CYP27B1

CYP27B1, as the synthesising enzyme of calcitriol, is normally expressed at low levels in the colon⁽ Reference Bises, Kallay and Weiland ^{25 ,} Reference Tangpricha, Flanagan and Whitlatch ^{26 )}. In well-differentiated and moderately differentiated colorectal cancer samples, expression of CYP27B1 is elevated, whereas in poorly differentiated colorectal cancer samples the expression is repressed⁽ Reference Bises, Kallay and Weiland ^{25 –} Reference Bareis, Bises and Bischof ^{28 )}. Ogunkolade et al. ⁽ Reference Ogunkolade, Boucher and Fairclough ^{29 )} reported that CYP27B1 mRNA expression levels are similar in colorectal cancer samples and in healthy colon samples, but are decreased in adjacent normal colon mucosa, 10 cm from the tumour border; this finding suggests that CYP27B1 expression in adjacent colon cells is regulated by the tumour, or that low expression of CYP27B1 in the colon is a risk for carcinogenesis. Bareis et al. ⁽ Reference Bareis, Kallay and Bischof ^{30 )} showed that the slowly dividing, highly differentiated colorectal cancer cell line Caco-2/15 responds in a dose-dependent manner to epidermal growth factor (EGF) or calcitriol by up-regulating the expressions of VDR and CYP27B1, whereas highly proliferative, less-differentiated cell lines (Caco-2/AQ, COGA-1A and COGA-1E) show a down-regulation of VDR and CYP27B1 after EGF or calcitriol treatment. Although definite in vivo evidence is lacking, local production of calcitriol in the colon has been indirectly suggested by human studies. The serum concentration of 25(OH)D, rather than that of calcitriol, was inversely associated with colonic epithelial cell proliferation in a chemoprevention study⁽ Reference Holt, Arber and Halmos ^{31 )}. Wagner et al. ⁽ Reference Wagner, Dias and Schnabl ^{32 )} showed a positive correlation between serum and colon calcitriol concentrations (r 0·58, P=0·0008), with a positive colon calcitriol intercept (21·5 pmol/kg, P<0·001) at zero serum calcitriol, supporting the notion of synthesis of calcitriol within the colon. To summarise, elevated CYP27B1 expression suggests possible benefit from treatment with vitamin D, especially in well-differentiated and moderately differentiated tumours, whereas the relatively low expression of CYP27B1 in poorly differentiated colorectal cancer indicates a mechanism of resistance of the cancer cells to calcitriol actions.

CYP24A1

As the main enzyme determining the biological half-life of calcitriol, CYP24A1 is found in low levels in normal human colon mucosa and in colorectal adenomas, but in elevated levels in the majority of adenocarcinomas⁽ Reference Horvath, Lakatos and Kosa ^{33 )}. CYP24A1 mRNA expression is also increased in poorly differentiated and late-stage colorectal cancers, compared with well-differentiated, early-stage tumours⁽ Reference Bareis, Bises and Bischof ^{28 )}. Anderson et al. ⁽ Reference Anderson, Nakane and Ruan ^{34 )} showed that CYP24A1 mRNA expression is not only significantly up-regulated in human HT29 cells but also profoundly stimulated by calcitriol treatment, abrogating the anti-proliferative effect of calcitriol. Kosa et al. ⁽ Reference Kosa, Horvath and Wolfling ^{35 )} also observed that CYP24A1 mRNA is induced by calcitriol treatment in Caco-2 – a human colon adenocarcinoma cell line. Cell viability and proliferation are not influenced by calcitriol alone, but are markedly reduced when calcitriol is co-administered with KD-35 – a CYP24A1 inhibitor. Together, these findings suggest that CYP24A1 exhibits a potent negative-feedback effect, and that inhibition of CYP24A1 may be a good strategy for enhancing the anti-tumour effect of calcitriol.

Vitamin D receptor

As the major receptor to mediate the biological effects of calcitriol, VDR is present in most cells of the human body, and is especially abundant in intestinal epithelial cells⁽ Reference Wang, Zhu and DeLuca ^{36 )}. VDR expression is increased in adenoma and in well-differentiated or moderately differentiated colorectal cancer tissues, but is decreased in poorly differentiated tumours, and is negligible in metastatic lymph nodes⁽ Reference Cross, Bareis and Hofer ^{27 ,} Reference Matusiak, Murillo and Carroll ^{37 )}. Palmer et al. ⁽ Reference Palmer, Larriba and Garcia ^{38 )} discovered that the transcription factors SNAI1 (snail family zinc finger 1) and SNAI2 (snail family zinc finger 2) repress VDR expression in SW480-ADH cells and block the anti-tumour action of the calcitriol analogue EB1089. RNA expressions of SNAI1 and SNAI2 are up-regulated in human colorectal cancers, and are inversely correlated with VDR mRNA expression⁽ Reference Palmer, Larriba and Garcia ^{38 ,} Reference Larriba, Bonilla and Munoz ^{39 )}. These findings suggest that high levels of SNAI1 and SNAI2 are a probable cause for VDR down-regulation and for vitamin D unresponsiveness in advanced colorectal cancer, and that vitamin D therapy may not be a good treatment choice for patients who overexpress SNAI1 and SNAI2.

