Complex Rab4-mediated regulation of endosomal size and EGFR activation

Abstract

Early sorting endosomes are responsible for the trafficking and function of transferrin receptor (TfR) and epidermal growth factor receptor (EGFR). These receptors play important roles in iron uptake and signaling and are critical for breast cancer development. However, the role of morphology, receptor composition, and signaling of early endosomes in breast cancer remains poorly understood. A novel population of enlarged early endosomes was identified in breast cancer cells and tumor xenografts but not in non-cancerous MCF10A cells. Quantitative analysis of endosomal morphology, cargo sorting, EGFR activation and Rab GTPase regulation was performed using super-resolution and confocal microscopy followed by 3D rendering. MDAMB231 breast cancer cells have fewer, but larger EEA1-positive early endosomes compared to MCF10A cells. Live-cell imaging indicated dysregulated cargo sorting, since EGF and Tf traffic together via enlarged endosomes in MDAMB231, but not in MCF10A. Large EEA1-positive MDAMB231 endosomes exhibited prolonged and increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A over-expression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knock-down of Rab4A lead to increased endosomal size in MCF10A but not in MDAMB231 cells. Nevertheless, Rab4A knock-down resulted in enhanced EGF-induced activation of EGFR-p1068 in MDAMB231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation.