INVESTIGATION OF THE ROLE OF THE ENDOCYTIC PROTEIN EPSIN3 IN EMT AND MAMMARY STEM CELL REGULATION IN BREAST CANCER

Endocytosis is a critical regulator of many cellular processes, including proliferation, migration, polarity, fate determination, epithelial-to-mesenchymal transition (EMT) and maintenance of the stem cell (SC) compartment. Subversion of endocytic routes plays an important role in cancer development, including breast cancer (BC). We recently uncovered an oncogenic role for the endocytic protein Epsin3 (Epn3) in BC. By screening a large cohort of BC patients, we found that the EPN3 gene is amplified in ∼10% of cases, while Epn3 protein is overexpressed in ∼50% of BCs. Epn3 overexpression correlated with poor prognosis and distant metastasis and with increased in vivo tumorigenic potential in orthotopic transplantation experiments in NOD (non-obese diabetic) - SCID (severe combined immunodeficiency) IL2RG null mice (NSG mice). The goal of the present Ph.D. thesis was to uncover the mechanisms through which Epn3 exerts its oncogenic properties in BC. To this aim, we overexpressed Epn3 in non-tumorigenic mammary epithelial cells, MCF10A, to mimic the overexpression occurring in BC. We showed that Epn3 overexpression causes a TGFβ-dependent EMT and expansion of the SC-like compartment, two events that are causally linked. Mechanistically, we found that Epn3 overexpression enhances E-Cadherin endocytosis, resulting in destabilization of cell-cell junctions and translocation of β-catenin in the nucleus. The β-catenin/TCF-LEF pathway leads to the activation of the EMT transcriptional program, consisting of the up-regulation of EMT master regulators (ZEB1, Snail, Twist), of mesenchymal markers (N-Cadherin, Vimentin, extracellular matrix (ECM) components, and proteases - Fibronectin and Serpine) and, importantly, of the transcription of TGFβ receptors and ligands. Consequent secretion of TGFβ ligands is a key event in the establishment of an autocrine TGFβ positive feedback loop, which is able to increase Epn3-dependent E-Cadherin endocytosis and TGFβ ligand production and to self-sustain EMT. This loop could be reverted only by the abrogation of the TGFβ pathway itself or of the β-catenin/TCF-LEF pathway. We also investigated the effects of Epn3 overexpression in two BC cell model systems, MCF10DCIS.com and HCC1569 and we observed that they partially recapitulate Epn3-dependent phenotypes observed in non-tumorigenic MCF10A-Epn3 cells. Indeed, although MCF10DCIS.com and HCC1569 are tumorigenic cells that have already undergone some form of EMT, they showed at least a partial cooperation between Epn3 overexpression and TGFβ stimulation in the induction of EMT. In addition, Epn3-overexpressing MCF10DCIS.com showed EMT reversion upon abrogation of the TGFβ signaling pathway (not tested for HCC1569). Importantly, Epn3 overexpression in tumorigenic HCC1569 cells was able to increase their in vivo tumorigenic potential upon orthotopic transplantation in NSG mice. In conclusion, our results identify new oncogenic properties for the endocytic protein Epn3 and suggest an endocytic-based mechanism of how it contributes to EMT, cancer stem cell expansion and breast tumorigenesis.