A Rab4‐Regulated Endosomal Compartment Prolongs EGFR Activation in Breast Cancer Cells

Abstract

Early sorting endosomes are responsible for the proper trafficking of membrane-bound receptors that can drive cancer progression. However, the morphology, cargo sorting and signaling of early endosomes remains poorly understood in breast cancer. We have identified a novel population of enlarged early endosomal vesicles in breast cancer cells and human tumor xenografts (MDAMB231 and T47D) but not in non-cancerous epithelial cells (MCF10A) using confocal and super-resolution 3D dSTORM microscopy. A detailed quantitative analysis of the whole-cell endosomal morphology, cargo sorting, maturation, and regulatory Rab-GTPases associated with this compartment in cancerous breast epithelial cell lines was performed using Airyscan microscopy and 3D rendering approaches. This analysis showed that highly aggressive triple negative MDAMB231 breast cancer cells have larger but significantly fewer EEA1-enriched early sorting endosomes compared to non-cancerous MCF10A cells. Furthermore, live-cell time-lapse imaging showed that, despite normal transferrin recycling, the epidermal growth factor (EGF) and transferrin are found together in these large endosomal vesicles following internalization in MDAMB231 but not in MCF10A cells. Most importantly, these large EEA1-positive MDAMB231 endosomes exhibited prolonged EGF-induced activation of EGFR (p1068). We investigated the role of Rab4-mediated changes on endosome structure and function and found that over-expression of wild-type Rab4 in non-cancerous MCF10A cells produced similar EEA1-positive enlarged early-endosomes with prolonged EGF induced EGFR activation (p1068). Rab4 mutants in dominant-negative or constitutively-active state did not elicit the same effects as the wild-type Rab4, indicating that the on-off cycling of Rab4 is critical for the regulation of endosomal size and number. These large endosomes contain adjacent Rab4 and Rab11 domains, are located primarily at the perinuclear region and can undergo actin-associated budding events to recycle cargo out towards the plasma membrane. Altogether, this extensive characterization of early-endosomes in breast cancer cells has identified a novel Rab4 regulated enlarged endocytic compartment leading to prolonged EGFR activation. Interestingly, our data suggests that a population of “pre-existent” large EEA1-containing endosomes is present in some breast cancer cells, challenging the current views of endosome biogenesis. This research is essential to determining receptor regulation in translational breast cancer models which can impact the optimization of anti-cancer human therapies. Alterations in endocytic regulation in cancer cells may be key to understanding the adaptability of cancer cells and lead to new therapeutic targets. Support or Funding Information This work was supported by the National Institute of Health (NIH) R01 BRG CA20772 Breast cancer cells have altered early-endocytic morphology Representative images from whole-cell Airyscan microscopy assay to quantitatively characterize early-endocytic morphology (with respect to whole-cell morphology) in non-cancerous breast epithelial cells (MCF10A)and breast cancer cell lines (MDAMB231 and T47D) with 3D rendering techniques. Example image from live-cell 4D imaging of endosomal dynamics that facilitates our comprehensive characterization of Rab4-regulated endosomes in breast cancer cells. Prolonged EGF-induced EGFR activation (p1068) in MDAMB231 compared to MCf10A This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.