Expression of the proto-oncogene Axl in renal cell carcinoma

Abstract

In this investigation, we examined the role of the Axl proto-oncogene in renal cell carcinoma (RCC). Axl is a tyrosine kinase receptor implicated in myeloid leukogenesis, and has been found to be overexpressed in lung cancers and breast cancers. Axl has been described to act as a mitogenic factor along with its ligand Gas-6. Axl has also shown to have a role in apoptosis, cell adhesion, and chemotaxis. The differential expression of the Axl RNA transcript was examined in 20 pairs of matched normal kidney and clear cell RCC patient samples. We found that there was a significant increase in the steady-state levels of Axl mRNA in the RCC compared with the normal kidney pair (Student's paired t-test P < 0.001). There was also a significant increase in Axl expression overall in RCC compared to normal kidney (P < 0.03). Western blotting was utilized to determine Axl protein levels in six out of the 20 pairs of the normal/RCC matched pairs. Overall, the level of expression was not significantly different between the paired normal kidneys and kidney tumors, but the detected Axl protein appeared to be at slightly different molecular weights. Primers were constructed for the two known Axl variant, RT-PCR performed, but no differences were observed in the expression of each variant. Next, we performed a gene silencing experiment utilizing double-stranded RNA constructed to silence the Axl gene in the 293 transformed kidney cell line. There was a 50% decrease in Axl gene expression in the RNAi transfected over control cells. In addition, flow cytometry performed to determine DNA content showed a 30% increase in G1/G0 cells, which were transfected with axl RNAi compared to control. Altogether, these findings suggest an overexpression of Axl as part of a proliferative phenotype in RCC.