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Elucidating Mechanisms of Bladder Cancer Metastasis

Periodic Reporting for period 1 - EMBRACE (Elucidating Mechanisms of Bladder Cancer Metastasis)

Periodo di rendicontazione: 2018-09-01 al 2020-08-31

Bladder cancer is one of the most prevalent cancers in the EU, frequently exhibiting invasion and metastasis and therefore associated with poor prognosis. Hepatocyte Growth Factor or HGF has been frequently associated with advanced stages of the disease, although targeting it or its receptors (c-Met) have failed in the clinic. This project aimed to delineate the precise downstream molecular mechanisms of HGF mediated metastasis in bladder cancer cells. Accordingly, this project had two major objectives. First to identify the downstream (targetable) intracellular signaling pathways that are responsible for HGF mediated bladder cancer metastasis. Thereafter, delineate the intra-cellular molecular mechanisms. The second objective was to develop biomarkers that can be potentially used to detect early invasive/metastatic behaviour of bladder cancer cells.
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.
The results obtained within the project have implications for the society. Bladder cancer is one of the most common cancers in the EU, and there has been a lack of proper understanding of the mechanisms of this disease. This project has uncovered the precise mechanisms and signaling pathways that are most active during bladder cancer progression, the knowledge has further allowed to target these pathways with success using mice studies. This project has uncovered a crucial link between the HGF/c-MET pathway and TGF-beta signaling pathway. The HGF/c-MET pathway is hard to target with its inhibitors failing in clinical trials for bladder cancer patients. Here, we have identified an alternative pathway which works in conjunction with the c-MET pathway that can be effectively targeted to block bladder cancer progression.

The next step of the project would be to test the combinatorial drug treatment strategies on human trials. Also, further testing the efficacy of SMURF2 as a potential biomarker is warranted. These studies will lead to precise and personalized treatment strategies for bladder cancer patients. The TRAF4 project also has a lot of potential as a novel biomarker for bladder cancer progression. We observed that TRAF4 expression is epigenetically silenced in advanced stages of bladder cancer patients and also observed a negative trend of its expression across the advancing stages of bladder cancer progression. We also uncovered an important link between TRAF4 and the BMP signaling pathway. TRAF4 is able to degrade SMURF1, which is a negative regulator of BMP signaling pathway. A positive correlation between BMP signaling pathway’s effector molecule pSMAD1/5/8 and TRAF4 expression was observed. Also, we observed a negative correlation between NF-KappaB signaling pathway and TRAF4 expression.
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