Ascites induces modulation of α6β1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma

Interactions between cancer cells and the surrounding medium are not fully understood. In this study, we demonstrate that ascites induces selective changes in the expression of integrins and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) in ovarian cancer cells. We hypothesise that this change of integrin and uPA/uPAR expression triggers signalling pathways responsible for modulating phenotype-dependent functional changes in ovarian cancer cells. Human ovarian surface epithelial (HOSE) cell lines and epithelial ovarian cancer cell lines were treated with ascites for 48 h. Ascites induced upregulation of α6 integrin, without any change in the expression of αv, β1 and β4 integrin subunits. Out of the four ovarian cancer cell lines studied, ascites induced enhancement in the expression of uPA/uPAR in the more invasive OVCA 433 and HEY cell lines without any change in the noninvasive OVHS1 and moderately invasive PEO.36 cell lines. On the other hand, no change in the expression of α6 integrin or uPAR, in response to ascites, was observed in HOSE cells. In response to ascites, enhancement in proliferation and in adhesion was observed in all four ovarian cancer cell lines studied. In contrast, no significant increase in proliferation or adhesion by ascites was observed in HOSE cells. Ascites-induced expression of uPA/uPAR correlated with the increased invasiveness of HEY and OVCA 433 cell lines but was not seen in OVHS1, PEO.36 and HOSE cell lines. Upregulation of α6 integrin and uPA/uPAR correlated with the activation of Ras and downstream Erk pathways. Ascites-induced activation of Ras and downstream Erk can be inhibited by using inhibitory antibodies against α6 and β1 integrin and uPAR, consistent with the inhibition of proliferation, adhesion and invasive functions of ovarian cancer cell lines. Based on these findings, we conclude that ascites can induce selective upregulation of integrin and uPA/uPAR in ovarian cancer cells and these changes may modulate the functions of ovarian carcinomas

RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF

Tumours with mutant BRAF are dependent on the RAF-MEK-ERK signalling pathway for their growth. We found that ATP-competitive RAF inhibitors inhibit ERK signalling in cells with mutant BRAF, but unexpectedly enhance signalling in cells with wild-type BRAF. Here we demonstrate the mechanistic basis for these findings. We used chemical genetic methods to show that drug-mediated transactivation of RAF dimers is responsible for paradoxical activation of the enzyme by inhibitors. Induction of ERK signalling requires direct binding of the drug to the ATP-binding site of one kinase of the dimer and is dependent on RAS activity. Drug binding to one member of RAF homodimers (CRAF-CRAF) or heterodimers (CRAF-BRAF) inhibits one protomer, but results in transactivation of the drug-free protomer. In BRAF(V600E) tumours, RAS is not activated, thus transactivation is minimal and ERK signalling is inhibited in cells exposed to RAF inhibitors. These results indicate that RAF inhibitors will be effective in tumours in which BRAF is mutated. Furthermore, because RAF inhibitors do not inhibit ERK signalling in other cells, the model predicts that they would have a higher therapeutic index and greater antitumour activity than mitogen-activated protein kinase (MEK) inhibitors, but could also cause toxicity due to MEK/ERK activation. These predictions have been borne out in a recent clinical trial of the RAF inhibitor PLX4032 (refs 4, 5). The model indicates that promotion of RAF dimerization by elevation of wild-type RAF expression or RAS activity could lead to drug resistance in mutant BRAF tumours. In agreement with this prediction, RAF inhibitors do not inhibit ERK signalling in cells that coexpress BRAF(V600E) and mutant RAS