AbstractCisplatin is an anti-cancer drug that acts by introducing toxic DNA interstrand crosslinks into proliferating cells, causing both cytostatic and cytotoxic effects. Although various models of the interstrand crosslink (ICL) response have been proposed, none of them are complete and much still remains unknown about how this process is coordinated. By understanding more about how cisplatin and ICLs cause cell death, we may be able to optimise the use of current drugs, or identify novel targets for new chemotherapeutic drugs. It is known that ubiquitination is important for the regulation of the ICL response and it is thus probable that some ubiquitin-related proteins have previously unidentified roles in this process.
This thesis describes the use of a robust high throughput siRNA screen to identify such novel ubiquitin-associated components of the response to cisplatin. Positive candidates were identified based on their ability to cause sensitivity to cisplatin and included both known and novel mediators of the ICL response. The siRNA screen formed an initial platform for further study of the roles of novel hits RNF113B, HOIP and ABIN1 in the DNA damage response.
RNF113B was a previously uncharacterized E3 ligase. Studies described herein initially suggested that RNF113B might have a role in DNA repair after cisplatin treatment. However, final validation of a role for RNF113B in the ICL response was not forthcoming.
HOIP is an E3 ubiquitin ligase that creates linear polyubiquitin chains as part of the linear ubiquitin assembly complex (LUBAC). LUBAC is required for innate and adaptive immunity, suppressing inflammation and the control of cell death through TNF and etoposide-induced signalling. HOIP was identified and validated as a top candidate of the siRNA screen. Within this thesis I show that cisplatin-induced caspase-3/7 activity is enhanced in HOIP-depleted cells, and corresponds with enhanced cisplatin-induced caspase-8 and -9 activities. Furthermore, HOIP depletion re-sensitises a cisplatin-resistant cancer line to cisplatin. Thus HOIP is an anti-apoptotic protein in the response to cisplatin and may be a potential target for anti-cancer therapeutics.
ABIN1 is a polyubiquitin-binding protein involved in autoimmunity, inflammation and protecting cells from TNF-induced apoptosis. In this thesis I present data implying that ABIN1 also protects cells from cisplatin-induced cell death. ABIN1-deficient cells, including ubiquitin-binding deficient Abin1D485N/D485N MEFs, are hypersensitive to interstrand crosslinkers. DNA damage checkpoint and repair responses are mostly intact in ABIN1-deficient cells, while cisplatin-induced apoptosis is vastly enhanced. Furthermore, GFP-ABIN1 forms ubiquitin-binding dependant macro-molecular structures in the cytosol that resemble previously described ‘speckles’ seen in caspase-8 mediated apoptotic signalling. As HOIP creates linear chains that ABIN1 can bind to, and both proteins protect cells from cisplatin-induced apoptosis, I also discuss whether ABIN1 and HOIP may act together in the same pathway.
In conclusion, the siRNA screen identified several potential targets as novel regulators of the response to cisplatin. Further investigations revealed that HOIP and ABIN1 protect cells from cisplatin-induced apoptosis, emphasising the importance of ubiquitin signalling in cell death and the DNA damage response.
|Date of Award||2014|
|Supervisor||Arno Alpi (Supervisor)|