Colorectal cancer is the third leading cause of
cancer-related mortality in the world; the main cause of death of
colorectal cancer is hepatic
metastases, which can be treated with
hyperthermia using isolated hepatic perfusion (IHP). In this study, we report that mild
hyperthermia potently reduced cellular FLIP(long), (c-
FLIP(L)), a major regulator of the
death receptor (DR) pathway of apoptosis, thereby enhancing humanized anti-DR4 antibody
mapatumumab (Mapa)-mediated mitochondria-independent apoptosis. We observed that overexpression of c-
FLIP(L) in CX-1 cells abrogated the synergistic effect of Mapa and
hyperthermia, whereas silencing of c-FLIP in CX-1 cells enhanced Mapa-induced apoptosis.
Hyperthermia altered c-
FLIP(L) protein stability without concomitant reductions in FLIP
mRNA. Ubiquitination of c-
FLIP(L) was increased by
hyperthermia, and
proteasome inhibitor MG132 prevented heat-induced downregulation of c-
FLIP(L). These results suggest the involvement of the
ubiquitin-
proteasome system in this process. We also found
lysine residue 195 (K195) to be essential for c-
FLIP(L) ubiquitination and proteolysis, as mutant c-
FLIP(L)
lysine 195
arginine (
arginine replacing
lysine) was left virtually un-ubiquitinated and was refractory to
hyperthermia-triggered degradation, and thus partially blocked the synergistic effect of Mapa and
hyperthermia. Our observations reveal that
hyperthermia transiently reduced c-
FLIP(L) by proteolysis linked to K195 ubiquitination, which contributed to the synergistic effect between Mapa and
hyperthermia. This study supports the application of
hyperthermia combined with other regimens to treat colorectal hepatic
metastases.