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Early childhood tumours are thought to arise from transformed embryonic cells, which often carry large copy number variants (CNVs). However, it remains unclear how CNVs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ human embryonic stem cell (hESC) differentiation to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNVs impair the specification of hESC-derived trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. Overexpression ofMYCN, whose amplification co-occurs with CNVs in NB, exacerbates the differentiation block, and enables tumourigenic cell proliferation. We find links between disrupted cell statesin vitroand tumour cells and connect these states with stepwise disruption of developmental transcription factor networks. Together, our results chart a possible route to NB and provide a mechanistic framework for the CNV-driven initiation of embryonal tumours.

Neuroblastoma (NB) is a pediatric cancer of the sympathetic nervous system and one of the most common solid tumors in infancy. Amplification of MYCN, copy number alterations, numerical and segmental chromosomal aberrations, mutations, and rearrangements on a handful of genes, such as ALK, ATRX, TP53, RAS/MAPK pathway genes, and TERT, are attributed as underlying causes that give rise to NB. However, the heterogeneous nature of the disease—along with the relative paucity of recurrent somatic mutations—reinforces the need to understand the interplay of genetic factors and epigenetic alterations in the context of NB. Epigenetic mechanisms tightly control gene expression, embryogenesis, imprinting, chromosomal stability, and tumorigenesis, thereby playing a pivotal role in physio- and pathological settings. The main epigenetic alterations include aberrant DNA methylation, disrupted patterns of posttranslational histone modifications, alterations in chromatin composition and/or architecture, and aberrant expression of non-coding RNAs. DNA methylation and demethylation are mediated by DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) proteins, respectively, while histone modifications are coordinated by histone acetyltransferases and deacetylases (HATs, HDACs), and histone methyltransferases and demethylases (HMTs, HDMs). This article focuses predominately on the crosstalk between the epigenome and NB, and the implications it has on disease diagnosis and treatment.

Early childhood malignancies are driven by sparse genetic aberrations in oncogenes that often co-occur with large copy number variants (CNVs). The combination of these mutations is thought to transform developmentally pliant embryonic cells to initiate tumorigenesis. However, the mechanistic interactions between CNVs, oncogenes, and differentiation have not been systematically studied due to several obstacles: (i) CNVs cannot be engineered efficiently yet; (ii) transient embryonic progenitors are absent in full-grown tumors; and (iii) inter-species differences in lineage specification limit the applicability of animal models. To overcome these challenges, we used isogenic human embryonic stem cell (hESC) lines carrying gains of chromosome 17q/1q, which are prevalent in the embryonal tumor neuroblastoma (NB). We differentiated these cells toward trunk neural crest (NC) and their sympathoadrenal derivatives, the putative cells-of-origin of NB, and performed single-cell RNA sequencing and cell-biological assays at key differentiation stages. We found that CNVs impaired the specification of sympathoadrenal cell types and instead potentiated early Schwann-cell-precursor-like phenotypes. Additional overexpression of the oncogene MYCN (which is frequently amplified together with CNVs in high-risk NB tumors) exacerbated these differentiation defects, enabled tumourigenic cell proliferation, and generated cell states in vitro that transcriptionally resembled NB tumor cells. Finally, using epigenome analysis, we connected these states to a stepwise disruption of gene-regulatory networks centered on developmental transcription factors. Together, our results chart a mechanistic route to NB tumorigenesis and provide a general framework for the CNV-driven initiation of embryonal tumors, in which CNVs ‘prime’ embryonic cells for oncogenic transformation. The tumor-like cells in our model may serve as proxies to experimentally test therapeutic interventions during tumorigenesis. Citation Format: Ingrid M. Saldana-Guerrero, Luis F. Montano-Gutierrez, Christoph Hafemeister, Dylan Stavish, Lisa E. Shaw, Irfete S. Fetahu, Andrea Wenninger-Weinzierl, Caterina Sturtzel, Celine Souilhol, Sophia Tarelli, Mohamed R. Shoeb, Marie Bernkopf, Polyxeni Bozatzi, Maria Guarini, Eva Bozsaky, Michelle C. Buri, Eva M. Putz, Peter W. Andrews, Ivana Barbaric, Helen E. Bryant, Martin Distel, Sabine Taschner-Mandl, Matthias Farlik, Anestis Tsakiridis, Florian Halbritter. A stem cell model dissects detrimental effects of neuroblastoma-linked chromosomal aberrations on cell differentiation during neural crest development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3542.

