Role of Epsin3-mediated endocytosis in cell plasticity, breast cancer progression and metastasis
Epithelial-to-mesenchymal transition (EMT) is one of the mechanisms regulating metastatic dissemination of breast cancer (BC) cells. In particular, partial EMT (pEMT) states, characterized by the coexistence of epithelial and mesenchymal markers, confer the highest level of plasticity to cells: i.e., the ability to switch between invasive/mesenchymal and proliferative/epithelial modes, which is required for the dissemination of cancer cells and their subsequent colonization of distant sites. For this reason, pEMT, rather than full EMT, has been proposed to render cells more prone to metastasize to distant organs. Interestingly, pEMT is often linked to the endocytic-dependent re-localization of epithelial markers, such as E-cadherin (ECAD), and not to their transcriptional downregulation.
In this scenario, we previously discovered that the endocytic protein epsin3 (EPN3) is an oncogene in BC. It is overexpressed and amplified in BC (in ~50% of cases it is co-amplified with the oncogene ERBB2), and its overexpression correlates with distant metastasis and poor prognosis. EPN3 is able to induce pEMT states and invasive phenotypes in breast epithelial cells through an endocytic-based mechanism, regulating ECAD trafficking and causing the establishment of TGF-beta autocrine loops. Based on these results, our hypothesis is that alterations of endocytic circuitries, with EPN3 representing a case in point, may provide plasticity to cancer cells, inducing "metastable" cellular phenotypes, such as pEMT states, that favor their ability to invade the surrounding microenvironment, disseminate and colonize distant sites.
The present project aims to: i) characterize the in vivo role of EPN3 in BC development, and its potential cooperation with the BC oncogene ERBB2; ii) establish the involvement of EPN3-induced endocytosis and pEMT in the appearance of circulating tumor cells (CTCs) and metastasis. To this aim, we will perform a series of in vivo and ex vivo studies using conditional knock-in mice overexpressing EPN3 in the mammary epithelium crossed with MMTV-NeuN mice, a known BC mouse model that overexpresses the oncogene ERBB2 in the mammary epithelia. In this model system, we will investigate EPN3-drived tumorigenesis and metastasis, and the appearance of CTCs in the bloodstream using an already set-up microfluidic device. Isolated CTCs will be analyzed by WB, IF, RT-qPCR, and/or RNA-seq experiments, in parallel to the primary tumor counterpart, looking for unbiased endocytic/pEMT/invasive signatures, which might unveil characteristics specifically linked to EPN3 overexpression and metastasis formation.
With this project, we will obtain new insights into the metastatic process in BC, which will serve to identify subsets of patients at high risk of developing metastasis and to possibly design, in the future, novel tailored-treatments and endocytosis-targeted therapies able to reduce the metastatic potential of BC.