FIGURE 7 from Signaling Pathway Alterations Driven by BRCA1 and BRCA2 Germline Mutations are Sufficient to Initiate Breast Tumorigenesis by the PIK3CA^H1047R Oncogene

Immortalized BRCA1- or BRCA2-mutant cells display elevated NFκB activity. A, PIK3CAH1047R distinctly influences differentiation properties of immortalized BRCA1 and BRCA2 mutation carriers. Vector control or PIK3CAH1047R-expressing breast epithelial cell lines from non-carriers (KTB34 and KTB39) and BRCA1 or BRCA2 mutation carriers were stained with indicated antibodies and characterized by flow cytometry (n = 3). Representative data are shown. PIK3CAH1047R robustly increased EpCAM expression and increased differentiated phenotype of BRCA1-mutant cells. CD49f+/EpCAM+ cells increased from 12% to 36% whereas CD49f+/EpCAM− cells decreased from 80% to 34% upon PIK3CAH1047R overexpression. B, BRCA1 mutant cells display elevated phosphorylation of p65, a NFκB subunit, which indirectly suggests activation of NFκB. Expression levels of PIK3CA in cells transduced with PIK3CA-mutant virus are shown. Regular growth media condition had to be used to detect PIK3CA-mutant overexpression because of robust induction of endogenous PIK3CA upon serum starvation. An antibody that preferentially recognizes PIK3CAH1047R mutant was used in the Western blot analysis. The same extract was used to measure pAKT(S473) and AKT to ensure that PIK3CAH1047R is functional in transduced cells. C, Approximately 30% of breast cancers in BRCA1 or BRCA2 mutation carriers carry PIK3CA mutations. Data were generated using cBioportal (54). D, Immortalized BRCA1 or BRCA2 mutant cells but not cells from non-carriers are sensitive to DMAPT. PIK3CAH1047R-overexpressing cells, irrespective of BRCA mutation status, were sensitive to DMAPT (statistical test used—one-way ANOVA). E, Immortalized cell lines from BRCA1 or BRCA2 mutation carriers express higher levels of MIR205HG (statistical test used—unpaired t test). Note that PIK3CAH1047R, which robustly induced differentiation of BRCA1-mutant cells, reduced MIR205HG levels in these cells. P values, *<0.05; **<0.01. Immortalized BRCA1/2-mutant cells express BRCA1 and BRCA2 transcripts at variable levels with BRCA2-mutant cells expressing significantly higher levels of BRCA1 transcripts compared with non-carrier cells (statistical test used—one-way ANOVA).