Pathogenic roles of alterations in vitamin D and vitamin D receptor in gastric tumorigenesis

Gastric cancer is currently the second leading cause of cancer-related death worldwide, especially in Japan, Korea and China, and the 5-year survival rate of gastric cancer is less than 30%. Thus, it is important to shed more lights on novel agents to prevent gastric cancer or to improve survival rate of the patients. Vitamin D not only maintains calcium and bone homeostasis, but also mostly inhibits tumor genesis, invasion, and metastasis through activation of vitamin D receptor. Although epidemiological results are not consistent, accumulating evidence from gastric cancer cells, animal models, and clinical trials suggest that vitamin D deficiency may increase the risk and mortality of gastric cancer, but vitamin D supplement might be a safe and economical way to prevent or treat gastric cancer. Here, we reviewed the current studies on vitamin D and its receptor and focused on the pathogenic roles of their alterations in gastric tumorigenesis.


INTRODUCTION
Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related death in the world. An estimated 1 million new cases of gastric cancer occurred and over 0.7 million patients died worldwide in 2012 only [1]. Currently, gastric cancer is difficult to prevent and cure because of the poor understanding of its pathogenesis and difficulty in its early diagnosis. Even worse, the future burden of gastric cancer is expected to rise with the increase in worldwide population and aging process [2]. Therefore, gastric cancer is regarded as a major public health problem in the world. Multiple therapies (surgery, chemotherapy, radiotherapy, immunotherapy, etc.) are applied to gastric cancer although surgical resection is considered the primary choice for the early stage [3,4]. Despite of much progress in the pathogenesis, diagnosis and treatment of gastric cancer, its 5-year survival rate is still less than 30% [5]. Therefore, it is urgent to investigate gastric tumorigenesis and to elucidate the underlying molecular mechanisms so that the best ways for preventment and treatment could be developed to decrease the current high morbidity and mortality of gastric cancer.
It is well known that vitamin D plays an important role in maintaining calcium and bone homeostasis and participates in a variety of biological processes in our body as well [6]. It has long been thought low vitamin D status and inadequate calcium intake are important risk factors for various types of human cancer. As early as in 1980, the ultraviolet-B (UVB)-vitamin D-cancer hypothesis was first proposed by Garland [7] who demonstrated that vitamin D is a protective factor against the development of colon cancer. Since then, numerous studies have shown that vitamin D could inhibit the tumorigenesis and prevent tumor progression of breast, colon, skin, pancreas and many other cancers [8][9][10][11][12]. The potential anti-tumor mechanisms of vitamin D may be relevant to its specific receptor, vitamin D receptor (VDR) [13]. VDR is a member of the steroid hormone receptor superfamily of ligand-activated transcription factors [14]. An interaction of vitamin D and VDR can induce a cascade of gene Review www.impactjournals.com/oncotarget regulation and cell signaling to play important roles in their anti-tumor mechanisms, such as suppression of proliferation, stimulation of apoptosis and autophagy, inhibition of angiogenesis, regulation of immune system and so on [15,16]. Although several reviews on vitamin D and VDR in some types of human cancer were published, there is no a systematic review on their roles in gastric cancer in the literatures so far. Therefore, in this review, we try to assess the association between vitamin D/VDR and gastric cancer, to explore their multiple anti-tumor mechanisms, and to analyze the safety and validity of vitamin D in the clinical therapy for gastric cancer.

VITAMIN D: SOURCES, METABOLISM AND RECEPTOR Vitamin D sources
Vitamin D is not really a vitamin but a prohormone of the steroid hormone calcitriol, which was first discovered and named by McCollum in 1922 [17]. Although at least ten kinds of vitamin D have been found, the most important forms of vitamin D relevant to human health are vitamin D 2 (ergocalciferol) and vitamin D 3 (cholecalciferol). However, vitamin D 2 and D 3 are shortage in our normal dietary [18]. Abundant vitamin D is synthesized in the skin when exposed to sunlight [19]. In brief, the UVB (290-315nm) transforms 7-dehydrocholesterol into previtamin D 3 in the skin, and then previtamin D 3 is further converted into vitamin D by thermal isomerization [20,21]. In addition, food supplement, such as normal dietary (the least source of vitamin D), fortified food (egg, milk, salmon, etc.) and concentrated natural food (e.g. cod liver oil), is another subordinate source of vitamin D [22].