Proliferation

Previous studies have established VDR as a biomarker for vitamin D-mediated inhibition of human colon cancer cell growth⁽ Reference Shabahang, Buras and Davoodi ^{43 )}. The anti-proliferative effect of vitamin D on colorectal cancer involves multiple pathways. In Caco-2 cells, calcitriol and its analogues (F6-D3, ZK 156718 and BGP-13) increase the expressions of the cyclin-dependent kinase (CDK) inhibitors CDKN1A (cyclin-dependent kinase inhibitor 1A (p21, Cip1)) and CDKN1B (cyclin-dependent kinase inhibitor 1B (p27, Kip1)), which inhibit CDK2 and CDK6, leading to G1 phase arrest⁽ Reference Scaglione-Sewell, Bissonnette and Skarosi ^{44 –} Reference Berkovich, Sintov and Ben-Shabat ^{46 )}. Calcitriol also results in the activation of latent transforming growth factor-β1 (TGFB1) in Caco-2 cells, and sensitises these cells to the growth inhibitory effects of TGFB1⁽ Reference Chen, Davis and Sitrin ^{47 )}. Synthetic low-calcemic vitamin D analogues (EB1089 and CB1093) decrease proliferation of HT29 human cancer cells by inhibiting the secretion of insulin-like growth factor 2 (IGF2) and by inducing the insulin-like growth factor-binding protein-6, which sequesters IGF2 with high affinity⁽ Reference Oh, Kim and Schaffer ^{48 )}. Calcitriol also counteracts EGF-stimulated Caco-2 cell growth by markedly decreasing epidermal growth factor receptor expression⁽ Reference Tong, Hofer and Ellinger ^{49 )}.

Differentiation

Calcitriol has multiple pro-differentiation effects in colorectal cancer cells. The classic marker for differentiation is the expression of alkaline phosphatase, which is found along the brush border of the colon mucosa but is poorly expressed in proliferating colorectal cancer cells. Calcitriol and its analogues (ZK 156718 and EB1089) increase the activity of alkaline phosphatase in colorectal adenoma cell lines (RG/C2 and AA/C1) and colorectal cancer cells (Caco-2, PC/JW, HT29 and SW620)⁽ Reference Gaschott, Steinmeyer and Steinhilber ^{45 ,} Reference Diaz, Paraskeva and Thomas ^{50 )}. Chen et al. ⁽ Reference Chen, Davis and Bissonnette ^{51 )} reported that calcitriol increases alkaline phosphatase activity in Caco-2 cells by stimulating activator protein-1 (FOS/JUN (jun proto-oncogene)) activation, which is accomplished via a protein kinase C α- and mitogen-activated protein kinase (MAPK)-dependent mechanism.

Apart from affecting the expression of alkaline phosphatase, calcitriol also induces the expression of E-cadherin (CDH1, cadherin 1) and other adhesion proteins, causing β-catenin (CTNNB1, catenin beta 1) to translocate from the nucleus to E-cadherin complexes at the plasma membrane in the human colon cancer SW480-ADH cell line⁽ Reference Palmer, Gonzalez-Sancho and Espada ^{52 ,} Reference Palmer, Sanchez-Carbayo and Ordonez-Moran ^{53 )}; similar effect on Cdh1 has been observed in an Apc ^min/+ (adenomatous polyposis coli) mouse model⁽ Reference Xu, Posner and Stevenson ^{54 )}. Meanwhile, ligand-activated VDR competes with the T cell-specific transcription factor 7-like 2 (TCF7L2) for CTNNB1 binding and represses downstream gene expression in SW480-ADH cells⁽ Reference Palmer, Gonzalez-Sancho and Espada ^{52 )}. Calcitriol–VDR also inhibits CTNNB1 activity in Caco-2 cells, and the inhibition is enhanced by wild-type APC⁽ Reference Egan, Thompson and Vitanov ^{55 )}. Finally, the WNT (wingless-type MMTV integration site family) antagonist DKK1 (dickkopf WNT signaling pathway inhibitor 1) is induced by calcitriol in association with E-cadherin in SW480-ADH cells⁽ Reference Aguilera, Pena and Garcia ^{56 )}. As a result, calcitriol and its analogues inhibit the WNT/CTNNB1 pathway and the activation of its target genes in colorectal cancer cells; this in turn contributes to the inhibition of cell proliferation and to the maintenance of the differentiated phenotype.