The host immune response is a fundamental mechanism for attenuating cancer progression. Here we report a role for the DNA demethylase and tumor suppressor TET2 in host anti-tumor immunity. Deletion of Tet2 in mice elevates IL-6 levels upon tumor challenge. Elevated IL-6 stimulates immunosuppressive granulocytic myeloid-derived suppressor cells (G-MDSCs), which in turn reduce CD8+ T cells upon tumor challenge. Consequently, systematic knockout of Tet2 in mice leads to accelerated syngeneic tumor growth, which is constrained by anti-PD-1 blockade. Removal of G-MDSCs by the anti-mouse Ly6g antibodies restores CD8+ T-cell numbers in Tet2-/- mice and reboots their anti-tumor activity. Importantly, anti-IL-6 antibody treatment blocks the expansion of G-MDSCs and inhibits syngeneic tumor growth. Collectively, these findings reveal a TET2-mediated IL-6/G-MDSCs/CD8+ T-cell immune response cascade that safeguards host adaptive anti-tumor immunity, offering a cell non-autonomous mechanism of TET2 for tumor suppression.

Background Alzheimer’s disease (AD) is the most common type of dementia, and patients with advanced AD frequently lose the ability to identify family members. The fusiform gyrus (FUS) of the brain is critical in facial recognition. However, AD etiology in the FUS of AD patients is poorly understood. New analytical strategies are needed to reveal the genetic and epigenetic basis of AD in FUS. Results A complex of new analytical paradigms that integrates an array of transcriptomes and methylomes of normal controls, AD patients, and “AD-in-dish” models were used to identify genetic and epigenetic signatures of AD in FUS. Here we identified changes in gene expression that are specific to the FUS in brains of AD patients. These changes are closely linked to key genes in the AD network. Profiling of the methylome (5mC/5hmC/5fC/5caC) at base resolution identified 5 signature genes (COL2A1, CAPN3, COL14A1, STAT5A, SPOCK3) that exhibit perturbed expression, specifically in the FUS and display altered DNA methylome profiles that are common across AD-associated brain regions. Moreover, we demonstrate proof-of-principle that AD-associated methylome changes in these genes effectively predict the disease prognosis with enhanced sensitivity compared to presently used clinical criteria. Conclusions This study identified a set of previously unexplored FUS-specific AD genes and their epigenetic characteristics, which may provide new insights into the molecular pathology of AD, attributing the genetic and epigenetic basis of FUS to AD development.

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6 × 10

Additional file 1: Figures S1-S6. Supplementary_figures_merge.pdf.

The discovery of AD epigenetic signatures lays a molecular basis for new methods to detect early AD using patient blood cells., Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is the most common untreatable form of dementia. Identifying molecular biomarkers that allow early detection remains a key challenge in the diagnosis, treatment, and prognostic evaluation of the disease. Here, we report a novel experimental and analytical model characterizing epigenetic alterations during AD onset and progression. We generated the first integrated base-resolution genome-wide maps of the distribution of 5-methyl-cytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), and 5-formyl/carboxy-cytosine (5fC/caC) in normal and AD neurons. We identified 27 AD region–specific and 39 CpG site–specific epigenetic signatures that were independently validated across our familial and sporadic AD models, and in an independent clinical cohort. Thus, our work establishes a new model and strategy to study the epigenetic alterations underlying AD onset and progression and provides a set of highly reliable AD-specific epigenetic signatures that may have early diagnostic and prognostic implications.

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

The calcium sensing receptor (CaSR) is a class C G-protein-coupled receptor that is crucial for the feedback regulation of extracellular free ionised calcium homeostasis. While extracellular calcium (Ca2+o) is considered the primary physiological ligand, the CaSR is activated physiologically by a plethora of molecules including polyamines and l-amino acids. Activation of the CaSR by different ligands has the ability to stabilise unique conformations of the receptor, which may lead to preferential coupling of different G proteins; a phenomenon termed ‘ligand-biased signalling’. While mutations of the CaSR are currently not linked with any malignancies, altered CaSR expression and function are associated with cancer progression. Interestingly, the CaSR appears to act both as a tumour suppressor and an oncogene, depending on the pathophysiology involved. Reduced expression of the CaSR occurs in both parathyroid and colon cancers, leading to loss of the growth suppressing effect of high Ca2+o. On the other hand, activation of the CaSR might facilitate metastasis to bone in breast and prostate cancer. A deeper understanding of the mechanisms driving CaSR signalling in different tissues, aided by a systems biology approach, will be instrumental in developing novel drugs that target the CaSR or its ligands in cancer. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

