Teresa Manhardt

The Vitamin D endocrine system of the gut—Its possible role in colorectal cancer prevention

While Vitamin D insufficiency in the US and European population is rising, epidemiological studies suggest an inverse correlation between low serum levels of 25-hydroxyvitamin D(3) (25-OH-D(3)) and colorectal cancer incidence. The antimitotic, prodifferentiating and proapoptotic active metabolite 1alpha,25-dihydroxyvitamin D(3) (1,25-(OH)(2)-D(3)) is synthesized also by colonocytes, since these possess Vitamin D synthesizing (CYP27B1) and catabolic (CYP24) hydroxylases similar to the kidney. Early during colon tumor progression, expression of CYP27B1 and of the Vitamin D receptor increases, suggesting an autocrine/paracrine growth control in colon tissue as a physiological restriction against tumor progression. However, in human adenocarcinomas expression of the catabolic CYP24 is also enhanced when compared with adjacent normal mucosa. Therefore, to maintain colonic accumulation of 1,25-(OH)(2)-D(3) its catabolism needs to be restricted. Our studies in mice show that low nutritional calcium causes hyperproliferation of colon crypts and significant elevation of CYP24 expression, which can be completely abrogated by soy feeding. We suggest that phytoestrogens in soy, known to be estrogen receptor modulators, are responsible for decreased CYP24 expression. These results and our observation that 17beta-estradiol can elevate CYP27B1 expression in rectal tissue of postmenopausal women, may underlie the observed protective effect of estrogens against colorectal cancer in females.

Regulation of the calcium-sensing receptor expression by 1,25-dihydroxyvitamin D3, interleukin-6, and tumor necrosis factor alpha in colon cancer cells.

Highlights • 1,25 Dihydroxyvitamin D3 induces the expression of CaSR in Caco2/AQ and Coga1A cells.• TNFα is the main driver of CaSR expression in Coga1A.• In Caco2/AQ cells 1,25 dihydroxyvitamin D3 counteracts the action of TNFα and IL-6.

Role of proinflammatory cytokines on expression of vitamin D metabolism and target genes in colon cancer cells

Highlights • TNFα decreases CYP27B1 mRNA expression.• TNFα inhibits transcription of the calcium ion channel TRPV6.• 1,25-D3 inhibits TNFα-induced upregulation of COX-2.

Epigenetic regulation of the 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) in colon cancer cells

Highlights ► Basal and calcitriol-induced expression of CYP24A1 is cell line dependent. ► Histone deacetylase and methyltransferase inhibitors increase CYP24A1 expression. ► Cell line specific drug response does not correlate with promoter methylation. ► Drug treatment may activate genes upstream of CYP24A1 and cause indirect induction.

A 24-Phenylsulfone Analog of Vitamin D Inhibits 1α,25-Dihydroxyvitamin D3 Degradation in Vitamin D Metabolism-Competent Cells

The antimitotic, prodifferentiating, and proapoptotic steroid hormone, 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3], at supraphysiological levels has potential for tumor therapy. However, epithelial cells from tumor-prone organs such as colon, prostate, and breast express not only the vitamin D receptor, but also vitamin D hydroxylases. In contrast to normal cells, malignant cells have high basal levels of the hydroxylase 25-hydroxyvitamin D3-24-hydroxylase (CYP24) and, in addition, have the potential to induce CYP24 in response to 1α,25-(OH)2D3. Because 24-hydroxylation by CYP24 would rapidly degrade the steroid hormone in the course of therapy, the enzyme activity in tumor cells should be inhibited. We demonstrate that a 24-phenylsulfone analog of 1α,25-(OH)2D3, KRC-24SO2Ph-1 (S-4a), rapidly and potently inhibits 24-hydroxylase activity in human tumor cells derived from colon, prostate, and mammary gland. Although enzymatic inhibition is a consequence of direct interaction, S-4a as a vitamin D analog apparently binds to the vitamin D receptor and induces CYP24 mRNA, which, however, is not translated into increased enzymatic activity. 25-Hydroxyvitamin D3-1α-hydroxylase expression is not affected at all by S-4a. When both 1α,25-(OH)2D3 and S-4a are added to the cell culture, transcription of CYP24 is increased, possibly because of an increase in the half-life of the hormone. The colon cell line COGA-13 has very high levels of CYP24 and is, therefore, resistant to the action of vitamin D. Yet, S-4a imparts antimitotic activity to 1α,25-(OH)2D3 and may therefore constitute a therapeutic to stimulate the antiproliferative potential of vitamin D-based antitumor activity.

