A PRO-INFLAMMATORY PROGRAM DRIVEN BY EGFR ACTIVATION IN EPITHELIAL OVARIAN CANCER

Epithelial Ovarian Cancer (EOC) is the leading cause of death from gynecology cancer. Initially EOCs are characterized by local growth mass. In advanced-stage, EOCs present an increased accumulation of ascites in the peritoneum. These ascites contain a variable numbers of tumor cells and inflammatory leukocytes, as well as, variable levels of cytokines and chemokines. The epidermal growth factor receptor (EGFR), a member of ErbB family of receptor tyrosine kinases (RTK), activates multiple signaling cascades that cause growth and invasion of tumor cells. In a homeostatic state, EGFR plays a key role in normal ovarian follicle development and cell growth regulation of the ovarian surface epithelium (OSE), whose cells might give rise to EOC. In EOC samples, EGFR is expressed in an estimated 10-70% of EOCs, and its altered expression is associated with advanced-stage disease and poor prognosis, but at the moment the EGFR signaling cascade has not yet been directly associated with induction of an inflammatory network. This thesis is aimed to assess in EOC whether the activation of EGFR could induce a pro-inflammatory program. High levels of interleukin-6 (IL-6) have been found in the ascites of EOC patients and correlate with shorter survival. In vitro analysis of EOC cell lines revealed that ligand-stimulated EGFR activated NFkB-dependent transcription and induced secretion of IL-6 and plasminogen activator inhibitor (PAI-1). Twelve of 23 primary EOC tumors from advanced-stage patients, with malignant ascites at surgery, co-expressed membrane EGFR, IL-6, and PAI-1 by immunohistochemistry (IHC), and both IL-6 and PAI-1 were present in 83% of the corresponding ascites. Analysis of a publicly-available gene expression dataset from 204 EOCs confirmed a significant correlation between IL-6 and PAI-1 expression, and patients with the highest IL-6 and PAI-1 co-expression showed a significantly shorter progression-free survival time. All these data suggests that EGFR/NFkB/IL-6-PAI-1 may have a significant impact on the therapy of a particular subset of EGFR-expressing EOCs, and that IL-6/PAI-1 co-expression may be a novel prognostic marker. Subsequently, to analyze the role of IL-6 in EOC, we decided to perform a bioinformatic analysis of seven publicly available datasets of gene expression profile from EOC patients. We identified an IL-6-correlated gene signature in EOCs, containing 40 genes mainly associated with proliferation. Thirty-three of 40 genes were also significantly correlated in low malignant potential (LMP) EOCs, while 7 genes were IL-6-correlated only in advanced stage EOCs. Further analyses allowed us to identify, among the 40 genes, a gene set associated with ‘early growth factor response’ and a biological network related to ‘thrombosis and cardiovascular disease’ for the 7-gene signature. Accordingly, selected genes from the identified signatures were validated in vitro by real time RT-PCR in serous EOC cell lines upon stimulation with EGF. In vitro analyses to assess the biologic role of IL-6 showed that IL-6 produced by EGFR/MEK/NF-kB pathway, triggers an autocrine loop of ligand/EGFR activation through STAT3/src signalling. Subsequently, the role of PAI-1 in EOC cells has been analyzed. Experiments on PAI-1-silenced SKOV3 and OAW42 cells showed that the loss of PAI-1 affected cell adhesion to different substrates, as well as inhibited cell migration and invasion likely as inducer of epithelial mesenchymal transition (EMT) and regulator of EOC cells plasticity.