Abstract 287: JUNB exhibits loss of “Paused” Pol II regulation and promotes survival in response to Flavopiridol in malignant MCF10CA1a cells

The long term goal of this research is to investigate the role of “Paused” RNA Polymerase II (Pol II) regulation in breast cancer. The term “Paused” Pol II describes the presence of an active “Paused” Pol II multi-protein complex that has initiated transcription and then “paused,” after transcribing a short ∼50 nucleotide transcript. “Paused” Pol II genes function in cell programs relevant to breast cancer, including growth, survival, migration and apoptosis. We investigated the profile and expression of published “Paused” Pol II regulated genes in nontransformed (MCF10A) and malignant (MCF10CA1a) human mammary epithelial cell lines. The presence of protein components of the “Paused” Pol II complex in association with gene-specific regions was detected by chromatin immunoprecipitation (ChIP) plus PCR amplification. Gene expression was assessed by RT-PCR in response to growing (GR), growth arrest (GA) and growth arrest plus the cytokine Oncostatin M (GA + OSM) treatments. In nontransformed MCF10A cells, JUNB expression was virtually undetectable under GR conditions; however, ChIP results detected the presence of “Paused” Pol II and Cyclin Dependent Kinase9 (CDK9), a component of the positive transcription elongation factorb (pTEFB) complex, in association with the JUNB gene promoter. In addition, Negative elongation factorb (NELFB), an inhibitor of Pol II transcriptional elongation, and an essential component of the “Paused” Pol II complex, was also detected in association with the JUNB promoter in MCF10A cells under GR conditions. In contrast to the MCF10A results, all candidate “Paused” Pol II regulated genes were expressed at detectable levels in response to the GR, GA or GA + OSM treatments in the malignant MCF10CA1a cell line. This suggests the possibility of a general loss of “Paused” Pol II regulation in malignant cells. ChIP assays detected Pol II and CDK9 in association with the JUNB promoter, but NELFB was virtually undetectable. NELFB protein levels were similar between the two cell lines. Although JUNB expression was not dramatically altered by GR, GA or GA + OSM treatments in MCF10CA1a cells, JUNB expression increased dramatically in MCF10CA1a cells treated with a lethal dose of Flavopiridol (FP) a chemotherapeutic drug that blocks CDK9 activity. JUNB siRNA treated MCF10CA1a cells exhibited a dramatic increase in FP-induced cell death compared to controls, suggesting a potential pro-survival role for JUNB. These results demonstrate defective “Paused” Pol II regulation of JUNB gene expression in malignant MCF10CA1a cells. In addition, the results indicate that JUNB plays a pro-survival role in FP treated MCF10CA1a cells and suggest that JUNB may play a previously unrecognized role in the resistance of breast cancer cells to chemotherapeutic agents. Supported by the U.S. Army Breast Cancer Research Program (JD) and P30 CA16058 (OSUCCC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 287. doi:1538-7445.AM2012-287