Vitamin D metabolism
Since 1α,25-dihydroxyvitamin D (1α,25(OH) 2 D 3 , calcitriol) nor vitamin D is the most active metabolite in our body, vitamin D needs two important cytochrome P450-mediated hydroxylation steps in the metabolism. Firstly, vitamin D obtained from both dietary and skin is converted to 25-hydroxyvitamin D 3 (25(OH)D 3 ) by the hepatic 25-hydroxylases (CYP27A1) after transporting to liver via the vitamin D binding protein. Secondly, 25(OH) D 3 is hydroxylated again in the kidney by the enzyme 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) to yield calcitriol ( Figure 1). Of course, vitamin D metabolism is alternatively proceeded in other organs and/or cells and is regulated by parathyroid hormone, fibroblast growth factor 23, and calcitriol itself [23][24][25][26].

Vitamin D receptor
As mentioned above, the biological function of calcitriol is primarily mediated by vitamin D receptor, which is composed of 427 amino acid residues and belongs to the superfamily of steroid/thyroid hormone receptor [13]. VDR regulates biological function of calcitriol by two mechanisms, one involves transcriptional regulation of nuclear VDR, and the other involves nongenomic signal transduction pathways of membrane VDR [27]. The first one is the most studied anti-tumor mechanism of vitamin D. When activated by calcitriol, the phosphorylated nuclear VDR forms homodimers or heterodimers VDR-RXR with one of the retinoid X receptors (RXR), then the calcitriol-VDR-RXR complex translocates into nucleus and attaches to the vitamin D response elements (VDREs) in the promoters of target genes, causing the recruitment of co-activators or co-repressors to regulate gene expression in target cells [28,29]. In addition to the genomic responses mediated by nuclear VDR, membrane VDR mediates rapid responses pathways within 1-2 min to 15-45 min, including intestinal absorption of Ca 2+ (transcaltachia) [30], secretion of insulin by pancreatic β-cells [31], opening of voltage-gated Ca 2+ and Clchannels in osteoblasts and sertoli cells [32,33], and migration of endothelial cells [34].

VITAMIN D/VDR AND GASTRIC CANCER: EPIDEMIOLOGICAL DATA Ultraviolet B (UVB) and vitamin D in gastric cancer
Abundant vitamin D is synthesized in the skin by UVB, which is commonly regarded as the major vitamin D . Since Frank Garland proposed the UVBvitamin D-cancer hypothesis in 1980 [7], more and more epidemiology studies support that UVB related vitamin D deficiency is an important risk factor of cancer incidence and mortality in recent years [35][36][37][38]. In the stomach, almost all epidemiological data support a strong inverse association between sunlight exposure and cancer incidence and/or mortality rates [34-44] ( Table 1).
Ecological studies in predominantly European populations reported higher cancer survival in areas of higher solar UVB irradiation [39], indicating that high vitamin D status can improve gastric cancer survival. Chen [40] also found the 5-year survival proportions for gastric cancer were inversely associated with ambient UVB in the developing countries. In particular, Boscoe [41] extended the analysis of this relationship to include cancer incidence as well as mortality. After studying over three million cancer cases between 1998-2002 in the United States and three million cancer deaths with daily satellite-measured solar UVB levels between 1993-2002, they found an inverse relationship between solar UVB exposure and cancer incidence and mortality for 10 types of cancer, including gastric cancer. Following this initial finding, the protection role of UVB in gastric cancer was continually reported worldwide [42][43][44]. The people with deficiency of synthesis vitamin D in the skin are also in the high risk and/or mortality of gastric cancer [40][41][42][43]. For example, the premature with inadequate doses of solar UVB radiation showed increased gastric cancer mortality in the U.S. [45], the Blacks with low vitamin D levels were found to have higher risk of cancer incidence and mortality especially in digestive system [46]. To further assess whether patients with skin cancer have an altered risk of developing other cancers, a study found that the patients with skin cancer really have a high risk of developing other cancers, and the standardized incidence ratio of gastric cancer in sunny countries was significant lower than in less sunny countries (SIR(S)/ SIR(L) 0.61, 95%CI 0.38-0.9). Moreover, the apparently protective effect of sun exposure against second primary cancer was more pronounced after non-melanoma skin cancers than melanoma [47]. This result was also reported in an ecologic study of cancer mortality rates in Spain [48]. However, another case control study from Sweden [49] showed divergent result, the cases with the diagnosis of basal cell carcinoma in skin had an increased risk of getting another form of cancer, and had no correlation with gastric cancer (OR 1.00, 95%CI 0. 85-1.17). One limitation of this study is the exposure factor bias, whereby the study completely neglects the factor of sun exposure to patients, which may lead to this contradiction. More convincingly, Grant [50] found that solar UVB and vitamin D can reduce the risk of gastric cancer using Hill's criteria for causality. But most of these epidemiological results are from ecologic studies, further investigations to directly measure vitamin D status in vivo are needed.

