Re: Vitamin D and Cancer: A review of molecular mechanisms

[문형철]

beyond reason

임상논문을 검색해보면 비타민 d는 거의 기적과 같은 물질임을 알 수 있다. 우리가 알고 있는 거의 모든 질환에서 비타민 d는 치료효과를 보인다. 심지어 암을 예방하고 치료하고 수명을 연장한다. 

그 기전의 이해

우리 몸의 모든 세포(stromal cell, endothelial cell, immune cell)의 cytoplasm에는 비타민 D 수용체(VDR)이 있다. 핵내의 XRX(retinoid X receptor)와 결합( 1,25(OH)2D3+VDR+XRX complex)하여 세포기능을 정상화한다. 

비타민  d은 간에서 1차변환(calcidiol)하고 신장에서 비타민 d의 활성물질 형태인 calcitriol이라는 물질로 변환되어 gene transcription을 조절하여 모든 세포의 기능을 정상화시킨다. 

암세포의 사멸을 유도하고(apoptosis of tumor cell), DNA 수리, 항산화, 면역조절효과를 보인다. 

암세포 혈관신생억제, 암세포 성장억제, 암세포 유전자 돌연변이 억제, 염증성 사이토카인 억제 등...

Biochem J. Author manuscript; available in PMC 2015 Sep 16.

Published in final edited form as:

Biochem J. 2012 Jan 1; 441(1): 61–76.

doi: 10.1042/BJ20110744

PMCID: PMC4572477

NIHMSID: NIHMS721722

PMID: 22168439

Vitamin D and Cancer: A review of molecular mechanisms

James C. Fleet,1,5 Marsha DeSmet,4 Robert Johnson,2 and Yan Li3

Author information Copyright and License information Disclaimer

The publisher's final edited version of this article is available at Biochem J

See other articles in PMC that cite the published article.

Go to:

Synopsis

The population-based association between low vitamin D status and increased cancer risk can be inconsistent but is now generally accepted. These relationships link low serum 25 hydroxyvitamin D levels to cancer while cell-based studies show that the metabolite 1,25 dihydroxyvitamin D is the biologically active metabolite that works through vitamin D receptor to regulate gene transcription.

Here we review the literature relevant to the molecular events that may account for the beneficial impact of vitamin D on cancer prevention or treatment. This data shows that while vitamin D-induced growth arrest and apoptosis of tumor cells or their non-neoplastic progenitors are plausible mechanisms, other chemoprotective mechanisms are also worthy of consideration. These alternative mechanisms include enhancing DNA repair, antioxidant protection, and immunomodulation. In addition, other cell targets such as the stromal cells, endothelial cells, and cells of the immune system may be regulated by 1,25 dihydroxyvitamin D and contribute to vitamin D mediated cancer prevention.

Keywords: 1,25 dihydroxyvitamin D; 25 hydroxyvitamin D; proliferation; apoptosis; transcription; vitamin D receptor

Introduction

Over the past decade, researchers have generated data demonstrating that vitamin D and its metabolites have actions that may be useful for the prevention or treatment of various cancers. This is a new role for vitamin D that is distinct from the traditional role it has in the control of calcium and bone metabolism [1].

The first suggestion that vitamin D might influence cancer came in 1980 when Garland and Garland proposed that the high rate of colon cancer seen in the Northern US compared to the Southern US was due to the UV light-induced production of vitamin D in the skin [2]. Ecological studies have since extended the “sunlight” hypothesis to 18 different types of cancer [3]. Although ecological studies like these are the weakest forms of scientific evidence, several other lines of evidence suggest that vitamin D or its metabolites have a direct inhibitory action on the development and progression of various cancers. Some population-based studies show that low serum 25 hydroxyvitamin D (25OH D) levels are associated with increased risk of cancers of the colon, [4], breast [5], and prostate [6] [7] as well as other cancers [8]. Studies in animals have shown that severe vitamin D deficiency [9–11] or deletion of the vitamin D receptor (VDR) gene [12–14] increase cancer risk. In addition there are a number of studies that show a reduction in cancer (tumor incidence or tumor size) in animals injected with chemical analogs of the vitamin D hormone 1,25 dihydroxyvitamin D (1,25(OH)2 D) [15–20]. While there is a growing consensus that vitamin D and its metabolites are important for the control of various cancers, the mechanistic foundation for this protection is still being determined. In this review we summarize the current thinking on the mechanisms used by vitamin D metabolites to influence the development of cancer.

Brief review of Vitamin D metabolism and the cellular actions of 1,25 dihydroxyvitamin D (1,25(OH)2 D)

Vitamin D3 can be produced from 7-dehydrocholesterol when skin is exposed to UVB light. However, regardless of whether vitamin D comes from the skin or the diet, vitamin D3 is transported in the circulation by the Vitamin D Binding Protein (DBP) [21]. Once delivered to the liver, vitamin D is hydroxylated on its side chain to form 25 hydroxyvitamin D (25OH D). This is a stable metabolite whose serum levels are used to assess vitamin D status. The kidney is the primary site where the active form of vitamin D, 1,25(OH)2 D, is produced. The circulating levels of 1,25(OH)2 D result from 1 alpha hydroxylase (CYP27B1) mediated conversion of 25OH D in the proximal tubule of the kidney [22]. Renal CYP27B1 gene expression‎ is activated by PTH and suppressed by 1,25(OH)2 D and the serum levels of these hormones are inversely related to dietary calcium intake [23].

Once produced in the proximal tubule, 1,25(OH)2 D is released into the serum and acts as an endocrine hormone on the intestine, bone, and kidney to control calcium metabolism. Although serum 1,25(OH)2 D levels do not associate with cancer risk in human studies, this is the metabolite responsible for the anticancer actions of vitamin D at the cellular level. In addition to the 1 alpha hydroxylase activity found in the kidney, low levels of CYP27B1 protein and message have been observed in many tissues, including cells of the skin, lymph nodes, colon, pancreas, adrenal medulla, brain, placenta [24], in primary prostate epithelial cells [25] in the MCF-7 breast cancer cell line [26], and in the colon cancer cell lines HT-29 and Caco-[27]. Extrarenal production of 1,25(OH)2 D is proposed to be driven by the serum 25OH D level, thereby accounting for why serum levels of this inactive metabolite could associate with cancer risk. This is consistent with studies that show CYP27B1 operates well below its Km [28], the fact that 1,25(OH)2 D production is seen in cultured non-renal cells treated with 25OH D [29;30], and the observation that targeted disruption of the CYP27B1 gene in RAS-transformed keratinocytes blocks the antiproliferative and prodifferentiating effects of 25OH D in vitro [31]. However, the weakness of this hypothesis is that no direct evidence currently exists to prove that meaningful local production occurs in vivo.

Molecular mechanism of 1,25(OH)2 D signaling

The primary molecular action of 1,25(OH)2 D is to initiate or suppress gene transcription by binding to the vitamin D receptor (VDR), a member of the steroid hormone receptor superfamily of ligand-activated transcription factors [32] (Figure 1A). The VDR can be found in both the cytoplasm and nucleus of vitamin D target cells but in many cells it is predominantly a nuclear protein. Binding of 1,25(OH)2 D to the VDR promotes association of VDR with the retinoid X receptor (RXR) and this interaction is essential for VDR transcriptional activity. Some data demonstrates that heterodimerization is required for migration of the RXR-VDR-ligand complex from the cytoplasm to the nucleus [33–36] where the 1,25(OH)2 D-VDR-RXR complex binds to vitamin D response elements (VDRE) in DNA to initiate gene transcription [32].

However, recent data from studies using chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to identify VDR binding sites throughout the genome show that RXR can be bound to VDR binding sites in DNA prior to VDR recruitment to those sites [37]. ChIP-seq analysis conducted in osteoblasts [37] and in lymphoblastoid cells [38] also reveal that the great majority of genomic sites occupied by VDR after 1,25(OH)2 D treatment are likely acting as enhancer elements – only 13 to 23% of VDR binding sites are within the classical promoter regions of genes just proximal to the transcription start site (Figure 1B). Regardless, once bound to DNA the VDR-RXR heterodimer recruits protein complexes that alter chromatin structure. For transcriptional activation these proteins form a complex with histone acetyl transferase (HAT) activity (e.g. CBP/p300, SRC1 [39;40]) as well as ATP-dependent remodeling activity (e.g. the BAF57 subunit of SWI/SNF directly interacts with SRC1 and steroid hormone receptors [41]) to release higher-order chromatin structure that limits gene transcription. After chromosomal unwinding, the VDR-RXR dimer recruits the mediator complex to the promoter and utilizes it to recruit and activate the basal transcription unit containing RNA polymerase II [42]. For transcriptional repression the VDR-RXR dimer recruits co-repressors like NCoR1, NCoR2/SMRT, and Alien that then recruit histone deacetylases and DNA methyltransferases that alter histone tails in ways that lead to a more compact chromatin structure [43] (Figure 1A). For those interested in more details regarding the molecular activation of VDR and its role in gene transcription, the subject was recently reviewed by Pike et al. [37;44].

