Abstract 4973: C/EBPβ increases expression of the vitamin D3 catabolizing enzyme, CYP24, in non-small cell lung cancer cells

Abstract

Abstract Introduction: We previously reported that the vitamin D3 catabolizing enzyme CYP24 is over-expressed in non-small cell lung cancer (NSCLC) cells, where it decreases the growth inhibitory effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Studies were conducted to establish the clinical relevance of this finding and the mechanism for CYP24 over-expression in NSCLC cells. Methods: CYP24 mRNA expression was quantified by real-time PCR in patient-matched histologically normal lung tissue and primary lung tumors. To elucidate factors that control CYP24 expression, a panel of NSCLC cell lines that exhibit a 70-fold variation in CYP24 mRNA levels under basal growth conditions was used (relative mRNA expression H292 =1.0; 201T = 7.0; 128.88T = 68; A549 =69). CYP24 gene copy number was assessed by fluorescence in situ hybridization. CYP24 promoter activity was measured following transfection of NSCLC cells with either a wildtype promoter-luciferase reporter plasmid or plasmids harboring CYP24 promoter mutations. Transcription factor binding to CYP24 promoter elements was evaluated by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assays. Results: CYP24 was significantly over-expressed in tumors (p = 0.0017), with 53% of NSCLC cases showing a minimum of 5-fold over-expression. CYP24 gene copy number was comparable among the NSCLC cell lines used. When NSCLC cells were transfected with a wildtype CYP24 promoter-luciferase reporter plasmid, significant differences in promoter activity were observed that paralleled endogenous CYP24 transcript levels. ChIP assays revealed that the CCAAT-enhancer binding protein C/EBPβ binds to the endogenous CYP24 promoter under basal growth conditions in 128.88T cells. To establish a role for C/EBP proteins in regulating CYP24 transcription, we introduced mutations into the C/EBP binding site within the CYP24 promoter. The mutations disrupted C/EBP binding and significantly reduced basal promoter activity in 128.88T and A549 cells. EMSAs that employed a consensus C/EBP site as probe demonstrated that C/EBP binding increases with CYP24 promoter activity: nuclear extracts from untreated H292 and 201T cells contained low levels of C/EBP binding activity, whereas 128.88T and A549 extracts contained high levels. Conclusions: CYP24 mRNA is frequently over-expressed in primary lung tumors. A C/EBPβ-mediated transcriptional control mechanism contributes to the increased expression of CYP24 in NSCLC cells. Signaling pathways that increase C/EBPβ expression/activation may contribute to lung cancer by upregulating CYP24 and allowing neoplastic cells to bypass the antiproliferative effects of 1,25(OH)2D3. This work was supported by the University of Pittsburgh Center for Environmental Oncology, the Hillman Foundation, the Pittsburgh Foundation and R01 CA132844. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4973.