Breast cancer driven by estrogen receptor (ER) is responsible for approximately 60-70% of cases among women. Selective inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) and ER signaling is now standard-of-care therapy for ER positive (ER+) metastatic breast cancer. CDK4/6 inhibitors, including palbociclib (PD), used in combination with endocrine therapy have shown improvement in progression-free survival compared to endocrine therapy alone in the metastatic setting. However, the inevitable development of acquired resistance significantly limits the efficacy of this targeted therapy. In ER+ breast cancer resistant cells, rewired signaling driven by different oncogenes, including the epidermal growth factor receptor (EGFR), overcomes the inhibition of CDK4/6 by PD, which, in turn, allows cell cycle progression contributing to PD-resistance. We investigated the dependency of ER+ breast cancer cell lines sensitive and resistant to PD on EGFR signaling in vitro. We evaluated the expression and activity of EGFR in two different matching pairs of ER+ palbociclib-sensitive (pS) and palbociclib-resistant (pR) breast cancer cell lines (MCF7 and T47D) and observed elevated EGFR expression in MCF7/pR cells. Increased EGFR expression in MCF7/pR cells correlated with elevated ER alpha phosphorylation at S118 and S167 sites, suggesting a direct cross-talk between EGFR and ER signaling in PD-resistant cells. Serum starvation significantly decreased EGFR activity, cdk2 and cyclin D1 expression in both MCF7 and T47D PD-resistant cells compared to the parental cell lines, confirming their dependency on the presence of growth factors. Notably, stimulation with EGF promoted ER phosphorylation and expression of cell cycle related targets: cdk2, cyclin E and wee1 in both MCF7 and T47D cell lines sensitive and resistant to PD. Furthermore, stimulation with estrogen promoted EGFR activation in MCF7/pS and MCF7/pR cells, indicating a direct cross-regulation between these molecular targets. Further, exposure to the EGFR inhibitor erlotinib dramatically inhibited the proliferation of MCF7/pR cells compared to the parental cells, suggesting that EGFR could be an important bypass signaling regulator contributing to resistance to PD. We found that dual treatment with erlotinib and PD significantly inhibited Rb phosphorylation in MCF7/pR cells improving the effect of PD on cell cycle arrest. Moreover, dual treatment of MCF7/pR cells attenuated ER expression suggesting the higher dependency of ER signaling on EGFR activity in resistant cells. Taken together, our findings suggest that EGFR signaling is a mechanism of resistance in ER+ cell lines with acquired resistance to PD in vitro and that EGFR targeting can be a promising approach to improve palbociclib efficacy in breast cancer patients.

Citation Format: Nadiia Lypova, Lilibeth Lanceta, Susan M. Dougherty, Jason A. Chesney, Yoannis Imbert-Fernandez. EGFR signaling as a mechanism of resistance to CDK4/6 inhibitors in Palbociclib-resistant ER+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1772.