Sir Peter MacCallum Department of Oncology - Research Publications

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    A DOMINANT-NEGATIVE MUTANT OF MSOS1 INHIBITS INSULIN-INDUCED RAS ACTIVATION AND REVEALS RAS-DEPENDENT AND RAS-INDEPENDENT INSULIN SIGNALING PATHWAYS
    SAKAUE, M ; BOWTELL, D ; KASUGA, M (AMER SOC MICROBIOLOGY, 1995-01)
    The role of the Grb2-SOS complex in insulin signal transduction was investigated with a deletion mutant of mSOS1 that lacks the guanine nucleotide exchange domain of the wild-type protein. Cells over-expressing either wild-type (CHO-IR/SOS cells) or mutant (CHO-IR/delta SOS cells) mSOS1 were established by transfection of Chinese hamster ovary cells that express human insulin receptors (CHO-IR cells) with the appropriate expression plasmid. The mutant mSOS1 protein did not contain the guanine nucleotide exchange activity in vitro and associated with Grb2 both in vivo and in vitro. In both CHO-IR and CHO-IR/SOS cells, insulin rapidly stimulated the formation of GTP-bound Ras and the phosphorylation of mitogen-activated protein (MAP) kinase; both these effects of insulin were markedly inhibited in CHO-IR/delta SOS cells. Insulin-induced glycogen synthase and 70-kDa S6 kinase activities were not affected by expression of either wild-type or mutant mSOS1. These results show that the mutant mSOS1 acts in a dominant-negative manner and suggest that the Grb2-SOS complex mediates, at least in part, insulin-induced activation of Ras in intact cells. The data also indicate that Ras activation is not required for insulin-induced stimulation of glycogen synthase and 70-kDa S6 kinase.
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    Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficient mice
    Murphy, MA ; Schnall, RG ; Venter, DJ ; Barnett, L ; Bertoncello, I ; Thien, CBF ; Langdon, WY ; Bowtell, DDL (AMER SOC MICROBIOLOGY, 1998-08)
    The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, the Caenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates, indicating an unanticipated role for c-Cbl in regulating mammary growth. Collectively, the hyperplastic histological changes seen in c-Cbl mutant mice are indicative of a normal role for c-Cbl in negatively regulating signalling events that control cell growth. Consistent with this view, we observed greatly increased intracellular protein tyrosine phosphorylation in thymocytes following CD3epsilon cross-linking. In particular, phosphorylation of ZAP-70 kinase in thymocytes was uncoupled from a requirement for CD4-mediated Lck activation. This study provides the first biochemical characterization of any organism that is deficient in a member of this unique protein family. Our findings demonstrate critical roles for c-Cbl in hemopoiesis and in controlling cellular proliferation and signalling by the Syk/ZAP-70 family of protein kinases.
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    Normal p53 function in primary cells deficient for Siah genes
    Frew, IJ ; Dickins, RA ; Cuddihy, AR ; Del Rosario, M ; Reinhard, C ; O'Connell, MJ ; Bowtell, DDL (AMER SOC MICROBIOLOGY, 2002-12)
    Overexpression studies have suggested that Siah1 proteins may act as effectors of p53-mediated cellular responses and as regulators of mitotic progression. We have tested these hypotheses using Siah gene knockout mice. Siah1a and Siah1b were not induced by activation of endogenous p53 in tissues, primary murine embryonic fibroblasts (MEFs) or thymocytes. Furthermore, primary MEFs lacking Siah1a, Siah1b, Siah2, or both Siah2 and Siah1a displayed normal cell cycle progression, proliferation, p53-mediated senescence, and G(1) phase cell cycle arrest. Primary thymocytes deficient for Siah1a, Siah2, or both Siah2 and Siah1a, E1A-transformed MEFs lacking Siah1a, Siah1b, or Siah2, and Siah1b-null ES cells all underwent normal p53-mediated apoptosis. Finally, inhibition of Siah1b expression in Siah2 Siah1a double-mutant cells failed to inhibit cell division, p53-mediated induction of p21 expression, or cell cycle arrest. Our loss-of-function experiments do not support a general role for Siah genes in p53-mediated responses or mitosis.