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., Calcitriol is the hormonally active form of vitamin D and has anti-proliferative and pro-apoptotic effects. Calcitriol and its precursor calcidiol (25(OH)D3) are degraded by the 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1). This enzyme is overexpressed in colorectal tumors, however, the mechanisms of this overexpression remain to be elucidated. CYP24A1 mRNA level differs among colorectal cancer cell lines and range from almost undetectable to high. Since DNA methylation and histone acetylation regulate CYP24A1 gene expression in prostate cancer cell lines, we investigated whether epigenetic mechanisms could explain the differences in basal expression of CYP24A1 in colon cancer cells. Methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) treatment resulted in an over 50-fold induction of CYP24A1 mRNA expression in Coga1A and HT-29 cells but in no response in Caco2/AQ and Coga13 cells. This finding is supported by a strong increase in CYP24A1 activity after DAC treatment in Coga1A (35%). In addition, calcitriol and DAC had synergistic effects on CYP24A1 gene transcription. Interestingly, the CYP24A1 promoter was not methylated in Coga1A and HT-29 (

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications(1,2). Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer(3–5). High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the antidiabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel ‘phospho-switch’ that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.

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.

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

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., Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) are proinflammatory cytokines that play a critical role in inflammatory bowel disease, as well as in colorectal tumorigenesis. We hypothesize that these cytokines modulate the expression and thus activity of the vitamin D system in colonic epithelial cells. We treated the colon cancer cell line COGA-1A for 6, 12, and 24 h with 1,25-dihydroxyvitamin D3 (1,25-D3), IL-6, TNFα, and with combinations of these compounds. Using quantitative RT-PCR, we analyzed mRNA expression of genes activating and catabolizing 1,25-D3 (1α-hydroxylase (CYP27B1), 24-hydroxylase (CYP24A1)), expression of several vitamin D target genes, as well as expression of cyclooxygenase 2 (COX-2) and 15-hydroxyprostaglandin dehydrogenase. As expected, treatment with 1,25-D3 resulted in an upregulation of CYP24A1, whereas expression of CYP27B1 was not affected. Treatment with TNFα and IL-6 led to decreased expression of the vitamin D activating enzyme CYP27B1. The strong inflammatory property of TNFα was mirrored by its activation of COX-2 and inhibition of prostaglandin E2 (PGE2) catabolism. Interestingly, expression of the calcium ion channel TRPV6 was markedly decreased by TNFα. We conclude from these results that the presence of proinflammatory cytokines might impair activation of 1,25-D3, limiting its anti-inflammatory action. This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’.

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. What’s new? Recently, it has been suggested that the association between colorectal cancer and reduced levels of circulating vitamin D may be related to overexpression of the vitamin D-catabolizing enzyme, CYP24A1 in the tumor. In this search for a mechanistic explanation, increased CYP24A1 gene copy number was associated with the enzyme’s overexpression in 60 percent of colorectal tumors, and expression was correlated strongly with proliferation markers. The findings suggest that CYP24A1 overexpression is likely to deplete tumor calcitriol (1,25-dihydroxyvitamin D3) levels, possibly increasing the proliferative potential of the tumors.

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 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 Höbaus, Irfete Sh. Fetahu, Ildikó Mesteri, Enikö Kállay. 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

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 Höbaus, 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

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 Höbaus, Abhishek Aggarwal, Doris M. Hummel, Ursula Thiem, Irfete Fetahu, Ildiko Mesteri, Enikö 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

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., Anti-proliferative effects of calcium in the colon are mediated, at least in part, via the calcium-sensing receptor (CaSR), a vitamin D target gene. The expression of CaSR decreases during colorectal tumor progression and the mechanisms regulating its expression are poorly understood. The CaSR promoter harbors vitamin D elements responsive to 1,25-dihydroxyvitamin D3 (1,25D3) and NF-κB, STAT, and SP1 binding sites accounting for responsiveness to proinflammatory cytokines. Therefore, in the current study we investigated the impact of 1,25D3, tumor necrosis factor alpha (TNFα), and interleukin (IL)-6 on CaSR expression in a differentiated (Caco2/AQ) and in a moderately differentiated (Coga1A) colon cancer cell line. 1,25D3 induced CaSR expression in both cell lines. Treatment with TNFα was accompanied by a 134-fold induction of CaSR in Coga1A (p