Vitamin D intake and serum vitamin D in the patients with gastric cancer
Although the protective role of vitamin D from solar UVB on gastric cancer is obvious, the relationship between vitamin D intake and serum vitamin D in the patients with gastric cancer is inconsistent. There are about ten cohort studies in all (Table 2). These studies measured serum concentrations of 25(OH)D 3 as serum vitamin D status, and assessed dietary vitamin D intake by a diet history questionnaire [51,52], or food frequency questionnaire [53,54]. The latest cohort study from China [55]investigated whether baseline serum 25(OH)D 3 concentrations were associated with all-cause mortality and cause-specific mortality rates over 24 years of follow-up (1986-2010). They found that serum 25(OH)D 3 concentrations were not associated with the mortality of upper gastrointestinal cancer in Chinese population. Similarly, Pelucchi [53] and Chen [56] did not find a significant relationship between dietary intake of vitamin D and risk of gastric cancer. However, another study [57] reported a non-statistically significant but suggestive inverse relationship between vitamin D status and risk of gastric cancer. In particular, the Cohort Consortium Vitamin D Pooling Project of Rarer Cancers (VDPP) brought together 10 cohorts to conduct a prospective study of the association between vitamin D status and upper gastrointestinal cancers. In multivariate adjusted models, circulating 25(OH)D 3 concentrations were not significantly associated with the risk of upper gastrointestinal cancer, but higher concentrations of 25(OH)D ( > 100 nmol/L) were inversely associated with the risk of gastric cancer (OR:0.65, 95% CI 0.26-1.62) [57]. However, some studies found a positive association between vitamin D and the risk of gastric cancer [52,58]. Such as in a case-control study from Italy [52], a significant positive association was reported between vitamin D intake and the risk of gastric cancer (OR: 1.35, 95% CI: 1.00-1.83).
On the contrary, some studies support the notion of vitamin D-reduced the incidence and mortality of gastric cancer [54][55][56]. A retrospective case-control study revealed that the prevalence of vitamin D deficiency in gastric adenocarcinoma group was significantly higher than that in the control group (OR: 3.8, 95% CI:1.42-10.18, P:0.0079), suggesting a positive correlation between vitamin D deficiency and incidence of gastric adenocarcinoma [59]. A Health Professionals Follow-Up study found an increment of 25 nmol/L in predicted 25(OH)D 3 level was associated with a 17% reduction in total cancer incidence, a 29% reduction in total cancer mortality, and a 45% reduction in digestive cancer mortality, particularly in gastric cancer. It was therefore recommended that at least 1500 IU/day may be necessary for the vitamin D supplementation to prevent digestive cancer mortality [60]. Ren [61] also reported an inverse association of serum 25(OH)D 3 concentrations with clinical stage and lymph node metastasis of gastric cancer, suggesting that serum vitamin D level is a significant independent prognostic factor and vitamin D deficiency is associated with poor prognosis in gastric cancer.
As mentioned earlier, dietary vitamin D intake is a minimum source for the levels of circulating serum vitamin D since only 3 ng/mL differences were found in measured serum vitamin D between high and low dietary intake [62]. Besides, serum vitamin D status in most studies were based on one-time blood collection only, it is obvious that a single measurement of serum vitamin D cannot reflect real exposure to vitamin D in an etiologically relevant period. Even more, vitamin D status not just refer 25(OH)D 3 but include 1α,25(OH) 2 D 3 and vitamin D binding to DBP since a very little vitamin D circulates as a free form of 25(OH)D 3 [63]. All these may make an inconsistent relationship between vitamin D status and gastric cancer.

