Abstract B015: Knock-in of PIK3CA mutations in MCF10A mammary epithelial cells modifies their proteomic profile to resemble basal-like breast cancer and stimulate EGFR-dependent cell proliferation
Authors: Young Christian D. , Zimmerman Lisa J. , Whitwell Corbin A. , Hanker Ariella B. , Stricker Thomas , Brantley-Sieders Dana M. , Park Ben Ho , Liebler Daniel C. , Cook Rebecca S. , Arteaga Carlos L. 
Published: May 2015

Abstract
PIK3CA, the gene encoding the p110α catalytic subunit of phosphatidylinositol-3 kinase (PI3K), is frequently mutated in breast cancer. Activating mutations in PIK3CA are known to transform mammary epithelial cells (MECs). Genomic knock-in of the two most frequent hot-spot PIK3CA mutations (E545K or H1047R) into MCF10A MECs resulted in growth factor-independent proliferation. Shotgun LC-MS/MS mass spectrometry analysis of wild type, E545K and H1047R MCF10A cell lysates revealed 73 proteins uniquely altered in the both mutant cell lines compared to wild type cells. KEGG pathway analysis demonstrated that PIK3CA mutant cells have elevated levels of proteins involved in focal adhesion, ECM-receptor interactions and actin cytoskeleton regulation. Nearly half the proteins upregulated in the mutant cells are secreted or involved in extracellular matrix (ECM) processing or signaling. The EGFR ligand amphiregulin was five-fold higher in the conditioned media harvested from PIK3CA mutant cells as compared to wild type cells. The conditioned media of PIK3CA mutant cells, but not that of wild-type cells, was sufficient to stimulate proliferation and EGFR phosphorylation in wild type MCF10A cells. Proliferation and EGFR activation were inhibited with an amphiregulin-neutralizing antibody or with EGFR neutralizing antibodies and kinase inhibitors. PIK3CA mutant cells downregulated PTPRF, a receptor tyrosine phosphatase. EGFR signaling and proliferation were stimulated in wild type cells when PTPRF was downregulated with siRNA, suggesting that PIK3CA mutant MCF10A cells activate EGFR-dependent proliferation by suppression of a negative regulatory phosphatase and increased secretion of amphiregulin.
The expression of transglutaminase 2, peroxidasin, fibronectin, integrin α5, laminin β3, laminin γ2, thrombospondin and EphA2, eight proteins upregulated in PIK3CA mutant MCF10A cells, was evaluated in a panel of breast cancer cell lines. All eight proteins were more highly expressed in basal-like compared to luminal-like breast cancer cell lines. The expression of PTPRF, which is decreased in PIK3CA mutant MCF10A cells, was lower in basal-like cell lines. Interrogation of microarray data demonstrated that the RNA signal of proteins upregulated in mutant PIK3CA MCF10A cells correlated with decreased relapse-free survival in basal-like, but not in luminal-like breast cancer. siRNA-mediated silencing of peroxidasin, laminin γ2, EphA2, integrin β1 or amphiregulin reduced the proliferation of PIK3CA mutant MCF10A cells, suggesting that these proteins are necessary for maintenance of the transformed phenotype. siRNA-mediated silencing of peroxidasin and EphA2 proteins also reduces the proliferation of basal-like breast cancer cells. Our proteomic analysis of PIK3CA mutant MCF10A cells revealed mechanisms of autocrine and paracrine induced proliferation and alterations mostly limited to basal-like breast cancer cells. These may serve as therapeutic targets in this subtype of breast cancer.
Citation Format: Christian D. Young, Lisa J. Zimmerman, Corbin A. Whitwell, Ariella B. Hanker, Thomas Stricker, Dana M. Brantley-Sieders, Ben Ho Park, Daniel C. Liebler, Rebecca S. Cook, Carlos L. Arteaga. Knock-in of PIK3CA mutations in MCF10A mammary epithelial cells modifies their proteomic profile to resemble basal-like breast cancer and stimulate EGFR-dependent cell proliferation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B015.