Analysis of the mechanisms that control PKCe up-regulation in breast cancer cells

WANG H; ROSEMBLIT C; HUAISHENG LU; KAZANIETZ MG

Steamboat Springs, Colorado

Congreso; FASEB meeting on non-genomic and genomic steroid receptor interactions; 2012

FASEB

   PKCe, a serine/threonine protein kinase, has been widely implicated in malignant transformation, cell survival, motility and invasion. Numerous studies established that PKCe is overexpressed in human cancer, and its expression correlates with tumor aggressiveness in prostate, lung, head and neck, and breast cancer. The underlying mechanisms that control PKCe up-regulation in cancer are still unclear. Analysis of PKCe expression in mammary cellular models revealed high protein and mRNA levels in MCF-7, T-47D, MDA-MB-453, MDA-MB-468, BT-474 and MDA-MB-231 breast cancer cells relative to ?normal? immortalized MCF-10A cells. Studies on PKCe mRNA stability showed essentially no differences among the different cellular models.    In order to investigate transcriptional mechanisms that control the expression of PKCe, we PCR amplified a region comprising +37 bp to -2115 bp of the human PKCe promoter from genomic DNA and subcloned it into the pGL3-enhancer luciferase reporter vector. When transfected into MCF-7, T-47D, or MDA-MB-231 cells, a luciferase activity was observed which was higher than in MCF-10A cells. Different truncation mutants of the PKCe promoter were generated and expressed in MCF-7 cells. This analysis revealed primarily two regions responsible for promoter activity (-289 bp to -959 bp, and -1001 bp to -1103 bp). Analysis of putative transcription factor elements using the PROMO software revealed several Sp1 sites from -289 bp to -959 bp and two STAT1/STAT3 sites from -1001 bp to -1103 bp. Mutation of individual STAT1/STAT3 sites decreased PKCe promoter activity, and the effect was larger when the two STAT1/STAT3 sites were mutated together. RNAi knock-down of either STAT1 or STAT3 in MCF-7 cells significantly reduced PKCe promoter activity. Treatment of MCF-7 cells with the JAK-STAT inhibitor cucurbitacin also reduced PKCe promoter activity, suggesting a potential role for the JAK-STAT signaling pathway in the regulation of PKCe expression in breast cancer cells. We also found that the pan-PKC inhibitor GF 109203X, the PKCe inhibitor peptide eV1-2, or PKCe depletion using RNAi reduced PKCe promoter luciferase activity, suggesting that PKCe controls its own expression. Our results establish a potential role for STAT1/STAT3 transcription factors in the control of PKCe expression in breast cancer cells.