Vitamin D and colorectal cancer: Chemopreventive perspectives through the gut microbiota and the immune system

Abstract Vitamin D and its receptor are involved in health and diseases through multiple mechanisms including the immune system and gut microbiota modulations. Gut microbiota variations have huge implications in intestinal and extra‐intestinal disorders such as colorectal cancer (CRC). This review highlights the preventive role of vitamin D in colorectal tumorigenesis through the effects on the immune system and gut microbiota modulation. The different associations between vitamin D, gut microbial homeostasis, immune system, and CRC, are dissected. Vitamin D is supposed to exert several chemopreventive effects on CRC including direct antineoplastic mechanisms, the effects on the immune system, and gut microbiota modulation. Large clinical studies with a randomized design, are required to confirm the role of vitamin D in CRC, confirming its key role in the complex interplay between the gut immune system and microbiota.


| INTRODUCTION
Vitamin D is a liposoluble vitamin available in two forms, D2 (ergocalciferol) and D3 (cholecalciferol). In humans, it is biosynthesized from a derivative of the cholesterol in the skin under the influence of solar ultraviolet (UV)-B radiation. 1,2 Vitamin D3 is also present in dietary sources including animal foods such as fatty fish, liver, milk, eggs, and dietary supplements. Both forms of vitamin D are converted to 25-hydroxyvitamin D in the liver; then, 25-hydroxyvitamin D passes through the blood to the kidneys, where it is further modified to 1,25-dihydroxyvitamin D (1,25-D), or calcitriol, the active form of vitamin D in the body. 3 As reported in research studies, vitamin D status is assessed by different variables such as serum vitamin D, tissue vitamin D, and vitamin D gene receptor (VDR). 4 Serum 25-hydroxyvitamin D level is widely recognized as a biomarker in determining the effect of vitamin D status in the clinical setting. 5,6 Vitamin D is first known for its traditional role in bone metabolism, but it has other important biological functions including modulation of the immune system and anti-carcinogenic effects. 7 Recent meta-analyses of casecontrol studies, nested case-control studies, and cohort studies assessed the association between serum vitamin D and the risk of various types of cancer. [8][9][10] Moreover, vitamin D and its VDR are known to participate in gut microbiota modulations in health and diseases. 11 The gut microbiota is an extremely rich and diverse ecosystem inside the gastrointestinal tract, containing more than 10 14 microorganisms. 12 Gut microbiota variations have huge implications in intestinal and extra-intestinal disorders such as metabolic diseases and colorectal cancer (CRC). 13 In this context, this review aims to highlight the role of vitamin D in colorectal tumorigenesis through gut microbiota modulation and the effects on the immune system. We dissect the different associations between vitamin D, gut microbial homeostasis, immune system, and CRC, to depict the potential chemopreventive role of vitamin D in CRC. 13,14 2 | GUT MICROBIOTA, IMMUNE SYSTEM, AND CRC The human microbiota is a complex ecosystem inhabiting the human body and harboring more than 100 trillion microorganisms, most of which colonize the gastrointestinal tract, and specifically the colon, where bacterial density ranges between 10 11 and 10 12 cells per milliliter. Gut microbiota shows several and yet poor-known functions such as nutrient metabolism, maintenance of the gut mucosal barrier, and immunomodulation. Alterations of this ecosystem are nowadays recognized in several diseases, among which cancer, and in particular colon cancer. 13,14 Compositional gut bacteria variations between fecal samples of CRC patients and healthy volunteers have been shown in numerous studies. 15 A recent metaanalysis of fecal metagenomes reveals global microbial signatures that are specific for CRC. 16 In particular, in CRC patients, the abundance of butyrate producers decreases while opportunistic pathogens increase. This constitutes a major structural imbalance of gut microbiota. Butyrate is a short-chain fatty acid (SCFA) produced by the bacterial fermentation of dietary oligosaccharides and it has a key role in the gut homeostasis; it stimulates mature colonocytes and inhibits malignant cells; moreover, it lowers luminal pH and maintains a low O2-tension, favorable to anaerobic commensal bacteria, reducing the risk of an expansion of the pathogen Enterobacteriaceae. In the lamina propria, butyrate induces differentiation of Foxp3+ T regulatory (reg) cells and the expression of interleukin (IL)-10, an anti-inflammatory cytokine maintaining immune tolerance and immune-homeostasis. 