We successfully identified the precise underlying molecular mechanisms and effector or modulatory signaling pathways. We validated our findings using in vitro cell culture systems using clinically relevant and highly selective inhibitors targeting these signaling pathways. We identified TGF-beta and MAPK signalling pathways to be active downstream of HGF signalling pathway. We have identified SMURF2, an E3 ubiquitin ligase to be phosphorylated in aggressive bladder cancer cells but not in normal bladder epithelial cells. Furthermore, we have successfully developed and validated with specific phospho-antibodies to SMURF2, and used these on bladder cancer specimens. We have submitted a Data Management Plan as a deliverable. This plan contains information on how we deposited proteomic and transcriptomic results obtained from the study in publicly available databases. These results can be exploited further to uncover novel drug targets and potential biomarkers.
We have also identified and validated another E3 ubiquitin ligase TRAF4 (TNF receptor associated factor 4) as biomarker for advanced stages of bladder cancer progression. TRAF4 has been largely implicated as a tumour promoting element in a wide range of cancers. Over-expression and amplification of this gene product has been a common observation in breast , lung, prostate and other metastatic tumours. In contrast, we observed that expression of TRAF4 negatively correlated with overall (bladder cancer) patient survival. We also performed transcriptomic data analysis, which revealed the changes in the intracellular signaling pathways upon TRAF4 mis-expression in bladder cancer cells. BMP (Bone morphogenetic protein) signaling and the NF-B signaling pathways were the most affected by TRAF4 mis-expression in bladder cancer cells. Furthermore, we identified and validated an ERK kinase phosphorylation site on TRAF4 which not only affects the stability but also its ability to localize to the plasma membrane. Our findings were further validated using bladder cancer patient material.
