Epsin3 (EPN3) belongs to the Epsin family of endocytic adaptors, which in humans comprises 3 members: Epsin1 (EPN1), Epsin2 (EPN2) and EPN3. Differently from the other members, EPN3 is expressed at very low levels in almost all tissues and its function is unknown. Previous data have shown that EPN3 is overexpressed in about 47% of breast cancers, correlating with an aggressive tumor phenotype and increased risk of distant metastasis. At the cellular level, in vitro studies performed on MCF10A, a non-tumoral immortalized breast epithelial cell line, showed that overexpression of EPN3 (but not of EPN1) induces the appearance of fibroblast-like properties. Here, we found that EPN3 overexpression is able to induce the so-called partial epithelial-to- mesenchymal transition, pEMT, a condition characterized by a mix of epithelial and mesenchymal features that makes cells capable of easily converting between an epithelial and a mesenchymal state. Mechanistically, we showed that EPN3 interacts with the endocytic machinery (clathrin heavy chain and AP2) and with components of adherens junctions (p120, β- catenin, α-catenin), and increases E-Cadherin endocytosis, suggesting an alteration of E-Cadherin turnover, known to be necessary to allow EMT. Indeed, the increased E-Cadherin endocytosis activates the pEMT through the β-catenin/TCF4 pathway. Moreover, EPN3 overexpression in MCF10A cells upregulates the TGFβ pathway and synergizes with it, both in TGFβ signaling and in induction of E-Cadherin endocytosis. Interestingly, EPN3-induced pEMT generates a TGFβ- dependent loop that can be reverted by TCF4 or TGFβR1 KD, but it is not addicted to EPN3 expression once established. Interestingly, endogenous EPN3 expression is also implicated in the regulation of the TGFβ pathway and E-Cadherin endocytosis, indicating that EPN3 function upon overexpression is an exaggeration of its physiological function. In vivo, we generated an EPN3 inducible knock-in mouse, which overexpresses the transgene specifically in epithelial cells. High EPN3 levels did not cause significant differences during mammary gland development in puberty, but instead increased secondary and tertiary branching morphogenesis in adult virgin mice, increasing N- Cadherin expression in the epithelial ducts, supporting in vitro findings in MCF10A cells. Together, these data show that EPN3 is a novel prognostic factor for breast cancer metastasis acquisition, acting directly on E-Cadherin endocytosis and turn-over, resulting in a pEMT highly plastic and dynamic and that could recapitulate also branching alterations in mouse mammary gland. Moreover, preliminary evidences suggest that the upstream endocytic event is able to induce a transcriptional activation that becomes independent of the first genetic lesion, a possibility that would have a profound impact in breast cancer treatment.
CHARACTERIZATION OF EPSIN3 FUNCTION IN THE ACQUISITION OF A PARTIAL EMT STATE IN BREAST CANCER THROUGH E-CADHERIN ENDOCYTOSIS / G. Giangreco ; added supervisor: S. Sigismund ; internal advisor: G. Scita ; supervisor: P. P. Di Fiore. - Milano : Università degli studi di Milano. DIPARTIMENTO DI ONCOLOGIA ED EMATO-ONCOLOGIA, 2019 Jan 28. ((30. ciclo, Anno Accademico 2018.
|Titolo:||CHARACTERIZATION OF EPSIN3 FUNCTION IN THE ACQUISITION OF A PARTIAL EMT STATE IN BREAST CANCER THROUGH E-CADHERIN ENDOCYTOSIS|
|Data di pubblicazione:||28-gen-2019|
|Parole Chiave:||EPN3; epsin3; epsin; endocytosis; clathrin; E-Cadherin; adherens junctions; beta-catenin; EMT; pEMT; TGF-beta; metastasis; MCF10A; breast cancer; mouse model; N-Cadherin; Vimentin|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||CHARACTERIZATION OF EPSIN3 FUNCTION IN THE ACQUISITION OF A PARTIAL EMT STATE IN BREAST CANCER THROUGH E-CADHERIN ENDOCYTOSIS / G. Giangreco ; added supervisor: S. Sigismund ; internal advisor: G. Scita ; supervisor: P. P. Di Fiore. - Milano : Università degli studi di Milano. DIPARTIMENTO DI ONCOLOGIA ED EMATO-ONCOLOGIA, 2019 Jan 28. ((30. ciclo, Anno Accademico 2018.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/giangreco-giovanni_phd2019-01-28|
|Appare nelle tipologie:||Tesi di dottorato|