Figure 1

Summary of vitamin D mediated gene transcription through the vitamin D receptor (VDR). (A) Schematic showing alternative models for binding of VDR-RXR dimers to DNA and the regulation of gene transcription by vitamin D. In model A, RXR (yellow hexagon) is resident. VDR (green rectangle) binds after being activated by ligand, releasing any co-repressor complexes if they are present. In model B, VDR and RXR dimerize then bind to DNA. Both model A and B lead to the recruitment of additional co-activators, e.g. p300/CBP (star) and SRC-1 (orange oval) for histone acetylation, ATP-dependent chromatin remodeling factors like SWI/SNF, the mediator complex (dark purple rectangle) for recruitment and activation of RNA polymerase II (light blue oval). Model C shows that the vitamin D-VDR complex can also recruit co-repressor complexes with histone deacetylase activity (purple oval) and DNA methyl transferase activity (green circle) to gene promoters. This will suppress gene transcription. (B) Schematic showing the distribution of VDR binding sites in the genome of osteoblasts [211] and lymphoblastoid cells [38] relative to the transcription start site (TSS) of a gene. Values are given as a percentage of total VDR binding sites and were determined by chromatin immunoprecipitation coupled to DNA arrays (ChIP-chip) or next-generation sequencing (ChIP-seq).

In addition to the transcriptional regulation mediated through the VDR, there is some evidence that 1,25(OH)2 D may work in other novel ways. These mechanisms are less well described but, where relevant, they will be presented in the following sections.

Effects of 1,25(OH)2 D on Cell Proliferation and Apoptosis

Many believe that the target cells for the anti-cancer action of vitamin D are tumor cells and the normal cell types within tissues that transform into tumor cells. In this context the best-studied role of 1,25(OH)2 D is its growth inhibitory effects on proliferating epithelial cells. Colston et al. first showed a dose-dependent decrease in growth rate of melanoma cells treated with 1,25(OH)2 D [45]. The growth inhibitory property of 1,25(OH)2 D has since been reported in tumor-derived cells from other tissues including the colon [46], breast [47] and prostate [48]. The VDR is essential for 1,25(OH)2 D-mediated growth inhibition. In both mouse epidermal keratinocytes [49] and in SaOS-2 osteosarcoma cells [50] 1,25(OH)2 D-induced growth arrest is lost when VDR is absent. Also, antisense-oligonucleotides that reduce cell VDR levels can block 1,25(OH)2 D -induced growth arrest in ALVA-31 prostate cancer cells [51] and over-expression‎ of VDR enhanced 1,25(OH)2 D induced growth arrest in several prostate cancer cell lines [52;53]. Recently we reported that prostate epithelial cell (PEC)-specific deletion of VDR in the mouse increased PEC proliferation and reduced PEC apoptosis [54]. In contrast, Costa et al. [55] found that although siRNA mediated VDR knockdown was nearly complete in MCF7 cells (90% lower), the growth inhibitory effect of 1,25(OH)2 D was not significantly reduced by anti-VDR siRNA, suggesting there also may be VDR independent mechanisms for this action.

Cell cycle regulators

Many investigators have looked for direct effects of 1,25(OH)2 D on the expression‎ of genes that control cell growth. An example of this is 1,25(OH)2 D-mediated transcriptional regulation of the gene encoding the cyclin-dependent kinase inhibitor p21 in the myelomonocytic cell line U937 [56]. A number of other studies have shown that cyclin-dependent kinase inhibitors like p21 or p27 increase, and that cell cycle regulatory proteins like cyclins decrease, coincident with 1,25(OH)2 D-induced growth arrest [57–60]. In addition, 1,25(OH)2 D-mediated growth arrest of prostate cancer cell lines can be inhibited by antisense RNA or siRNA against p21 [58;61]. In contrast, 1,25(OH)2 D has a minimal effect on p21 mRNA levels in MCF-7 cells even though the cells growth arrest in response to treatment [62]. In addition, 1,25(OH)2 D increased p21 protein but not p21 mRNA in LNCaP cells [63]. This suggests the growth inhibitory effect of 1,25(OH)2 D is not mediated by VDR-mediated transcription activation of the p21 gene promoter in some cell types. However, Thorne et al. [64] recently found that histone modification patterns at three distinct VDRE containing sites in the p21 gene promoter (at −7 kb, −4.5 kb, and −2.1 kb) are important for vitamin D-regulated expression‎ of this gene in immortalized, but non-transformed RWPE1 prostate epithelial cells. In particular, they found that histone signatures associated with vitamin D-mediated gene activation were enriched in G1 and S phase cells, suggesting a more robust role for induction of p21 in the early phases of cell cycle.

FoxO proteins are tumor suppressors that control cell proliferation [65]. The function of several FoxO family members is inhibited by MAPK-mediated phosphorylation. Recently An et al. [66] showed that 1,25(OH)2 D treatment regulates binding of FoxO3a and FoxO4 to DNA regulatory regions by stimulating a direct interaction between VDR, FoxO3a or FoxO4, and the FoxO regulators Sirt1 (a class III histone deacetylase) and protein phosphatase 1. Sirt1 and protein phosphatase 1 promote nuclear retention of FoxO proteins by counteracting MAPK-mediated phosphorylation. Consistent with an essential role for these interactions, 1,25(OH)2 D-mediated growth arrest is blocked in SCC25 cells treated with siRNA against FoxO3a.

Microarray analyses have revealed other potential vitamin D gene targets related to cell cycle control. In primary prostate cancer cells, expression‎ of CDK1 mRNA (encoding a protein required for cell cycle progression) was suppressed after 24 hr of 1,25(OH)2 D treatment [67] and transcripts for cell cycle inhibitor proteins like RBL2 (Rb-like protein p130) and RBBP6 (Rb binding protein 6) were up-regulated by 1,25(OH)2D in the MCF-7 and MDA-MB-231 breast cancer cell lines [68]. A number of transcripts encoding cell cycle control proteins appear to be indirectly regulated by 1,25(OH)2D. For example, in RWPE1 prostate epithelial cells, cell cycle control genes generally do not change until 24 or 48 after treatment [69]. Consistent with this, cyclin A, cyclin B, and cyclin F mRNA levels were downregulated in SCC25 cells [70] and cyclin F mRNA was reduced in SW480-ADH cells only after 48 hr of treatment with 1,25(OH)2 D or the vitamin D analog EB1089.

Insulin-like Growth Factor (IGF) Signaling

1,25(OH)2 D may also indirectly influence the growth rate of cells by interfering with the action of growth factors that stimulate proliferation or by increasing the production of those that promote cell differentiation. In MCF-7 cells, insulin-like growth factor 1 (IGF1)-stimulated cell growth was inhibited by vitamin D analogs and this effect was associated with increased release of IGF binding protein 3 (IGFBP3) into the medium [71]. IGFBP3 is known to limit the pro-proliferative, anti-apoptotic actions of IGF1 and IGF2 by binding to them and limiting their ability to interact with cell surface receptors. 1,25(OH)2 D and vitamin D analogs also induce accumulation of IGFBP3 in prostate cancer cells and primary prostate epithelial cells and this subsequently inhibits IGF2-action [72;73]. Upregulation of IGFBP3 mRNA levels by 1,25(OH)2 D has also been seen in microarray analysis of LNCaP prostate cancer cells [74] and the immortalized prostate epithelial cell line RWPE1 [69]. A putative VDRE was identified in the IGFBP3 gene promoter and characterized by EMSA and ChIP assays, indicating that the 1,25(OH)2 D-induced increase in IGFBP3 levels is likely to be direct [75]. Further evidence that IGFBP3 is a critical mediator of the growth inhibitory properties of 1,25(OH)2 D comes from studies showing that antisense oligonucleotides against IGFBP3 abolish 1,25(OH)2 D-mediated growth arrest in LNCaP cells [76]. Nickerson and Huynh [77] have also shown that a 14-day treatment with the vitamin D analog EB1089 increased the prostate expression‎ of many IGFBP isoforms, including IGFBP3, and this was associated with reduced prostate size in rats. In addition to an effect on IGFBP3 mRNA levels, microarray analyses of 1,25(OH)2 D treated SW480-ADH cells also show upregulation of IGFBP2 and IGFBP6 transcripts but the functional impact of this regulation has not been explored [78].