17,18 Thus, butyrateproducing bacteria contribute to gut barrier function, reducing gut inflammation. 19 In turn, a reduction in butyrate-producers may induce an imbalance of gut microbiota, endotoxin, and bacterial products translocation, activating an immune response in the colonic mucosa. 20 More in-depth, the gut microbiota of the CRC patients is characterized by enrichment in Bacteroides fragilis, Enterococcus, Escherichia/ Shigella, Klebsiella, Streptococcus, and Peptostreptococcus and a decrease in Roseburia and other butyrate-producing bacteria of the family Lachnospiraceae, compared with healthy subjects. 15 On the other hand, the gut microbiota of the healthy subjects has higher levels of Bacteroides vulgatus and Bacteroides uniformis compared with CRC subjects. Also, Shen et al. 21 demonstrated a higher abundance of Proteobacteria and a lower abundance of Bacteroidetes, in colorectal adenomas cases compared to the normal colonic mucosa, suggesting a peculiar inflammatory microbial milieu in CRC precursors. Interestingly, the gut microbiota of CRC patients is enriched in Fusobacterium spp, and in particular Fusobacterium nucleatum, a periodontal pathogen, whose role in CRC has been extensively debated. 14,22 In detail, preclinical studies reported a heavy enrichment of CRC tissues in FN, which is associated with genetic and epigenetic alterations such as CpG island methylator phenotype (CIMP) status, microsatellite instability (MSI), and mutations in BRAF, KRAS, TP53, TGF-β, CHD7, and CHD8. 23,24 From a molecular point of view, F. nucleatum may express its oncogenic potential through the adhesin FadA, activating the E-cadherin/β-catenin and Wnt (7a, 7b, 9a) oncogenic pathways, and promoting the synthesis of proinflammatory cytokines such as IL-6, IL-8, and IL-18 as well as TNF-α and reactive oxygen species (ROS) with subsequently DNA damage. 25,26 Also, F. nucleatum colonizes the tumor environment through the lectin Fap2, binding the host polysaccharide Gal-GalNAc, which is overexpressed in colorectal adenocarcinoma and metastases. 27 Fap2 mediates the impairment of host-antitumor immunity binding and activating TIGIT, an immunoregulatory signaling receptor suppressing T cells and NK cells activation, thus reducing the control of CRC cells by the immune system. 28,29 Fap2 also induces tumor proliferation through lymphocyte apoptosis. 30 All the overmentioned findings reveal that alterations in CRC microbiota may contribute to CRC development. Wong et al. went further by giving germ-free C57BL/6 mice with stool from five patients with CRC or five controls. They collected intestinal tissues and performed histology analysis and 16S ribosomal RNA gene sequencing analysis of feces from mice, showing that gavages of fecal samples from patients with CRC promoted intestinal carcinogenesis. 31 According to several animal studies, antibiotics eliminate carcinogenic bacteria in mice models. 32,33 Hence, antibiotic administration could theoretically protect against tumor proliferation. Nevertheless, in humans, antibiotic use is closely linked with an increased risk of CRC incidence. 34, 35 We can hypothesize that in more complex ecosystems, antibiotic treatment exacerbates dysbiosis, by reducing gut microbiota diversity and richness of specific species leading to an increased risk of pathogenic bacterial colonization. 36

| VITAMIN D, IMMUNE SYSTEM, AND THE GUT MICROBIOTA
Given the long-term exposure of the colonic mucosa to external agents and the gut microbiota, the elevated load in pathogen-associated molecular patterns (PAMPs) continuously elicits the immune response. The gut (and systemic) immune homeostasis is thus preserved by regulatory factors such as microbial richness, antiinflammatory cytokines, and immune regulatory cells. 37 In this sense, 1,25-D, binding to its receptor, could represent a regulatory biofactor, affecting the immune system both directly and through gut microbiota modulation. VDR belongs to a superfamily of nuclear receptors that transduce hormonal signals from the immediate environment and participate in the activation of several genes. 