Abstract 970: Mass spectrometry analysis of PIK3CA mutant mammary epithelial cells identifies EGFR as a paracrine effector of PI3K in basal-like breast cancer
Authors: Young Christian D. , Zimmerman Lisa J. , Gatza Michael L. , Morrison Meghan M. , Whitwell Corbin A. , Bhola Neil E. , Hanker Ariella B. , Stricker Thomas , Patel Premal , Brantley-Sieders Dana M. , Perou Charles M. , Park Ben H. , Liebler Daniel C. , Cook Rebecca S. , Arteaga Carlos L. 
Published: May 2015

Abstract
PIK3CA, the gene encoding the p110α catalytic subunit of phoshatidylinositol-3 kinase (PI3K), is frequently mutated in luminal- and basal-like breast cancer. We assessed proteomic changes induced by knock-in of PIK3CA activating mutations in MCF10A mammary epithelial cells (MECs). Shotgun LC-MS/MS mass spectrometry revealed 72 proteins uniquely altered in PIK3CA mutant cell lines versus isogenic wild type cells. Nearly half the upregulated proteins are secreted or involved in extracellular matrix (ECM) processing or signaling. The EGFR ligand amphiregulin (AREG) was 5-fold higher in the media conditioned by PIK3CA mutant cells compared to wild type cells. While the conditioned media of PIK3CA mutant cells, but not that of wild-type cells, stimulated proliferation, EGFR phosphorylation and ERK signaling in recipient wild type MCF10A cells, this was inhibited when recipient cells were treated with an AREG-neutralizing antibody, EGFR inhibitor (cetuximab), or MEK inhibitor (AZD6244). PIK3CA mutant cells abundantly secreted the ECM protein fibronectin, and fibronectin enhanced AREG-induced proliferation of wild type MCF10A cells. PIK3CA mutant cells downregulated PTPRF, a receptor tyrosine phosphatase. PTPRF down-regulation enhanced EGFR signaling and proliferation in wild type cells. Conversely, PTPRF overexpression decreased EGFR signaling and proliferation in PIK3CA mutant cells. These data suggest that PIK3CA mutant MCF10A cells activate EGFR-dependent proliferation by suppression of a negative regulatory phosphatase and increased secretion of AREG and fibronectin.
Eight proteins upregulated in PIK3CA mutant MECs (transglutaminase 2, peroxidasin, fibronectin, integrin α5, laminin β3, laminin γ2, thrombospondin and EphA2) were evaluated in a panel of breast cancer cells. All 8 proteins were expressed at highest levels in basal-like breast cancer cell lines over luminal and HER2-amplified cell lines. Interrogation of microarray data demonstrated that transcripts encoding proteins upregulated in PIK3CA mutant MCF10A cells correlated with decreased relapse-free survival in basal-like, but not luminal breast cancer. Interrogation of human tumor data demonstrated increased EGFR activation in PIK3CA mutant basal-like breast cancer as compared to PIK3CA wild type. Finally, a p110α-specific inhibitor, BYL719, induced EGFR ligand expression and activation of EGFR in PIK3CA mutant, basal-like breast cancer cells such that dual inhibition of EGFR and p110α with cetuximab and BYL719 induced greater anti-tumor cell activity. Our proteomic analysis of PIK3CA mutant MCF10A cells revealed mechanisms of autocrine and paracrine induced proliferation and proteomic alterations common in basal-like breast cancer cells which may serve as therapeutic targets in this subtype of breast cancer.
Citation Format: Christian D. Young, Lisa J. Zimmerman, Michael L. Gatza, Meghan M. Morrison, Corbin A. Whitwell, Neil E. Bhola, Ariella B. Hanker, Thomas Stricker, Premal Patel, Dana M. Brantley-Sieders, Charles M. Perou, Ben Ho Park, Daniel C. Liebler, Rebecca S. Cook, Carlos L. Arteaga. Mass spectrometry analysis of PIK3CA mutant mammary epithelial cells identifies EGFR as a paracrine effector of PI3K in basal-like breast cancer. [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 970. doi:10.1158/1538-7445.AM2014-970