Transforming Growth Factor Beta (TGFβ) Signaling Pathway

TGFβ2 is essential for the maintenance of tissue homeostasis and is an anti-proliferative factor in normal epithelial cells and at early stages of carcinogenesis [79]. For example, the TGFβ-SMAD4 signaling axis constrains prostate cancer growth and metastatic progression in Pten-null mice [80]. Short term 1,25(OH)2 D or vitamin D analog treatment (< 12 h) increased expression‎ of TGFβ2 mRNA in MCF-7 cells, MDA-MB-231 cells [68], MCF10CA1a cells [81], and primary prostate cancer cells [67]. Consistent with this induction, Wu et al. [82] used deletion/mutation analysis in reporter gene assays and EMSA to identify and characterize two VDREs in the TGFβ2 promoter.

In addition to TGFβ2 expression‎, 1,25(OH)2 D and its analog EB1089 induce expression‎ of TGFβ1 and TGFβ receptors in MCF7 breast cancer cells and immortalized mammary epithelial cells (185A1 cells) through a mechanism that appears to require SMAD3 as a co-activator [83]. Also, negative regulators of TGFβ availability, LTBP1 (latent TGFβ binding protein 1) and LTBP2 were significantly suppressed by 1,25(OH)2 D-treatment in OVCAR3 cells [84] and primary prostate cancer cells, respectively [67]. The early response of these genes to 1,25(OH)2 D suggests that some genes whose protein products control TGFβ signaling may be direct targets of 1,25(OH)2D/VDR.

Another TGFβ superfamily member is growth differentiation factor 15 (GDF15). Forced expression‎ of GDF15 in PC-3 cells decreased cell proliferation, soft agar clone formation, and xenograft tumor growth [85]. GDF15 mRNA level is upregulated by 1,25(OH)2 D in LNCaP cells [74] and GDF15 has been shown to be a direct VDR target gene that is indispensible for 1,25(OH)2 D-mediated growth inhibition [85]. In contrast, the effects of 1,25(OH)2 D on TGFβ family member mRNA levels (e.g. TGFBR1, SMAD6, TGFβ1) are only seen after prolonged treatment in other cell types and this suggests that the effect may be indirect [70;78].

Bone morphogenic proteins (BMP) are another group of growth factors belonging to the TGFβ superfamily that play pivotal roles in regulating tissue morphogenesis; BMP signaling is often dysregulated in cancer (e.g. colon cancer [86]). The mRNA level of several BMP forms are regulated by 1,25(OH)2 D or vitamin D analog treatment in primary prostate cancer cells (BMP6 [67]), MCF10AT1 cells, (BMP2 and BMP6, [81], and squamous cell carcinoma lines (TGFβ1 and BMP2A [70]).

Wnt-β Catenin Signaling

An alternative hypothesis has emerged to explain how vitamin D mediates cell growth arrest - disruption of β-catenin function, the terminal mediator of Wnt signaling. In the cytoplasm, β-catenin is found in association with APC. Activation of Wnt signaling leads to the accumulation of β-catenin and its release from APC. This free β-catenin translocates to the nucleus, binds with the transcription factor TCF4 on DNA, and activates transcription of genes whose protein products control proliferation (e.g. c-myc and cyclin D1) [87]. Mutations in the APC gene that disrupt APC-β-catenin interactions are common in colon cancer [88]. 1,25(OH)2 D can block β-catenin-mediated gene transcription in cultured SW480-ADH [89], Caco-2, and HT-29 colon cancer cells [90] by inducing binding of VDR to β-catenin, an event that subsequently reduces the formation of the TCF4/β-catenin transcriptional complex [89]. Consistent with these cell-based observations, Xu et al. [91] found that thrice weekly injections of 1,25(OH)2 D and 1,25(OH)2 D analogs for 12 weeks reduced polyp number and load in APCmin mice and this was associated with reduced expression‎ of β-catenin target genes in small intestine and colon. Shah et al. used a mammalian two-hybrid assay in HEK293 kidney cells to show that the AF-2 domain of VDR interacts with the C-terminus of β-catenin; this interaction may also be enhanced by acetylation of lysines 671/672 on β-catenin [92]. 1,25(OH)2 D-mediated events may also indirectly influence β-catenin function through increased production of E-cadherin, a membrane protein that can bind β-catenin and prevent its nuclear accumulation. However, 1,25(OH)2 D treatment can repress β-catenin-mediated gene transcription even in SKBR-3 cells lacking the E-cadherin gene [92]. Thus, these data demonstrate that E-cadherin up-regulation is not the only mechanism for 1,25(OH)2 D-mediated repression of β-catenin signaling.

In addition to its impact on VDR-β-catenin interactions, 1,25(OH)2 D can also influence expression‎ of known regulators of Wnt-signaling: e.g. inhibition of the Wnt activator dickkopf-4 (DKK-4) [93] and upregulation of the Wnt antagonist dickkopf-1 (DKK-1) [94]. Evidence for crosstalk between vitamin D and the Wnt signaling pathway has also been observed in vivo. In ApcMin/+ mice 1,25(OH)2 D injections decreased nuclear β-catenin, TCF1, CD44, and c-Myc levels in tumor-free colonic and small intestine tissue [91].

Collectively these data suggest that vitamin D may decrease colon tumor cell proliferation by interfering with the Wnt signaling pathway. Still, many gaps exist in our understanding of this VDR-mediated growth arrest mechanism. For example, it is not clear whether 1,25(OH)2 D can interfere with β-catenin function through VDR in cells other than colonocytes and mammary gland cells lines [95]. In addition, it is not clear if all β-catenin-DNA binding sites are equally inhibited by VDR. Recent ChIP-chip studies show that at some TCF4/β-catenin binding sites, CDX2 is required for TCF4 binding and activation of gene transcription [96]. This shows that there are subgroups of β-catenin binding sites in DNA but it is not clear whether binding of β-catenin to these different types of sites are equally affected by 1,25(OH)2 D and VDR.

Apoptosis

Several groups have reported that 1,25(OH)2 D influences apoptosis in MCF-7 breast cancer cells [97] and a variety of colon cancer cell lines [98]. Pan et al.[99] found that 1,25(OH)2 D treatment promoted apoptosis in the undifferentiated gastric cancer cell line HGC-27 through a mechanism that depends upon VDR-mediated PTEN upregulation. PTEN is a tumor suppressor gene that negatively regulates the anti-apoptotic Akt signaling pathway. Also, in the colorectal cancer cell line MIP101, 1,25(OH)2 D treatment increased basal and chemotherapy-induced apoptosis by a mechanism that was sensitive to SPARC (secreted protein acidic and rich in cysteine)-induced VDR synthesis [100], suggesting the regulation may be transcriptionally mediated. Blutt et al observed that 6 days of 1,25(OH)2 D treatment induced apoptosis in LNCaP cells and this was accompanied by downregulation of the anti-apoptotic proteins Bcl-2 and Bcl-XL[101].

Consistent with an effect of 1,25(OH)2 D on the regulation of pro- and anti-apoptotic proteins, a number of studies have found that transcripts for genes encoding proteins that control apoptosis are regulated by 1,25(OH)2 D treatment. GoS2 is a pro-apoptotic protein whose expression‎ is frequently suppressed in cancer [102]. Expression‎ of GoS2 mRNA was induced after 1,25(OH)2 D treatment in SW480-ADH colon cancer cells [78] and by the 1,25(OH)2 D analog EB1089 in SCC25 squamous carcinoma cells [70]. In the chronic myeloid leukemia cell line K562 1,25(OH)2 D-induced growth arrest and apoptosis is accompanied by lower expression‎ of Bcl2 and Bcl-XL mRNA (anti-apoptotic) and increased Bax mRNA levels (pro-apoptotic)[103] while in SW480-ADH cells, 1,25(OH)2 D increased mRNA levels for the pro-apoptotic proteins Baxα, Baxγ and Baxδ [78]. Transcript profiling studies show that the mRNA levels for the pro-apoptotic proteins DAP-3 (Death associated protein 3), CFKAR (CASP8 and FADD-like apoptosis regulator), and a number of caspases (e.g. Caspase 3, 4, 6 and 8) were induced in 1,25(OH)2 D treated MCF-7 and/or MDA-MB-231 cells [68], while in MCF10AT1 and MCF10CA1a cells, the vitamin D analog Ro3582 induced PDCD4 (programmed cell death 4) mRNA levels [81]. While these transcript level changes suggest that 1,25(OH)2 D induces apoptosis by transcriptionally activating or repressing various genes, such direct regulation has not yet been reported for most of these genes.

