Abstract 5471: Contextual synthetic lethality in human triple negative breast cancer cells involving a novel interaction between DNA repair proteins EGFR, PARP1, and BRCA1
Authors: Stanley Jennifer Anne , Nowsheen Somaira , Cooper Tiffiny , Forero Andres , LoBuglio Alfred , Yang Eddy 
Published: May 2015

Background/Objectives:

Few therapeutic options are effective for triple negative breast cancers (TNBCs) necessitating novel approaches. We previously reported a novel synthetic lethality with combined EGFR and PARP inhibition due to a homologous recombination (HR) repair defect induced EGFR inhibition. The mechanism of the HR deficiency involved disruption of EGFR/BRCA1 interaction and sequestration of EGFR and BRCA1 to the cytosol. In this study, we have extended these findings to encompass multiple subtypes of triple negative breast cancer and EGFR inhibitors. Additionally, we further investigated the mechanisms by which EGFR regulates HR DNA repair.

Methods:

TNBC cells, including MDA-MB-231 (mesenchymal stem-like), MDA-MB-468 (basal-like), HCC1187 (immunomodulatory), BT549 (mesenchymal), MDA-MB-453 (luminal androgen receptor), were assessed for cytotoxicity and cell viability following EGFR inhibition (lapatinib, erlotinib, or cetuximab), PARP inhibition, or both by colony formation or ATPlite assays. MALDI-TOF mass spectroscopy was utilized to ascertain protein-protein interaction. To confirm interaction data and determine subcellular location we utilized immunoprecipitation and subcellular fractionation. To determine if interactions were dependent on DNA, lysates were treated with Ethidium Bromide and DNAse prior to immunoprecipitation.

Results:

EGFR inhibition with lapatinib, cetuximab, or erlotinib induced a contextual synthetic lethality with the PARP inhibitor, veliparib, in vitro in multiple TNBC subtypes, including MDA-MB-231 (mesenchymal stem-like), MDA-MB-468 (basal-like), HCC1187 (immunomodulatory), BT549 (mesenchymal), MDA-MB-453 (luminal androgen receptor). Interestingly, mass spectroscopy reaffirmed EGFR interaction with BRCA1 but also identified a novel interaction between EGFR and PARP1. These interactions were not found upon EGFR inhibition with lapatinib. Furthermore, we validated with immunoprecipitation and in vitro protein binding assays EGFR, BRCA1, and PARP1 protein-protein interactions that were disrupted upon EGFR inhibition. Additionally, these proteins were found to interact in the nucleus, independent of DNA. Lastly, EGFR inhibition also reduced nuclear EGFR and BRCA1 without significant changes in PARP1 localization.

Conclusions:

These results reveal a contextual synthetic lethality between combined EGFR and PARP inhibition in multiple subtypes of TNBC, suggesting potential generalizability of this combination in patients with TNBC. Furthermore understanding the mechanism by which EGFR regulates DNA repair may provide novel targets to confer other contextual synthetic lethalities that may be exploited to treat the aggressive TNBCs.

Citation Format: Jennifer Anne Stanley, Somaira Nowsheen, Tiffiny Cooper, Andres Forero, Alfred LoBuglio, Eddy Yang. Contextual synthetic lethality in human triple negative breast cancer cells involving a novel interaction between DNA repair proteins EGFR, PARP1, and BRCA1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5471. doi:10.1158/1538-7445.AM2014-5471