RAB5A IN THE CONTROL OF MAMMARY EPITHELIAL MORPHOGENESIS AND MOTILITY

Endocytosis has been originally considered as a fundamental mechanism involved in the internalization of nutrients and membrane-bound molecules into the cell. However, recent studies demonstrate that endocytosis is a more complex network that regulates the delivery of specific time- and space-resolved signals to the cell. The small GTPase RAB5A, a master regulator of endocytosis, is emerging as a key player in tumor progression and metastatic dissemination. Consistently, the expression of RAB5A has been shown to promote a tumor mesenchymal invasive program and to correlate with the metastatic potential of different tumors. In Drosophila, however, loss-of-function mutant of RAB5 transforms imaginal disc epithelia into highly proliferative tissues, pointing to a tumor suppressor function of RAB5. To dissect the complex role of this GTPase in tumor development, we investigated the impact of RAB5A deregulation on MCF-10A, an immortalized non-transformed mammary epithelial cell line that forms hollow acinar-like spheroids recapitulating the morphogenesis of the human mammary gland, when cultured on 3D reconstituted basement membrane. We generated stable and inducible MCF-10A cells expressing either RAB5A WT or its dominant negative form (RAB5AS34N). We found that the expression of RAB5AS34N is sufficient to sustain MCF-10A cells proliferation in the absence of EGF. We identified Amphiregulin, a known EGFR ligand, as the secreted diffusible factor responsible for EGF-independent proliferation. Conversely, the expression of RAB5A WT delayed cell 15 cycle progression of cells grown in 2D, albeit it promoted the formation of hyper- proliferative acini when these cells were grown in 3D, without affecting acini morphological architecture or polarity establishment. Thus, RAB5A may either be implicated in growth factor independent growth, or may promote proliferation in 3D. Both clinical data and in vitro studies showed that RAB5A is required for invasion and metastasis formation, suggesting its involvement in tumor progression. In particular, the overexpression of RAB5A has been shown to be predictive of aggressive behavior and metastatic ability in human breast cancer. To further explore this function, we investigated the role of RAB5A in MCF-10A cells motility. We demonstrated that RAB5A expression does not affect single cell migration, but specifically enhances collective locomotion, the typical motility mode frequently observed in invasive cancer of epithelial origin, such as breast carcinoma. Indeed, RAB5A expression promotes increased speed and coordination of the epithelial cell sheet motility, related both to the increase in the area and persistence of migratory protrusions in cells at the leading front, and possibly to a tightening of cell-to- cell contacts, which improves cohesiveness of the migrating epithelial sheet. In conclusion, our data suggest a temporally distinct dual role of RAB5A in tumor development. On one hand, RAB5A may exploit a tumor suppressor function controlling epithelial tissue morphogenesis and homeostasis, which impairment may induce tumor initiation. On the other hand, RAB5A promotes not only a mesenchymal program of individual cell invasion, as previously shown by our group, but also it enhances coordinated collective epithelial migration, thus promoting cancer progression and dissemination.