Abstract LB-219: Nuclear EGFR serves as a functional molecular target in triple-negative breast cancer.

Abstract Triple negative breast cancer (TNBC) is a subclass of breast cancer (i.e. estrogen receptor negative, progesterone receptor negative, and HER-2 negative) that has poor prognosis and very few identified molecular targets. Strikingly the majority of TNBC's overexpress the epidermal growth factor receptor (EGFR), yet EGFR inhibition has had very little clinical benefit in this setting. This suggests a gap in our knowledge of EGFR function in TNBC. Over the last decade, advances in the basic biology of EGFR in human cancer have established that the EGFR functions in two distinct signaling pathways: 1) Classical membrane bound signaling (classical EGFR pathway) and 2) nuclear signaling (nEGFR pathway). In the nEGFR pathway, published data from our laboratory suggests that the EGFR is phosphorylated by Src family kinases (SFKs) at tyrosine 1101 (Y1101) and this phosphorylation is a necessary and early event for trafficking EGFR from the membrane to the nucleus. Nuclear EGFR has been correlated with poor overall survival in breast cancer and has been shown to enhance resistance to the EGFR targeted therapies cetuximab and gefitinib. In the current study we found that TNBC cell lines were: 1) dependent on EGFR expression for proliferation as indicated by RNAi techniques, 2) resistant to cetuximab therapy and 3) harbored high levels of nEGFR and SFK activity. Further, a TNBC tissue microarray indicated that a portion of human TNBCs express nEGFR. Using molecular approaches to investigate if nEGFR and SFKs play a role in resistance to cetuximab we demonstrate that the overexpression of constitutively active Src (ca-Src) enhanced the nuclear localization of EGFR, while the overexpression of a negative regulator of Src, src-like adaptor protein (SLAP) prevented EGFR nuclear localization. SLAP not only associated with the EGFR but also led to a decrease in its phosphorylation at Y1101. Further, stable overexpression of several SFK members (Yes, Lyn, Fyn, Hck, Blk, and Fgr) in the breast cancer cell line MCF-7 led to both the activation of EGFR at Y1101 and potent increases in EGFR nuclear localization, indicating that SFKs exhibit functional redundancy in their ability to influence the nuclear translocation of the EGFR. Treatment of various cetuximab resistant TNBC cell lines with the small molecule SFK inhibitor dasatinib resulted in: 1) loss of nuclear localized EGFR, 2) increased plasma-membrane levels of the EGFR and 3) statistically significant, increases in cetuximab sensitivity. Taken together, these findings suggest targeting nEGFR, by abrogating its translocation to the nucleus via SFK inhibition, increases plasma-membrane levels of EGFR where it is susceptible to targeting by cetuximab. The combinatorial therapy of cetuximab and dasatinib results in the simultaneous targeting of both the classical and nEGFR signaling pathways, and therefore may enhance the therapeutic response of nEGFR expressing TNBCs to cetuximab. Citation Format: Toni M. Brand, Mari Iida, Neha Luthar, Matthew Wleklinski, Kellie Kostopoulos, Deric L. Wheeler. Nuclear EGFR serves as a functional molecular target in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-219. doi:10.1158/1538-7445.AM2013-LB-219