Calcitriol (vitamin D or 1,25 dihydroxycholecalciferol), a central factor in bone and mineral metabolism, has significant antitumor activity in vitro and in vivo. Induction of CYP24, the enzyme primarily responsible for calcitriol catabolism, may be a factor in the anti-proliferative activity pre-clinically and clinically. In addition, the endothelial cells in tumors are sensitive to calcitriol and uniquely modulate CYP24 expression through epigenetic changes. Epigenetic events affect gene expression without alteration in DNA gene sequence and lead to transcriptional gene silencing and inactivation of tumor suppressor genes in human cancer. While many studies document epigenetic changes in tumor cells, only limited data support a role for epigenetic changes in the """"""""normal"""""""" cells found in the tumor microenvironment. Calcitriol also inhibits proliferation of endothelial cells and can inhibit angiogenesis in a number of tumor model systems. We have established a method for the isolation of fresh, tumor-derived endothelial cells (TDEC) that maintain phenotypic characteristics which are distinct from endothelial cells isolated from normal tissues and from Matrigel plugs (MDEC). In TDEC, calcitriol induces G0/G1 arrest, modulates p27 and p21, and induces apoptotic cell death and decreases P-Erk and P-Akt. In contrast, endothelial cells isolated from normal tissues and MDEC are unresponsive to calcitriol-mediated anti-proliferative effects despite intact signaling through the vitamin D receptor (VDR). Differences may be due to the over-expression of CYP24 in MDEC where mRNA, protein and enzymatic activity for CYP24 are markedly increased. In TDEC, which is sensitive to calcitriol, the CYP24 promoter is hypermethylated in two CpG island regions located at the 5' end, which may contribute to gene silencing of CYP24 in TDEC. The extent of methylation in these two regions is significantly less in MDEC. Treatment of TDEC with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restores calcitriol-mediated induction of CYP24 and resistance to calcitriol. Lastly, when MDEC are incubated with conditioned media from tumor cells for 14-21 days, these cells regain sensitivity to the antiproliferative effects of calcitriol, CYP24 induction is silenced and regions 1 and 2 of the CYP24 promoter are hypermethylated. The characteristics of these cells are similar to the endothelial cells found in tumors or TDEC. These unique differences in the epigenetic silencing of calcitriol-induced CYP24 gene expression in endothelial cells from the tumor microenvironment directly impact on calcitriol-mediated signaling pathways and ultimately on therapeutic application. Therefore, we propose 1) to elucidate the epigenetic signal transduction mechanism(s) that lead to the epigenetic silencing of CYP24; 2) to determine whether epigenetic silencing of CYP24 occurs in human endothelial cell and tumor cell populations isolated from human prostate cancer; and 3) To identify other potential targets of DNA methylation in TDEC vs. MDEC by investigating the scope of the epigenetic defect from the tumor microenvironment on the endothelial cells. We developed a model system for isolation of endothelial cells freshly from tumors and demonstrate that CYP24, the catabolic enzyme involved in vitamin D (1,25 dihydroxycholecalciferol or calcitriol) signaling, becomes epigenetically silenced selectively in tumor-derived endothelial cells. These unique differences in the epigenetic silencing of calcitriol-induced CYP24 gene expression in endothelial cells from the tumor microenvironment directly impact on calcitriol-mediated signaling pathways and ultimately on therapeutic application. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA095045-06
Application #
7372804
Study Section
Tumor Microenvironment Study Section (TME)
Program Officer
Xie, Heng
Project Start
2002-09-01
Project End
2013-07-31
Budget Start
2008-09-15
Budget End
2009-07-31
Support Year
6
Fiscal Year
2008
Total Cost
$270,000
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
824771034
City
Buffalo
State
NY
Country
United States
Zip Code
14263
Doig, Craig L; Battaglia, Sebastiano; Khanim, Farhat L et al. (2016) Knockdown of AKR1C3 exposes a potential epigenetic susceptibility in prostate cancer cells. J Steroid Biochem Mol Biol 155:47-55
Long, Mark D; Sucheston-Campbell, Lara E; Campbell, Moray J (2015) Vitamin D receptor and RXR in the post-genomic era. J Cell Physiol 230:758-66
Ma, Yingyu; Hu, Qiang; Luo, Wei et al. (2015) 1?,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells. J Steroid Biochem Mol Biol 148:166-71
Thorne, James L; Campbell, Moray J (2015) Nuclear receptors and the Warburg effect in cancer. Int J Cancer 137:1519-27
Singh, Prashant K; Long, Mark D; Battaglia, Sebastiano et al. (2015) VDR regulation of microRNA differs across prostate cell models suggesting extremely flexible control of transcription. Epigenetics 10:40-9
Long, Mark D; Campbell, Moray J (2015) Pan-cancer analyses of the nuclear receptor superfamily. Nucl Receptor Res 2:
Long, Mark D; van den Berg, Patrick R; Russell, James L et al. (2015) Integrative genomic analysis in K562 chronic myelogenous leukemia cells reveals that proximal NCOR1 binding positively regulates genes that govern erythroid differentiation and Imatinib sensitivity. Nucleic Acids Res 43:7330-48
Long, Mark D; Thorne, James L; Russell, James et al. (2014) Cooperative behavior of the nuclear receptor superfamily and its deregulation in prostate cancer. Carcinogenesis 35:262-71
Ma, Yingyu; Yu, Wei-Dong; Su, Bing et al. (2013) Regulation of motility, invasion, and metastatic potential of squamous cell carcinoma by 1ýý,25-dihydroxycholecalciferol. Cancer 119:563-74
Luo, Wei; Yu, Wei-Dong; Ma, Yingyu et al. (2013) Inhibition of protein kinase CK2 reduces Cyp24a1 expression and enhances 1,25-dihydroxyvitamin D(3) antitumor activity in human prostate cancer cells. Cancer Res 73:2289-97

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