Abstract 4751: 25-hydroxyvitamin D3 induces vitamin D signaling independent of CYP27B1 in non-small cell lung cancer cells

Abstract

Abstract Introduction 25-hydroxyvitamin D3 (25(OH)D3) levels are associated with increased overall survival in early stage non-small cell lung cancer (NSCLC). Studies suggest that 25(OH)D3 is converted locally in tissues by CYP27B1 into the active metabolite, 1,25-dihydroxyvitamin D (1α,25(OH)2D3). Once produced, 1α,25(OH)2D3 exerts anti-tumor effects by binding to the vitamin D receptor (VDR) and regulating target gene expression. It is unknown whether CYP27B1 is necessary for NSCLC cells to respond to 25(OH)D3. We therefore tested the requirement for VDR and CYP27B1 in mediating the anti-tumor effects of 25(OH)D3 in NSCLC. Methods Immunoblot and qRT-PCR were used to evaluate VDR expression. Small interfering RNA was used to knock down the VDR. Colony formation assays and qRT-PCR were used to assess effects of 25(OH)D3 and 1α,25(OH)2D on growth and transcriptional responses. To study the requirement for CYP27B1, pharmacological inhibition was achieved using ketoconazole, and 1α,25(OH)2D3 production was measured by LC/MS-MS. A xenograft study was performed to determine the in vivo effect of modulating 25(OH)D3 levels. Results NSCLC cells expressing high levels of VDR (VDRhigh) are more responsive to vitamin D metabolites. In these cells, 25(OH)D3 and 1α,25(OH)2D3 induce target gene transcription and inhibit colony formation. These effects are not observed in VDRlow cells. When VDRhigh cells were transfected with VDR siRNA, the ability of 25(OH)D3 to induce target gene transcription was diminished, and no VDR protein was observed. VDRhigh cells were exposed to ketoconazole to determine if effects of 25(OH)D3 required conversion to 1α,25(OH)2D3. Ketoconazole effectively abrogated production of 1α,25(OH)2D3 from 25(OH)D3. However, VDR target gene expression remained induced by 25(OH)D3. Thus, NSCLC cells may not require CYP27B1 to promote vitamin D signaling. To more specifically inhibit CYP27B1, zinc finger nucleases (ZFNs) were used to create CYP27B1 knockout cells. The ZFNs have been transfected into cells, assays optimized, and positive clones are being expanded. Lastly, a mouse xenograft experiment was performed to determine if 25(OH)D3 promotes a decrease in tumor volume. Mice were fed diets containing 100, 1,000 or 10,000 IU/kg vitamin D3 prior to implantation of VDRhigh NSCLC cells. Mice fed the 10,000 IU/kg diet had a statistically significant decrease in tumor volume. Conclusions NSCLC cells expressing high levels of VDR display increased responsiveness to vitamin D metabolites. Although NSCLC cells express CYP27B1, our results demonstrate that the enzyme may not be required to achieve vitamin D signaling. Rather, 25(OH)D3 may act via the VDR to elicit an anti-tumor responses. The implication of these findings is that dietary supplementation to increase circulating 25(OH)D3 may be beneficial in a subset of NSCLC patients. Funding provided by NIH RO1 CA132844, training grant 5T32CA009072-37 and P30CA47904 Citation Format: Alissa R. Verone, Suzanne Shoemaker, Robert Parise, Jan H. Beumer, Pamela A. Hershberger. 25-hydroxyvitamin D3 induces vitamin D signaling independent of CYP27B1 in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4751. doi:10.1158/1538-7445.AM2014-4751