title: Hyperthermia enhances mapatumumab-induced apoptotic death through ubiquitin-mediated degradation of cellular FLIP(long) in human colon cancer cells.
abstract: 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.
authors.affiliation: Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: X
authors.initials: X
authors.lastname: Song
authors.forename: S-Y
authors.initials: SY
authors.lastname: Kim
authors.forename: Z
authors.initials: Z
authors.lastname: Zhou
authors.forename: E
authors.initials: E
authors.lastname: Lagasse
authors.forename: Y T
authors.initials: YT
authors.lastname: Kwon
authors.forename: Y J
authors.initials: YJ
authors.lastname: Lee
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006358:Hot Temperature; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D059748:Proteolysis; D015398:Signal Transduction / Q000187:drug effects; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D007976:Leupeptins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C507357:mapatumumab
references.citation: Oncogene. 2001 Aug 23;20(37):5225-31
references.pmid: 11526513
references.citation: Cell Death Differ. 2012 Jan;19(1):36-41
references.pmid: 22075988
references.citation: Mol Cancer Ther. 2008 Nov;7(11):3556-65
references.pmid: 19001438
references.citation: Cell Death Differ. 2010 Aug;17(8):1354-67
references.pmid: 20300110
references.citation: Ann Surg Oncol. 2005 Feb;12(2):138-44
references.pmid: 15827794
references.citation: J Biol Chem. 2012 Feb 17;287(8):6053-61
references.pmid: 22219201
references.citation: Eur J Cancer. 2002 May;38(7):1023-33
references.pmid: 11978527
references.citation: Cell. 2006 Feb 10;124(3):601-13
references.pmid: 16469705
references.citation: Mol Cell Biol. 2001 Aug;21(16):5299-305
references.pmid: 11463813
references.citation: Cancer Res. 2008 Sep 1;68(17):6932-41
references.pmid: 18757407
references.citation: Nature. 2009 Mar 26;458(7237):422-9
references.pmid: 19325621
references.citation: Mol Cell Biol. 2001 Dec;21(24):8247-54
references.pmid: 11713262
references.citation: Oncogene. 2001 Feb 1;20(5):571-80
references.pmid: 11313989
references.citation: Nat Rev Cancer. 2006 Oct;6(10):776-88
references.pmid: 16990855
references.citation: J Cell Biochem. 2010 Aug 1;110(5):1073-81
references.pmid: 20544795
references.citation: J Biol Chem. 2001 Jun 8;276(23):20633-40
references.pmid: 11279218
references.citation: Clin Cancer Res. 2009 Sep 1;15(17):5584-90
references.pmid: 19690193
references.citation: Int J Hyperthermia. 2006 Dec;22(8):713-28
references.pmid: 17391000
references.citation: Oncogene. 2006 Feb 9;25(6):838-48
references.pmid: 16247474
references.citation: J Immunol. 2008 Mar 1;180(5):3072-80
references.pmid: 18292530
references.citation: EMBO J. 2002 Jul 15;21(14):3704-14
references.pmid: 12110583
references.citation: J Biol Chem. 2002 Jun 21;277(25):22320-9
references.pmid: 11940602
references.citation: Cancer Res. 2007 Jun 15;67(12):5754-62
references.pmid: 17575142
references.citation: J Cell Biochem. 2012 May;113(5):1547-58
references.pmid: 22174016
references.citation: Ann Surg Oncol. 2010 Jul;17(7):1870-7
references.pmid: 20221901
references.citation: Surg Oncol. 1994 Apr;3(2):103-8
references.pmid: 7952389
references.citation: Physiol Rev. 2006 Apr;86(2):669-707
references.pmid: 16601271
references.citation: J Cell Biochem. 2008 Jan 1;103(1):98-109
references.pmid: 17520700
references.citation: Blood. 2008 Jan 15;111(2):829-37
references.pmid: 17932249
references.citation: Clin Cancer Res. 2007 Sep 1;13(17):5070-5
references.pmid: 17785559
references.citation: Carcinogenesis. 2010 Nov;31(11):1956-63
references.pmid: 20876284
references.citation: Cell Death Differ. 2009 Sep;16(9):1215-26
references.pmid: 19343040
references.citation: Cancer Res. 2011 Oct 1;71(19):6270-81
references.pmid: 21868755
references.citation: Cancer Gene Ther. 2005 Mar;12(3):228-37
references.pmid: 15550937
references.citation: Br J Cancer. 2010 Feb 2;102(3):506-12
references.pmid: 20068564
references.citation: J Biol Chem. 2005 Jul 22;280(29):27345-55
references.pmid: 15886205
references.citation: Br J Cancer. 2010 Dec 7;103(12):1783-7
references.pmid: 21081929
references.citation: J Cell Biol. 2010 Aug 9;190(3):377-89
references.pmid: 20696707
references.citation: Nature. 1997 Jul 10;388(6638):190-5
references.pmid: 9217161
references.citation: J Immunol. 2007 Mar 15;178(6):3944-53
references.pmid: 17339495
references.citation: Nature. 2003 Dec 18;426(6968):895-9
references.pmid: 14685250
references.citation: Ann Surg Oncol. 2009 Feb;16(2):385-94
references.pmid: 19034580
references.citation: J Cell Biochem. 2007 Jun 1;101(3):619-30
references.pmid: 17212362
references.citation: Cancer Res. 2008 Jun 15;68(12):4511-7
references.pmid: 18559494
references.citation: Oncogene. 2008 Oct 20;27(48):6216-27
references.pmid: 18931689