Irfete S. Fetahu

Vitamin D and the epigenome

Epigenetic mechanisms play a crucial role in regulating gene expression. The main mechanisms involve methylation of DNA and covalent modifications of histones by methylation, acetylation, phosphorylation, or ubiquitination. The complex interplay of different epigenetic mechanisms is mediated by enzymes acting in the nucleus. Modifications in DNA methylation are performed mainly by DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) proteins, while a plethora of enzymes, such as histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), and histone demethylases (HDMs) regulate covalent histone modifications. In many diseases, such as cancer, the epigenetic regulatory system is often disturbed. Vitamin D interacts with the epigenome on multiple levels. Firstly, critical genes in the vitamin D signaling system, such as those coding for vitamin D receptor (VDR) and the enzymes 25-hydroxylase (CYP2R1), 1α-hydroxylase (CYP27B1), and 24-hydroxylase (CYP24A1) have large CpG islands in their promoter regions and therefore can be silenced by DNA methylation. Secondly, VDR protein physically interacts with coactivator and corepressor proteins, which in turn are in contact with chromatin modifiers, such as HATs, HDACs, HMTs, and with chromatin remodelers. Thirdly, a number of genes encoding for chromatin modifiers and remodelers, such as HDMs of the Jumonji C (JmjC)-domain containing proteins and lysine-specific demethylase (LSD) families are primary targets of VDR and its ligands. Finally, there is evidence that certain VDR ligands have DNA demethylating effects. In this review we will discuss regulation of the vitamin D system by epigenetic modifications and how vitamin D contributes to the maintenance of the epigenome, and evaluate its impact in health and disease.

Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25‐dihydroxyvitamin D 24‐hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25‐dihydroxyvitamin D3 (1,25‐D3). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1‐specific transcription factors. We quantified CYP24A1 gene copy‐number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25‐hydroxyvitamin D 1α‐hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy‐number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25‐D3 levels. We conclude that high copy‐number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

Calcium-sensing receptor silencing in colorectal cancer is associated with promoter hypermethylation and loss of acetylation on histone 3

The calcium‐sensing receptor (CaSR) is suggested to mediate the antiproliferative effects of calcium in colon. However, in colorectal cancer (CRC) the expression of the CaSR is silenced and the underlying mechanisms leading to its loss are poorly understood. We investigated whether loss of the CaSR expression in colorectal tumors is caused by DNA hypermethylation and imbalance of transcriptionally permissive/repressive histone alterations. We observed significantly lower CaSR mRNA expression (n = 65, p < 0.001) in colorectal tumors compared with the adjacent mucosa from the same patient. Immunofluorescence staining confirmed downregulation of the CaSR protein also. The CaSR promoter was methylated to a greater extent in tumors compared with adjacent mucosa as determined by bisulfite sequencing (n = 20, p < 0.01) and by pyrosequencing (n = 45, p < 0.001), and methylation correlated inversely with mRNA expression (n = 20, ρ = −0.310, p < 0.05 and n = 45, ρ = −0.588, p < 0.001). Treatments with 5‐aza‐2′‐deoxycytidine (DAC), a DNA methyltransferase inhibitor and/or with two different histone deacetylase inhibitors, trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) restored the expression of CaSR in colon cancer cells. Restored CaSR expression in Coga1A and HT29 cells was functional. Inhibition of lysine‐specific demethylase 1 (LSD1) to prevent demethylation of mono‐ and dimethylated H3K4, increased CaSR expression only marginally. Our data show that hypermethylation of the CaSR promoter and H3K9 deacetylation, but not H3K4me2 demethylation are important factors that cause silencing of the CaSR in colorectal cancer.

miR‐135b‐ and miR‐146b‐dependent silencing of calcium‐sensing receptor expression in colorectal tumors

Studies have shown that the calcium‐sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco‐2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain‐ and loss‐of‐function studies with the top candidates: miR‐9, miR‐27a, miR‐135b, and miR‐146b. Modulation of miR‐135b or miR‐146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR‐135b and miR‐146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR‐135b and miR‐146b, and this correlated inversely with CaSR expression (miR‐135b: r = −0.684, p < 0.001 and miR‐146b: r = −0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR‐135b and miR‐146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.

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.

Abstract 5383: Overexpression of the vitamin D catabolizing enzyme CYP24A1 is caused by gene amplification and results in highly proliferative colorectal tumors.

Vitamin D insufficiency increases risk of colorectal cancer. Vitamin D is produced photochemically in the skin, thus, low sunlight exposure results in vitamin D insufficiency. Vitamin D is further processed in the liver to its storage form calcidiol (25-OH vitamin D3). Calcidiol can be activated by 1α-hydroxylation to the secosteroid hormone calcitriol (1α,25-OH vitamin D3). Although systemic levels of bioactive calcitriol are regulated by the kidneys, almost every tissue can synthesize and degrade calcitriol. Tissue calcitriol acts in an autocrine/paracrine manner and controls proliferation, apoptosis, and differentiation. In colorectal cancer, the calcidiol and calcitriol-degrading enzyme CYP24A1 is substantially overexpressed both on mRNA and protein level. High CYP24A1 levels markedly reduce the half-life of vitamin D metabolites, likely reducing the anti-tumorigenic effects of calcitriol in the tumor. The causes and consequences of this overexpression are not fully understood. Here, we investigated gene amplification of the CYP24A1 locus (20q13.2) as a possible cause of CYP24A1 overexpression and increased proliferation as a consequence thereof. Quantitative real time PCR assays showed that approximately 60% of colorectal tumors carry CYP24A1 gene amplification (n=127). This gene amplification correlated with increased mRNA expression (ρ=0.38, p In conclusion, our data suggest that CYP24A1 gene amplification results in increased mRNA expression in colorectal tumors. Further, high CYP24A1 expression correlates with increased proliferation, possibly caused by an inhibition of the anti-proliferative effects of calcitriol. Tumor specific inhibition of CYP24A1 may provide a future strategy to restore local vitamin D levels and its anti-tumorigenic activities. Citation Format: Julia Hobaus, Abhishek Aggarwal, Doris M. Hummel, Ursula Thiem, Irfete Fetahu, Ildiko Mesteri, Eniko Kallay. Overexpression of the vitamin D catabolizing enzyme CYP24A1 is caused by gene amplification and results in highly proliferative colorectal tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5383. doi:10.1158/1538-7445.AM2013-5383

