Despite the increase in patient survival rates promoted by increased screening and prevention efforts, much faster tumor genome sequencing and developed smart targeted therapies, de novo or acquired chemoresistance remains to be a significant factor for treatment failure in breast cancer therapeutics. Conventional chemotherapy, radiotherapy as well as targeted therapies activate mitochondrial cell death machinery to eliminate cancer cells. BCL‐2 protein family members regulate mitochondrial cell death pathway by controlling mitochondrial outer membrane permeabilization. Neratinib and dacomitinib are potent and irreversible pan‐EGFR inhibitors, which block their autophosphorylation and downstream signaling. Moreover, neratinib and dacomitinib have been shown to activate cell death in HER2‐overexpressing cell lines. The aim of our study was to identify molecular pathways responsible for panHER2 inhibitor resistance in HER2+ breast cancer cells.
The expression of EGFR and BCL‐2 protein family members was determined by immunoblotting and qPCR. CellTiter‐Glo was used to measure cell viability and AnnexinV/PI staining and flow cytometer was used to evaluate apoptotic response. BH3 profiling was used to determine the apoptotic blocks and mitochondrial cell death priming in breast cancer cells.
Here we showed that increased MCL‐1 and decreased BIM mediate resistance to neratinib in ZR‐75‐30 and SKBR3 cells while increased BCL‐XL and BCL‐2 and decreased BIM promoted neratinib resistance in BT474 cells. Cells were also cross-resistant to dacomitinib. BH3 profiles of HER2+ breast cancer cells efficiently predicted anti‐apoptotic protein dependence and development of resistance to panHER2 inhibitors. Adding specific ERK1/2 inhibitor SCH772984 to neratinib or dacomitinib led to increased apoptotic response in SKBR3 and ZR‐75‐30 cells, but we did not detect a similar response in BT‐474 cells. Reactivation of ERK1/2 was primarily responsible for acquired resistance in SKBR3 and ZR‐75‐30 cells. Intriguingly, both ERK1/2 and Akt/NFkappaB pathways were responsible for neratinib resistance in BT474 cells.
Our results showed that different mitochondrial apoptotic blocks mediated acquired panHER2 resistance in HER2+ breast cancer cell lines as well as highlighted the potential of BH3 profiling assay in prediction of panHER2 resistance in breast cancer cells.
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All authors have declared no conflicts of interest.