Vitamin D receptor in gastric cancer
The biological function of vitamin D, especially its anticancer effects, are largely through activation of VDR [64,65], which is required to suppress tumorigenesis and may be a new target for cancer chemoprevention and/or chemotherapy [66]. It has been reported that a higher VDR expression is associated with reduced mortality, favorable tumor characteristics and an improved prognosis in breast, prostate and colon cancer [67][68][69][70]. One study from China [71] reported similar results that VDR expression was significantly lower in gastric cancer tissues, and that among cancer tissues VDR was higher expressed in well and moderate differentiated tissues and in small tumors, indicating that VDR could be a prognostic factor for gastric cancer.
Most VDR gen polymorphisms were identified since 1997 [72], and about six gen polymorphisms were found to be associated with cancers [73][74][75]. A casecontrol study [76] revealed a strong relationship between VDR TaqI(T/T) and the susceptibility of Chinese Han population to gastric cancer. Another study in Chinese Han population [77] revealed the patients of gastric cancer with the ƒ allele (Fƒ+ƒƒ) had higher risk of a poorly differentiated type of gastric cancer. This finding has been reproduced in Uygur [78].

Vitamin D binding protein in gastric cancer
Vitamin D binding protein (DBP), a key protein in vitamin D metabolism, also mediates the biological function of vitamin D [79]. Several studies [80] have investigated serum DBP levels and DBP polymorphisms in association with cancer risk. Humphries [81] has validated DBP as one of the novel biomarkers of human gastric cancer. Two common coding single nucleotide polymorphisms (SNP) were identified in DBP gene, Glu416Asp (rs7041) and Thr420Lys (rs4588) [82]. Zhou [83]found that DBP Thr420Lys and Glu416Asp polymorphism had significant impact on the risk of developing gastrointestinal cancers in Chinese population. So far, the evidence is too little to confirm the relationship between DBP and gastric cancer, but it sheds some light to further study on DBP in gastric cancer. www.impactjournals.com/oncotarget

VITAMIN D/VDR AND GASTRIC CANCER: LABORATORIAL RESEARCH Vitamin D in animal models of gastric cancer
The anti-tumor effects of vitamin D have been extensively studied in animal models. Since vitamin D has the side effect of hypercalcemia, its analogs are widely used. Hiroki [84] found that 1α(OH)D 3 , a synthetic analogue of vitamin D 3 , markedly inhibited the inductions of ornithine decarboxylase (ODC) activity by promoters of carcinogenesis in the stomach, suggesting an antitumor effect of vitamin D on gastric carcinogenesis. Vitamin D analogue 1α(OH)D 3 largely reduced the incidence of gastrointestinal tumors induced by N-methyl-N'-nitro-N-nitrosoguanidine in male Wistar rats [85]. 24R, 25-dihydroxyvitaminD 3 , a vitamin D 3 derivative, also had chemopreventive effects on glandular stomach carcinogenesis in rats possibly by influencing calcium pharmacodynamics [86]. All of these results suggest that vitamin D and its analogs can inhibit the occurrence and development of gastric cancer in animal models.