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    Generation and analysis of Siah2 mutant mice
    Frew, IJ ; Hammond, VE ; Dickins, RA ; Quinn, JMW ; Walkley, CR ; Sims, NA ; Schnall, R ; Della, NG ; Holloway, AJ ; Digby, MR ; Janes, PW ; Tarlinton, DM ; Purton, LE ; Gillespie, MT ; Bowtell, DDL (AMER SOC MICROBIOLOGY, 2003-12)
    Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.
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    The ubiquitin ligase component Siah1a is required for completion of meiosis I in male mice
    Dickins, RA ; Frew, IJ ; House, CM ; O'Bryan, MK ; Holloway, AJ ; Haviv, I ; Traficante, N ; de Kretser, DM ; Bowtell, DDL (AMER SOC MICROBIOLOGY, 2002-04)
    The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including beta-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division.
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    The Prognostic Effect of Immune Cell Infiltration Depends on Molecular Subtype in Endometrioid Ovarian Carcinomas.
    Heinze, K ; Cairns, ES ; Thornton, S ; Harris, B ; Milne, K ; Grube, M ; Meyer, C ; Karnezis, AN ; Fereday, S ; Garsed, DW ; Leung, SCY ; Chiu, DS ; Moubarak, M ; Harter, P ; Heitz, F ; McAlpine, JN ; DeFazio, A ; Bowtell, DDL ; Goode, EL ; Pike, M ; Ramus, SJ ; Pearce, CL ; Staebler, A ; Köbel, M ; Kommoss, S ; Talhouk, A ; Nelson, BH ; Anglesio, MS (American Association for Cancer Research (AACR), 2023-09-01)
    PURPOSE: Endometrioid ovarian carcinoma (ENOC) is the second most-common type of ovarian carcinoma, comprising 10%-20% of cases. Recently, the study of ENOC has benefitted from comparisons to endometrial carcinomas including defining ENOC with four prognostic molecular subtypes. Each subtype suggests differential mechanisms of progression, although tumor-initiating events remain elusive. There is evidence that the ovarian microenvironment may be critical to early lesion establishment and progression. However, while immune infiltrates have been well studied in high-grade serous ovarian carcinoma, studies in ENOC are limited. EXPERIMENTAL DESIGN: We report on 210 ENOC, with clinical follow-up and molecular subtype annotation. Using multiplex IHC and immunofluorescence, we examine the prevalence of T-cell lineage, B-cell lineage, macrophages, and populations with programmed cell death protein 1 or programmed death-ligand 1 across subtypes of ENOC. RESULTS: Immune cell infiltrates in tumor epithelium and stroma showed higher densities in ENOC subtypes with known high mutation burden (POLEmut and MMRd). While molecular subtypes were prognostically significant, immune infiltrates were not (overall survival P > 0.2). Analysis by molecular subtype revealed that immune cell density was prognostically significant in only the no specific molecular profile (NSMP) subtype, where immune infiltrates lacking B cells (TILB minus) had inferior outcome (disease-specific survival: HR, 4.0; 95% confidence interval, 1.1-14.7; P < 0.05). Similar to endometrial carcinomas, molecular subtype stratification was generally superior to immune response in predicting outcomes. CONCLUSIONS: Subtype stratification is critical for better understanding of ENOC, in particular the distribution and prognostic significance of immune cell infiltrates. The role of B cells in the immune response within NSMP tumors warrants further study.
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    Multiomic analysis of homologous recombination-deficient end-stage high-grade serous ovarian cancer
    Burdett, NL ; Willis, MO ; Alsop, K ; Hunt, AL ; Pandey, A ; Hamilton, PT ; Abulez, T ; Liu, X ; Hoang, T ; Craig, S ; Fereday, S ; Hendley, J ; Garsed, DW ; Milne, K ; Kalaria, S ; Marshall, A ; Hood, BL ; Wilson, KN ; Conrads, KA ; Pishas, K ; Ananda, S ; Scott, CL ; Antill, Y ; McNally, O ; Mileshkin, L ; Hamilton, A ; Au-Yeung, G ; Devereux, L ; Thorne, H ; Bild, A ; Bateman, NW ; Maxwell, GL ; Chang, JT ; Conrads, TPP ; Nelson, BH ; Bowtell, DDL ; Christie, ELL (NATURE PORTFOLIO, 2023-03)
    High-grade serous ovarian cancer (HGSC) is frequently characterized by homologous recombination (HR) DNA repair deficiency and, while most such tumors are sensitive to initial treatment, acquired resistance is common. We undertook a multiomics approach to interrogate molecular diversity in end-stage disease, using multiple autopsy samples collected from 15 women with HR-deficient HGSC. Patients had polyclonal disease, and several resistance mechanisms were identified within most patients, including reversion mutations and HR restoration by other means. We also observed frequent whole-genome duplication and global changes in immune composition with evidence of immune escape. This analysis highlights diverse evolutionary changes within HGSC that evade therapy and ultimately overwhelm individual patients.