Novel Molecular Events Regulated by 1,25(OH)2 D that May Contribute to its Anti-Cancer Activity

In the past decade 1,25(OH)2 D has been shown to regulate a much wider array of cellular events than previously thought possible. Below we will discuss several of these processes and relate them to the prevention of cancer.

Autophagy

Autophagy is a process used by cells to degrade cytosolic macromolecules and organelles in lysosomes. While autophagy is generally considered a survival tactic to protect cells during stress (e.g. starvation, pro-oxidant conditions), this process can also be used to trigger the death of cancer cells and to block tumor growth [104]. The first suggestion that 1,25(OH)2 D may induce autophagy was indirect. Mathiasen et al. [105] found that 1,25(OH)2 D induced cell growth arrest and cell death by a caspase and p53 independent pathway that could be inhibited by the anti-apoptotic protein Bcl-2. Hoyer-Hansen [106] later found that the vitamin D analog EB1089 induced autophagy in MCF-7S1 cells that could be enhanced by the Atg protein beclin-1. In HL-60 leukemia cells 1,25(OH)2 D treatment suppresses anti-autophagic mTOR protein and activity levels as well as increases the level of the pro-autophagic protein beclin-1. This treatment also increases the interactions between beclin-1 and PI3K (a pro-autophagic event) or the anti-apoptotic protein Bcl-XL (leading to reduced apoptosis). Vitamin D-induced autophagy may require a complex interplay with cyclin-dependent kinase inhibitors [107]; in p19-deficient SCC25 cells 1,25(OH)2 D can induce autophagy that can be suppressed by suppression of p27. Despite these promising mechanistic relationships, there is currently no in vivo research to directly connect activation of autophagy to the anti-cancer actions of vitamin D compounds. However, the logic of the argument in favor of vitamin D-induced autophagy as a mechanism for cancer treatment or prevention was recently discussed in an opinion piece by Hoyer-Hansen et al. [108].

Antioxidant Defense and DNA Repair

Oxidative stress-induced damage of DNA and loss of DNA repair mechanisms contribute to carcinogenesis [109] but these effects can be prevented by induction of antioxidant defense mechanisms that reduce the biological impact of reactive oxygen species. Oxidative DNA damage (measured by the level of 8 hydroxy-2’deoxyguanosine) is elevated in the distal colonic epithelium of VDR knockout mice [110] and is reduced in the colon epithelium of humans receiving a daily supplement of 800 IU vitamin D3 [111]. 1,25(OH)2 D has been shown to induce the expression‎ of several enzymes involved in the antioxidant defense system. In primary prostate cancer cells, SW480-ADH, MCF-7, MDA-MB-231, and MCF10AT1 cells, 1,25(OH)2 D or vitamin D analogs induce the expression‎ of TXNRD1 (thioredoxin reductase 1) a protein that keeps thioredoxin in the reduced state needed for its role as an antioxidant [67;68;78;81]. In addition, mRNA levels for the essential antioxidant proteins SOD1 and SOD2 (superoxide dismutase) are induced by 1,25(OH)2 D in primary prostate epithelial cells [67] and LNCaP cells [85], respectively. 1,25(OH)2 D-induced SOD1 activity has also been seen in the liver of diethylnitrosamine-treated rats and is associated with reduced DNA damage (assessed by comet assay) [112]. 1,25(OH)2 D induced G6PD (glucose-6-phosphate dehydrogenase) after treatment in ovarian cancer cells [84], in RWPE1 cells [69], and in cells from benign prostatic hypertrophy, but not in malignant prostate cancer cells (DU 145, CWR22R) [113]. G6PD is an enzyme involved in maintaining reduced glutathione levels in cells. Consistent with a critical role for G6PD in vitamin D-mediated antioxidant protection, Bao et al. [113] showed that G6PD expression‎ is controlled by 1,25(OH)2 D in prostate epithelial cells through a VDRE located in the first intron of the gene, that 1,25(OH)2 D protected RWPE1 cells against H2O2-induced apoptosis, and this protection was lost in the presence of a non-competitive G6PD inhibitor. It is also possible that vitamin D-mediated protection from pro-oxidant stress is indirect due to the induction of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), a transcription factor that controls expression‎ of genes for many antioxidant enzyme systems [114]. NFE2L2 expression‎ is down-regulated in prostate cancer and suppression of NFE2L2 promotes prostate tumor development in TRAMP mice [115]. Consistent with a role for NFE2L2 in vitamin D-mediated cancer prevention, a number of NFE2L2 target genes were increased in RWPE1 cells after 1,25(OH)2 D treatment, e.g. GPX3, HMOX1, AKR1C2, and TXNRD1 [69]. Finally, GPX1 (glutathione peroxidase) was induced by 1,25(OH)2 D in SW480-ADH cells [78] and by EB1089 in SCC25 cells [70].

There is some evidence that 1,25(OH)2 D regulates genes for proteins that protect the genome. Akutsu et al [116] found that the 1,25(OH)2 D analog EB 1089 up-regulated Growth Arrest and DNA-Damage-inducible alpha (GADD45α) mRNA and protein levels in SSC cells. GADD45α is a p53 target gene whose product is involved in DNA repair. It was later shown that the GADD45 gene contains an exonic enhancer element that binds VDR after 1,25(OH)2 D treatment leading to increased GADD45 mRNA levels in ovarian cancer cells [117]. 1,25(OH)2 D-mediated G2/M arrest in ovarian cancer cells is lost upon deletion of GADD45, suggesting the critical importance of GADD45 induction in vitamin D effects [117]. Other 1,25(OH)2 D regulated transcripts whose protein products may contribute to DNA-repair and pro-apoptotic effects of vitamin D have been revealed by microarray analyses. In MCF-7 cells, 1,25(OH)2 D induced the expression‎ of mRNAs for p53, RAD23B (RAD23 homolog B), PCNA [68] and DAP-1α (45) [78]. Taken together, it is possible that 1,25(OH)2 D directly regulates the expression‎ of a variety of genes whose protein products are involved in DNA damage repair and programmed cell death, thereby offering protection against carcinogenesis.

Prostaglandin Metabolism and Action

A variety of studies have shown that prostaglandin signaling stimulates cancer cell growth and cancer progression [118–121]. In this context, cyclooxygenases 1 and 2 (COX1 and COX2) are the rate limiting enzymes in prostaglandin synthesis. In particular, COX-2 expression‎ is induced by a variety of mitogens, cytokines, and tumor promoters [122] making this pathway a drug target for cancer treatment, e.g. drugs specifically targeting COX-2 have been shown to lower the risk of prostate cancer [120]. 1,25(OH)2 D has been proposed as a negative regulator of prostaglandin synthesis and signaling and this is supported by research on the advanced prostate cancer cell lines, LNCaP and PC-3 [123]. In these cells, 1,25(OH)2 D suppressed COX2 expression‎, reduced expression‎ of the prostaglandin receptors EP2 and FP, and induced expression‎ of 15-PGDH (hydroxyprostaglandin dehydrogenase 15-NAD), the enzyme that inactivates prostaglandins [74;123]. More importantly, 1,25(OH)2 D reduced prostaglandin E2 levels, blocked prostaglandin-mediated induction of c-fos mRNA, and reduced the growth stimulatory effects of prostaglandins and prostaglandin precursors in LNCaP cells [124]. These results suggest the direct regulation of the prostaglandin metabolism and signaling by 1,25(OH)2 D and the subsequent attenuation of their signaling activities. In contrast to the findings from advanced prostate cancer cells, COX2 mRNA was induced by 1,25(OH)2D in the immortalized but non-tumorigenic RWPE1 cell line and neither 15-PGDH nor prostaglandin receptor mRNA levels were altered [69]. Consistent with this observation, Moreno et al.[124] found that COX2 mRNA was suppressed and 15-PGDH mRNA was increased in some, but not all, primary prostate epithelial cell lines.

