P4-01-08: The Role of Ser991 PELP1 Phosphorylation in Therapy Resistance and Metastasis of Breast Cancer.

BACKGROUND: Growth factor induced activation of MAPK pathway is suggested to play a critical role both in invasive and hormone therapy resistant breast cancer via ligand independent activation of ERα. Proline Glutamic acid Leucine Rich Protein (PELP1) is a well established ER-coregulator that plays a critical role in ER9s nuclear and extra-nuclear functions. PELP1 functions as a proto-oncogene and deregulation of PELP1 expression is linked to increased proliferation, metastasis and therapy resistance. The objective of this study is to examine, whether growth factor signaling modulates PELP1 mediated ER coactivation functions via MAPK pathway. METHODS: Bioinformatics and phosphopeptide specific antibodies were employed in this study. In vitro kinase assays and MAPK inhibitors were used to confirm MAPK phosphorylation of PELP1. Site directed mutants and ERE reporter gene assays were used to demonstrate the significance of MAPK site in PELP1. Growth factor and estrogen regulation of PELP1 phosphoryaltion at Ser991 was validated in therapy sensitive (MCF7, ZR75) and resistant (MCF-7 HER2, MCF7-Tam and MCF-7Ca-LTLT cells using PELP1 specific phosphoantibody. Breast cancer model cells stably expressing PELP1 mutant or PELP1 peptides encompassing MAPK phosphorylation was used to test the significance of this MAPK site in PELP1 using proliferation, migration, and invasion assays. IHC analysis using PELP1 phospho antibodies was performed using murine xenograft tumors and human breast tumor arrays (TMA). RESULTS: Growth factors (EGF, Heregulin) induced Ser991 phosphorylation of PELP1 in murine mammary epithelial cells and also in human breast cancer cell lines. Growth factor mediated Ser991 PELP1 phosphorylation was abrogated by ERK1/2 MAPK pathway inhibitors including PD98059 and U0126. In ERE-reporter luciferase assays using ZR75 cells, PELP1 (WT) but not the ser991Ala mutant of PELP1 corroborated growth factor-ER crosstalk. Therapy resistant model cells (MCF-7-Tam, MCF-7-LTLT-ca and MCF-7-HER2) exhibited enhanced Ser991 PELP1 phosphoryation compared to parental MCF7. TAT-Peptide encompassing Ser991 inhibited growth factor mediated phosphorylation of PELP1 and revealed a potent ability to inhibit the growth of therapy resistant cells. Xenograft tumors of hormone therapy model cell lines showed increased Ser991 PELP1 phosphorylation and enhanced nuclear localization compared to hormonal sensitive xenograft tumors. IHC analysis of human breast invasive TMA revealed enhanced PELP1 ser991 phosphorylation in advanced metastatic tumors with prominent nuclear/nucleolar localization compared to normal and benign tumors. CONCLUSIONS: In this study, we identified that growth factor-MAPK pathway modulate ER coregulator PELP1 functions via phosphorylation and demonstrated that Ser991 phosphorylation of PELP1 has the potential to govern the growth Factor-ER cross talk leading to therapy resistance and metastatic phenotype of breast cancer. Peptides encompassing Ser991 PELP1 site and Phosphoantibodies targeting Ser991 represent a novel therapeutic target and diagnostic markers in breast cancer. Supported by DOD Fellowship W81XWH-09-1-0010 (BCN) and DOD W81XWH-08-1-0604 (RKV) Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-08.