Exploring the role of FLIP and Itch in mechanisms of resistance to apoptosis

Abstract

A considerable number of cancers are inherently resistantto apoptosis demonstrating the need to develop novel strategies for combatingthis resistance. FLIP is the key negative regulator of the TRAIL-mediatedapoptotic pathway and is frequently overexpressed in cancer. It elicits itsanti-apoptotic function by preventing procaspase-8 homodimerization andauto-activation at the DISC. FLIP is a possible therapeutic target which hasbeen difficult target due to structural similarities with procaspase-8. Toprogress the understanding of FLIP as a therapeutic targeted we investigatedthe regulation of FLIP through Itch-mediated ubiquitination and by disruptingits interaction with the pro-apoptotic FADD using newly developed inhibitors.

Here we identify the E3 ubiquitin ligase Itch as a regulatorof TRAIL sensitivity. OE33 Itch knockdown cells were more resistant toTRAIL-mediated apoptosis despite no observable change in the expression FLIPsplice forms. We identified a novel role for Itch in regulating TRAIL-mediatedapoptosis and chemotherapy.

This study identified that levels of FLIP were adetermining factor in mediating the induction of apoptosis in response to twonovel FLIP inhibitors. These compounds induced procaspase-8 autoactivationwhich promoted downstream caspase signalling and induction of apoptosis. Weobserved that recruitment of FLIP to the death inducing signalling complex(DISC) at the plasma membrane was impaired by treatment with FLIP inhibitorsthus facilitating the induction of apoptosis. Additionally, it identified thatthese inhibitors could enhance the activity of TRAIL in a FLIP-dependent mannerin the HCT116 and U2OS cell lines. Procaspase-8 has a critical role in thefunction of these inhibitors. Its absence prevented DISC formation andinduction of apoptosis in response to TRAIL. Knockout of its paralog,procaspase-10 was shown to enhance apoptotic signalling and DISC formation inresponse to izTRAIL and FLIP inhibition but could not compensate for the lossof procaspase-8. It was frequently observed that FLIP protein expression wasnegatively affected by the FLIP inhibitors. The expression of theanti-apoptotic protein MCL-1 was negatively affected by the FLIP inhibitors.

Through two independent mechanisms this studydemonstrated the importance of developing new approaches to overcome resistanceto apoptosis in cancer. Further investigations are required to elucidate theresistance mechanisms governed by Itch and by FLIP in cancer cells. The FLIPinhibitors used in this study have promising in vitro effects which warrantfurther investigation in vivo.

Thesis is embargoed until 31 December 2028.