Relative Expression of Vitamin D Hydroxylases, CYP27B1 and CYP24A1, and of Cyclooxygenase-2 and Heterogeneity of Human Colorectal Cancer in Relation to Age, Gender, Tumor Location, and Malignancy: Results from Factor and Cluster Analysis

Previous studies on the significance of vitamin D insufficiency and chronic inflammation in colorectal cancer development clearly indicated that maintenance of cellular homeostasis in the large intestinal epithelium requires balanced interaction of 1,25-(OH)2D3 and prostaglandin cellular signaling networks. The present study addresses the question how colorectal cancer pathogenesis depends on alterations of activities of vitamin D hydroxylases, i.e., CYP27B1-encoded 25-hydroxyvitamin D-1α-hydroxylase and CYP24A1-encoded 25-hydroxyvitamin D-24-hydroxylase, and inflammation-induced cyclooxygenase-2 (COX-2). Data from 105 cancer patients on CYP27B1, VDR, CYP24A1, and COX-2 mRNA expression in relation to tumor grade, anatomical location, gender and age were fit into a multivariate model of exploratory factor analysis. Nearly identical results were obtained by the principal factor and the maximum likelihood method, and these were confirmed by hierarchical cluster analysis: Within the eight mutually dependent variables studied four independent constellations were found that identify different features of colorectal cancer pathogenesis: (i) Escape of COX-2 activity from restraints by the CYP27B1/VDR system can initiate cancer growth anywhere in the colorectum regardless of age and gender; (ii) variations in COX-2 expression are mainly responsible for differences in cancer incidence in relation to tumor location; (iii) advancing age has a strong gender-specific influence on cancer incidence; (iv) progression from well differentiated to undifferentiated cancer is solely associated with a rise in CYP24A1 expression.

Expression profiling of colorectal cancer cells reveals inhibition of DNA replication licensing by extracellular calcium.

Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca2+]o) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24 h with 2 mM [Ca2+]o identified significant changes in expression of 1571 probe sets (ANOVA, p < 10− 5). The main biological processes affected by [Ca2+]o were DNA replication, cell division, and regulation of transcription. All factors involved in DNA replication-licensing were significantly downregulated by [Ca2+]o. Furthermore, we show that the calcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca2+]o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor.

Abstract 1136: Gene amplification and epigenetic modifications: Mechanisms behind unbalanced expression of the proto-oncogene CYP24A1 in colorectal tumors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL There is an inverse association between colorectal cancer and pre-diagnostic Vitamin D levels (25-OHD3), underlining the importance of Vitamin D in the prevention of colon cancer. 1α,25(OH)2D3 (1,25D3) is the most active form of Vitamin D and exhibits anti-proliferative, pro-differentiating, anti-angiogenic functions. Both, calcitriol and the calcitriol precursor 25(OH)D3 can be catabolized by the 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1). CYP24A1 is expressed at very low basal levels in the colon but is strongly induced by 1,25D3. This induction is due to a complex formation of the 1,25D3 and the Vitamin D Receptor and binding of this complex to Vitamin D Responsive Elements (VDRE) located within the CYP24A1 promoter region. This feedback loop shortens the biological half-life of 1,25D3. A high expression of the Vitamin D catabolizing enzyme CYP24A1 implies faster degradation of 1,25D3, thereby restricting its anti-tumorigenic functions. A genomic copy number gain and an overexpression of CYP24A1 on mRNA level have been observed in different malignancies, such as colon and breast. Our aim is to test whether the observed overexpression of CYP24A1 is due to gene amplification and/or epigenetic modulations. In human colon cancer cell lines we found an increase in genomic copy number of CYP24A1 compared to human placenta using real time-PCR. In human colorectal cancer patients (n=89) we observed a gain of CYP24A1 copy number in approximately 40% of the tumors compared to their respective adjacent mucosa indicating a role of CYP24A1 gene amplification in tumorigenesis. Further, on mRNA level we could show a trend toward increased CYP24A1 expression in tumors compared with their adjacent mucosa using reverse transcription qRT-PCR. However, the effect of copy number gain can be impaired by epigenetic regulation. Aberrant DNA methylation is a characteristic of various tumors. The promoter region of the CYP24A1 gene contains several CpG islands. Therefore we have examined if there are any differences in the methylation status of the CYP24A1 promoter between the tumor tissue and the adjacent “normal” mucosa. First we have compared the methylation status in 4 cell lines. Bisulfit-sequencing of the genomic DNA of the highly differentiated colon cancer cell line Caco2Aq showed heavy methylation of the CYP24A1 promoter including the VDREs located within this promoter area. In comparison, the colon cell lines Coga1A, LS174, and Coga13 (derived from less differentiated tumors) appeared largely unmethylated. Using methylation specific PCR we found that human colorectal tumors are often less methylated than their adjacent mucosa. In conclusion, our data indicate that both gene amplification and DNA-hypomethylation could be mechanisms leading to the overexpression of CYP24A1 in colorectal tumor patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1136. doi:10.1158/1538-7445.AM2011-1136