Vitamin D regulation of specific signaling pathways in gastric cancer cells
Although abundant evidences from epidemical studies and animal models suggest vitamin D could obviously inhibit gastric cancer in vivo, its antitumor mechanisms are unclear. Some evidence indicate that vitamin D could block cell cycle, induce apoptosis and inhibit cell invasion and metastasis (Figure 2.) [87,88].
Selective inhibitors of mammalian DNA polymerase α, vitamin D 2 and D 3 could halt NUGC-3 human gastric cancer cells at the G1 phase in the cell cycle [89]. Park [90] found that 19-nor-1,25-dihydroxyvitamin D 2 , a vitamin D analog, could block cell cycle of MKN45 gastric cancer cells by decreasing the expression of cyclindependent kinase(CDK), CDK2, CDK4, CDK6 and Cyclin D1. Functional VDR elements have been identified in the promoter of PTEN, suggesting that vitamin D may play a role in the regulation of PTEN expression as a nuclear transcription factor [91]. A study [92] demonstrated that vitamin D induced apoptosis through PTEN upregulation in HGC-27 gastric cancer cells, and that vitamin D receptor, Egr-1 and p300 induced PTEN expression in a synergistic fashion. Another study indicated that EB1089, a vitamin D analog induced gastric cancer cells apoptosis through a VDR and mitochondrial apoptosis pathway, which was blocked by treating the cells with VDR siRNA or butin, an inhibitor of the mitochondrial apoptosis pathway [93].
Since Hedgehog signaling pathway plays an important role in the pathogenesis and the prognosis of gastric cancer, targeting this pathway is a new potential therapeutic opportunity in gastric cancer [94]. Vitamin D 3 may act as an antagonist of hedgehog signaling to suppress viability of gastric cancer cells, and it also has a synergistic effect with other anticancer drugs by reducing mRNA expression of the target genes of hedgehog signaling ( Ptch1, Gli1, cyclin D1 and bcl2) [95]. Co-treatment with cisplatin and 1α,25(OH) 2 D 3 enhanced cisplatin-mediated cell growth inhibition and cell apoptosis of human gastric cancer cells with an upregulation of Bax, a decrease in ERK and AKT phosphorylation levels, and an increase in p21 and p27 levels [96].

Vitamin D up-protein1
Vitamin D 3 upregulated protein 1 (VDUP1) is a 46 kDa protein upregulated by 1α,25(OH) 2 D 3 [97]. VDUP1 has an antitumor activity by forming a transcriptional repressor complex, which induced cell-cycle arrest at the G0/G1 phase and suppressed cell invasiveness and tumor metastasis [98,99]. In clinic, VDUP1 expression is significantly lower in gastric cancer tissue than in their adjacent normal tissue and the downregulation of VDUP1 expression is associated with poor prognosis [100]. Kwon [101] found that VDUP1 negatively regulates Helicobacter pylori-associated gastric carcinogenesis in mice by disrupting cell growth and inhibiting the induction of TNFa, NF-kB and COX-2, suggesting that VDUP1 may serve as a potential target for the development of anticancer agents for gastric cancer.

Viatmin D with H. pylori and microRNA
Helicobacter pylori (Hp) infection plays an important pathogenic role in most gastric cancer cases [102,103]. International Agency for Research on Cancer (IARC) classified Hp as a group 1 carcinogen in 1994, and reconfirmed this classification in 2009 [2]. A crosssectional study found a significant positive correlation between the levels of serum 25-OH vitamin D and serum Hp specific IgG antibody titers, indicating that vitamin D analog may have antibacterial action against Hp [104]. Kouichi Hosoda [105] further confirmed Vitamin D 3 decomposition product (VDP1) can exert an antibacterial action against Hp by inducing a collapse of cell membrane structures of Hp and ultimately lysing the bacterial cells. These findings suggest that VDP1 may become a new antibacterial substance against Hp.
MicroRNAs (miRNAs) are short, single strands of noncoding RNA with important functions in mRNA translation and regulation of cell cycle and apoptosis [106]. It was reported that miR145 induced by 1α, 25(OH) 2 D 3 through VDR could inhibit colony formation, gastric cancer cell viability and induce cell arrest at S-phase by targeting E2F3 and CDK6. This might hold www.impactjournals.com/oncotarget promise for prognosis and therapeutic strategies for gastric cancer [107].

