Abstract A76: EGFR activation signaling cascade leads to phosphorylation on serine 732 of FAK and mitosis in a subset of epithelial ovarian cancer
Authors: Tomassetti Antonella , Rea Katia , Sensi Marialuisa , Canevari Silvana 
Published: August 2014

The epidermal growth factor receptor (EGFR) is expressed in up to 70% of epithelial ovarian cancer (EOC) where it correlates with poor prognosis. Despite the evidence for EGFR expression in the majority of EOCs, to date a uniform picture of the biological and clinical consequences of EGFR expression and activation has not emerged. Indeed, treatment of EOC patients with anti-EGFR results in a very poor response and is not correlated to EGFR expression. We aimed to characterize EGFR-expressing EOCs in the aim to better define EGFR-expressing EOC subtypes. We have recently showed that co-expression as well as the concomitant presence of IL-6 and PAI-1 in EOC ascites could characterize a subset of EGFR-expressing EOCs with shorter progression-free survival after chemotherapy. In vitro we have also found that the EOC cell line resembling this subset of EOC is more sensitive to anti-EGFR compounds suggesting that the association of anti-EGFR agents with taxol and cisplatin could be more appropriate for treating this subset of EOCs. We are now further investigating EGFR signaling cascade in EOC cell lines not only in traditional 2D cultures but also in 3D culture. Indeed, we found that in EOC E-cadherin (cadh) may be still present at cell-cell contacts during tumor progression and that EOC cells present in the ascites preferentially express E-cadh at cell-cell contacts. Therefore, we have set up a 3D culture system, resembling the multicellular aggregates (MCAs) found in the ascites, by using a commercially available alginate matrix to which human cells do not attach. Immunoblotting on 3D EOC cell line showed a significant increase of E-cadh expression together with that of cytokeratin 8/18 and vimentin. Interestingly, in this system we found a tremendous increase of EGFR phosphorylation and that EGF stimulation induced EGFR phosphorylation and triggered PI3K/AKT and MEK/ERK activations. In line with these results, down-modulation of E-cadh expression by specific siRNA, was associated with a significant decrease of EGFR phosphorylation with consequent decrease of both AKT and MAPK phosphorylations. Interestingly, EGF stimulation led to phosphorylation (P-) of FAK on tyrosine residues and also on FAK at Ser-732 (FakSer732). In vivo, in E-cadh-expressing EOC cells present in the ascites P-FAKser732 was also present. In vitro P-FAKSer732 was barely detectable during interphase while its levels strongly increased in mitotic cells upon activation of the EGFR/MEK/ERK axis in an integrin-independent manner. P-FAKSer732 presence was crucial for the maintenance of the proliferation rate and its level was inversely related to the level of acetylated α-tubulin. P-FAKSer732 localized at the microtubules (MT) of the spindle, biochemically associated with MT and contributed to MT depolymerization. The lack of the phosphorylation on Ser732 as well as the inhibition of CDK5 activity by roscovitine impaired mitotic spindle assembly and correct chromosome alignment during mitosis. In conclusion, we have identified that the EGF-dependent EGFR activation led to increased P-FAKSer732 and polymerized MT. Our data shed light on the multifunctional roles of FAK in neoplastic cells, being involved not only in integrin-dependent migratory signaling, but even in integrin-independent MT dynamics and mitosis control. These findings provide a new potential target for inhibiting the growth of EOC cells in which the EGFR/MEK/ERK/CDK5 pathway is active.

Partially supported by Associazione Italiana per la Ricerca sul Cancro (AIRC)

Citation Format: Antonella Tomassetti, Katia Rea, Marialuisa Sensi, Silvana Canevari. EGFR activation signaling cascade leads to phosphorylation on serine 732 of FAK and mitosis in a subset of epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A76.