Abstract B16: Role of RasGRF2 in AnxA6-mediated growth of TNBC cells

Abstract

Abstract Introduction: The mechanisms by which annexin A6 (AnxA6) inhibits anchorage-independent tumor cell growth but promotes tumor cell motility remain unclear. Using whole-genome gene expression profiling, we identified RasGRF2 (GRF2), a Ca2+-activated RasGEF, as one of the most upregulated genes following downregulation of AnxA6 in breast cancer (BC) cells. Here, we examined the role of GRF2 as a critical RasGEF in AnxA6-meditated BC cell growth. Methods and Results: We show that the cellular levels of GRF2 are relatively low in AnxA6-high mesenchymal-like BC cells but higher in AnxA6-low basal-like BC cells. We also show that loss of AnxA6 in AnxA6-high BC cells is associated with early onset and rapid growth of xenograft tumors, while upregulation of AnxA6 in AnxA6-low BC cells is associated with a decrease in growth of xenograft tumors. To explore the reciprocal expression of GRF2 and AnxA6, we demonstrate that ionomycin-induced surge in intracellular Ca2+ led to GRF2 downregulation and that AnxA6, GRF2 and Ras proteins are within the same complex. We demonstrate that upon activation of EGFR, GRF2 is rapidly degraded and that the basal GRF2 levels were restored as the receptor activity decreased. The activation/degradation of GRF2 was followed by the activation of ERK1/2, was not affected by chelation of intracellular Ca2+ with BAPTA, but GRF2 was stabilized by chelation of extracellular Ca2+ with EGTA. Lapatinib treatment efficiently blocked the activation of EGFR but did not block the degradation of GRF2. Finally, we show that downregulation of GRF2 and inhibition of EGFR led to a greater inhibition of tumor cell growth. Conclusion: Together, these data identify RasGRF2 as a major effector of AnxA6-dependent breast tumor growth and that reduced cellular levels of GRF2 imply active RasGEF. Combined inhibition of GRF2 and EGFR may be a viable alternative treatment for triple-negative BC. Citation Format: Diva S. Whalen, Sarrah E. Widatalla, Olga Y. Korolkova, Stephen D. Williams, Heather K. Beasley, Amos M. Sakwe. Role of RasGRF2 in AnxA6-mediated growth of TNBC cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B16.