Influence of the erbB receptor family on ovarian cancer cell lines

The erbB family consists of four structurally related, transmembrane, receptors - the epidermal growth factor receptor (EGFr; erbBl), erbB2, erbB3 and erbB4. In association with the EGF-like growth factors, they create a potent and complex ligandreceptor network, which has been associated with a number of cell phenotypes, including proliferation and motility. The ERK kinase, PI-3 kinase and PLCy pathways have been widely implicated as downstream mediators of these effects. Deregulation of the network is common in human cancers, including ovarian tumours, and is frequently associated with poor patient survival. These observations have generated interest in the underlying mechanisms of the network, and how its disruption manifests as oncogenic malignancy. This project investigates the roles of different erbB receptors and their downstream signalling pathways in ovarian cancer systems. A panel of six cell lines (SKOV-3, PEOl, OVCAR5, A2780, 41M and PE01cddp) were chosen to represent a range of erbB receptor expression levels, and these were characterised within growth and migration assays. Growth assays were based upon cell counts, whereas migration assays were developed using transwell inserts coated with collagen IV, fibronectin and laminin.To assess the contribution of individual erbB family members to cell growth and migration, specific ligand-receptor interactions were exploited within functional assays; transforming growth factor-alpha (TGFa) binds and activates the EGFr, while neuregulin (NRG) growth factors activate erbB3 and erbB4. Such growth factor stimulation induces receptor homo- or heterodimerisation within the family, which initiates intracellular signalling. At equal concentrations (InM), NRGip was a more potent mitogen than TGFa, although both significantly increased cell growth in a number of cell lines; NRG la effects however were trivial. Although the neuregulin growth factors both associate with the same receptors, the a-isoform is known to be less potent. Therefore these data suggest that erbB3 and/or erbB4 receptors have the most significant influences upon cell proliferation and increased cell survival. Correlative analysis revealed some association between erbB3 expression levels cell growth rates (TGFa stimulated growth p=0.0069; NRGp stimulated growth p=0.0456). This is consistent with the suggestion that erbB3, probably in combination with erbB2, is the most potently mitogenic receptor complex. In migration assays, growth factor iii concentrations were varied to equalise mitogenic effects, TGFa was used at InM, whereas NRG1(3 was used at O.lnM. TGFa demonstrated the most potent effects in these experiments, and a strong correlation was found between migration on collagen and cell line expression levels of erbB2 (p=0.004), suggesting an association between EGFr-erbB2 dimers and cell motility. Blockade of the EGFr, using the small molecule inhibitor Iressa (ljaM), significantly reduced cell growth (in SKOV-3, PEOl and OVCAR5 lines) and migration (in SKOV-3 and PEOl cells). Herceptin downregulation of erbB2 was ineffective and thus had minimal impact on cell function.A subset of four cell lines (SKOV-3, PEOl, OVCAR5 and A2780) was used to analyse TGFa and NRGlp signalling effects within the ERK, PI-3 kinase and PLCy pathways. Three cell lines (SKOV-3, PEOl and OVCAR5) showed pathway induction in response to either one or both ligands, whereas A2780 cells showed no response, but appeared to have some constitutive pathway activation. Growth factor activation of the ERK and PI- 3 kinase cascades was observed only in the lines that showed growth stimulation. Ligand activation of all three pathways occurred where cells were stimulated to migrate, possibly in a signal duration dependant manner. Specific inhibitors of MEK (PD98059 and U0126), PI-3 kinase (LY294002) and PLCy (U73122) were utilised within functional assays. Blockade of the ERK and PI-3 kinase pathways decreased cell growth in all of the cell lines. However, only blockade of PLCy was capable of inhibiting migration in all of the lines. These results are consistent with findings in other cell systems.These studies have demonstrated that the erbB3 receptor has a potent effect on cell mitogenesis and/or increased cell survival, probably through activation of the ERK and PI-3 kinase pathways. They have also shown that the EGFr in combination with erbB2 is an important drive to cell migration, predominantly utilising the PLCy pathway. These finding are relevant to ovarian cancers expressing erbB receptors, particularly those which have established an autocrine loop with stimulating ligands, and may be important for determination of patient treatment strategies in the future