Abstract P5-04-04: Significance of PELP1/HDAC2/microRNA-200 regulatory network in EMT and metastasis of breast cancer
Authors: Roy SS , Gonugunta VK , Bandyopadhyay AM , Rao M , Goodall G , Sun L , Tekmal RR , Vadlamudi RK 
Published: August 2014

Tumor metastasis remains a significant clinical problem and is the leading cause of death among breast cancer patients. Estrogen receptor (ER)-coregulators play an essential role in cancer progression and metastatic tumors express increased levels of coregulators. Proline glutamic acid rich protein (PELP1) is an ER coregulator, its expression is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. MicroRNA (miR) mediated regulation of tumorigenesis is emerging as a new paradigm in cancer biology and widespread misexpression of miRs has been reported in breast cancer. The objective of this study is to examine the mechanism and therapeutic significance of PELP1 regulation of miRs leading to breast cancer metastasis. We have used both ER+ve (ZR75, MCF7) and ER-ve (MDAMB231, MDAMB468) models that either stably overexpress PELP1 or PELP1shRNA. Boyden chamber, and invasion assays demonstrated that PELP1 down regulation significantly affect migration of both ER+ve and ER-ve cells. Epithelial to Mesenchymal Transition (EMT) real time qPCR Array studies identified PELP1 modulate expression of EMT genes Snail, Twist, ZEB1, ZEB2, Vimentin and MMPs. Importantly, whole genome microRNA array analysis using PELP1 model cells revealed that miR200a and miR141 were significantly upregulated in cells expressing PELP1-shRNA compared to control cells. Accordingly, over expression of PELP1 in low metastatic model cells decreased expression of miR200a and miR141. PELP1 regulation of miRs was further confirmed by ZEB1 and ZEB2 3′ UTR luciferase reporter assays. ChIP analysis revealed recruitment of PELP1 to the proximal promoter region of miR-200a and miR141 and promoter reporter assays further confirmed PELP1 regulation of miRs. Interestingly, PELP1 down regulated expression of miR200a and miR141 by promoting repressive chromatin modifications via HDAC2. Supporting this, HDAC inhibitors reversed PELP1 driven repressive effects. Further, ectopic expression of miR200a and miR141 mimetic decreased PELP1 mediated invasion/metastatic functions. Prognostic significance of PELP1-miRNA axis was determined using Tissue micro-array (TMA) and in situ hybridization (ISH assays) of Locked Nucleic Acid (LNA™)-based microarray approach in 102 human breast tumors. To test therapeutic potential in vivo, we have generated ZR-PELP1- and MCF7-PELP1-shMIMIC of miR200a and miR141 stable cells. In vitro gene expression and Boyden chamber assays using these model cells revealed that shMIMIC of miR200a and miR141 reversed PELP1 mediated alterations in gene expression and reduced PELP1 driven migration/invasion. Proof of principle studies using IVIS imaging of nude mice based assays of GFP-Luc labeled cells demonstrated therapeutic efficacy of miRIDIAN shMIMIC of miR200a and miR141 on PELP1 driven in vivo metastasis. Collectively, these novel findings demonstrate for the first time a previously unknown role for PELP1 in epigenetically controlling the functions of tumor metastasis suppressor miR-200a and miR141. These results suggest that PELP1-miR axis may be crucial stimulus for promoting EMT and breast cancer metastasis. This study is funded by NIH T32CA148724 Postdoc Fellowship Grant.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-04.