Are Adult or Cancer Stem Cells Targets of Vitamin D Action?

In the past decade adult stem cells have been discussed as potential target cells for accumulating mutations that contribute to carcinogenesis and therefore as cells that would benefit from exposure to cancer prevention agents [125]. Adult stem cells in tissues serve the primary function of replacing cells lost during the normal lifespan of an organ or following tissue injury. The hallmarks of the adult stem cell are its ability for self-renewal and multipotency. Unfortunately, this capacity for self-renewal means that adult stem cells can accumulate first-hit mutations that, while not harmful themselves, could be combined with subsequent mutations to cause cancer. Trosko argued that rather than the induction of “immortalization” of a normal differentiated cell, we should view carcinogenesis as a process that begins by blocking “mortalization” (or the ability to growth arrest and differentiate) of an adult stem cell [126].

In addition to adult stem cells, a small subset of cells within tumors has the ability to recapitulate the morphologic diversity of a neoplasm when isolated from the parent neoplasm and xenotransplanted into nude mice. These cells have been called “cancer stem cells” and are thought to be the cells from which primary cancers arise or which survive cytotoxic treatments and cause tumor recurrence. Cancer stem cells are also attractive targets for cancer prevention and treatment [127].

The question to be asked in the context of vitamin D signaling and carcinogenesis is “Are adult stem cells or cancer stem cells targets for 1,25(OH)2 D action?” As discussed above, 1,25(OH)2 D regulates a number of important biological processes. These effects would be beneficial for protecting an adult stem cell (e.g. DNA repair and protection from oxidative cellular injury) or for limiting the expansion of cancer stem cells (e.g. cell cycle arrest, apoptosis). To date, the vast majority of the experimental data exploring the molecular function of 1,25(OH)2 D has been collected from in vitro cell culture systems using cancer cell lines or primary cell cultures. Only recently have researchers begun to directly eval‎uate the impact of 1,25(OH)2 D on stem cells. For example, as part of a team headed by Dr. Scott Cramer (Wake Forest University) we have shown that the growth of mouse prostate stem cells [128] is suppressed by 1,25(OH)2 D through a VDR-dependent induction of IL-1α production [129]. In the mouse colon, VDR message is predominantly found in the differentiated, luminal colonocytes [130]. However, Fedirko et al. recently found that hTERT labeling in the upper part of the colon crypt was suppressed in people given vitamin D3 and calcium supplements [131]. hTERT is the catalytic subunit of telomerase that marks a slowly cycling population of intestinal stem cells [132], thus vitamin D may suppress the expansion of this cell population and protect them from potential cancer-causing gene mutations. Finally, 1,25(OH)2 D and its analogues have been show to regulate the expression‎ of a putative cancer stem cell marker (CD44) in MCF10DCIS.com human breast cancer cells in vitro and when they are implanted into immunodeficient mice [133]. Research on the impact of vitamin D on adult and cancer stem cells is certain to expand in the future.

Are Non-epithelial Cells the Targets of Vitamin D-Mediated Cancer Prevention?

Although most cell-based vitamin D research has focused on the impact of 1,25(OH)2 D on either tumor cells or their non-neoplastic progenitors, there is also evidence that other cells that exist in the tissue or tumor microenvironment are targets of vitamin D action. For example, in prostate the communication between epithelial cells and the stromal cells surrounding them is critical for the progression of cancer in that organ [134]. Lou et al. [135] reported that 1,25(OH)2 D can suppress the growth of prostate stromal cell lines but it is not yet clear if it can alter stromal-epithelial cell communication. In the next section we will discuss two additional examples of non-epithelial cell targets of vitamin D action – vascular cells and immune cells.

Inhibition of Angiogenesis

Angiogenesis is essential for the expansion of tumor growth and for tumor cell metastasis. It is a multistep, multicellular process that depends upon a variety of pro-angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet derived growth factor BB homodimer (PDGF BB). 1,25(OH)2 D may inhibit the development of the tumor vasculature that is required for the progression of solid tumors and this may occur due to effects on either endothelial or epithelial cells [136–140]. 1,25(OH)2 D can directly inhibit the proliferation of aortic and tumor-derived endothelial cells and can stop endothelial cell sprouting and elongation induced by VEGF [136;141]. Consistent with a direct role of vitamin D signaling in endothelial cells, Chung et al. [138] found that prostate tumors from TRAMP mice grew larger, had a greater vascular volume, and had larger vessels when implanted into VDR knockout mice. Effects of vitamin D on epithelial cells may also influence angiogenesis. One proposed mechanism for the anti-angiogenic effects of 1,25(OH)2 D is that it suppresses expression‎ of VEGF family members, the major pro-angiogenic cytokines. 1,25(OH)2 D reduces expression‎ of VEGF in normal prostate epithelial cells [142], reduces VEGF mRNA and increases mRNA levels for the anti-angiogenic protein thrombospondin-1 in SW480-ADH colon cancer cells [140], reduces hypoxia-induced VEGF expression‎ in a variety of cancer cell lines [143], and suppresses mRNA levels for VEGFC and the VEGF receptors KDR and NRP1 in RWPE1, a prostate epithelial cell line [69]. The impact of 1,25(OH)2 D is partially due to reduced levels and activity of the transcription factor hypoxia inducible factor-1 (HIF-1); the ability of 1,25(OH)2 D to suppress VEGF expression‎ is lost in HCT116 colon cancer cells lacking HIF-1α [143]. VEGF signaling can be suppressed by competitive binding of semaphorins to the VEGF receptor NRP1. In RWPE1 cells, 1,25(OH)2 D induced expression‎ of several semaphorin isoforms including SEMA3B, 3F, and 6D and VDR binding to the SEMA3B gene promoter was enriched after 1,25(OH)2 D treatment [69]. In prostate cancer cells 1,25(OH)2 D can also reduce the mRNA level of the pro-angiogenic cytokine IL-8 by reducing translocation of the p65 subunit of NFkB into the nucleus, thereby limiting NFkB−mediated IL-8 gene transcription [139]. However, the impact of 1,25(OH)2 D on VEGF gene regulation has not been entirely consistent. In mouse embryo fibroblasts and human vascular smooth muscle cells 1,25(OH)2 D induces VEGFA expression‎ through a VDRE in its promoter [144;145].

Regulation of Immune Cell Function by 1,25(OH)2 D

The first line of protection against pathogens and environmental insults is the barrier epithelium of the lung, skin, and intestine. There is evidence that 1,25(OH)2 D signaling through the VDR helps maintain intestinal barrier function. In VDR knockout mice, but not wild-type mice, dextran sulfate sodium (DSS) treatment causes the redistribution of the tight junction proteins occludin and zona occludin-1 away from the membrane and severely disrupts tight junctions in the colon [146]. This is consistent with earlier work showing that prolonged treatment of SW480-ADH colon cancer cells with 1,25(OH)2 D (100 nM, 2–7 days) induced expression‎ of tight junction and adhesion proteins (E-cadherin, occludin, ZO-1, vinculin) and promoted translocation of ZO-1 to the plasma membrane [89]. In vitamin D deficient rats, microarray analysis revealed that 1,25(OH)2 D treatment increased the mRNA level of junction proteins like claudin 3, claudin 17, and RhoA [147]. Ordonez-Moran et al. [148] found that 1,25(OH)2 D treatment increased cytosolic calcium leading to the activation of RhoA in SW480-ADH cells. By using dominant negative RhoA, they were subsequently able to block 1,25(OH)2 D-induced localization of occludin to the plasma membrane. Collectively, these data support a mechanism whereby vitamin D signaling regulates the level and localization of tight junction proteins leading to a more secure intestinal barrier. It is not clear whether similar protections are present in other barrier tissues like skin and lung nor is it clear what level of vitamin D status is necessary to protect this function.

There are three distinct roles for the immune system in cancer prevention [149]. First, it protects the host against viral infection and suppresses virus-induced tumors. Second, it suppresses inflammation that facilitates tumorigenesis by effectively eliminating pathogens and limiting the period of inflammation that can promote carcinogenesis [150;151]. Finally, it performs immunosurveillance that eliminates nascent tumor cells in tissues by activating receptors on innate immune cells and lymphocytes of the adaptive immune system. As a result of these functions, the cells of the immune system may be important targets for limiting cancer. However, in the context of cancer, the role of the immune system is not straightforward. Interactions can occur between cancer cells and host immune cells in the tumor microenvironment to create an immunosuppressive network that promotes tumor growth and protects the tumor from immune attack [152]. Thus, agents that can suppress inflammation and immune responses prior to tumor formation may be harmful in established tumors where tumor-associated immunosuppression already exists.

