Abstract P4-21-22: Oncogenic potential of ERBB3 mutations in human mammary epithelial cells

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Abstract Activation of the HER (ErbB) family of receptor tyrosine kinase signaling is common in patients with metastatic breast cancer. HER2, a member of ErbB family is over-expressed in 25% of breast cancers. A recent large-scale genomics study reported that HER3 (ErbB3) mutations play a significant role in the development of acquired resistance in breast cancer patients. We are examining the role of HER3 mutations in the context of HER2-driven and ER-driven breast cancers, both in vitro and in vivo. We introduced a series of HER3 mutations (F94L, G284R, D297Y, D313H, K329T, T355I, L792V, E1261A), identified in breast cancer patients, using site-directed mutagenesis. Mutant HER3 constructs were subcloned into the Gateway-compatible lentiviral expression vector pDONR223-ErbB3 which were subsequently subcloned into the Gateway-compatible lentiviral pLX302 destination vector. Stable cell lines were generated in MCF10A and MCF10A/HER2 and ER+ MCF-7 and T47D breast cancer cells using lentiviral transduction. We confirmed the presence of HER3 constructs by digestion with BsrGI restriction enzyme and v5-tagged protein expression. We evaluated the oncogenic potential of these cell lines using various cell based assays. Out of the above listed mutations, T355I mutation was transforming in the breast cancer cell lines. In parallel experiments, we tested the effect of knocking down HER3 in tumor cell lines that harbor endogenous HER3 mutations. The HER3 K742E mutation occurs endogenously in the IGROV-1 (ovarian) and P262H & V104M in SNU-407 (colorectal), A232V in SNU-1040 (colorectal), N126K and R667H in HCT-15 (colorectal) cancer cell lines. We transfected the cells with either HER3 or control siRNA and determined that each cell line had a reduction in proliferation with knockdown of HER3. These data further suggest the potential for these HER3 mutations to be oncogenic.Furthermore, we investigated the signal transduction pathways in wild-type (wt) and mutant (mut) HER3 in ER+ MCF-7 and T47D as well as MCF10A and MCF10A/HER2 breast cancer cells using clinically relevant inhibitors against ER and HER2. In ongoing experiments, the wt and mut ER+ MCF-7 and T47D stable cells are subjected to ER specific inhibitors, fulvestrant or 4-hydroxytamoxifen. In separate experiments, MCF10AHER2 cells are being treated with the HER2 inhibitor, lapatinib and HER2-mediated transformation is being assessed by colony formation, cell migration/invasion and three-dimensional growth assays. We are also investigating the signaling pathways responsible for the oncogenic transformation of these cell lines. Accordingly, cell lysates prepared from MCF-7 and T47D or MCF10A/HER2 are being analyzed for Akt, Erk, and ErbB activation. These studies will provide the first systematic assessment of how mutations in HER3 affect response of HER2+ or ER+ breast cancers to clinically relevant inhibitors, using a library of naturally occurring HER3 mutants. Citation Format: Mishra R, Yuan L, Solomon T, Garrett JT. Oncogenic potential of ERBB3 mutations in human mammary epithelial cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-22.