Abstract PD09-07: Therapeutic potential of targeting ATM-PELP1-p53 axis in triple negative breast cancer
Authors: Krishnan SR , Nair BC , Sareddy GR , Mann M , Roy SS , Vadlamudi RK 
Published: August 2014

Abstract
Triple negative breast cancer (TNBC) comprises approximately 15% of all breast cancers, lacks expression of ER, PR, and HER2 and is clinically aggressive with shorter disease free survival. TNBC patients do not benefit from antiestrogen and herceptin-based therapies. Evolving evidence suggests that overexpression of mutant p53 is significantly associated with TNBC progression. DNA damage response (DDR) is critical for the maintenance of genome stability and serves as an anti-cancer barrier during tumorigenesis. However, the role of DDR in tumor progression and metastasis is less known. Recent studies suggest that the ATM kinase is hyperactive in late stage breast tumor tissues with lymph node metastasis. Our ongoing studies have identified proline, glutamic acid, leucine rich protein 1 (PELP1), a proto-oncogene overexpressed in breast cancer, as a novel substrate of ATM. The objective of this study is to determine the mechanism and significance of ATM mediated phosphorylation of PELP1 and its crosstalk with the p53 pathway in TNBC cells. To test this we have used three ER-negative mutant p53 breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT20), with ER-positive (ZR-75 and MCF7) cell lines as controls. Using PELP1 specific shRNAs, we generated model cells that have stable expression of either control or PELP1 shRNA. Our results with PELP1 knockdown models indicate that PELP1 promotes stability of p53 and functions as a co-regulator of p53. PELP1 has the potential to modulate CBP/p300 mediated acetylation of the Lysine 382 residue of p53. Immunoprecipitation and chromatin immunoprecipitation (ChIP) assays were performed to examine PELP1 interaction with p53 and its recruitment to p53 target genes respectively. ChIP assays revealed that PELP1 knockdown significantly reduces the recruitment of p53 to the target genes. The p53 regulatory role of PELP1 is mediated by the phosphorylation of PELP1 at the conserved SQ motif by ATM. We generated model cells that overexpress WT or mutant (S1033A) PELP1 and demonstrated the significance of ATM mediated phosphorylation in PELP1 mediated p53 co-activation functions. Based on the sequence of the site of phosphorylation, we have developed a novel cell permeable peptide (TAT-1033PELP1 inhibitor) as well as a phospho-PELP1 antibody that uniquely recognizes S1033 phosphorylated PELP1. We confirmed ATM mediated phosphorylation of PELP1 in vivo using phospho PELP1 antibody (1033p-PELP1). The TAT 1033 inhibitor peptide significantly reduced ATM mediated phosphorylation of endogenous PELP1. Treatment of ER− positive breast cancer cells with this peptide resulted in resistance to genotoxic stress compared to cells that are treated with control TAT peptide. However, in TNBC cells that has a mutant p53, PELP1 knockdown or treatment with TAT-PELP1 inhibitor resulted in significant loss of cell viability and increase in apoptosis in response to genotoxic stress. IHC analysis of tumor tissue array (n = 100) revealed increased PELP1 phosphorylation in advanced ER-negative tumors and its status correlated with ATM. Collectively, our results suggest that hyperactive ATM-PELP1-p53 pathway contributes to TNBC progression and that the TAT-1033PELP1 inhibitor represents a novel therapeutic to block PELP1 oncogenic functions in TNBC.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD09-07.