38 The VDR is expressed in at least 30 different target tissues including bone, kidney, blood, breast, prostate, gut, activated B-and T-lymphocytes, monocytes, and keratinocytes; this widespread expression may largely explain the pleiotropic effects of vitamin D. 38 In the colonic mucosa, it has been assessed that VDR stabilizes cell tight junctions between the intestinal epithelial cells inducing the expression of cell junction proteins such as ZO-1, E-cadherin, and occludin. 39,40 Moreover, nuclear VDR is directly involved in the regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, a pathway essential for inflammatory response 41 ; indeed, VDR absence leads to reduced levels of I-kappa-B-alpha protein, the endogenous inhibitor of NF-κB. 42 Vitamin D has a key role in both the innate and the adaptive immune systems. 43 1,25-D can stimulate the expression of β-defensins and cathelicidins, 44,45 the main classes of antimicrobial peptides (AMPs) 46 -key components of innate host defense 47 -by interacting with VDR. In addition, 1,25-D enhances immune homeostasis decreasing Th1/Th17 CD4+ T cells activity, enhancing Treg activity, downregulating T cell-driven IgG production, and inhibiting dendritic cell differentiation 48 in the intestinal lamina propria. VDR gene acts as a key host modulator of the gut microbiome, even if it is not expressed by prokaryotes cells. 49 Hence, the effects of vitamin D and its receptor on gut microbiota are considered as indirect through the host immune system. 50 A recent genome-wide host-microbiota association study 51 identified the VDR gene among the most significant loci that were associated with overall gut microbial variation and abundance of individual bacteria, as well as functions of gastrointestinal and immune-related tissues and cells. In VDR knock-out (KO) (VdrÀ/À) mice, feces are depleted in Lactobacillus and enriched in Clostridium and Bacteroides; Alistipes and Odoribacter abundances are significantly decreased whereas Eggerthella quantity is increased. Moreover, the intestinal-specific deletion of VDR could be associated with a decrease in butyrateproducing bacteria. In mice, vitamin D deficiency (combined with a high-fat diet) may cause dysbiosis, increased gut permeability, mucosal collapse, and systemic inflammation leading to insulin resistance and hepatic steatosis. 52 In humans, a recent randomized controlled trial enrolling 26 healthy overweight adults evaluated the effects of vitamin D supplementation on the fecal microbiota of two groups (treatment vs. placebo). 53 The vitamin D group had a higher abundance of genus Lachnospira, and a lower abundance of genus Blautia than the placebo group. Moreover, individuals with 25-hydroxyvitamin D > 75 nmol/L had a higher abundance of genus Coprococcus and lower abundance of genus Ruminococcus compared to those with 25-hydroxyvitamin D < 50 nmol/L, suggesting potential beneficial effects of vitamin D supplementation. 53 A recent study of 80 otherwise healthy vitamin D-deficient women 54 measured serum 25-hydroxyvitamin D levels in the blood before and after vitamin D supplementation. Vitamin D supplementation significantly increased gut microbial diversity. Specifically, the Bacteroidetes to Firmicutes ratio increased. At the genus level, significant variations in Bacteroides and Prevotella, along with the abundance of the health-promoting probiotic taxa Akkermansia and Bifidobacterium, indicated a beneficial variation in enterotypes following supplementation. Interestingly, comparing samples, researchers found more pronounced changes in abundance of major phyla in those of responders. A recent cross-sectional study conducted in 567 North Americans found that men with higher levels of 1,25-D had greater α-diversity (the diversity of microbial species within an individual) and β-diversity (the difference in microbial composition between two samples) than counterparts with lower levels, even after adjusting for other covariates of microbial diversity such as age, geographical origin, race, PPI, and antibiotic use. 55 Another systematic review of animal and human studies confirmed a significant association between vitamin D status and β-diversity. 56 The beneficial role of vitamin D on gut microbial modulation is also confirmed by the evidence that men with elevated 1,25-D plasmatic levels are more likely to possess butyrateproducing bacteria. 55

| CHEMOPREVENTIVE EFFECTS OF VITAMIN D AND THE COMPLEX NETWORK OF GUT MICROBIOTA AND IMMUNE SYSTEM
Vitamin D is supposed to exert several chemopreventive effects on CRC (Figure 1).

