Abstractc-FLIP (cellular FADD-like-interleukin-ip-converting enzyme (FLICE) inhibitory protein) is a major anti-apoptotic protein that prevents apoptosis mediated by the death receptors TNF-R1, Fas, DR4 (TRAIL-R1) and DR5 (TRAIL-R2). c-FLIP binds to the death inducing signalling complex (DISC) via its tandem death effector domains (DEDs) and the DED of Fas-associated death domain-containing protein (FADD), thereby inhibiting processing of procaspases 8 and 10 at the DISC. Overexpression of c-FLIP results in the evasion of apoptosis, a key hallmark of cancer and a major factor in drug resistance.
Previous research in our group demonstrated that c-FLIP is critical regulator of cell death, regulating apoptosis induced not only by death ligands, but also by a range of chemotherapeutic agents in a variety of disease models. Importantly, normal cells do not appear to rely on c-FLIP for survival to the same extent as cancer cells. c-FLIP is overexpressed in colorectal cancer, and high c-FLIP expression has been demonstrated to be an independent adverse prognostic marker in stage ll/lll colorectal cancer. Collectively, these and a number of other preclinical studies have indicated that c-FLIP is an important determinant of cancer cell survival and drug resistance and that inhibition of c-FLIP constitutes a promising therapeutic strategyfor the treatment of cancer.
We conducted a yeast-2-hybrid screen to identify novel binding partners of c-FLIP. The DNA repair protein Ku70 was identified in this screen, and the aim of this study was to characterise the interaction between c-FLIP and Ku70, with a particular focus on how Ku70 regulates c-FLIP expression. It was found that Ku70 interacts with both long and short forms of c-FLIP. The region of Ku70 that interacts with c-FLIP was identified as amino acids 430-496. This region is also important in mediating Ku70 DNA repair functions by facilitating the heterodimerisation with Ku86 to form the Ku complex. Furthermore, the nuclear localisation signal (NLS) that is involved in the retention of Ku70 in the nucleus is also located in this region. It was also demonstrated that the second DED (DED2) of c-FLIP is important in mediating its interaction with Ku70, and more specifically arginine 122 of c-FLIP was found to be involved. However, this arginine residue did not affect the interaction between c-FLIP and FADD and procaspase 8, indicating that c-FLIP uses distinct but overlapping regions to interact with FADD/procaspase 8 and Ku70 respectively.
Using siRNAs targeted against Ku70, it was demonstrated that when Ku70 is silenced, c-FLIP expression is decreased in a panel of colorectal cancer cells. Further investigation determined that siRNA-mediated Ku70 silencing results in decreased protein stability, increased ubiquitination of c-FLIP and increased protein turnover. In addition, silencing Ku70 induces caspase 8-dependent apoptotic cell death that requires down-regulation of c-FLIP. These results demonstrate that Ku70 plays a role in regulating c-FLIP expression by inhibiting its ubiquitination.
Ku70 is a target for acetylation, and this acetylation is increased following treatment with HDAC inhibitors. The interaction between c-FLIP and Ku70 was demonstrated to be highly dependent on acetylation of Ku70, with acetylation of lysines K539 and K542 of Ku70 important in regulating the c-FLIP-Ku70 interaction. Furthermore, it was demonstrated that the pan-FIDAC inhibitor SAFIA could increase the acetylation of Ku70, dissociate the c-FLIP-Ku70 interaction and cause the degradation of c-FLIP through the ubiquitin-proteasome system. Moreover, degradation of c-FLIP by SAHA induced caspase 8-dependent apoptosis that was also dependent on the expression of death receptors, particularly DR5. In addition, it was found that SAHA significantly down-regulated c-FLIP expression and retarded the growth of colorectal xenografts. Also, the overexpression of c-FLIP attenuated the in vivo growth retardation caused by SAHA treatment. Through the use of the more selective HDAC inhibitors, Droxinostat (HDAC3, 6 and 8) and Tubacin (HDAC6), it was determined that HDAC6 is a key HDAC in regulating the acetylation of Ku70 and the expression of c-FLIP in colorectal cancer cells.
In summary, this study identified an interaction between the anti-apoptotic protein c- FLIP and the DMA repair protein Ku70 that is regulated by HDAC6-dependent acetylation of Ku70 and which regulates the expression of c-FLIP. In addition, due to the importance of c-FLIP down-regulation during the apoptotic response to SAHA and the frequency of c-FLIP overexpression in a variety of cancer subtypes, it is possible that c-FLIP, caspase 8 and Ku70 may be biomarkers of response to HDACinhibitor treatment.
|Date of Award||Jul 2012|
|Supervisor||Daniel Longley (Supervisor)|