgins¹⁸¹⁾. These compounds are SCFA produced during fermentation by endogenous intestinal bacteria and have the capacity to act as histone deacetylases inhibitors. Stein and co-workers have identified the effects in colon cancer cells of 1α,25(OH)₂D₃+sodium butyrate co-treatments to include the coordinate regulation of the VDR itself. The authors' studies, in the time-frame studied (0–24 h), have shown no evidence for changes in VDR mRNA levels on co-treatment with 1α,25(OH)₂D₃ plus trichostatin A. However, together these studies underscore further the importance of the dietary-derived milieu in the regulation of epithelial proliferation and differentiation beyond sites of action in the gut.