| Direct antineoplastic effects
Vitamin D provides a direct preventive action in CRC onset and progression: its binding to VDR induces several intracellular and intranuclear pathways linked to growth, differentiation, and apoptosis. Transcriptomic studies in CRC cell lines identified hundreds of genes involved in DNA synthesis, apoptosis, and intracellular signals activated by VDR. 57,58 In detail, the antiproliferative effects of Vitamin D in CRC includes the VDR binding to β-catenin (preventing the activation of TCF7L2 complexes in the nucleus), the upregulation of the protein CDH1 (E-cadherin), a transmembrane protein whose intracellular tail attracts β-catenin, and the inhibition of the proto-oncogene MYC (upregulated by the WNT/βcatenin pathway). Moreover, 1,25-D regulates and inhibits the activity of the epidermal growth factor receptor (EGFR) gene and the signaling activity of insulin growth factor (IGF) 2 protein. 58,59 Vitamin D has also a regulatory epigenetic action, controlling the histone demethylases KDM6B and activating the cystatin D (CST5) gene. Cystatin D, in turn, inhibits several cysteine proteases of the cathepsin family, reducing migration and growth in CRC cells. 60 Another indirect activity of 1,25-D is the regulation of miR-22 -a short RNA molecule targeting histone deacetylase 4 (HDAC4) and myc binding protein (MYCBP)-enhancing its antiproliferative and anti-migratory effect. 61 Vitamin D activity could also mediate the potential chemopreventive effect of estrogen replacement therapy in postmenopausal women. VDR is a known estrogen-responsive gene: estradiol administration results in significant changes of estrogen-responsive genes and seems to increase VDR signaling in the colorectal epithelium of postmenopausal women. 62 A recent study confirmed that vitamin D intake could have a clinical impact in advanced or metastatic CRC patients undergoing chemotherapy, in a median follow-up of 2 years. 18 During the study, half of the patients were given a daily dose of 400 IU of vitamin D. The other group first took two 4000 IU capsules per day for 2 weeks, then one 4000 IU capsule daily. In both groups, the course of the disease slowed significantly, with greater progression-free survival in the arm receiving the highest supplementation. Moreover, in 2 years, CRC was 36% less likely to spread or be fatal in subjects who consumed more vitamin D. 63 An updated meta-analysis of randomized controlled trials confirmed that vitamin D supplementation significantly reduced cancer-related mortality even though it did not reduce total cancer incidence. 63 F I G U R E 1 The chemopreventive effects of vitamin D: vitamin D could affect CRC both directly and through the immune system. Gut microbiota and immune system are also interconnected, both of them influencing CRC development and progression 4.2 | Effects through the gut microbiota and immune system Gut microbiota is a key driver of CRC development and progression: invasive pathobionts (e.g., Escherichia coli) may contribute to cancer onset counteracting autophagy and antimicrobial responses in epithelial colonic cells, 64 and CRC patients have a pro-inflammatory gut microbial signature, as stated above. Microbiota is, in turn, modulated by several nutrients such as the micronutrient vitamin D, through the immune system. Gut microbiota and immune system are strictly interconnected. The gut microbiota supports the development and response of the immune system, which in turn, regulates intestinal eubiosis balancing tolerance and immunity on the gut microbiota. Vitamin D plays a pivotal role in this complex network, since VDR is expressed (and activated under-stimulation) in several immune cell lineages including CD4 and CD8 T cells, B cells, neutrophils, macrophages, and dendritic cells. 65 In a human interventional study, vitamin D supplementation (a weekly dose of 980 IU/kg body weight of vitamin D3 for 8 weeks) significantly changed gut microbiome composition, reducing opportunistic pathogens and increasing bacterial richness. Specifically, the class of Gammaproteobacteria (including Pseudomonas spp and Escherichia/Shigella spp), significantly decreased. The effects on gut bacteria are supposed to be mediated by mucosal CD8+ T cells. These immune cells have a high expression of VDR: under vitamin D supplementation a switch from naïve to effector form occurs 66 ; CD8+ T effector cells reduce the inflammatory environment through the calcitriol synthesis and allowing beneficial bacteria (such as Bacteroidetes) to outweigh opportunistic pathogens. 67 These gut microbial modulations could counteract the gut barrier dysfunctions and dysbiosis seen during CRC development and progression. Conversely, vitamin D deficiency exacerbates gut microbiota dysfunctions triggered by CRC such as dysbiosis, decreases bacterial butyrate producers, and increases chronic inflammation leading to immunosuppression. 68 A recent case-control study analyzed fecal microbiota composition of CRC and controls subjects evaluating the role of microbiome and diet (including vitamin D) in CRC etiology and regulation of inflammation markers. 