Apoptosis

Calcitriol induces apoptosis in colorectal adenoma and colorectal cancer by up-regulating the pro-apoptotic proteins BAK1 (BCL2-antagonist/killer 1) and BAX (BCL2-associated X protein) and by down-regulating the expressions of anti-apoptotic proteins BAG1, BIRC5 (baculoviral IAP repeat containing 5) and BCL2 (B-cell CLL/lymphoma 2). In two colorectal adenoma and three colorectal cancer cell lines, calcitriol and the vitamin D analogue EB1089 induced p53-independent apoptosis in a dose-dependent manner, and the levels of the pro-apoptotic protein BAK1 were consistently increased in all the cell lines examined⁽ Reference Diaz, Paraskeva and Thomas ^{50 )}. Barnes et al. ⁽ Reference Barnes, Arhel and Lee ^{57 )} showed that EB1089 induces apoptosis in a colorectal adenoma S/RG/C2 cell line by re-distributing the anti-apoptotic protein BAG1 from the nucleus to the cytoplasm. Liu et al. ⁽ Reference Liu, Hu and Chakrabarty ^{58 )} discovered that calcitriol suppresses the expression of BIRC5 (survivin) and promotes a cytotoxic response to 5-fluorouracil in human colon cancer cells (CBS, Moser, Caco-2 and HCT116) in a Ca-sensing receptor (CASR)-dependent manner, possibly by binding the VDRE in CASR promoters⁽ Reference Canaff and Hendy ^{59 ,} Reference Chakrabarty, Wang and Canaff ^{60 )}. In an Apc^1638N/+ mouse model of intestinal cancer, a western-pattern diet decreased the expression of the pro-apoptotic protein BAX and increased the expression of the anti-apoptotic protein BCL2; treatment with vitamin D and Ca reverses these effects of the western-style diet and markedly inhibits tumour growth⁽ Reference Yang, Lamprecht and Shinozaki ^{61 )}. In a human colorectal cancer xenograft model in nude mice, treatment with the vitamin D analogues BGP-13 and BGP-15 activated cell apoptosis⁽ Reference Berkovich, Sintov and Ben-Shabat ^{46 )}. However, the pro-apoptotic effect of calcitriol appears to be not always true: Stambolsky et al. ⁽ Reference Stambolsky, Tabach and Fontemaggi ^{62 )} reported that mutant TP53 (tumor protein p53) is recruited to VDR-regulated genes and converts calcitriol into an anti-apoptotic agent in SW480 cells. Thus, TP53 mutation status might be a predictive marker for vitamin D treatment response.

Angiogenesis

Calcitriol also inhibits angiogenesis. Mantell et al. ⁽ Reference Mantell, Owens and Bundred ^{63 )} showed that calcitriol significantly inhibits the sprouting and elongation of vascular endothelial growth factor A (VEGFA)-induced endothelial cells in a dose-dependent manner. In human colorectal cancer SW480 cells, calcitriol treatment for 24 h at 0·1 and 1 µm decreases the expression of hypoxia-inducible factor-1α and at 1 µm inhibits the secretion of VEGFA under conditions of hypoxia⁽ Reference Ben-Shoshan, Amir and Dang ^{64 )}. However, Fernandez-Garcia et al. ⁽ Reference Fernandez-Garcia, Palmer and Garcia ^{65 )} reported that calcitriol increases the levels of VEGFA and the anti-angiogenic factor thrombospondin 1, leading to a minimal balanced change in the angiogenic potential of SW480-ADH cells. Calcitriol also represses the expression of DKK4 (dickkopf WNT signaling pathway inhibitor 4) in SW480-ADH cells; DKK4 is induced by the TCF7L2/CTNNB1 pathway and enhances the migratory, invasive and pro-angiogenic potential of colorectal cancer⁽ Reference Pendas-Franco, Garcia and Pena ^{66 )}. In a rat model of colon tumourigenesis induced by azoxymethan, intraperitoneal administration of calcitriol significantly reduced the incidence of colon tumours and also decreased the level of VEGFA and microvessel counts in tumours, suggesting that anti-angiogenesis is a mechanism for the anti-tumourigenic effect of vitamin D⁽ Reference Iseki, Tatsuta and Uehara ^{67 )}.

