Costabile, Valeria (2017) Epithelial to mesenchymal transition (EMT) and cell plasticity in human colorectal cancer. [Tesi di dottorato]

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Abstract

Colorectal cancer (CRC) was ranked third and second of all cancers affecting men and women, respectively, with metastases representing the first cause of death and defining the stage IV of the disease, characterized by a relatively short overall survival. It has been recently demonstrated that epithelial to mesenchymal transition (EMT), a physiological mechanism involved in embryonic development and tissue remodelling, could also play a critical role in invasion and metastasis of many types of cancer, including CRC. During EMT, epithelial cancer cells acquire not only mesenchymal traits, but also a stem cell-like phenotype, with ability of self-renewal, unlimited proliferation and resistance to apoptosis. On this point of view, it is of significant and primary interest for cancer biomedical research to identify genes and proteins mainly involved in EMT and stemness, since they could represent good potential targets for therapy and predictive/prognostic molecular biomarkers. During this work we isolated primary mesenchymal colorectal cancer cells from CRC patients, and demonstrated that they expressed epithelial (E-Cadherin and Cytokeratins), mesenchymal (N-Cadherin and Vimentin) and stemness markers, together with high level of epithelial-mesenchymal transition-transcription factors (EMT-TFs) (Snail and Twist). These primary cell cultures were isolated from adenocarcinomas, which are epithelial tumours, thus our data suggested that these cells were epithelial cells undergone EMT. GSK-3β is a multifunctional serine/threonine kinase and an important regulator of cell survival by regulating PI3K, MAPK and WNT pathways. It may act as anti- or pro-apoptotic factor in a cell‑specific manner; in colon and pancreatic cancer cells, GSK-3β activation confers a selective growth advantage to these cells, thus acting as a tumor promoter. Therefore, we investigated GSK-3β as a new druggable target and LiCl as new drug for cancer therapy. We demonstrated that LiCl was able to induce mesenchymal-epithelial reverting transition (MET) and cellular differentiation, affecting migration in our cell cultures. Spherical cancer models represent one of the major 3D in vitro models that have been described over the past 4 decades. These models have gained popularity in cancer stem cell research using tumorospheres. They contribute to chemoresistance, radioresistance, tumorigenicity, invasion and migration studies. We demonstrated that our cell cultures were able to aggregate to form spheroids in suspension and this capability was affected when cells were incubated with LiCl. Furthermore, the inhibition of GSK-3β by LiCl, strongly down-regulated the expression of stem cell markers, such as Oct4, Sox2, Nanog, ALDH1 and LGR5, in cancer spheroids. Finally, we demonstrated that LiCl affected cellular plasticity, an important feature in tumor progression and metastasis; indeed, after LiCl treatment cancer cells were unable to switch from one phenotype to another. In conclusion, we set up an experimental system of primary mesenchymal colorectal cancer cell cultures to study the role of EMT in CRCs, and molecular basis of cell plasticity during cancer progression and acquisition of resistance to the therapy. Furthermore, we observed that LiCl induced MET and altered dynamic of cancer spheres formation, indicating differentiated state that correlates with expression of undifferentiation/differentiation markers, suggesting that GSK-3β and LiCl could represent an eligible target and a potential drug to set up therapy able to interfere with CRCs progression.

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