Abstract 4973: C/EBPβ increases expression of the vitamin D3catabolizing enzyme, CYP24, in non-small cell lung cancer cells

Introduction: We previously reported that the vitamin D 3 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 D 3 (1,25(OH) 2 D 3 ). 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) 2 D 3 . 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.