Vitamin D/VDR in immunity
In the last few years, accumulating evidence indicates an important modulatory role of vitamin D/VDR in adaptive and innate immune cells [108][109][110], which is distinct from their classical anti-tumor roles. After identification of VDR in series immune cells, numerous cellular and molecular targets of VDR in the immune system have been elaborated [111,112]. VDR regulates all stages of a T cells life, ranging from development to differentiation and elicitation of effector functions [113]. Likewise, VDR is essential for Th2 cell function, and vitamin D could increase the activities of regulatory T cells and Th2 cells while suppressing Th1 cell activity [114,115]. In antigen-presenting cells, vitamin D is believed to program dendritic cells (DC) for tolerance, dampen their ability to activate effector T-cell generation, and enhance their potential to induce anti-inflammatory regulatory T (Treg) cells. Vitamin D also interacts with DC to influence their migration and their capacity to instruct T cells and hence to initiate, fine tune or dampen immune reactions [116]. On the other hand, vitamin D-treated DCs are significantly more potent in driving differentiation of IL-22-producing T cells and are markedly enhanced to secrete TNF-α, IL-6, IL-1β and IL-23 [117]. However, despite compelling evidence for the roles of vitamin D/ VDR in immunity, there are no studies on vitamin D/VDR in gastric cancer immunity.

Vitamin D and gastric cancer: clinical trial
To date, it is scarce for the clinical trial directly exploring vitamin D to potentially treat gastric cancer [118,119]. In a 4 year, population-based, double-blind, randomized placebo-controlled trial [119], Joan et al. found that the relative risk of developing cancer was 0.232 for the calcium plus vitamin D group and 0.587 for the calcium alone group, and that serum 25-hydroxyvitamin D concentrations were significant, independent predictors of cancer risk. As Hp is regarded as an independent risk factor of gastric cancer, Kawaura et al. [118] tested whether long term 1α(OH)D 3 administration could inhibit Hp infection, and they found that Hp infection rate was significantly lower in subjects with 1α(OH)D 3 treatment than those without treatment. This study reconfirms vitamin D analog has antibacterial action against Hp.

CONCLUSIONS
Vitamin D has received extensive attention in recent years, especially after Mark [120] found vitamin D could promote protein homeostasis and longevity in nematodes. Most current evidence suggests that vitamin D is inversely associated with the morbidity and mortality of gastric cancer. Not only laboratorial studies at the levels of cells, tissues and animal models but also clinical trial support an anti-cancer role of vitamin D. However, the epidemiological data are still paradoxical. The studies utilizing ultraviolet B exposure as a main measurement consistently show the increased risk of gastric cancer with vitamin D deficiency, but some studies measuring serum 25(OH)D 3 levels in human body do not support this notion. As discussed above, imprecise and inconsecutive assessment of serum vitamin D status may lead to the obscure relationship between 25(OH)D 3 levels and gastric cancer risk. The laboratorial studies demonstrate that vitamin D and its metabolites activate VDR to inhibit viability, proliferation and metastasis of gastric cancer cells, and also explore the underlying molecular mechanisms against gastric tumorigenesis and progression. Furthermore, vitamin D metabolites or analogues might also inhibit Hp infection and Hpassociated gastric cancer. Although basic research supports the protective effects of vitamin D against gastric cancer, further studies are needed to elucidate its anti-tumour mechanisms, especial its interaction with VDR. At last but not least, large-scale and long-term clinical randomized controlled trials (RCTs) are necessary to make a definite conclusion whether vitamin D can really offer preventive and/or therapeutic benefits to gastric cancer.