A variety of studies have shown that 1,25(OH)2 D regulates cells of both the innate and adaptive immune system [153]. However, few of these studies have been conducted in the context of cancer. As a result, it remains to be shown that the effects of vitamin D on immunity are effective in the prevention of cancer or in the tumor environment. However, non-cancer examples give us insight into the impact that vitamin D could have on inflammation and immunity in carcinogenesis.

The innate immune system interacts with vitamin D in several interesting ways. First, a primary role of macrophages is to engulf and kill bacteria. Invading microbes can induce inflammation when barrier tissues become leaky, e.g. during the intestinal condition Crohn’s disease or experimental colitis induced by DSS [154]. 1,25(OH)2 D can regulate a number of genes in cells of the innate immune system whose protein products are crucial for autophagy and anti-microbial actions. The human cathelicidin and beta defensin genes are activated by 1,25(OH)2 D treatment through vitamin D response elements (VDRE) in their promoters but these VDREs are not conserved in mice [155;156]. Beta defensin can also be up-regulated by an NFkB-mediated transcriptional event after NFkB has been activated through the pattern recognition receptor NOD2 (nucleotide-binding oligomerization domain 2). 1,25(OH)2 D can stimulate expression‎ of the NOD2 gene in primary human monocytic cells through a VDRE in a distal enhancer [157]. In addition, the mRNA levels for the antimicrobial protein angiogenin-4 are significantly reduced in the colon of vitamin D deficient mice and this may increase the severity of DSS-induced colitis [158].

Several cytokines can modulate vitamin D metabolism in macrophages, monocytes, and dendritic cells. Proinflammatory cytokines like IFNγ [159] and TNFα [160] stimulate production of 1,25(OH)2 D by increasing CYP27B1 expression‎ in monocytes. Inflammatory cytokines and toll like receptor agonists also increase CYP27B1 and VDR expression‎ in dendritic cells [161]. In contrast, IL-4 produced by Th2 cells activates CYP24 expression‎ in monocytes leading to formation of the inactive metabolite 24, 25 dihydroxyvitamin D [159]. These regulatory events can influence local vitamin D metabolite levels and these vitamin D metabolites may then modulate the function other cells in the microenvironment. For example, in vitro 1,25(OH)2 D reprograms dendritic cells to become tolerogenic [161].

In the adaptive immune system, many T-lympocyte subpopulations express the VDR and are vitamin D target cells [162]. However, mice lacking VDR have no gross abnormalities in either the number or type of T cells present [163] nor is the function of their mature T-cells strongly influenced by VDR deletion [164;165]. This suggests that VDR does not have a primary function for normal T-cell function but it does not preclude the possibility that 1,25(OH)2 D may be a modulator of T-cell mediated immune responses. Consistent with the “modulator” hypothesis, 1,25(OH)2D promotes T-cell profiles reflective of immunotolerence and immunosuppression. In vivo, VDR deficient non-obese diabetic (NOD) mice develop a more aggressive form of autoimmune prostatitis with more severe lesions, a greater lymphoproliferative response against prostate antigen, higher levels of INFγ secretion, and higher mononuclear cell infiltration into the prostate gland [166]. Similarly, VDR is necessary in CD4+ cells for 1,25(OH)2 D to prevent autoimmune T-cell responses that cause experimental autoimmune encephalomyelitis in mice [167]. In vitro, 1,25(OH)2 D can suppress NFkB signaling necessary for T helper cell activation [168], increase the activity of regulatory T cells necessary for immunosuppression [169], and block development of Th17 and Th9 cells implicated in the pathogenesis of different types of autoimmunity and inflammatory diseases [170]. The effect of 1,25(OH)2 D on Th9 cell development is mediated through IL-10, a cytokine that can be induced by 1,25(OH)2 D in B cells [171] and naïve T-cells [170]. Treatment of Th17 cells with 1,25(OH)2 D suppresses IL-17 production indirectly by inducing the expression‎ of C/EBP homologous protein (CHOP), a molecule involved in endoplasmic reticulum stress and translational inhibition.

Other cells outside of the traditional immune system have the capacity to respond to and produce immuno-modulatory factors. CD14 mRNA is strongly up-regulated by 1,25(OH)2 D in the prostate epithelial cell line RWPE1 [69], keratinocytes [172], U937 monocytes [173] and the myeloid cell line HL-60 [174]. CD14 and toll-like receptor 4 (TLR4) are important for detection of pathogens that have lipopolysaccharide on their cell surface [175] and CD14 has been identified as a crucial factor for vitamin D induced expression‎ of the antimicrobial peptide cathelicidin in human keratinocytes [172]. Nonn et al. have shown that in normal prostate epithelial cells, 1,25(OH)2 D inhibits TNFα-induced IL-6 production through a mechanism that requires direct transcriptional regulation of the MAPK phosphatase 5 gene (DUSP-10) [176].

Although these data suggest that vitamin D should suppress immune and inflammatory conditions that promote cancer, very little data explicitly supports this hypothesis, particularly as it relates to the ranges of vitamin D status being discussed as relevant to cancer prevention in humans. However, injections of the vitamin D analog Ro26-2198 delayed the onset of clinical colitis and reduced the number of dysplastic foci present in mice treated with azoxymethane and DSS [177].

1,25(OH)2 D Action May not be Uniform Across all Stages of Cancer

Although we generally focus on the impact that vitamin D has on the development of cancer, it is just as important to understand the impact that cancer has on vitamin D action. In this section we will review studies that indicate vitamin D signaling is altered in various cancers. This concept is summarized in Figure 2.

Figure 2

A summary of the negative effects of cancer on vitamin D metabolism and action.

VDR

There are conflicting findings regarding the impact of cancer on VDR protein levels. For example, using immunohistochemistry (IHC) Matusiak et al. [27] found that VDR protein levels decline as a function of colon tumor dedifferentiation while Kure et al. [178] observed that VDR protein levels were overexpressed in colorectal cancer tumors with KRAS mutations. Unfortunately, a recent study highlights the inadequacy of many anti-VDR antibodies for IHC [179], and therefore IHC-based studies should be view with caution. None-the less, using IHC VDR levels have been reported to decline with the development of cancer in pigmented skin lesions [180], invasive breast tumors [181], and ovarian cancer [182] while high VDR is associated with reduced risk of lethal prostate cancer [183] and improved overall survival for non-small cell lung carcinoma [184]. Consistent with the hypothesis that cancer reduces VDR protein levels, Xu et al. [91] showed that VDR mRNA and protein expression‎ (by Western blot analysis) were lower in the small intestine and colon tissue of ApcMin/+ mice compared to wild-type mice. Other mutations that cause cancer can also lead to reduced VDR expression‎; H-Ras transformation in HC-11 mammary cells [185], SV40 large T antigen in HME human mammary epithelial cells [186], and in KRAS transformation in RWPE-2 human prostate epithelial cells [187].

A number of factors have been proposed to influence VDR expression‎ in cancer. First, over-expression‎ of SNAIL transcription factors can reduce VDR gene expression‎ and blunt 1,25(OH)2 D or vitamin D analog-induced cell growth arrest and/or gene expression‎ of colon cancer cell lines (e.g. SW-480-ADH, HCT116, Caco-2, LS174T, HT29) [188–190]. SNAIL1 is absent in normal tissue and expressed in colon tumors while SNAIL2 is expressed in normal tissue but its levels are increased in colon tumors [190;191]. In colon cancer cell lines and breast cancer cell lines SNAIL1 and 2 can bind to E-boxes in the proximal promoter of the VDR gene and increase recruitment of co-repressors that down-regulate VDR gene expression‎ [190;192]. Another protein regulating VDR gene expression‎ is p53. Over-expression‎ of wild-type p53 in Saos-2 and H1299 cells (both p53 null) increases VDR protein levels by enhancing p53 association with conserved p53 binding sites within intron 1 of the VDR gene [193]. In addition to suppression of VDR gene expression‎, the mutant form of p53 found in many tumors can interact with VDR, be recruited to VDR-regulated genes, and modulate the expression‎ of these genes in breast and lung cancer cell lines [194]. In this context, mutant p53 converts 1,25(OH)2 D treatment from a pro-apoptotic agent into an anti-apoptotic agent and suggesting in cancers with p53 mutations the therapeutic potential of vitamin D or vitamin D analogs could be limited.

