Linking a deubiquitinating enzyme to the regulation of a human AlkB demethylation complex in cancer progression

• Applicant: Privatdozent Dr. Sebastian Dango
• Subject Area: General and Visceral Surgery
• Term: from 2013 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 245513254

Maintaining DNA integrity by dealkylation is a crucial step in the maintenance of genomic stability. Since the majority of alkylation damage occurs endogenously, alkylation repair is of great relevance to maintain genomic integrity, in particular for high proliferating tissues like in cancer. Alkylation damage is mainly repaired directly by demethylases/dealkylases. Our previous findings suggest that Abh3, a human demethylase/dealkylase complex, promotes cancer progression via alkylation repair. TAP-purified Abh3 complex purification in prostate tumor cells reveals presence of a deubiquitinase, named Ovarian Tumor Domain OTUD4. Strikingly, treatment over time with the proteasome-inhibitor MG132 revealed an increase of Abh3, which is not seen in the controls. Western blot analysis of whole cell extracts demarks co-expression of Abh3 and OTUD4 in cancer cells and shRNAi-mediated knock down of OTUD4 is associated with decreased Abh3 expression in vivo, at least in cancer cells. Knock down of Abh3 and OTUD4 results in decreased proliferation as well as increased alkylation damage sensitivity. Therefore, our findings implicate that regulation of Abh3 may be performed via the ubiquitin pathway, which may be cancer specific, and interplay of Abh3 and OTUD4 may promote tumor progression. This grant proposal revolves around our primary hypothesis that OTUD4 promotes tumor progression by linking its deubiquitinase activity to the human Abh3 demethylase complex. We first plan to invest co-localization of Abh3 and OTUD4 using tumor tissue arrays and characterize further biochemical interaction of Abh3 and OTUD4. Second, we will elucidate the function of OTUD4 in DNA alkylation repair and tumor growth in vivo. Third, we will investigate if OTUD4 deubiquitinates Abh3 in vitro and in vivo and characterize the nature of OTUD4 deubiquitinase activity. Fourth, we plan to analyze the impact of ubiquitin conjugation on Abh3 and the specific ubiquitination site(s) and characterize the nature of ubiquitin linkage. Fifth, we will show that OTUD4 deubiquitinase activity regulates the cellular stability of Abh3 protein half-life by modulation of proteasome-mediated degradation, and analyze the impact of OTUD4 on Abh3 catalytic activity and on Abh3-mediated alkylation damage repair. Sixth, we will investigate a possible impact of our two enzymes on chemoresistance in our cancer model in the presence of various alkylating agents.Taken together, understanding Abh3 and OTUD4 cooperativity may help us to understand how cellular expression of demethylases/dealkylases is regulated and maintained. Expression of dealkylating enzymes could be a possible explanation how cancer cells mechanistically adopt alkylation damage resistance e.g. during chemotherapy with alkylating agents. The proposed project may allow us to connect DNA alkylation repair to epigenetic events and may promote Abh3 and OTUD4, two oncogene candidates, to potential anticancer targets in the future.

• DFG Programme: Research Grants