Immune modulation

Calcitriol modulates innate and adaptive immunity in the colon⁽ Reference van Harten-Gerritsen, Balvers and Witkamp ^{68 )}. Calcitriol induces the expression of the cathelicidin anti-microbial peptide, a major component of the innate immune system, in HT29 cells⁽ Reference Gombart, Borregaard and Koeffler ^{69 )}. Lithocholic acid, a secondary bile acid and a vitamin D analogue, decreases NF-κB activity via the VDR in colonic cancer cells (Caco-2 and HT29C19A)⁽ Reference Sun, Mustafi and Cerda ^{70 )}. CYP27B1-knockout mice show increased IL1 and IL17 expressions in the colon and are more susceptible to colitis compared with heterozygote controls⁽ Reference Liu, Nguyen and Chun ^{71 )}. In a Smad3 ^–/– mouse model of bacteria-induced colitis, increased dietary vitamin D suppressed MAPK and NF-κB activation, severity of colitis and incidence of intestinal cancer⁽ Reference Meeker, Seamons and Paik ^{72 )}. In addition, calcitriol has effects on several immune cell types including dendritic cells, B cells and T cells throughout the human body⁽ Reference Veldhoen and Brucklacher-Waldert ^{73 )}. Specifically, the Vdr-knockout mouse model showed that VDR is required for the maturation and proliferation of intestinal CD8αα ⁺ intra-epithelial lymphocytes⁽ Reference Bruce and Cantorna ^{74 )}, which might have a regulatory role within the gut⁽ Reference van Wijk and Cheroutre ^{75 )}. On the other hand, the effect of calcitriol and the level of expression of VDR may both be affected by the immune environment of the colon: in human colon ductal epithelium, VDR expression is considerably decreased in patients with ulcerative colitis and is even lower in patients with colitis-associated colorectal cancer⁽ Reference Wada, Tanaka and Maeda ^{76 )}. In line with this, treatment with TNF and IL-6 leads to decreased expression of CYP27B1 in colonic epithelial COGA-1A cells⁽ Reference Hummel, Fetahu and Groschel ^{77 )}.

Recent studies have shown interactions between gut microbiota and immunity in colon carcinogenesis⁽ Reference Johnson, Dejea and Edler ^{78 –} Reference O’Keefe, Li and Lahti ^{80 )}, and vitamin D has been reported to regulate the gut microbiome. In a dextran sodium sulphate-induced colitis model, mice on vitamin D-deficient diet showed more prominent symptoms of colitis and elevated concentrations of bacteria compared with mice on vitamin D-sufficient diet⁽ Reference Lagishetty, Misharin and Liu ^{81 )}. Similarly, in the same colitis model, Ooi et al. ⁽ Reference Ooi, Li and Rogers ^{82 )} showed that Cyp27b1-knockout mice had higher concentrations of the Helicobacter species in the faeces and more severe symptoms of colitis compared with wild-type littermates. In addition, calcitriol supplementation (1·25 µg/100 g diet) to Cyp27b1-knockout mice reduced Helicobacter numbers and colitis severity⁽ Reference Ooi, Li and Rogers ^{82 )}. Given the data from mouse models, it would be interesting to investigate changes in the human gut microbiome after vitamin D supplementation.

MicroRNA

MicroRNAs (miR) are implicated in the anti-neoplastic influence of vitamin D⁽ Reference Christakos, Dhawan and Verstuyf ^{12 )}. Alvarez-Diaz et al. ⁽ Reference Alvarez-Diaz, Valle and Ferrer-Mayorga ^{83 )} reported that miR-22 is induced by calcitriol in a time-, dose- and VDR-dependent manner in multiple human colorectal cancer cell lines. Specifically, in SW480-ADH and HCT116 cells that express VDR, miR-22 is required for the anti-proliferative and anti-migratory effects of calcitriol, and regulates the expression of several target genes of calcitriol. Consistently, miR-22 expression is associated with VDR expression in human colorectal cancer samples, suggesting that miR-22 has a role in the VDR-mediated anti-tumour effect of vitamin D.

Padi et al. ⁽ Reference Padi, Zhang and Rustum ^{84 )} found that calcitriol up-regulates miR-627, which in turn mediates the anti-growth effect of calcitriol in HT29 cells; they reported that miR-627 down-regulates the expression of KDM3A (lysine demethylase 3A, a histone demethylase), increases methylation of histone H3K9, and thereby suppresses the expression of proliferative factors such as GDF15 (growth differentiation factor 15). This same effect of miR-627 is also found in the HCT116 xenograft model of nude mice⁽ Reference Padi, Zhang and Rustum ^{84 )}. Collectively, these findings suggest that enhancing the effect of miR-627, or suppressing its target KDM3A, has the same anti-tumour effect as does vitamin D, and may bypass the side-effects of hypercalcaemia.

