Receptor Tyrosine Kinase AXL is a Potential Therapeutic Target in Muscle-Invasive Bladder Cancer

Bladder cancer (BC) accounts for more than 104 deaths per year in the UK. 80% of bladder tumors are non-muscle-invasive comprising superficial and carcinoma in situ (CIS) lesions. CIS commonly progress towards muscle-invasive form of bladder cancer (MIBC) that is characterised by distant metastases and short patients’ survival. During tumour progression, MIBC cells acquire a number of properties that enhance tumour growth and invasion. There is an urgent need to develop novel treatment schemes for this group of patients. AXL is a receptor tyrosine kinase activated by the GAS6 ligand or through the heterodimerization with ErbB proteins resulting in the amplification of EGF signalling. Because of the association of AXL with the progression of several human cancers, development of different AXL inhibitors has been initiated. Some of these inhibitors are currently in clinical trials. In contrast to other malignancies, such as breast or ovarian cancers, the role of AXL in BC remains elusive, and the potential therapeutic efficacy of AXL targeting therapies remains to be resolved. According to the preliminary data obtained in our group, a significant association of AXL immunopositivity with distant metastases and poor patients’ survival has been found in BC patients. To characterise the molecular mechanisms involved in AXL regulation in BC, expression and localization of AXL was investigated in a panel of cell lines. As in other cancers, AXL expression was correlated with a mesenchymal phenotype. Zebrafish xenograft model was established to analyse the role of AXL in invasiveness of BC cells. It was confirmed by RNAi approach, that AXL promotes migratory and invasive ability of BC cells. In this study, the molecular mechanisms implicated in up-regulation of AXL in BC cells were analysed. Data indicate that at least two transcription factors, namely STAT3 and YAP/TEAD, coordinate AXL expression and contribute to the invasiveness of bladder cancer cells in zebrafish xenografts. Application of a dominant-negative mutant of E-cadherin and siRNAs targeting several structural proteins of adherens junctions highlighted the role of cell-cell contacts and Hippo signalling pathway in the regulation of AXL expression. In order to develop new approaches to AXL inhibition, small molecule inhibitors (R428, ONO) or agents interfering with AXL gene transcription (Verteporfin, Metformin) were applied. Data showed that AXL expression in BC cells can be efficiently decreased by Verteporfin or Metformin, and that this treatment affected also tumorigenicity (3D cultures) and migratory ability of BC cells (zebrafish xenografts). Surprisingly, treatment of cells with R428 or ONO inhibitors stimulated AXL expression. Upon prolonged treatment with R428, BC cells developed resistance associated with the enhanced AXL expression and presence of the phosphorylated form. Importantly, R428-resistant BC cells became more sensitive to Verteporfin treatment compared to the control cultures. This observation may indicate that development of R428 resistance in BC cells is associated with their addiction to the hyperactivated YAP/TEAD pathway. Taken together, our results demonstrate that AXL activates invasion in BC cells in an in vivo assay, and suggest that combined treatment of BC cells with small molecule inhibitors and Verteporfin may represent an effective methodology for blocking AXL in BC.