Dissecting the role of CDK17 in Epithelial Ovarian Cancer

High Grade Epithelial Ovarian Cancers (HGEOCs) is a heterogeneous group of tumours. Late diagnosis and drug-resistant recurrences make HGEOCs the most lethal gynaecological malignancy. The central role played by Cyclin-Dependent Kinases (CDKs), in a plethora of cellular mechanisms, such as control of cell cycle progression, DNA repair, transcription, and apoptosis, make them promising targets to overcome drug-resistance in HGEOCs. In this thesis we have focus on the possible role of CDK17 in the response to platinum (PT) starting from an unbiased loss of function shRNA screening, suggesting that it might play a role in this context. We confirmed by specific targeting that silencing of CDK17 significantly increased PT-induced cell death, in several EOC cell lines. CDK17 (PCTAIRE2) is a member of the largely unexplored CDKs subfamily PCTAIRE whose interactome was unknown. Un unbiased proteomic approach looking at CDK17-interactin proteins coupled with functional enrichment analysis identified endocytosis as the most enriched pathway. Network analysis predicted AP2A2 and EPS15, both belong to endocytosis pathway, among the top score proteins. A subsequent proteomic study to compare the proteins bound to WT CDK17 with those interacting with the clinically relevant CDK17 R312G mutant, demonstrated that WT and mutant CDK17 had the same affinity for EGFR, whose recycling is governed by the binding to AP2A2 and EPS15. Co-immunoprecipitation experiments on endogenous proteins validated CDK17 interaction with AP2A2, EPS15 and EGFR and demonstrated that CDDP-treatment further promotes these interactions. Abrogation of CDK17 expression interferes with EGFR internalization and is associated with reduced ligand-activation of EGFR signalling. Overexpression of CDK17 WT and especially CDK17 R312G mutant maintained a persistent EGFR active phosphorylation and was accompanied by a higher activation of pro-survival signals. Accordingly, EOC cells expressing the CDK17 mutants were more resistant to PT, respect to control and WT counterpart. Moreover, results obtained in PT resistant EOC cells, suggested that CDK17 might contribute to PT-resistance, since its silencing re-sensitize these cells to PT, EGFR inhibition by Gefitinib and their combination. By kinase assay, we showed that CDK17 active complex is able to phosphorylate at different extents EGFR, EPS15 and a subunit of the AP2 complex. Accordingly, CDK17 silencing, concomitantly to PT-treatment, reduced EGFR phosphorylation levels at Threonine residues, further supporting a direct possible involvement of CDK17 kinase activity in the control of stress-induced EGFR activation. Overall, we have identified a new and unexpected role for CDK17 in the regulation of EGFR pathway that could influence PT-response and cell survival capabilities, that could have a relevant translational impact since CDK17 could represent an novel actionable target of anti-EGFR therapies and a therapeutic target alone or in combination, in the context of PT-resistant EOC patients for which effective treatments are still lacking.

High Grade Epithelial Ovarian Cancers (HGEOCs) is a heterogeneous group of tumours. Late diagnosis and drug-resistant recurrences make HGEOCs the most lethal gynaecological malignancy. The central role played by Cyclin-Dependent Kinases (CDKs), in a plethora of cellular mechanisms, such as control of cell cycle progression, DNA repair, transcription, and apoptosis, make them promising targets to overcome drug-resistance in HGEOCs. In this thesis we have focus on the possible role of CDK17 in the response to platinum (PT) starting from an unbiased loss of function shRNA screening, suggesting that it might play a role in this context. We confirmed by specific targeting that silencing of CDK17 significantly increased PT-induced cell death, in several EOC cell lines. CDK17 (PCTAIRE2) is a member of the largely unexplored CDKs subfamily PCTAIRE whose interactome was unknown. Un unbiased proteomic approach looking at CDK17-interactin proteins coupled with functional enrichment analysis identified endocytosis as the most enriched pathway. Network analysis predicted AP2A2 and EPS15, both belong to endocytosis pathway, among the top score proteins. A subsequent proteomic study to compare the proteins bound to WT CDK17 with those interacting with the clinically relevant CDK17 R312G mutant, demonstrated that WT and mutant CDK17 had the same affinity for EGFR, whose recycling is governed by the binding to AP2A2 and EPS15. Co-immunoprecipitation experiments on endogenous proteins validated CDK17 interaction with AP2A2, EPS15 and EGFR and demonstrated that CDDP-treatment further promotes these interactions. Abrogation of CDK17 expression interferes with EGFR internalization and is associated with reduced ligand-activation of EGFR signalling. Overexpression of CDK17 WT and especially CDK17 R312G mutant maintained a persistent EGFR active phosphorylation and was accompanied by a higher activation of pro-survival signals. Accordingly, EOC cells expressing the CDK17 mutants were more resistant to PT, respect to control and WT counterpart. Moreover, results obtained in PT resistant EOC cells, suggested that CDK17 might contribute to PT-resistance, since its silencing re-sensitize these cells to PT, EGFR inhibition by Gefitinib and their combination. By kinase assay, we showed that CDK17 active complex is able to phosphorylate at different extents EGFR, EPS15 and a subunit of the AP2 complex. Accordingly, CDK17 silencing, concomitantly to PT-treatment, reduced EGFR phosphorylation levels at Threonine residues, further supporting a direct possible involvement of CDK17 kinase activity in the control of stress-induced EGFR activation. Overall, we have identified a new and unexpected role for CDK17 in the regulation of EGFR pathway that could influence PT-response and cell survival capabilities, that could have a relevant translational impact since CDK17 could represent a novel actionable target of anti-EGFR therapies and a therapeutic target alone or in combination, in the context of PT-resistant EOC patients for which effective treatments are still lacking.