Measurement of vitamin D in human populations

Determination of the vitamin D status of individuals in population-based studies needs a consideration of both biology and logistics. The plasma concentration of total 25(OH)D, the major circulating metabolite of vitamin D, is commonly used to determine vitamin D status⁽ Reference Holick ^{100 )}. For instance, a 25(OH)D concentration of <20 ng/ml (50 nmol/l) is considered vitamin D insufficiency⁽ Reference Ross, Manson and Abrams ^{101 )}, and 25(OH)D concentration of >150 ng/ml (375 nmol/l) may cause vitamin D intoxication⁽ Reference Holick ^{100 )}. However, the association of 25(OH)D with colorectal cancer may be confounded by other risk factors. For example, both obesity and low physical activity have been associated with lower plasma 25(OH)D concentrations, as well as with increased colorectal cancer risk⁽ Reference Davis and Dwyer ^{102 )}. Inflammation has been postulated as another confounder based on the assumption that inflammation reduces 25(OH)D concentrations⁽ Reference Autier, Boniol and Pizot ^{103 )}, although there is some evidence against this theory⁽ Reference Song, Wu and Chan ^{104 )}. Moreover, especially for cohorts, the time of blood sampling will likely precede the diagnosis of colorectal cancer for a variety of years for different patients, and it might be helpful to have an additional 25(OH)D measurement that is within a comparable time from diagnosis among all patients⁽ Reference Hofmann, Yu and Horst ^{105 ,} Reference Grant ^{106 )}. However, serial blood sampling may not be feasible in many large-scale cohort studies.

Alternatively, dietary or supplementary intakes of vitamin D can be assessed repeatedly with questionnaires. Nevertheless, recall of diet and supplement use is imprecise. Moreover, as skin exposed to sunlight also produces vitamin D, vitamin D intake does not necessarily represent overall vitamin D status or the plasma concentration of 25(OH)D. In 3345 subjects of the Women’s Health Initiative (WHI) observational study, total vitamin D intake calculated based on information from questionnaires explained 9 % variance in serum 25(OH)D concentrations⁽ Reference Cheng, Millen and Wactawski-Wende ^{107 )}.

Recently, a predicted 25(OH)D score using dietary and lifestyle information collected from questionnaires has been used as a surrogate of vitamin D status⁽ Reference Bertrand, Giovannucci and Liu ^{108 ,} Reference Jung, Qian and Yamauchi ^{109 )}. Using multivariate linear regression, Bertrand et al. ⁽ Reference Bertrand, Giovannucci and Liu ^{108 )} derived this score based on known determinants of circulating 25(OH)D, including age, race, UV radiation exposure, vitamin D intake, BMI, physical activity, alcohol intake, postmenopausal hormone use and season of blood collection from more than 4500 participants with available blood samples in three US nationwide cohorts. The predicted score explained 25–33 % variance in plasma 25(OH)D concentrations in different cohorts. This approach of using information from questionnaires estimates vitamin D status data in cohorts where plasma concentrations are not available, and incorporates not only dietary vitamin D intake but also non-dietary exposures, which are associated with increased plasma 25(OH)D concentrations. Of note, the predicted score was derived from the original cohorts, and its application to other cohorts will require further validation.

Plasma concentrations of 25-hydroxyvitamin D and incidence of colorectal cancer

Table 1 summarises previous studies investigating plasma 25(OH)D concentrations and incidence of colorectal cancer with at least 300 cases⁽ Reference Jung, Qian and Yamauchi ^{109 –} Reference Song, Nishihara and Wang ^{122 )}. Evidence for the association of plasma 25(OH)D concentration or 25(OH)D score with lower colorectal cancer incidence is quite strong. To further support this, two meta-analyses reported inverse associations between plasma 25(OH)D concentration and risk of colorectal adenoma, a well-established precancerous lesion for colorectal cancer⁽ Reference Lee ^{123 ,} Reference Yin, Grandi and Raum ^{124 )}.

Table 1 Major studies (no. of cases ≥300) investigating plasma 25-hydroxyvitamin D (25(OH)D) concentrations and incidence of colorectal cancer (Odds ratios, hazard ratios (HR) and 95 % confidence intervals)

WHI, Women’s Health Initiative; NHS, Nurses’ Health Study; HPFS, Health Professionals Follow-up Study; JPHC Study, Japan Public Health Center-based Prospective Study; EPIC, European Prospective Investigation into Cancer and Nutrition; Ref., referent values; MCCS, Melbourne Collaborative Cohort Study; ATBC, Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study; VDR, vitamin D receptor; DBP, vitamin D-binding protein; PLCO, Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial; MPE, molecular pathological epidemiology.

* P _{heterogeneity} is for colon cancer v. rectal cancer.

† P _{heterogeneity} is for VDR (–) v. VDR (+).

‡ P _{heterogeneity} is for low v. high DBP.

§ P _{heterogeneity} is for high v. mild v. absent reaction.

