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Title: Post translational modifications of FOXM1 expression
Author: Karunarathna, H. R. Upekha
ISNI:       0000 0004 6422 8653
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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FOXM1 plays an important role during mitosis and DNA damage response, and its overexpression has been implicated in breast cancer initiation and drug resistance. However, only a limited knowledge is available as to how FOXM1 expression is regulated by chemotherapeutic drugs and how these regulatory mechanisms are overcome during acquisition of resistance. Accumulating evidence shows that FOXM1 is regulated post-translationally in response to different cues that are received by the cell, eg: during the cell cycle and drug resistance. My study shows that FOXM1 is also modified post-translationally by SUMOylation, ubiquitination and deubiquitination. Initially, it was shown that FOXM1 is primarily modified by SUMO1 but not by SUMO2/3 at multiple sites in MCF-7 breast cancer cells and that the SUMOylation of FOXM1 is increased in response to epirubicin treatment. Analysis of wild-type (WT) and the SUMOylation-deficient mutant, indicated that SUMOylation inhibits FOXM1 activity, promotes its cytoplasmic re-localisation and ultimately mediates its degradation via the APC-Cdh1 complex. Further investigation showed that once SUMOylated, FOXM1 is targeted by E3-ubiquitin ligases and STUbLs proteins thus initiating FOXM1 ubiquitination. In particular, co-immunoprecipitation, western blot analysis and RT-qPCR analysis, indicated a post-translational regulation of FOXM1 by RNF8. In addition, the epirubicin treatment on MCF-7 cells resulted in a decline in FOXM1 expression, but to an increase in RNF8 and K-48 poly-ubiquitin chains, indicating that FOXM1 is targeted for degradation. Moreover, with the use of WT FOXM1 and a SUMOylation deficient FOXM1, it was revealed that FOXM1 SUMOylation is indispensable for RNF8 interaction. In addition, it was shown that RNF8 is required for the recruitment and interaction of RNF168 with FOXM1 and that this association is lost in the absence of RNF8. This possibly indicates that the primary ubiquitination is recognised by RNF168, and might be involved in extending the initial ubiquitin to form a poly-ubiquitin chain, thus marking FOXM1 for its degradation. As the activity of E-3 ubiquitin ligases is controlled by deubiquitinating enzyme (DUB) proteins I also investigated the DUB which is involved in regulating FOXM1. To this end, upon the identification of an interaction between the DUB protein OTUB1 and FOXM1, via proteomic data, using co-immunoprecipitation and western blots I showed that OTUB1 can specifically limit the K-48 ubiquitination of FOXM1 thus can antagonise the action of RNF8. In agreement, the overexpression of OTUB1 but not the catalytically inactive mutant (C19S) was shown to increase the half-life of FOXM1 and also was shown to enhance the proliferation rate and epirubicin resistance through targeting FOXM1. Collectively the data presented show that the molecular players involved in SUMOylation, ubiquitination and deubiquitination of FOXM1 may play an important role in breast cancer drug resistance and could be targeted as novel therapies in overcoming drug resistance.
Supervisor: Lam, Eric ; Matthews, Steve Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral