EGFR-to-Src family tyrosine kinase switching in proliferating-DTP TNBC cells creates a hyperphosphorylation-dependent vulnerability to EGFR TKI

Abstract

Triple Negative Breast Cancer (TNBC) is the most aggressive type of breast malignancy, with chemotherapy as the only mainstay treatment. TNBC patients have the worst prognoses as a large fraction of them do not achieve complete pathological response posttreatment and develop drug-resistant residual disease. Molecular mechanisms that trigger proliferation in drug-resistant chemotherapy residual TNBC cells are poorly understood due to the lack of investigations using clinically relevant cellular models. In this study, we have established TNBC subtype-specific cellular models of proliferating drug-tolerant persister (PDTP) cells using different classes of chemotherapeutic agents that recapitulate clinical residual disease with molecular heterogeneity. Analysis of total phospho-tyrosine signals in TNBC PDTPs showed an enhanced phosphotyrosine content compared to the parental cells (PC). Interestingly, using mass-spectrometry analysis, we identified a dramatic decrease in epidermal growth factor receptor (EGFR) expression in the PDTPs, while the presence of hyperactivated tyrosine phosphorylation of EGFR compared to PC. Further, we show that EGFR has enhanced lysosomal trafficking in PDTPs with a concomitant increase in NMyc Downstream Regulated-1 expression that colocalizes with EGFR to mediate receptor degradation. More surprisingly, we found that reduced protein levels of EGFR are coupled with a robust increase in Src family kinases, including Lyn and Fyn kinases, that creates a hyper-phosphorylation state of EGFR-Src tyrosine kinases axis in PDTPs and mediates downstream over-activation of STAT3, AKT and MAP kinases. Moreover, paclitaxel derived PDTPs show increased sensitivity to EGFR TKI Gefitinib and its combination with paclitaxel selectively induced cell death in PDTP-P TNBC cells and 3D spheroids by strongly downregulating phosphorylation of EGFR and Src with concomitant downregulation of Lyn and Fyn tyrosine kinases. Collectively, this study identifies a unique hyper-phosphorylation cellular state of TNBC PDTPs established by switching of EGFR to Src family tyrosine kinases creating a vulnerability to EGFR TKI.