There is also some evidence that the VDR gene may be subject to epigenetic silencing in cancer. Smirnoff et al. [195] first observed that in colonic tissue from ovariectomized rats treated with dimethylhydrazine (DMH), CpG island methylation in the VDR gene increased. This was associated with reduced VDR protein levels; both CpG island methylation in the VDR gene and reduced VDR expression‎ could be prevented by estradiol treatment. In breast tumors and in breast cancer cell lines CpG island methylation in the −700 bp VDR promoter region was associated with lower VDR expression‎ [196]. Demethylation of the VDR promoter with 5-azacytidine increased both VDR protein levels and potentiated the growth inhibitory response to 1,25(OH)2 D in normal and breast cancer cell lines.

VDR mRNA levels are also a target of the microRNA miR125b [197]. Over-expression‎ of miR125b reduced VDR protein levels and attenuated 1,25(OH)2 D-induced expression‎ of CYP24 in MCF7 cells [197]. Similarly, melanoma cell lines with low miR125b levels have higher VDR levels and are more sensitive to 1,25(OH)2 D-induced growth suppression than cells with high miR125b levels [198]. miR125b levels are upregulated in patients with metastatic prostate cancer [199] but are downregulated in subjects with HER2-overexpressing breast cancers [200] but it remains to be determined whether miR125b expression‎ can influence the therapeutic potential of vitamin D for specific cancers.

In addition to the negative impact of cancer on VDR levels, there is also evidence that Ras activation, a common mutation in many cancers, can impair vitamin D-mediated transcriptional activity. This was first shown by Solomon et al. [201] who found that H-Ras transformed kerotinocytes have reduced VDR transcriptional activity due phosphorylation of the VDR heterodimeric partner RXRα on serine 260. In the K-Ras transformed human prostate epithelial cell line RWPE2, we have shown that 1,25(OH)2 D mediated transcription is blunted due to phosphorylation of RXRα within the AF-1 domain [187], a process that impairs the ability of the coactivator SRC-1 to bind to RXRα. Collectively, this suggests that the development of cancer may lead to lower responses to 1,25(OH)2 D by impairing signaling through the VDR.

CYP27B1

The current hypothesis for how high vitamin D status reduces cancer risk is that 1,25(OH)2 D is produced and acts locally, i.e. the CYP27B1 enzyme known to produce 1,25(OH)2 D from 25OH D in the kidney is present and active in cancer target tissues [202]. Several groups have used cultured cells to show that CYP27B1 activity, and the ability to produce 1,25(OH)2 D from 25OH D, is lost as cells develop a more severe cancer phenotype. Hsu [25] found that CYP27B1 was present in normal prostate epithelial cells but that its activity was reduced in cells isolated from subjects with benign prostatic hypertrophy and nearly absent in cells isolated from subjects with prostate cancer. As a result, while normal cells could respond to treatment with 25OH D by growth arresting, cancer cells with low CYP27B1 expression‎ could not. This observation was confirmed by Chen et al. [30] who also showed that transgenic expression‎ of CYP27B1 confers a 25OH D-mediated growth inhibitory response to LNCaP cells with low CYP27B1 activity. CYP27B1 expression‎ is also absent in metastases from colon tumors in humans [27;203]. However, cancer-associated reductions in CYP27B1 levels are not uniformly observed for all cancers. Friedrich et al. [26] found that CYP27B1 mRNA was increased in breast cancer as compared to normal tissue. More recently Lopes [181] analyzed CYP27B1 protein levels by immunohistochemistry in breast tissue samples and found that CYP27B1 expression‎ did not significantly change between normal and cancer tissue.

CYP24

Some have also hypothesized that the enzyme responsible for the degradation of vitamin D metabolites, 25 hydroxyvitamin D-24 hydroxylase (CYP24), is influenced by cancer. CYP24 has been proposed as a putative oncogene that is amplified in breast tumors [204]. Consistent with this, Anderson et al. [205] reported that CYP24 mRNA levels are higher in colorectal cancer than in adjacent normal colon tissue. Matusiak and Benya [203] subsequently found that CYP24 protein was present in the nuclei of normal tissue, that CYP24 levels were increased in aberrant crypt foci and polyps, and that CYP24 protein was shifted to the cytoplasm in tumors and metastatic colon cancer. Overexpression‎ of CYP24 has also been reported in ovarian, cervical, lung, cutaneous basal cell, and squamous cell carcinoma [205–207]. This suggests that increased 1,25(OH)2 D metabolism may be a feature of advanced cancer. This is supported by studies showing that KTZ, a CYP24 inhibitor, can restore 1,25(OH)2 D-mediated growth arrest to prostate cancer cells that are insensitive to the action of 1,25(OH)2 D [208]. Recently, Komagata et al. [209] found that CYP24 protein levels are post-transcriptionally suppressed by the miR-125b, a microRNA that can bind to the 3’ UTR of the CYP24 mRNA. In addition, they found that CYP24 levels were inversely related to miR-125b levels in breast cancer, suggesting that the loss of this regulatory RNA may account for elevated CYP24 levels in cancer. In contrast to other cancers, a microarray dataset in the ONCOMINE database shows that CYP24 expression‎ can be lower in prostate cancer (www.oncomine.org). Consistent with this, CYP24 mRNA levels are inversely correlated with CYP24 promoter methylation in prostate cancer cell lines with the promoter being unmethylated, and vitamin D induction of CYP24 expression‎ being greatest, in DU145 cells [210].

The overall impact of these cancer-induced changes to vitamin D metabolism and signaling could affect cancer prevention in several ways. The protection provided by high serum 25OH D levels depends upon the level of CYP27B1 in cells. Therefore, if CYP27B1 activity is lost, so will the protection due to high vitamin D status. On the other hand, when CYP24 activity is elevated and/or when VDR level/signaling is reduced, higher cellular levels of 1,25(OH)2 D will be needed to induce chemoprotective molecular events. However, it isn’t clear whether local production of 1,25(OH)2 D is sufficient to overcome this cancer-associated insensitivity. Because of the strong effect it has on calcium metabolism, increases in serum 1,25(OH)2 D levels are not likely to be an effective means to correct tumor associated vitamin D resistance. 1,25(OH)2 D-treatment-associated hypercalcemia is a major motivation to produce vitamin D analogs that separate the calcemic and non-calcemic effects of 1,25(OH)2 D. However, even the effectiveness of vitamin D analogs will be limited in cancers with reduced VDR expression‎.

Conclusions

There is now a large amount of population-based evidence showing that higher vitamin D status can protect against a variety of cancers. Unfortunately, the mechanisms supporting this association have not yet been resolved. None-the-less, mechanistic explanations for how vitamin D can reduce cancer risk or disrupt the biology of transformed cells are increasing. While the role of 1,25(OH)2 D in cancer has traditionally been linked to the suppression of proliferation and the stimulation of apoptosis, there is now compelling evidence that 1,25(OH)2 D regulates other cancer-relevant cellular processes. For example, several lines of evidence suggest that the immunoregulatory actions of vitamin D metabolites are worth exploring for cancer prevention. The breadth of potential mechanisms used by vitamin D to prevent or suppress cancer is given in Figure 3. Still, many gaps remain in our understanding of how vitamin D regulates cell biology relevant to cancer prevention and treatment. Many of the mechanisms proposed for vitamin D and cancer prevention have been studied only in the context of one tissue or one type of cancer and so further studies must be conducted to determine if these mechanisms can be generalized (e.g. VDR-mediated inhibition of β catenin function has been studied primarily in colon). Also, while many studies show that 1,25(OH)2 D can alter various transcript levels, it is not clear which genes are direct targets for VDR-mediated regulation. Recent advances in global profiling of transcription factor binding sites with techniques like ChIP-seq should overcome this uncertainly and help researchers differentiate between the direct and indirect effects of vitamin D in the context of cancer.

Figure 3

A summary of the potential molecular events regulated by 1,25(OH)2 D (VD) relevant to cancer.