By integrating exposure data such as vitamin D status and tumour molecular/immune features of colorectal cancer tissue, MPE⁽ Reference Ogino, Chan and Fuchs ^{5 ,} Reference Ogino and Stampfer ^{6 ,} Reference Ogino, Lochhead and Chan ^{125 ,} Reference Ogino, Campbell and Nishihara ^{126 )} research provides new insights into the relationship between vitamin D and colorectal cancer. Jung et al. ⁽ Reference Jung, Qian and Yamauchi ^{109 )} studied the risk of colorectal cancer in relation to the predicted score for 25(OH)D concentration (with 1059 incident cases during the follow-up of 140 418 participants). A higher predicted 25(OH)D score was inversely associated with colorectal cancer risk (P<0·001), regardless of VDR expression levels in tumour cells (P _{heterogeneity}=0·75). Considering the role of vitamin D in the immune system, another MPE study showed that high plasma 25(OH)D concentration was associated with lower risk of colorectal cancer with high-level immune reaction (P _trend<0·001), but not with risk of tumour with lower-level reaction (P _trend>0·50, P _{heterogeneity}=0·001)⁽ Reference Song, Nishihara and Wang ^{122 )}. This statistical analysis of heterogeneity is critical, as the hypotheses address differential effects of vitamin D on subtypes⁽ Reference Wang, Kuchiba and Ogino ^{127 ,} Reference Wang, Spiegelman and Kuchiba ^{128 )}. These data support the hypothesis that effect of vitamin D might be strong in tumours enriched with immune cells⁽ Reference Song, Nishihara and Wang ^{122 )}, because immune cells in tumour can activate vitamin D, and thereby increase local levels of active vitamin D⁽ Reference Edfeldt, Liu and Chun ^{129 –} Reference Sigmundsdottir, Pan and Debes ^{131 )}. Although a replication by additional studies is needed, these findings suggest an interplay of vitamin D status and the immune system in inhibiting the tumourigenesis of colorectal cancer. In addition, a possible interaction may exist between vitamin D status and tumour immunity status in colorectal cancer patient survival analyses, requiring further investigation. With complex immune and inflammatory processes suggested to be involved in colorectal cancer progression and regulated by vitamin D, it has been recommended that future epidemiological studies should measure both vitamin D and inflammatory markers, preferably multiple times, and perform mediation analysis⁽ Reference Nishihara, VanderWeele and Shibuya ^{132 )} to study the role of inflammation as a mediator between vitamin D and colorectal cancer⁽ Reference van Harten-Gerritsen, Balvers and Witkamp ^{68 )}.

Plasma 25-hydroxyvitamin D concentration and survival of colorectal cancer

Table 2 shows previous studies with at least 300 cases on plasma 25(OH)D concentration and survival of patients with diagnosed colorectal cancer⁽ Reference Ng, Meyerhardt and Wu ^{133 –} Reference Ng, Venook and Sato ^{139 )}. Of note, to reduce potential reverse causation associated with undiagnosed tumours at the time of blood sampling that might lower plasma 25(OH)D concentrations, the Nurses’ Health Study⁽ Reference Ng, Meyerhardt and Wu ^{133 ,} Reference Ng, Wolpin and Meyerhardt ^{134 )}, the Health Professionals Follow-up Study⁽ Reference Ng, Meyerhardt and Wu ^{133 ,} Reference Ng, Wolpin and Meyerhardt ^{134 )} and the European Prospective Investigation into Cancer and Nutrition study⁽ Reference Fedirko, Riboli and Tjonneland ^{136 )} measured plasma 25(OH)D concentration before diagnosis of colorectal cancer, and excluded cases diagnosed within 2 years after blood collection. In contrast, the Study of Colorectal Cancer in Scotland⁽ Reference Zgaga, Theodoratou and Farrington ^{137 )} and the Cancer and Leukemia Group B/Southwest Oncology Group 80405⁽ Reference Ng, Venook and Sato ^{139 )} studies measured 25(OH)D shortly after diagnosis, a timing more subject to reverse causation. Despite the different timing of blood collection, there is a consistent prognostic association of plasma 25(OH)D concentration with colorectal cancer patient survival.

Table 2 Major studies (no. of cases ≥300) investigating plasma 25-hydroxyvitamin D (25(OH)D) concentrations and survival of patients with diagnosed colorectal cancer (CRC) (Hazard ratios (HR) and 95 % confidence intervals)

NHS, Nurses’ Health Study; HPFS, Health Professionals Follow-up Study; NCCTG, North Central Cancer Treatment Group; mCRC, metastatic colorectal cancer; PFS, progression-free survival; EPIC, European Prospective Investigation into Cancer and Nutrition; SOCCS, Study of Colorectal Cancer in Scotland; CALGB, Cancer and Leukemia Group B; SWOG, Southwest Oncology Group.

Vitamin D intake and incidence of colorectal cancer

Table 3 lists previous studies exploring the relationship between vitamin D intake and risk of colorectal cancer with at least 500 cases⁽ Reference Jenab, Bueno-de-Mesquita and Ferrari ^{113 ,} Reference Ma, Zhang and Wang ^{115 ,} Reference Martinez, Giovannucci and Colditz ^{140 –} Reference McCullough, Robertson and Rodriguez ^{150 )}. In contrast to the consistent and strong evidence from the studies measuring plasma 25(OH)D, the association between vitamin D intake and incidence of colorectal cancer is conflicting. Nevertheless, a 2011 meta-analysis⁽ Reference Ma, Zhang and Wang ^{115 )} of prospective studies reported an inverse association between vitamin D intake and colorectal cancer incidence.

