FOCAL ADHESION KINASE INVOLVEMENT IN MODULATING THE PROLIFERATION OF TUMOR CELLS.

The focal adhesion kinase (FAK) is well characterized non-receptor tyrosine kinase, although it undergoes also to phosphorylation on serine residues whose role is not fully understood. FAK is ubiquitously expressed and plays a crucial role as integrator of signaling from either integrins or Receptor Tyrosine Kinases (RTKs) and has been linked to cell transformation and progression in many solid tumors. Among those, the Epithelial Ovarian Cancer (EOC) has a different behaviour during progression: it rarely metastasizes to distant sites but disseminate within the peritoneal cavity. Our attention was focused on the characterization of the role of FAK in different type of tumor cells and in particular on the phosphorylation (P) on the serine 732 of FAK (P-FAKSer732). We found that P-FAKSer732 was present at variable levels in cancer cell lines but its activation was independent from integrin engagement and from the kinase activity of FAK. P-FAKSer732 was not localized at the focal adhesion site during migration but it was accumulated in dividing cells, co-localizing with the tubulin of the spindle during mitosis and regulating microtubule (MT) polymerization and mitotic spindle formation. CDK5 was identified as the specific kinase responsible for FAK serine phosphorylation whose activation was induced by EGF stimulation of the EGFR/ERK signalling. Thus, we have identified for the first time the axis EGFR/MEK/ERK/CDK5/P-FAKSer732 as a novel pathway leading to mitosis of tumor cells. We next aimed to identify the mechanism governing the crosstalk between adhesion molecules and RTKs thus leading to proliferation, migration and invasion of tumor cells. I focused my analysis on EOC which is the main cancer histotype studied in our laboratory. We therefore investigated in EOC cells a possible role of E-cadherin (cadh) to contribute to the novel signalling pathway described above. Indeed, we found that E-cadh was the adhesion molecule necessary for the formation of the EOC multi-cellular aggregates (MCAs) present in the ascites. The E-cadh and CDK5 resulted to be necessary to the generation of FAKSer732 and this protein complex was necessary for the EGF-mediated EGFR activation likely due to the stabilization of EGFR protein on the cell membrane. In line with these results, E-cadh expressing EOC cells were more susceptible to the CDK5 inhibitor, roscovitine. These data demonstrated a pivotal role of E-cadh in contributing to the proliferation of EOC MCAs. To clarify the processes responsible for the switch from a proliferative to a more invasive phenotype of EOC MCAs necessary for tumor dissemination, we were studied another RTK, Axl whose expression has been already reported to be associated to a more invasive phenotype. We found that the stimulation of Axl by its ligand Gas6 required the engagement of β3-integrin with components of the extra-cellular matrix (ECM). The crosstalk between Gas6/Axl and the integrin signallings was independent by FAK but required the contribution of p130Cas, a scaffold protein involved in the transmission of the ECM-integrin signalling, for adhesion, migration and invasion of EOC cells. Indeed, the lack of p130Cas decreased the level of Gas6-dependent Axl activation and inhibited Gas6-induced EOC cells adhesion, migration and invasion. In 56 out of 72 EOC biopsies, Axl and p130Cas were significantly co-expressed indicating of a collaborative signaling between Axl and p130Cas also in vivo. We have identified an Axl signalling which requires a p130Cas-mediated crosstalk with integrins for the dissemination of EOCs. Altogether, our investigation allowed the definition of two novel adhesion-dependent signalling pathways which drive the proliferation and the invasion of EOC cells, respectively. The inhibition of these pathways will likely affect EOC MCA growth as well as the invasion of EOC solid primary and secondary lesions.