Abstract 855: Targeting triple negative breast cancer using novel cell permeable inhibitor to block ATM-PELP1-p53 axis.
Authors: Samaya Krishnan, Binoj C. Nair, Gangadhara R. Sareddy, Monica Mann, Ratna K. Vadlamudi
Journal: Cancer Research (2013)

Abstract Triple negative breast cancer (TNBC) is clinically aggressive and TNBC patients do not benefit from antiestrogen and herceptin-based therapies; so new therapeutic drugs are urgently needed. DNA damage response (DDR) is critical for the maintenance of genome stability and serves as an anti-cancer barrier. Recent evidence suggests that deregulation of key components of the DDR pathway such as p53 and ATM is associated with TNBC progression and 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. PELP1 overexpression in breast cancer is an indicator of poor prognosis. The objective of this study is to determine the mechanism and significance of the ATM-PELP1-p53 pathway in TNBC cells. For this purpose, we used ER-negative mutant (MT) p53 (MDA-MB-231, BT20, MDA-MB-468) and ER-positive wildtype (WT) p53 (ZR-75 and MCF7) breast cancer cell lines. We generated model cells that have stable expression of control or PELP1shRNA. Our results with PELP1 knockdown models indicate that PELP1 functions as a co-regulator of p53. Immunoprecipitation and chromatin immunoprecipitation (ChIP) assays revealed that PELP1 interacts with p53 and is recruited to p53 target genes, respectively. The p53 regulatory role of PELP1 is mediated by the phosphorylation of PELP1 (p-PELP1) by ATM at the conserved SQ motif. Through model cell lines that overexpress WT or MT (S1033A) PELP1 we demonstrated the significance of p-PELP1 in WT p53 co-activation functions. Utilizing the unique sequence surrounding the ATM phosphorylation site in PELP1, we developed a novel cell permeable peptide (TAT-1033PELP1 peptide) as well as a p-PELP1 antibody that uniquely recognizes S1033 p-PELP1. We confirmed ATM mediated phosphorylation of PELP1 in vivo using S1033 p-PELP1 antibody. The TAT-1033PELP1 inhibitor peptide significantly reduced ATM mediated phosphorylation of endogenous PELP1. Interestingly, in TNBC cells that have MTp53, PELP1 knockdown or treatment with TAT-1033PELP1 peptide resulted in a significant loss of cell viability in response to genotoxic stress. However, treatment of ER- positive breast cancer cells with this peptide or PELP1 knockdown resulted in resistance to genotoxic stress, suggesting unique therapeutic activity of TAT-1033PELP1 peptide towards TNBC cells. Mechanistic studies showed that PELP1 functionally interacts with MTp53 and regulates the expression of NF-Y and p63/p73 target genes. The TAT-1033PELP1 peptide also reduces the migratory potential of TNBC cells. IHC analysis of a tumor tissue array revealed increased PELP1 phosphorylation in TNBC tumors. Collectively, our results suggest that a hyperactive ATM-PELP1-p53MT pathway contributes to TNBC progression and that the TAT-1033PELP1 peptide represents a novel therapeutic to block PELP1 oncogenic functions in TNBC. Citation Format: Samaya Krishnan, Binoj C. Nair, Gangadhara R. Sareddy, Monica Mann, Ratna K. Vadlamudi. Targeting triple negative breast cancer using novel cell permeable inhibitor to block ATM-PELP1-p53 axis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 855. doi:10.1158/1538-7445.AM2013-855