Abstract P5-09-12: Novel role of proto-oncogene PELP1 in A-NHEJ pathway of double strand break repair: Implications for treatment of hormonal therapy resistance
Authors: SR Krishnan, BC Nair, M Mann, GR Saareddy, J Stark, V Gorbunova, RK Vadlamudi
Journal: Cancer Research (2013)

Abstract Background: DNA damage response (DDR) is critical for the maintenance of genome stability and serves as an anti-cancer barrier. Deregulation of key components of the DDR pathway such as p53 and ATM is associated with breast cancer progression. Recent studies implicated Alternative Non-homologous End-Joining (A-NHEJ) pathway in hormonal therapy resistance. Expression of proline, glutamic acid, leucine rich protein 1 (PELP1), a proto-oncogene, is an indicator of poor prognosis and therapy resistance in ER+ breast cancer. PELP1 was recently identified as a novel substrate of DNA damage kinases such as ATM. The objective of this study is to determine the role of PELP1 in the regulation of DNA repair and to study its effects on therapy resistance Methods: We have used ER+ therapy sensitive MCF7, MCF7-Aro, ZR75 and therapy resistant MCF7-Tam, MCF7-LTLT, MCF7-HER2 model cells in our assays. UV-laser micro-irradiation, immunofluorescence staining and confocal microscopy were used to examine Ser-1033 phospho-PELP1 localization upon DNA damage. Homologous recombination (HR) reporter cell lines and NHEJ pathway reporter plasmids were used to determine the role of PELP1 in DNA repair pathways. U2OS-EJ2-GFP reporter cell line was used to evaluate the role of PELP1 in A-NHEJ pathway. MEFs lacking PELP1 and PELP1 specific siRNA were used to determine the role of PELP1 in DNA repair. Immunoprecipitation and western blotting were used to determine PELP1 interacting proteins. Yeast based peptide library screen was performed to identify PELP1 binding inhibitory peptides. MTT and clonogenic assays were used to determine the therapeutic effect of PELP1 inhibitor on therapy resistant breast cancer model cells. Results: Results from this study showed that Ser-1033 phospho PELP1 was recruited to the site of DSB and co-localize with γ-H2A.X in breast cancer cells. Using reporter plasmid assays that distinguish various DNA repair pathways, we found that PELP1 regulates the NHEJ pathway and not the HR pathway. Further, using U2OS-EJ2-GFP reporter cell lines, we demonstrated that PELP1 is important for A-NHEJ mediated double strand break repair. Mechanistic studies revealed PELP1 interacts with E3ubiquitin ligase, SNF2 Histone Linker PHD RING Helicase (SHPRH, a yeast homolog of Rad5p). We have developed cell permeable peptide inhibitor (PIP3) that bind PELP1 and interferes with its interactions with SHPRH. Accordingly, treatment of U2OS-EJ2-GFP reporter cell lines with the (PIP3) peptide reduced the A-NHEJ repair frequency. Further, therapy resistant breast cancer cells which are addicted to A-NHEJ pathway were highly sensitive to PIP3 treatment. Conclusions: These data provide novel insights into role of PELP1 in DNA repair, underscore the importance of PELP1 in the A-NHEJ pathway of DNA repair and demonstrates the significance of targeting PELP1 to sensitize therapy resistant breast cancer cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-12.