Another feature of the vitamin D/cancer paradigm that requires additional investigation is that vitamin D metabolism and signaling through the VDR may be disrupted as cancers progress. Until we understand this relationship more clearly, our ability to use vitamin D or vitamin D analogs for cancer prevention or treatment will be limited. Similarly, few controlled animal studies exist that confirm‎ the validity of proposed molecular mechanisms for vitamin D-mediated cancer prevention using the ranges of vitamin D status that are observed in human populations. The lack of such “proof of principle” studies has hampered a broader acceptance of the vitamin D/cancer relationship in the research and medical community. For example, while higher serum 25OH D is associated with reduced cancer risk, and CYP27B1 is expressed in non-renal cells, there is no evidence that the low-level expression‎ of CYP27B1 in tissues leads to meaningful local production of 1,25(OH)2 D, and that this mediates the protection from cancer provided by high vitamin D status. Still, even with all of these gaps in our understanding, this is an exciting time for research on vitamin D and cancer.

Acknowledgements

This work was supported by NIH award CA10113 and American Institute for Cancer Research award 09A098 to JCF.

Go to:

Footnotes

Disclosures

JCF has acted as a scientific consultant for Johnson & Johnson

Go to:

Reference List

1. Fleet JC. In: In Calcium in Human Health. Weaver CM, Heaney RP, editors. Totowa, NJ: Humana Press; 2006. pp. 163–190. [Google Scholar]

2. Garland CF, Garland FC. Do sunlight and vitamin D reduce the likelihood of colon cancer? Int.J.Epidemiol. 1980;9:227–231. [PubMed] [Google Scholar]

3. Grant WB, Mohr SB. Ecological studies of ultraviolet B, vitamin D and cancer since 2000. Ann Epidemiol. 2009;19:446–454. [PubMed] [Google Scholar]

4. Garland CF, Comstock GW, Garland FC, Helsing KJ, Shaw EK, Gorham ED. Serum 25-hydroxyvitamin D and colon cancer: eight-year prospective study. Lancet. 1989;2:1176–1178. [PubMed] [Google Scholar]

5. Engel P, Fagherazzi G, Boutten A, Dupre T, Mesrine S, Boutron-Ruault MC, Clavel-Chapelon F. Serum 25(OH) vitamin D and risk of breast cancer: a nested case-control study from the French E3N cohort. Cancer Epidemiol Biomarkers Prev. 2010;19:2341–2350. [PubMed] [Google Scholar]

6. Ahonen MH, Tenkanen L, Teppo L, Hakama M, Tuohimaa P. Prostate cancer risk and prediagnostic serum 25-hydroxyvitamin D levels (Finland) Cancer Causes Control. 2000;11:847–852. [PubMed] [Google Scholar]

7. Tretli S, Hernes E, Berg JP, Hestvik UE, Robsahm TE. Association between serum 25(OH)D and death from prostate cancer. Br.J Cancer. 2009;100:450–454. [PMC free article] [PubMed] [Google Scholar]

8. Giovannucci E, Liu Y, Rimm EB, Hollis BW, Fuchs CS, Stampfer MJ, Willett WC. Prospective study of predictors of vitamin D status and cancer incidence and mortality in men. J.Natl.Cancer Inst. 2006;98:451–459. [PubMed] [Google Scholar]

9. Sitrin MD, Halline AG, Abrahams C, Brasitus TA. Dietary calcium and vitamin D modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat. Cancer Res. 1991;51:5608–5613. [PubMed] [Google Scholar]

10. Llor X, Jacoby RF, Teng BB, Davidson NO, Sitrin MD, Brasitus TA. K-ras mutations in 1,2-dimethylhydrazine-induced colonic tumors: effects of supplemental dietary calcium and vitamin D deficiency. Cancer Res. 1991;51:4305–4309. [PubMed] [Google Scholar]

11. Tangpricha V, Spina C, Yao M, Chen TC, Wolfe MM, Holick MF. Vitamin D deficiency enhances the growth of MC-26 colon cancer xenografts in Balb/c mice. J.Nutr. 2005;135:2350–2354. [PubMed] [Google Scholar]

12. Mordan-McCombs S, Brown T, Wang WL, Gaupel AC, Welsh J, Tenniswood M. Tumor progression in the LPB-Tag transgenic model of prostate cancer is altered by vitamin D receptor and serum testosterone status. J Steroid Biochem Mol Biol. 2010;121:368–371. [PMC free article] [PubMed] [Google Scholar]

13. Zinser GM, Suckow M, Welsh J. Vitamin D receptor (VDR) ablation alters carcinogen-induced tumorigenesis in mammary gland, epidermis and lymphoid tissues. J Steroid.Biochem Mol Biol. 2005;97:153–164. [PubMed] [Google Scholar]

14. Zheng W, Wong KE, Zhang Z, Dougherty U, Mustafi R, Kong J, Deb DK, Zheng H, Bissonnette M, Li YC. Inactivation of the vitamin D receptor in APC(min/+) mice reveals a critical role for the vitamin D receptor in intestinal tumor growth. Int J Cancer e-pub ahead of print. 2011 [PMC free article] [PubMed] [Google Scholar]

15. Chen TC, Holick MF, Lokeshwar BL, Burnstein KL, Schwartz GG. Eval‎uation of vitamin D analogs as therapeutic agents for prostate cancer. Recent.Results.Cancer Res. 2003;164:273–288. [PubMed] [Google Scholar]

16. Oades GM, Senaratne SG, Clarke IA, Kirby RS, Colston KW. Nitrogen containing bisphosphonates induce apoptosis and inhibit the meval‎onate pathway, impairing Ras membrane localization in prostate cancer cells. J Urol. 2003;170:246–252. [PubMed] [Google Scholar]

17. Huerta S, Irwin RW, Heber D, Go VL, Koeffler HP, Uskokovic MR, Harris DM. 1alpha,25-(OH)(2)-D(3) and its synthetic analogue decrease tumor load in the Apc(min) Mouse. Cancer Res. 2002;62:741–746. [PubMed] [Google Scholar]

18. Mehta RG. Stage-specific inhibition of mammary carcinogenesis by 1alpha-hydroxyvitamin D5. Eur.J Cancer. 2004;40:2331–2337. [PubMed] [Google Scholar]

19. Otoshi T, Iwata H, Kitano M, Nishizawa Y, Morii H, Yano Y, Otani S, Fukushima S. Inhibition of intestinal tumor development in rat multi-organ carcinogenesis and aberrant crypt foci in rat colon carcinogenesis by 22-oxa-calcitriol, a synthetic analogue of 1 alpha, 25-dihydroxyvitamin D3. Carcinogenesis. 1995;16:2091–2097. [PubMed] [Google Scholar]

20. Seubwai W, Wongkham C, Puapairoj A, Okada S, Wongkham S. 22-oxa-1,25-dihydroxyvitamin D3 efficiently inhibits tumor growth in inoculated mice and primary histoculture of cholangiocarcinoma. Cancer. 2010;116:5535–5543. [PubMed] [Google Scholar]

21. White P, Cooke N. The multifunctional properties and characteristics of vitamin D-binding protein. Trends Endocrinol Metab. 2000;11:320–327. [PubMed] [Google Scholar]

22. Hewison M, Zehnder D, Bland R, Stewart PM. 1alpha-Hydroxylase and the action of vitamin D. J Mol.Endocrinol. 2000;25:141–148. [PubMed] [Google Scholar]

23. Song Y, Peng X, Porta A, Takanaga H, Peng JB, Hediger MA, Fleet JC, Christakos S. Calcium transporter 1 and epithelial calcium channel messenger ribonucleic acid are differentially regulated by 1,25 dihydroxyvitamin D3 in the intestine and kidney of mice. Endocrinology. 2003;144:3885–3894. [PubMed] [Google Scholar]

24. Zehnder D, Bland R, Williams MC, McNinch RW, Howie AJ, Stewart PM, Hewison M. Extrarenal expression‎ of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase. J Clin.Endocrinol Metab. 2001;86:888–894. [PubMed] [Google Scholar]

25. Hsu JY, Feldman D, McNeal JE, Peehl DM. Reduced 1alpha-hydroxylase activity in human prostate cancer cells correlates with decreased susceptibility to 25-hydroxyvitamin D3-induced growth inhibition. Cancer Res. 2001;61:2852–2856. [PubMed] [Google Scholar]

26. Friedrich M, Diesing D, Cordes T, Fischer D, Becker S, Chen TC, Flanagan JN, Tangpricha V, Gherson I, Holick MF, Reichrath J. Analysis of 25-hydroxyvitamin D3-1alpha-hydroxylase in normal and malignant breast tissue. Anticancer Res. 2006;26:2615–2620. [PubMed] [Google Scholar]