Abstract P6-04-14: The role of PELP1-TFAP2C crosstalk in mediating endocrine-therapy-resistance in breast cancer

Background: A significant proportion of estrogen receptor (ER) positive (ER+) breast cancer (BC) initially respond to endocrine therapies, such as antiestrogens or aromatase inhibitors. However, ER+ BC can build up resistance to treatment progressing into therapy resistant-BC (TR-BC). Development of effective therapeutics for endocrine-therapy-resistant BC represents a significant unmet need in BC treatment options. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is oncogenic nuclear receptor coregulator, commonly overexpressed in BC. PELP1 overexpression is correlated with poorer patient survival and is associated with development of TR-BC. TFAP2C (AP2Gamma) is a known regulator of ER activity. In addition, high expression of TFAP2C is associated with a decreased response to the steroidal antiestrogen, Fulvestrant. However, it remains unknown whether PELP1 and TFAP2C crosstalk and if the PELP1/TFAP2C axis behaves synergistically to contribute to the development of therapy resistance in TR-BC. Methods: To gain insight into PELP1 signaling mechanisms, we used yeast two-hybrid screen to identify proteins that interact with PELP1. The interaction between PELP1 and TFAP2C was confirmed by immunoprecipitation using both endogenous and GFP tagged proteins. GST fusions of various domains of PELP1 were used to identify the TFAP2C interacting domain. Functional significance of the crosstalk was tested using Celltiter Glo, MTT, apoptosis, and invasion assays. Mechanistic studies were conducted using shRNA, overexpression, Western Blot, reporter gene assays, RT-qPCR, ChIP and RNA-Seq analysis. Biological significance of PELP1 and TFAP2C in endocrine-therapy-resistance was examined using overexpression and under-expression models of PELP1 and TFAP2C in multiple ER+ BC and TR-BC model cells (MCF-7, ZR-75, T-47D, MCF-7-TamR, MCF7-LTLT and ZR75-ERMT537S. Results: Screening of a mammary gland cDNA expression library using PELP1 as the bait identified TFAP2C as a novel interacting protein of PELP1. Immunoprecipitation assays utilizing multiple BC cell lysates confirmed interaction of PELP1 with TFAP2C. We also confirmed PELP1 and TFAP2C interactions using GFP and GST epitope tagged proteins. Using GST fusion of various domains of PELP1, we identified the PELP1 N-terminal domain (aa 400-600) as the major interaction site for TFAP2C. Using PELP1 knockdown BC model cell lines with RNA-Seq analysis, we identified a set of genes regulated by PELP1. The GSEA results from the RNA-Seq data predicted TFAP2C as an enriched transcription factor in a subset of PELP1 regulated genes. RT-qPCR analysis confirmed that PELP1 is needed for optimal regulation of the ER and associated ER target genes by TFAP2C. Reporter gene assays confirmed that PELP1 functions as a coregulator of TFAP2C. Overexpression of TFAP2C contributed to endocrine-therapy-resistance in BC model cells, while knockdown of PELP1 abolished TFAP2C mediated therapy resistance. Conclusions: Collectively, our studies identified PELP1 functions as a coregulator of TFAP2C in modulating ER target genes. TFAP2C functions as a transcription factor of PELP1 regulated genes and blocking the PELP1-TFAP2C axis will have therapeutic utility for treating TR-BC Citation Format: Junhao Liu, Suryavathi Viswanadhapalli, Mengxing Li, Uday P Pratap, Yiliao Luo, Kristin A Altwegg, Xiaonan Li, Gangadhara R Sareddy, Rajeshwar R Tekmal, Ratna K Vadlamudi. The role of PELP1-TFAP2C crosstalk in mediating endocrine-therapy-resistance in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-14.