Potential role of estrogen receptor coregulator in mitosis.

Abstract #5036 Background: Estrogen stimulates breast tissue to increase cell divisions (mitosis) and is implicated in breast cancer progression. ER action is complex and requires functional interactions with coregulators. Proline-, glutamic acid-, and leucine-rich protein (PELP)-1, also known as modulator of nongenomic actions of estrogen receptor (MNAR), is a novel nuclear receptor (ER) coregulator with multitude of functions. Emerging evidence suggest that PELP1 expression is deregulated in breast cancer and serves as a scaffolding protein that couples various signaling complexes with estrogen receptor. In this study we found that ER coregulator PELP1 plays a novel role in mitosis. Material and Methods: To understand the mechanism by which ER coregulator PELP1 contribute to breast cancer progression, we have utilized small RNA interference methodology and established breast cancer model cells that stably express PELP1-shRNA (MCF7-PELP1shRNA). FACS analysis was used to determine the cell cycle status of the model cells. Using confocal microscopy, immunoprecipitation, in vitro kinase assays, site directed mutagenesis and Western analysis using phospho-antibodies we studied the mechanism and significance of PELP1 signaling in mitotic progression. We also developed PELP1 siRNA nanoparticles and used them as well as CDK1 inhibitors in cell proliferation studies. Results: Down regulation of PELP1 expression resulted in decreased estrogen mediated cell proliferation, delayed mitotic progression and induced accumulation of mitotic cells. Interestingly, PELP1 depleted cells also exhibited multinucleation. Western analysis of various markers of mitotic progression revealed a delay in the kinetics of G2M initiation and progression. Confocal analysis revealed colocalization of CDK1 and PELP1 in G2M. Immunoprecipitation assays demonstrate that endogenous CDK1 form functional complex with PELP1 and Src kinase during mitosis. Using deletion and mutagenesis approach, we have mapped the putative CDK1 phosphorylation sites on PELP1. Down regulation of PELP1 or overexpression of PELP1 mutants (that cannot be phosphorylated by CDK1), reduces the magnitude of Src activation, which is an essential driving force for timely progression of M phase. PELP1 siRNA nanoparticles alone or in combination with CDK1 inhibitors have shown to significantly reduce the proliferation of breast cancer cells and showed increased response in tamoxifen resistant breast cancer cells. Discussion: These results suggest that ER coregulator PELP1 play a novel role in G2M progression. Since PELP1 expression is deregulated in breast cancer, PELP1 ability to regulate mitosis could contribute to the progression of cancer by causing genomic instability through the deregulation of mitosis. Taken together our findings suggest that estrogen can promote neoplasia using coregulators by two distinct mechanisms (1) Coregulator induction of target genes and (2) Coregulator mediated actions in mitosis. ER coregulator PELP1 play multiple roles in Estrogen mediated neoplasia, and thus represent a target for novel therapeutic breast cancer strategies by forming the “next generation” of antimitotic drugs. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5036.