Disrupting oncogenic signaling in triple negative breast cancer: The interplay of Cx43, Pyk2, and Src in MDA-MB-231 cells.

Triple-negative breast cancer (TNBC) lacks targeted therapies and is driven by dysregulated signaling networks that promote migration, invasion, and survival. Connexin43 (Cx43), a gap junction protein essential for maintaining normal mammary epithelial homeostasis, becomes aberrantly phosphorylated and mislocalized in breast cancer, contributing to disease progression. Because the tyrosine kinases Pyk2 and Src regulate Cx43 and multiple pro-tumorigenic pathways, we investigated whether their combined inhibition could suppress malignant behaviors in TNBC. In MDA-MB-231 cells, the Pyk2 inhibitor PF4618433 and Src inhibitor Saracatinib modestly reduced metabolic activity at high concentrations; however dual treatment produced a dose-dependent and synergistic reduction in viability. In migration and invasion assays, each inhibitor reduced motility, however dual inhibition produced the strongest suppression. Cx43 knockdown impaired baseline migration and invasion and altered the response to Pyk2/Src inhibition, indicating that Cx43 modulates sensitivity to these agents. PF4618433 increased Cx43 plaque formation without changing total protein levels. Mechanistically, Pyk2 inhibition reduced phosphorylation of Cx43 at Y265 and decreased levels of TAZ, p-Erk1/2, p130Cas, and Notch1, whereas Src inhibition only reduced p-Erk1/2. Dual treatment did not further decrease these signaling nodes but nonetheless produced stronger functional inhibition of viability and motility, and the shared regulation of p-Erk1/2 by Pyk2 and Src may help explain how compensatory Pyk2 activation limits the effectiveness of Src-targeted therapies. Together, these findings show that coordinated Pyk2 and Src inhibition restores Cx43 organization and disrupts multiple malignant traits in TNBC cells, supporting this combination as a promising therapeutic strategy.