Abstract 2141: Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells

In this study we investigated cellular and molecular mechanisms for breast cancer signaling by EGFR and Ras proteins, independent of activating mutations. Epidermal Growth Factor (EGF) ligation to its receptor, EGFR, at the plasma membrane (PM) activates Ras small GTPases, propagating Ras mitogenic signaling. However, activating Ras mutations are not associated with breast cancers. EGFR and palmitoylated (Pa-) Ras localize to lipid rafts at the PM. Pa-Ras isotypes (H- and N-Ras, but not K-Ras) cycle through vesicle trafficking pathways between the Golgi and PM. We have found that anterograde H-Ras vesicles are sorted in the endocytic recycling compartment (ERC) into vesicles which contain the epithelial-specific transport mediator Rab25 (Rab11c), and Rab11/FIP1C (Rab-coupling protein, RCP). RCP couples Rab25 to vesicles to mediate vesicle anterograde transport. Rab25 and RCP expression are highly correlated with breast cancer progression, aggressiveness, and invasion and metastasis. Rab25, unique among the large family of human Rab genes, has been identified as a major marker and potential stimulator of breast cancers. RCP was recently shown to be a breast cancer driver oncogene, supporting the suggestion that RCP and Rab25 status may be significant clinical markers. Moreover, RCP stimulates H-Ras activation. Meta-analysis of human breast cancer cell line panels shows that while most do not harbor H- or N-Ras mutations, both RCP and Rab25 are highly over-expressed and levels correlate with resistance to EGF and Ras signaling inhibitors. We demonstrate that Rab25, RCP, H-Ras and EGFR co-localized in the ERC and in transport vesicles in MCF10A mammary epithelial cells, and that dominant negative (DN) Rab25 sequestered H-Ras and EGFR from the plasma membrane. DN Rab25 or Rab25 knockdown by shRNA inhibited EGF-induced EGFR phosphorylation, H-Ras activation, and ERK phosphorylation (ppERK), demonstrating a functional link between Rab25 activity and EGFR and H-Ras signaling. Both ER/PR-positive and -negative human breast invasive ductal carcinoma tissues showed elevated ppERK correlated with elevated Rab25 expression over normal breast tissue, whereas HER2+ tumor tissues had elevated ppERK even with low Rab25 expression. Thus, Rab25 and RCP regulate EGFR and Ras trafficking to the plasma membrane, required for mitogenic signaling and associated with tumorigenic effects, even in Ras (WT) cells. Citation Format: Allison B. Herman, Jeremy G.T. Wurtzel, Lawrence E. Goldfinger. Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2141. doi:10.1158/1538-7445.AM2015-2141