Abstract 866: Delineation of novel CYP24A1 transcriptional regulators
Authors: Luo Wei , Ma Yingyu , Chernov Mikhail , Trump Donald L. , Johnson Candace S. 
Published: July 2016

The anti-proliferative and pro-apoptotic effects of 1á,25 dihydroxycholecalciferol (1,25D3) are well-documented in various tumor model systems in vitro and in vivo. However, limited anti-tumor effects of 1,25D3 have been observed in clinical trials. One potential reason may be the over-expression of CYP24A1, the primary 1,25D3 degrading enzyme, in tumors, and the subsequent rapid local inactivation of 1,25D3. A CYP24A1 inhibitor might be added to improve the anti-tumor activity of 1,25D3. However, most CYP24A1 inhibitors are non-specific, and their use is accompanied by a striking increase in the CYP24A1 expression level. In this study, we screened a small molecule library to identify novel CYP24A1 transcriptional inhibitors using a luciferase reporter assay in a human prostate cancer PC3 cell line stably expressing CYP24A1 promoter-driving luciferase reporter. Screening of DIVERSet™, a diverse library of 55,230 compounds, resulted in the identification of 150 hits, each of which had over 50% inhibition of 1,25D3-induced CYP24A1 promoter activity. After confirming initial observations, four hits (CPI3, CPI8, CPI11 and CPI17) displaying the strongest inhibitory effect were chosen to further examine their effects on endogenous and 1,25D3-regulated CYP24A1 expression in cancer cells. Prostate cancer (PC3) cells, bladder cancer (RT112, RT112/D21) cells, kidney cancer (A498, ACHN and 786-O) cells were treated with CPI3, CPI8, CPI11 and CPI17 alone or followed by 100 nM of 1,25D3. qRT-PCR analysis showed that 1,25D3 induced CYP24A1 mRNA expression in these cancer cells and the induction of CYP24A1 was significantly inhibited by the treatment with CPI8 and CPI17 (P &lt; 0.01). Western blot showed the reduction of 1,25D3-induced CYP24A1 protein expression in CPI8 and CPI17 treated cancer cells. These results indicate that CPI8 and CPI17 inhibit CYP24A1 expression at the transcriptional level. To investigate whether these compounds affect the transcriptional expression of vitamin D receptor (VDR) and other vitamin D target genes, the expression of VDR and p21Waf1 were measured by qRT-PCR in kidney cancer A498, ACHN and 786-O cells treated with CPI8, which displayed the strongest inhibitory effect on CYP24A1 expression. Treatment with CPI8 did not affect VDR expression in kidney cancer cells. In contrast with the reduction of CYP24A1 expression by CPI8, treatment with CPI8 markedly increased p21Waf1 mRNA and protein expression compared to 1,25D3 alone. These results indicate that CPI8 differentially affects the expression of vitamin D target genes. In summary, we identified novel CYP24A1 inhibitors, which differentially regulate 1,25D3 target genes. Inhibiting CYP24A1 expression, while increasing the expression of other anti-proliferative 1,25D3-target genes, may be a useful approach to enhance 1,25D3 antitumor activity.

Supported by NIH/NCI grant CA67267.

Citation Format: Wei Luo, Yingyu Ma, Mikhail Chernov, Donald L. Trump, Candace S. Johnson. Delineation of novel CYP24A1 transcriptional regulators. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 866.