P300 regulates the human RLIP76 promoter activity and gene expression

Abstract

A 76-kDa Ral-interacting protein (RLIP76) has been implicated in the pathogenesis of cancer and diabetes. It is often overexpressed in human malignant cell lines and human tumor samples and has been associated with metastasis and chemoresistance. RLIP76 homozygous knockout mice exhibit increased insulin sensitivity, hypoglycemia, and hypolipidemia, and resist cancer development. Little is known about the mechanism by which the expression of RLIP76 is regulated. In the present study, we functionally characterized the RLIP76 promoter using deletion mapping and mutational analysis to investigate the regulation of RLIP76 transcription. We have identified the promoter regions important for RLIP76 transcription, including a strong cis-activating element in the proximal promoter containing overlapping consensus cMYB and cETS binding sites. Transcription factor cMYB and the coactivator p300 associated with RLIP76 gene promoter as shown by CHIP assay. Knockdown of p300 in HEK293 cells reduced the activity of the promoter fragment containing wild type cMYB/cETS binding site in comparison to that with deleted or mutated cMYB/cETS binding site. Knockdown of p300 also decreased the RLIP76 expression as indicated by immunoblotting, immunocytochemistry and flow cytometry analysis. Thus, we report for the first time that p300 associates with the RLIP76 promoter via an overlapping cMYB and cETS binding site and regulates RLIP76 promoter activity and its expression.

Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

“…Immunocytological localization SMC1 was performed on MDA-MB-231 fixed cells by method described previously with slight modifications [37], [38], [40], [41]. Cells were grown on glass cover slips and fixed with ice-cold methanol and acetic acid (3∶1).…”

“…Slides were analyzed by confocal laser microscope (Zeiss LSM510 META, Germany) at 40× magnification ( Panel A ). Surface localization of SMC1 was determined by flow cytometry using indirect flow cytometry protocol [40]. Briefly, MDA-MB-231 cells were harvested and resuspended to approximately 1×10 6 cell/ml in ice-cold PBS, 10% FBA and 1% sodium azide.…”

“…Cell-surface localization of SMC1 was performed using indirect flow cytometry protocol as described [40]. Briefly, MDA-MB-231 cells were harvested and suspended in approximately 1×10 6 cells/mL in ice cold PBS, containing 10% FBS and 1% sodium azide.…”

RLIP76 regulates HIF‐1 activity, VEGF expression and secretion in tumor cells, and secretome transactivation of endothelial cells

“…VEGF has long been recognized as the major factor in promoting angiogenesis in carcinomas and other tumors and is well known to be overexpressed at the mRNA and protein levels in many tumors and tumor cell lines, including lung carcinomas and melanomas, leading to promotion of neovascularization and tumor growth (3, 21, 22, 44‐46). Although VEGF in the tumor environment is derived from many cells, including tumor stromal cells, vascular cells, and peripheral blood mononuclear cells (47‐50), tumor cells are the major source of VEGF at least in murine tumor models (21, 38, 51, 52). However, VEGF derived from the tumor stroma also supports tumor angiogenesis and progression, as evidenced by findings that VEGF blockade in the tumor cells alone significantly reduces, but not does eliminate, tumor growth (3, 21).…”

“…Like RLIP76, HIF‐1 is also overexpressed in many tumor cells, and targeting the HIF‐1/VEGF pathway has been shown to inhibit angiogenesis and growth in tumors (29, 58). Notably, HIF‐1 couples to p300/CBP to drive transcription, and recently RLIP76 expression was shown to be regulated by p300; furthermore, p300 and RLIP76 correlate in many tumor types, suggesting the possibility of a transcriptional regulatory loop (52). HIF‐1 in tumor cells stimulates the transcription of many genes downstream of the PI3K/mTOR and Ras/Raf/MEK/ERK pathways, both of which converge on elF4E1 to regulate HIF‐1 expression (27, 30, 31).…”