Table 3 Major studies (no. of cases ≥500) investigating vitamin D intake and incidence of colorectal cancer (CRC) (Odds ratios, relative risks (RR) and 95 % confidence intervals)

NHS, Nurses’ Health Study; SMC, Swedish Mammography Cohort; CPS II, Cancer Prevention study II; JPHC Study, Japan Public Health Center-based Prospective Study; EPIC, European Prospective Investigation into Cancer and Nutrition.

* Indoor, subjects engaged in sedentary or standing work (including no job) and no outdoor physical activity at leisure; outdoor, subjects engaged in work with labour or walking or outdoor physical activity at leisure at least of 120 min/week.

Vitamin D intake and survival of colorectal cancer

Observational studies on the impact of vitamin D intake in patients with diagnosed colorectal cancer are limited. In a paper published in 2014, Yang et al. ⁽ Reference Yang, McCullough and Gapstur ^{151 )} included 1111 participants in the Cancer Prevention Study II Nutrition Cohort who were diagnosed with invasive, non-metastatic colorectal cancer. The researchers evaluated associations of Ca, vitamin D and diary product intakes after colorectal cancer diagnosis with all-cause and colorectal cancer-specific mortality. After a mean follow-up of 7·6 years, both Ca and milk intakes were inversely associated with all-cause mortality and colorectal cancer-specific mortality, but vitamin D intake was not associated with either mortality outcomes⁽ Reference Yang, McCullough and Gapstur ^{151 )}.

Completed clinical trials of vitamin D intake and incidence of colorectal cancer

To date, four completed randomised controlled trials of vitamin D have a reasonable number of cancer cases (Table 4)⁽ Reference Wactawski-Wende, Kotchen and Anderson ^{110 ,} Reference Trivedi, Doll and Khaw ^{152 –} Reference Avenell, MacLennan and Jenkinson ^{154 )}. In a substudy of the WHI, 36 282 postmenopausal women were given 5µg of vitamin D and 500 mg of Ca twice daily (10µg of vitamin D and 1000 mg of Ca daily), or a matching placebo, for an average of 7 years⁽ Reference Wactawski-Wende, Kotchen and Anderson ^{110 )}. The incidence of invasive colorectal cancer in this study did not differ significantly between women assigned to Ca plus vitamin D and those assigned to placebo (168 v. 154 cases, hazard ratio 1·08; 95 % CI 0·86, 1·34; P=0·51), and tumour characteristics were similar in the two groups. This study has several limitations. First, the modest dose of vitamin D used in the trial led to only a small rise in plasma 25(OH)D concentrations⁽ Reference Ng, Scott and Drake ^{155 )}, which was measured only in a small sample of the study population. Second, the limited compliance in the treatment group and the allowance for the placebo group to take supplements could have further reduced the actual contrast of 25(OH)D between groups. In fact, as shown in a post hoc analysis of WHI, in 15 646 women (43 %) who were not taking personal Ca or vitamin D supplements at randomisation, Ca and vitamin D treatment non-significantly reduced the risk of colorectal cancer by 17 %⁽ Reference Bolland, Grey and Gamble ^{156 )}. Third, the 7-year follow-up may not be sufficient to show a benefit for the prevention of colorectal cancer, which has a long natural history and a relatively low incidence.

Table 4 Major completed randomised trials (n ≥1000) investigating vitamin D supplementation and cancer (Hazard ratios (HR) and 95 % confidence intervals)

CRC, colorectal cancer; WHI, Women’s Health Initiative; RECORD, Randomised Evaluation of Calcium Or vitamin D.

A second completed randomised trial was carried out in the UK, with 2686 participants (2037 men and 649 women)⁽ Reference Trivedi, Doll and Khaw ^{152 )}. An oral supplement of 2500µg vitamin D or a matching placebo was given every 4 months for 5 years. Over the 5-year period, twenty-eight and twenty-seven cases of colon cancer were documented in the treatment and control group, respectively, with no association with vitamin D treatment (relative risk 1·02; 95 % CI 0·60, 1·74; P=0·94). This study applied a dosage of vitamin D that had a moderate effect upon the measured plasma 25(OH)D concentration (74·3 nmol/l in the treatment group v. 53·4 nmol/l in the control group, P<0·001); nevertheless, it was limited by the small sample size and the short follow-up period.

