Analysis of Axl Protein Isoforms Functions in Bladder Cancer Cells

Abstract

Among TAM receptors, Axl has emerged as a key oncogenic regulator of tumor cell proliferation, epithelial-to-mesenchymal transition (EMT), immune evasion, and metastasis. It is highly associated with tumor aggressiveness, poor prognosis, and low survival rate in bladder cancer. It has suggested that Axl exists in two isoforms resulting from alternative splicing, that may vary in their cellular localization, proteolytic cleavage, and signaling potential. This study aims to analyse the Axl isoform-specific regulation of downstream signaling pathways, including Akt and Erk1/2 in bladder cancer. Cell lines (EJ28 and UMUC3) with Axl gene knockout (KO) were introduced with Axl isoform 1 and 2 tagged with OFP and c-Myc. Transfected clones were selected by using hygromycin B. Transfection efficiency was validated by fluorescence microscopy for GFP and OFP. Western blot was performed to analyse the protein expression of Axl, Akt, p-Akt-473, p-Akt-308, Erk1/2, p-Erk1/2. Axl expressions varied significantly across all variants as compared to wild-type (WT). Both isoforms (1 & 2) differentially regulates Akt-473/308 activation, but precise difference is still unclear. However, p-Erk1/2 levels were not significantly differed. Results suggest that expression dynamics of Akt and Erk1/2 could be affected because of cell specificity, isoform-specific activity, and changes in cellular environment i.e., oxidative/cellular/apoptotic stress. To delineate isoform specific functions, ligand (Gas6) stimulated response and time-course experimental analysis for Akt and Erk1/2 activation should be investigated. Quantitative analysis of mRNA and protein expression should also be assessed to evaluate difference due to transcriptional modulations and protein stability. Furthermore, impact of alternative splicing on the formation of soluble and nuclear forms of Axl has to be analysed in further research localization and their functional differences could be explored.