LAUSR

We sought to determine if patient-identified mutations in HER3 enhance ER-mediated transformation in human mammary epithelial cells (MCF-7 and T47D). Furthermore, we examined if HER3 mutations confer resistance to ER-specific inhibitors (fulvestrant and 4-hydroxytamoxifen) in ER+ breast cancer cells. A series of HER3 mutations identified in patient breast tumors (F94L, G284R, D297Y, D313H, K329T, T355I, L792V, and E1261A) were introduced and stable cell lines were generated in ER+ T47D and MCF-7 cells using lentiviral transduction. We identified the HER3 T355I mutant that had significantly higher cell proliferation than wild-type (wt) HER3 in ER+ MCF-7 and T47-D cells using several cell-based assays. We sought to determine whether this oncogenic T355I mutant renders resistance to antiestrogen therapy. These mutations counteracted the effect of the ER inhibitor 4-hydroxytamoxifen but not fulvestrant, indicating a possible role in antiestrogen therapy. ER+ cells overexpressing T355I have increased p-HER3 and p-ERK1/2 expression compared to wt or empty vector (ev) control. However, there was no major change in Akt signaling in HER3 T355I mutant as compared to control. Phospho-RTK array results indicate that ER+ T47D T355I whole-cell lysate had increased p-ERBB4 and ER+ MCF-7 T355I cells had enhanced p-EGFR expression, suggesting possible downstream activation of MAPK signaling via two distinct activation routes in ER+ T47D and MCF-7 cells. ER+ MCF-7 and T47D cells overexpressing T355I HER3 mutant were subjected to the ERBB family inhibitor lapatinib in presence or absence of ER inhibitor fulvestrant and specific ERK1/2 inhibitor, SCH772984. The data indicated that combined treatment of lapatinib and fulvestrant had reduced cell proliferation as compared to individual treatment groups both in ER+ cells overexpressing wt and HER3 T355I mutant. We also observed a significant reduction in cell proliferation when these cells were subjected to co-treatment of lapatinib and SCH772984 as compared to individual treatments. These data indicate that induced transforming activity observed in HER3 T355I mutant is via ERBB4/MAPK pathway in T47D cells and EGFR/MAPK in MCF-7 cells. Similar results were obtained in 3D-Matrigel assay under above treatment conditions. The Western blot data indicated that Cyclin D1 and p-ERK1/2 expression is altered in response to combined treatment of lapatinib with or without fulvestrant and SCH772984 in ER+ T47D and MCF-7 cells, indicating that Cyclin D1 mediates signaling downstream of the MAPK pathway. However, Akt signaling is not affected when cells were treated with EGFR family and ERK1/2 inhibitors or combination of both. We also observed ERα expression is upregulated in ER+ T47D and MCF-7 cells overexpressing HER3 mutant. Experiments are ongoing to determine possible crosstalk between HER3 and ER signaling in ER+ cells overexpressing HER3 mutant. Additionally, we tested the effect of knocking down HER3 in cell lines harboring endogenous HER3 mutations. Overall, this study provides the first systematic assessment of how mutations in HER3 affect response of ER+ breast cancers to clinically relevant inhibitors. Citation Format: Rosalin Mishra, Long Yuan, Thomas Solomon, Samar Alanazi, Joan T. Garrett. Role of EGFR/ERBB4 and MAPK signaling in modulating oncogenic potential of ER+ breast cancer cells overexpressing HER3 mutant [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B44.