Two other studies have investigated the association of vitamin D and Ca supplement intake with cancer incidence. The Nebraska trial⁽ Reference Lappe, Travers-Gustafson and Davies ^{153 )} detected lower incidence of cancer in patients treated with vitamin D plus Ca than with placebo (P<0·03), whereas the Randomised Evaluation of Calcium Or vitamin D trial⁽ Reference Avenell, MacLennan and Jenkinson ^{154 )} found no association. However, neither study was designed to detect the association of supplement use with colorectal cancer incidence as the primary end point.

In the recently published Vitamin D/Calcium Polyp Prevention trial (Table 4)⁽ Reference Baron, Barry and Mott ^{157 )}, patients with recently diagnosed adenomas were randomly assigned 25µg of vitamin D daily or no vitamin D in a factorial design. After 3 or 5 years of treatment, participants given vitamin D had a mean net increase in serum 25(OH)D concentrations of 7·83 ng/ml, relative to participants given placebo. Overall, 43 % of the participants had one or more adenomas diagnosed during follow-up, and the adjusted risk ratio for recurrent adenoma was 0·99 (95 % CI 0·89, 1·09) with vitamin D v. no vitamin D.

Two points are worth noting for comparison of this null finding with preexisting epidemiological evidence. First, as the authors admitted, the vitamin D dose in the Polyp Prevention trial (25µg daily) was lower than the dose many experts now recommend⁽ Reference Bischoff-Ferrari, Giovannucci and Willett ^{158 ,} Reference Bischoff-Ferrari, Shao and Dawson-Hughes ^{159 )}, and it was used for a limited time⁽ Reference Baron, Barry and Mott ^{157 )}. This resulted in a net increase of 7·83 ng/ml of serum 25(OH)D, in contrast with a generally >20 ng/ml difference between the high and low quartiles or quintiles of 25(OH)D in observational studies⁽ Reference Giovannucci ^{160 )}. Thus, the moderate dose of vitamin D might not cause a change in adenoma incidence that was detectable by the power of this trial. Second, the risk of incidence for recurrent adenoma is not a direct translation of the risk for incident adenoma or colorectal cancer. For instance, in a colorectal cancer screening trial, elevated dietary fibre intake was associated with reduced risk of incident colorectal adenoma and colorectal cancer (OR 0·76 and 0·85, respectively), but not with the risk of recurrent adenoma (OR 1·08)⁽ Reference Kunzmann, Coleman and Huang ^{161 )}. Similarly, a meta-analysis has also shown different associations of higher serum 25(OH)D with incident or recurrent colorectal adenoma (OR 0·82 or 0·87 for a 20 ng/ml increase, respectively)⁽ Reference Yin, Grandi and Raum ^{124 )}. Therefore, the null finding should not be generalised to persons without a recent history of colorectal adenoma. On the basis of the clinical literature included in this review, high vitamin D status might have the greatest anti-neoplastic effects early in colorectal carcinogenesis and later in disease progression, but less so in the metastatic stage or adenoma recurrence.

Ongoing clinical trials of vitamin D intake and incidence of colorectal cancer

Several randomised controlled trials are under way to study whether vitamin D supplementation reduces the risk of cancer (Table 5)⁽ Reference Manson and Bassuk ^{162 )}. These trials are applying higher dosages of vitamin D than previous trials, and are measuring baseline and/or follow-up plasma 25(OH)D concentrations. For example, the VITamin D and OmegA-3 TriaL study collects baseline blood samples on 17 000 participants and follow-up samples on 6000⁽ Reference Pradhan and Manson ^{163 )}. In aggregate, these trials have already recruited over 53 000 participants, and the first results are expected to be available in 2015.

Table 5 Major ongoing randomised trials (n ≥1000) investigating vitamin D supplementation and cancer

VITAL, VITamin D and OmegA-3 TriaL; FIND, Finnish Vitamin D Trial; CAPS, Clinical Trial of Vitamin D₃ to Reduce Cancer Risk in Postmenopausal Women; VIDAL, Vitamin D and Longevity Trial.

Clinical trial of vitamin D intake and survival of colorectal cancer

Accumulating evidence of the involvement of vitamin D in cancer progression demands clinical trials for patients diagnosed with colorectal cancer. The study of mortality, rather than incidence, of colorectal cancer will likely require fewer subjects and shorter follow-up. To date, only one clinical trial is registered on ClinicalTrials.gov addressing this question (NCT01516216); it is recruiting 120 participants with previously untreated metastatic colorectal cancer and randomising them into two arms. Together with the standard chemotherapy with FOLFOX and bevacizumab, arm 1 gets 10 µg/d of vitamin D, whereas arm 2 gets a loading dose of 200 µg/d for 2 weeks followed by a maintenance dose of 100 µg/d. Although the sample size is small, the study does collect plasma 25(OH)D concentrations, and thus analyses of the relationships between high-dose vitamin D treatment, 25(OH)D status and prognosis are possible.