Abstract 5314: Calcium mediated growth control in normal and transformed colon cells: A possible role of the calcium sensing receptor.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Calcium (Ca2+) plays an important role in regulating proliferation of colonic epithelium. Epidemiological studies suggest an inverse correlation between dietary Ca2+ intake and colorectal cancer (CRC) risk. However in CRC, transformed cells lose their sensitivity to the tumour inhibiting effects of Ca2+. We have shown that the extracellular calcium-sensing receptor (CaSR) expression is lost in CRC and therefore hypothesized that the CaSR mediates the effect of Ca2+ in regulating proliferation. Methods: We investigated the expression of the CaSR and of a cluster of genes responsible for ‘triggering off’ proliferation (CDT1, CDC6, CDC45, MCM2-7, Geminin, known as replication licensing genes) at mRNA level by qRT-PCR in CRC samples and in their respective ‘normal’ adjacent mucosa (n=57). To study the effect of Ca2+ in normal colon physiology, we fed male and female C57BL/6 mice with a diet containing high (0.9%) or low (0.1%) levels of Ca2+ and assessed the influence of dietary Ca2+ on proliferation (immunohistochemistry staining and mRNA expression) and CaSR mRNA expression. Results and Discussion: In tumours we found the expression of CaSR significantly downregulated when compared with respective adjacent mucosa (p<0.001). All genes involved in replication licensing are significantly higher expressed (p<0.001) in these samples. Our data suggest that the loss of CaSR gives the tumour cells growth advantage. In the tumour samples we found negative correlation between CaSR and licensing factors (CDC45 (p<0.05, ρ=−0.265) and MCM6 (p<0.05, ρ=−0.262) reaching significance). Surprisingly, CaSR showed positive correlation with the licensing factors in the ‘normal’ mucosa (CDT1 (p<0.05, ρ=0.269), MCM2 (p<0.003, ρ=0.382) and MCM5 (p<0.001, ρ=0.490) reaching significance). These results are similar to our observation in the mouse study, where low dietary Ca2+ enlarged the proliferative zone of the crypts with no significant change in CaSR expression. Conclusion: We conclude that the CaSR might regulate the calcium-dependent inhibition of proliferation in normal colonocytes. Upregulation of CaSR is probably a defence mechanism of the normal colon to control epithelial growth and counteract hyper-proliferative signals. This control mechanism is lost in cancer where CaSR expression is downregulated. Citation Format: Abhishek Aggarwal, Julia Hobaus, Irfete Sh. Fetahu, Ildiko Mesteri, Eniko Kallay. Calcium mediated growth control in normal and transformed colon cells: A possible role of the calcium sensing receptor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5314. doi:10.1158/1538-7445.AM2013-5314

Abstract 685: Epigenetic modulators and calcium-sensing receptor expression in colorectal cancer cells.

Introduction: Epidemiological studies suggest a role for calcium in prevention of colorectal cancer. The antiproliferative action of calcium in colon might be mediated by the calcium-sensing receptor (CaSR). The CaSR expression is lost in human colorectal cancer. We hypothesized that DNA methylation and histone modifications may be the cause of CaSR silencing in tumors. Material and methods: We analyzed CaSR mRNA and protein expression in colorectal tumors and cell lines by real time qRT-PCR and immunofluorescence. We determined the methylation pattern in the second promoter of the CaSR that contains a large CpG island by bisulfite sequencing. To induce the expression of the CaSR we treated colon tumor cell lines with 5-aza-2-deoxycytidine (5-aza-dC) and RG108, two DNA methyltransferase inhibitors, three different histone deacetylase inhibitors (HDACIs) Trichostatin A, suberoylanilide hydroxamic acid, sodium butyrate in the presence or absence of a lysine specific demethylase 1 (LSD1) inhibitor to prevent demethylation of mono- and dimethylated histone H3 lysine 4. Results: In our patient cohort we observed significantly less CaSR mRNA expression (n=57, P Conclusion: In summary, DNA methylation and histone deacetylation seem to be important factors in silencing the expression of CaSR in colorectal cancer, although additional mechanism or factors might be also involved. Citation Format: Irfete Sh. Fetahu, Julia Hobaus, Abhishek Aggarwal, Samawansha Tennakoon, Ildiko Mesteri, Sabina Baumgartner-Parzer, Enikoe Kallay. Epigenetic modulators and calcium-sensing receptor expression in colorectal cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 685. doi:10.1158/1538-7445.AM2013-685