Epithelial ovarian cancer: a study on the autophagic and microenvironmental regulation of cancer stem cells and the pro-tumorigenic role of casein kinase 1 delta

Epithelial ovarian cancer (EOC), the most lethal gynecological malignancy, is normally treated with surgery followed by platinum and taxane-based chemotherapy. Despite an initial high response rate, the majority of patients eventually relapse and die. The cause could be ascribed to a subpopulation of cancer cells, named cancer stem cells (CSC), endowed with self-renewal, high tumorigenic and metastatic potential, and drug resistance. Therefore, CSC eradication could be envisioned as a successful treatment. Part of the work included in this thesis stemmed from the idea that ovarian CSC could enjoy some growth advantage, deriving from intrinsic properties or from autocrine/paracrine circuits operating in tumor microenvironment. Our group and others demonstrated that CD44+CD117+ cells are likely EOC CSC. Recent studies indicated autophagy as a mechanism to survive chemotherapy and that CSC in particular could rely on this metabolic process. For this reason, we decided to evaluate autophagy activation and the effects of its perturbation in CSC from EOC ascitic effusions and from patient-derived xenografts (PDX) and we found that they presented a higher basal autophagy level compared to non-CSC. Autophagy blockade with chloroquine impaired CSC viability, in vitro spheroid growth, and in vivo tumorigenic potential. In addition, autophagy inhibition and carboplatin treatment had a synergistic effect both in vitro and in vivo. Our results suggest a possible clinical application of the combined therapy in EOC as this approach would counteract tumor growth and impair the CSC compartment at the same time, thus reducing tumor relapse. As the microenvironment contribution to CSC maintenance is revealing its growing importance, we focused on SCF, the ligand of CD117 (or c-Kit). Indeed, the SCF/c-Kit axis regulates cell viability, proliferation and differentiation both in physiological conditions and in cancer. Notably, SCF exists both as a soluble and transmembrane protein. We found that the EOC ascites contained high amounts of the cytokine. Soluble SCF was detected only in tumor-associated fibroblasts and macrophages supernatants, whereas the membrane isoform was also expressed by cancer cells. Both SCF isoforms were effective in activating Akt pathway in CD117+ cells, while the tyrosine kinase inhibitor imatinib abolished this effect. Accordingly, results obtained in spheroid assays suggest that SCF stimulation could enhance the canonical CSC properties and imatinib pre-treatment inhibited them. Overall, SCF stimulation might help CSC to survive in selective culture conditions and, more extensively, could support CSC survival in patient ascitic fluid. Besides the role played by CSC in tumor outgrowth, the malignant cells forming the tumor bulk are sustained by pro-tumorigenic protein activation. Thus, we investigated the role of casein kinase 1 delta (CK1δ), member of a kinase family involved in many cellular processes, which is genetically amplified in ovarian cancer and overexpressed in other cancer types. Since CK1δ inhibition induced cell cycle arrest and apoptosis in a variety of tumor cell lines and delayed tumor growth in vivo, we evaluated the effects of CK1δ knockdown on two EOC cell lines (OVCAR3 and IGROV1) both in vitro and in vivo. Silenced cells grew more slowly and were less tumorigenic in vivo. Moreover, CK1δ knockdown sensitized ovarian cancer cells to carboplatin treatment. Interestingly, CK1δ-depleted OVCAR3 cells resulted more prone to migrate in vitro and more metastatic in vivo, but these results need further confirmation in IGROV1 cells. Since numerous small molecule inhibitors targeting CK1δ have been synthesized and tested, it would be important to unveil the molecular mechanism linking CK1δ to metastatic potential before these compounds enter the clinic for EOC management. This thesis encompasses three different topics, characterized by the common aim of investigating EOC biology from different perspectives, in order to highlight novel weak points of this deadly disease