Axl which belongs to the

Axl, which belongs to the Tyro-Axl-Mer (TAM) family of RTKs, has been reported to regulate a variety of physiological processes including cell survival, proliferation, migration and adhesion, through the activation of the phosphoinositide 3-kinase (PI3K)/V-akt murine thymoma viral oncogene homolog (Akt) and MAPK/extracellular signal-regulated kinase (ERK) pathways [13]. Axl activation occurs through the binding of growth arrest specific 6 (Gas6) to the receptor extracellular domain, which results in the dimerization and the subsequent trans-autophosphorylation of tyrosine residues within the receptor cytoplasmic domain [14]. Consequently, substrates are phosphorylated and factors able to transduce signals within clofibrate are recruited [13], [14]. Various pathological conditions, including cancer, have been shown to be characterized by an aberrant and constitutive activation of Axl, which can promote the proliferation and survival of tumor cells through the regulation of factors involved in apoptosis and cell migration [15].
Materials and methods
Results
Discussion Despite ovarian carcinoma is rather responsive to platinum-based treatment, tumor cells may acquire drug resistance and disseminate in the peritoneal cavity [21]. Drug resistance has been often associated to a more aggressive behaviour of tumor cells, specifically an increase of the metastatic ability. Various members of the RTK family have been implicated in the regulation of such a feature of tumor cells [9], [22]. Based on the pattern of RTK expression observed in genome-wide analysis, the present study was designed to examine the role of the RTK Axl in aggressiveness and drug resistance of ovarian carcinoma cells. We investigated the expression of Axl in a panel of platinum-sensitive and -resistant cells, and afterwards focused on the IGROV-1/Pt1 resistant variant, which exhibits reduced sensitivity to cisplatin and to target-specific agents [11], [23]. Drug-resistant IGROV-1 sublines were found to display enhanced mRNA Axl levels compared to the parental cell line both by gene expression analysis and using the qRT PCR quantitative approach. In platinum-resistant cells, increased levels of Axl were also found at the protein level, being higher in the IGROV-1/Pt1 than in IGROV-1/OHP cells. The variable expression of Axl in drug-resistant variants other than IGROV-1 variants suggests a possible impact of the molecular cell background. We observed that IGROV-1/Pt1 cells exhibited increased migratory and invasive capability with respect to the parental cell line. Thus, given the improved Axl expression and their drug-resistant phenotype, IGROV-1/Pt1 cells were selected for following studies. Using a loss of function approach based on RNA interference with two siRNAs targeting different regions of the Axl transcript, we found that Axl-silenced cells exhibited reduced growth and decreased migratory, invasive and clonogenic abilities as compared to cells transfected with negative control siRNA. An interesting observation of the present study was the persistent downregulation of Axl protein evident up to 144h, similarly to what previously found by us using siRNAs directed to Pin1 [24]. A reduced clonogenic ability upon molecular targeting of Axl was also observed in other ovarian cancer cell lines. A decreased cell proliferation following Axl silencing was previously reported in Non Small Cell Lung Cancer cells resistant to the EGF-R inhibitor cetuximab [25]. The role of Axl in steps favouring the metastatic spread of ovarian carcinoma has been investigated by Rankin and colleagues [26], who employed ovarian carcinoma cells characterized by intrinsic drug resistance. These authors reported that the expression of different matrix metalloproteinases (i.e., MMP1, MMP2, and MMP9) was down-regulated in Axl-deficient cells. Our investigation of the expression of specific factors implicated in cell survival and invasive abilities in Axl silenced cells indicated decreased p38 and STAT3 activation, as shown by reduction of phosphorylation [27]. In contrast, the modest decrease in Akt phosphorylation after Axl silencing suggested that compensatory mechanisms may still activate Akt in IGROV-1/Pt1 cells. In such a context, receptors of the epidermal growth factor (EGF) family may play a role, as suggested by phospho-ErbB3 increase in Axl-silenced cells. Under our conditions, the canonical MAPK pathway, i.e., ERK1/2, did not appear to be involved in Axl-mediated signaling.