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    Methylation Signature Implicated in Immuno-Suppressive Activities in Tubo-Ovarian High-Grade Serous Carcinoma
    Wang, C ; Block, MS ; Cunningham, JM ; Sherman, ME ; McCauley, BM ; Armasu, SM ; Vierkant, RA ; Traficante, N ; Talhouk, A ; Doherty, JA ; Pejovic, N ; Kobel, M ; Jorgensen, BD ; Garsed, DW ; Fereday, S ; Ramus, SJ ; Ariyaratne, D ; Anglesio, MS ; Widschwendter, M ; Pejovic, T ; Bosquet, JG ; Bowtell, DD ; Winham, SJ ; Goode, EL (AMER ASSOC CANCER RESEARCH, 2023-04)
    BACKGROUND: Better understanding of prognostic factors in tubo-ovarian high-grade serous carcinoma (HGSC) is critical, as diagnosis confers an aggressive disease course. Variation in tumor DNA methylation shows promise predicting outcome, yet prior studies were largely platform-specific and unable to evaluate multiple molecular features. METHODS: We analyzed genome-wide DNA methylation in 1,040 frozen HGSC, including 325 previously reported upon, seeking a multi-platform quantitative methylation signature that we evaluated in relation to clinical features, tumor characteristics, time to recurrence/death, extent of CD8+ tumor-infiltrating lymphocytes (TIL), gene expression molecular subtypes, and gene expression of the ATP-binding cassette transporter TAP1. RESULTS: Methylation signature was associated with shorter time to recurrence, independent of clinical factors (N = 715 new set, hazard ratio (HR), 1.65; 95% confidence interval (CI), 1.10-2.46; P = 0.015; N = 325 published set HR, 2.87; 95% CI, 2.17-3.81; P = 2.2 × 10-13) and remained prognostic after adjustment for gene expression molecular subtype and TAP1 expression (N = 599; HR, 2.22; 95% CI, 1.66-2.95; P = 4.1 × 10-8). Methylation signature was inversely related to CD8+ TIL levels (P = 2.4 × 10-7) and TAP1 expression (P = 0.0011) and was associated with gene expression molecular subtype (P = 5.9 × 10-4) in covariate-adjusted analysis. CONCLUSIONS: Multi-center analysis identified a novel quantitative tumor methylation signature of HGSC applicable to numerous commercially available platforms indicative of shorter time to recurrence/death, adjusting for other factors. Along with immune cell composition analysis, these results suggest a role for DNA methylation in the immunosuppressive microenvironment. IMPACT: This work aids in identification of targetable epigenome processes and stratification of patients for whom tailored treatment may be most beneficial.
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    A comparison of DNA sequencing and gene expression profiling to assist tissue of origin diagnosis in cancer of unknown primary
    Posner, A ; Prall, OW ; Sivakumaran, T ; Etemadamoghadam, D ; Thio, N ; Pattison, A ; Balachander, S ; Fisher, K ; Webb, S ; Wood, C ; DeFazio, A ; Wilcken, N ; Gao, B ; Karapetis, CS ; Singh, M ; Collins, IM ; Richardson, G ; Steer, C ; Warren, M ; Karanth, N ; Wright, G ; Williams, S ; George, J ; Hicks, RJ ; Boussioutas, A ; Gill, AJ ; Solomon, BJ ; Xu, H ; Fellowes, A ; Fox, SB ; Schofield, P ; Bowtell, D ; Mileshkin, L ; Tothill, RW (WILEY, 2023-01)
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    Beating the odds: molecular characteristics of long-term survivors of ovarian cancer
    Garsed, DW ; Bowtell, DDL (Nature Research, 2022-12-01)
    High-grade serous ovarian cancer, the most common form of the disease, is often fatal. This study investigated the genomic and immune characteristics of tumors from women who survived more than 10 years after their initial diagnosis, and compared them with short-term and moderate-term survivors.