title: Ubiquitination profiling identifies sensitivity factors for IAP antagonist treatment.
abstract: Evasion of cell death is one crucial capability acquired by tumour cells to ward-off anti-tumour therapies and represents a fundamental challenge to sustaining clinical efficacy for currently available agents. Inhibitor of apoptosis (IAP) proteins use their ubiquitin E3 ligase activity to promote cancer cell survival by mediating proliferative signalling and blocking cell death in response to diverse stimuli. Using immunoaffinity enrichment and MS, ubiquitination sites on thousands of proteins were profiled upon initiation of cell death by IAP antagonists in IAP antagonist-sensitive and -resistant breast cancer cell lines. Our analyses identified hundreds of proteins with elevated levels of ubiquitin-remnant [K-GG (Lys-Gly-Gly)] peptides upon activation of cell death by the IAP antagonist BV6. The majority of these were observed in BV6-sensitive, but not-resistant, cells. Among these were known pro-apoptotic regulators, including CYC (cytochrome c), RIP1 (receptor-interacting protein 1) and a selection of proteins known to reside in the mitochondria or regulate NF-κB (nuclear factor κB) signalling. Analysis of early time-points revealed that IAP antagonist treatment stimulated rapid ubiquitination of NF-κB signalling proteins, including TRAF2 [TNF (tumour necrosis factor) receptor-associated factor 2], HOIL-1 (haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1), NEMO (NF-κB essential modifier), as well as c-IAP1 (cellular IAP1) auto-ubiquitination. Knockdown of several NF-κB pathway members reduced BV6-induced cell death and TNF production in sensitive cell lines. Importantly, RIP1 was found to be constitutively ubiquitinated in sensitive breast-cancer cell lines at higher basal level than in resistant cell lines. Together, these data show the diverse and temporally defined roles of protein ubiquitination following IAP-antagonist treatment and provide critical insights into predictive diagnostics that may enhance clinical efficacy.
authors.affiliation: *Departments of Early Discovery Biochemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Eugene
authors.initials: E
authors.lastname: Varfolomeev
authors.affiliation: †Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Anita
authors.initials: A
authors.lastname: Izrael-Tomasevic
authors.affiliation: †Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Kebing
authors.initials: K
authors.lastname: Yu
authors.affiliation: †Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Daisy
authors.initials: D
authors.lastname: Bustos
authors.affiliation: *Departments of Early Discovery Biochemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Goncharov
authors.affiliation: ‡Discovery Oncology, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Belmont
authors.affiliation: †Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Masselot
authors.affiliation: †Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Corey E
authors.initials: CE
authors.lastname: Bakalarski
authors.affiliation: †Protein Chemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Donald S
authors.initials: DS
authors.lastname: Kirkpatrick
authors.affiliation: *Departments of Early Discovery Biochemistry, Genentech, Inc. South San Francisco, CA 94080, U.S.A.
authors.forename: Domagoj
authors.initials: D
authors.lastname: Vucic
mesh_terms: D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051034:Inhibitor of Apoptosis Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D047992:TNF Receptor-Associated Factor 2 / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D025801:Ubiquitin / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: C119724:AGFG1 protein, human; C582180:BV6 peptide; C491731:IKBKG protein, human; D051034:Inhibitor of Apoptosis Proteins; D024101:Mitochondrial Proteins; D016328:NF-kappa B; D028861:Nuclear Pore Complex Proteins; D009842:Oligopeptides; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D047992:TNF Receptor-Associated Factor 2; D014157:Transcription Factors; D025801:Ubiquitin; D045304:Cytochromes c; C504507:RBCK1 protein, human; D044767:Ubiquitin-Protein Ligases; D051550:I-kappa B Kinase