69 Researchers found an inverse association between CRC risk and high consumption of fatty fish (rich in omega-3 polyunsaturated fatty acids and vitamin D). Conversely, a diet poor in fatty fish and rich in carbohydrates was significantly associated with CRC risk. The gut microbiome (Bifidobacterium/ Escherichia genera ratio) was found to significantly mediate the effect of diet on CRC risk. Moreover, the gut microbial profile of CRC patients was enriched in pro-inflammatory species such as Parvimonas micra, F. nucleatum, and B. fragilis species whereas controls specimens were associated with a higher abundance of Bacteroidetes and Bifidobacterium species. The link between gut inflammation and cancer has been well established. 70 The risk of CRC increases with the duration of colitis in patients affected by inflammatory bowel diseases (IBD), correlating with the grade of inflammation and extend of the disease. 71 Interestingly, a vitamin D deficit has been also related to IBDs onset, making this link ever closer. 72 A human study correlated seasonal changes of circulating 25-hydroxyvitamin D levels with the gut microbiome composition in IBD patients, observing a reduction in the abundance of bacterial genera typical for inflammation such as Eggerthella lenta, Fusobacterium spp, Bacteroides spp, Collinsella aerofaciens, and Helicobacter spp in summer/autumn period, when light exposure (and 1,25-D synthesis) is higher. 73 The immune cells are directly involved in the pathogenesis of both IBD and colitis-associated CRC. 74 In colon cancer cells, macrophage-derived IL-1β induces Wnt signaling. 75 In this context, the 1,25-D-VDR binding inhibits the Wnt/β-catenin activity in macrophages making them unable to activate Wnt signaling in colon cancer cells. 57,63 Moreover, chronic inflammation increases the turnover of epithelial cells and the release of ROS contributing to the development of low, high-grade, and finally carcinoma lesions through the stepwise mutation of key genes in carcinogenesis such as DNA-repair genes, p53, and KRAS. 76 A recent mice model of inflammation-associated colon cancer demonstrated that increased dietary vitamin D supplementation decreases inflammation, dysplasia, and tumor incidence, and is associated with decreased MAPK and NF-κB expression during the acute inflammatory stage of disease. 77 Cancer cells often contain abundant p53-upregulated modulators of apoptosis (PUMA) interacting with VDR genes. 78 VDR signaling is involved in the prevention of apoptosis by downregulating PUMA and blocking NF-κB thus preventing the spread of damaged cells. 79 In this context, the activity of VDR is also under the control of SCFAs, especially butyrate. 80 This makes even closer the triple link between gut microbiota, Vitamin D, and the immune system in CRC. A mouse model study of colitis-associated colon cancer 81 went further, investigating the anti-inflammatory, anti-proliferative, and chemopreventive effects of vitamin D analog and assessing a down-regulation of growth-promoting c-Myc gene, pro-inflammatory COX-2, and inhibition of ERK activation pathway in the premalignant phase. As regards CRC and the immune intestinal homeostasis, 1,25-D promotes both antimicrobial activity and a tolerogenic response, as seen in experimental models of colitis. 50 In detail, 1,25-D inhibits in vitro and in vivo the interferon (IFN)-γ and IL-17 production from T cells, 82 induces the anti-inflammatory cytokine IL-10 from Foxp3+ Treg cells 83 and the antimicrobial IL-22 from type 3 innate lymphoid (ILC3) cells. 84 All these findings and statements allow us to attribute to vitamin D a preventive and anti-proliferative role in CRC carcinogenesis (Figure 2), even if clinical studies with a prospective study design are still lacking in this setting.

| CONCLUSIONS
In conclusion, vitamin D has been associated with CRC prevention and better clinical outcomes. 85 Vitamin D acts as a regulatory prohormone with genetic and epigenetic targets, producing pleiotropic effects by the binding to its VDR, almost ubiquitous in the human body. In colonic mucosa, vitamin D may exert its action through an immune system modulation and microbial composition shaping, which, in turn, influences mucosal immunity. These effects influence the most known mechanisms of CRC development. To date, only preclinical and few clinical studies investigated the role of vitamin D as a chemopreventive agent in CRC. Further clinical studies with a large sample size and randomized design, are required to confirm the close interplay between vitamin D, microbiota, inflammation, and CRC, dissecting the role of vitamin D as a potential agent in CRC prevention and therapy.

ACKNOWLEDGMENTS
Open Access Funding provided by Universita Cattolica del Sacro Cuore within the CRUI-CARE Agreement.

CONFLICT OF INTEREST
The authors declare no conflict of interest. F I G U R E 2 The impact of vitamin D intake in CRC process through gut microbiota modulation DATA AVAILABILITY STATEMENT Data sharing is not applicable to this article as no new data were created or analyzed in this study.