Sir Peter MacCallum Department of Oncology - Research Publications
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ItemDeregulation of MYCN, LIN28B and LET7 in a Molecular Subtype of Aggressive High-Grade Serous Ovarian Cancers(PUBLIC LIBRARY SCIENCE, 2011-04-13)Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention.
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ItemComparison of Expression Profiles in Ovarian Epithelium In Vivo and Ovarian Cancer Identifies Novel Candidate Genes Involved in Disease Pathogenesis(PUBLIC LIBRARY SCIENCE, 2011-03-15)Molecular events leading to epithelial ovarian cancer are poorly understood but ovulatory hormones and a high number of life-time ovulations with concomitant proliferation, apoptosis, and inflammation, increases risk. We identified genes that are regulated during the estrous cycle in murine ovarian surface epithelium and analysed these profiles to identify genes dysregulated in human ovarian cancer, using publically available datasets. We identified 338 genes that are regulated in murine ovarian surface epithelium during the estrous cycle and dysregulated in ovarian cancer. Six of seven candidates selected for immunohistochemical validation were expressed in serous ovarian cancer, inclusion cysts, ovarian surface epithelium and in fallopian tube epithelium. Most were overexpressed in ovarian cancer compared with ovarian surface epithelium and/or inclusion cysts (EpCAM, EZH2, BIRC5) although BIRC5 and EZH2 were expressed as highly in fallopian tube epithelium as in ovarian cancer. We prioritised the 338 genes for those likely to be important for ovarian cancer development by in silico analyses of copy number aberration and mutation using publically available datasets and identified genes with established roles in ovarian cancer as well as novel genes for which we have evidence for involvement in ovarian cancer. Chromosome segregation emerged as an important process in which genes from our list of 338 were over-represented including two (BUB1, NCAPD2) for which there is evidence of amplification and mutation. NUAK2, upregulated in ovarian surface epithelium in proestrus and predicted to have a driver mutation in ovarian cancer, was examined in a larger cohort of serous ovarian cancer where patients with lower NUAK2 expression had shorter overall survival. In conclusion, defining genes that are activated in normal epithelium in the course of ovulation that are also dysregulated in cancer has identified a number of pathways and novel candidate genes that may contribute to the development of ovarian cancer.
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ItemTandem duplication of chromosomal segments is common in ovarian and breast cancer genomes(WILEY, 2012-08)The application of paired-end next generation sequencing approaches has made it possible to systematically characterize rearrangements of the cancer genome to base-pair level. Utilizing this approach, we report the first detailed analysis of ovarian cancer rearrangements, comparing high-grade serous and clear cell cancers, and these histotypes with other solid cancers. Somatic rearrangements were systematically characterized in eight high-grade serous and five clear cell ovarian cancer genomes and we report here the identification of > 600 somatic rearrangements. Recurrent rearrangements of the transcriptional regulator gene, TSHZ3, were found in three of eight serous cases. Comparison to breast, pancreatic and prostate cancer genomes revealed that a subset of ovarian cancers share a marked tandem duplication phenotype with triple-negative breast cancers. The tandem duplication phenotype was not linked to BRCA1/2 mutation, suggesting that other common mechanisms or carcinogenic exposures are operative. High-grade serous cancers arising in women with germline BRCA1 or BRCA2 mutation showed a high frequency of small chromosomal deletions. These findings indicate that BRCA1/2 germline mutation may contribute to widespread structural change and that other undefined mechanism(s), which are potentially shared with triple-negative breast cancer, promote tandem chromosomal duplications that sculpt the ovarian cancer genome.
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ItemAmplicon-Dependent CCNE1 Expression Is Critical for Clonogenic Survival after Cisplatin Treatment and Is Correlated with 20q11 Gain in Ovarian Cancer(PUBLIC LIBRARY SCIENCE, 2010-11-12)Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.
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ItemNo Preview AvailableResistance to CDK2 Inhibitors Is Associated with Selection of Polyploid Cells in CCNE1-Amplified Ovarian Cancer(AMER ASSOC CANCER RESEARCH, 2013-11-01)PURPOSE: Amplification of cyclin E1 (CCNE1) is associated with poor outcome in breast, lung, and other solid cancers, and is the most prominent structural variant associated with primary treatment failure in high-grade serous ovarian cancer (HGSC). We have previously shown that CCNE1-amplified tumors show amplicon-dependent sensitivity to CCNE1 suppression. Here, we explore targeting CDK2 as a novel therapeutic strategy in CCNE1-amplified cancers and mechanisms of resistance. EXPERIMENTAL DESIGN: We examined the effect of CDK2 suppression using RNA interference and small-molecule inhibitors in SK-OV-3, OVCAR-4, and OVCAR-3 ovarian cancer cell lines. To identify mechanisms of resistance, we derived multiple, independent resistant sublines of OVCAR-3 to CDK2 inhibitors. Resistant cells were extensively characterized by gene expression and copy number analysis, fluorescence-activated cell sorting profiling and conventional karyotyping. In addition, we explored the relationship between CCNE1 amplification and polyploidy using data from primary tumors. RESULTS: We validate CDK2 as a therapeutic target in CCNE1-amplified cells by showing selective sensitivity to suppression, either by gene knockdown or using small-molecule inhibitors. In addition, we identified two resistance mechanisms, one involving upregulation of CDK2 and another novel mechanism involving selection of polyploid cells from the pretreatment tumor population. Our analysis of genomic data shows that polyploidy is a feature of cancer genomes with CCNE1 amplification. CONCLUSIONS: These findings suggest that cyclinE1/CDK2 is an important therapeutic target in HGSC, but that resistance to CDK2 inhibitors may emerge due to upregulation of CDK2 target protein and through preexisting cellular polyploidy.
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ItemNonequivalent Gene Expression and Copy Number Alterations in High-Grade Serous Ovarian Cancers with BRCA1 and BRCA2 Mutations(AMER ASSOC CANCER RESEARCH, 2013-07-01)PURPOSE: High-grade serous carcinoma (HGSC) accounts for the majority of epithelial ovarian cancer deaths. Genomic and functional data suggest that approximately half of unselected HGSC have disruption of the BRCA pathway and defects in homologous recombination repair (HRR). Pathway disruption is regarded as imparting a BRCAness phenotype. We explored the molecular changes in HGSC arising in association with specific BRCA1/BRCA2 somatic or germline mutations and in those with BRCA1 DNA promoter methylation. EXPERIMENTAL DESIGN: We describe gene expression and copy number analysis of two large cohorts of HGSC in which both germline and somatic inactivation of HRR has been measured. RESULTS: BRCA1 disruptions were associated with the C2 (immunoreactive) molecular subtype of HGSC, characterized by intense intratumoral T-cell infiltration. We derived and validated a predictor of BRCA1 mutation or methylation status, but could not distinguish BRCA2 from wild-type tumors. DNA copy number analysis showed that cases with BRCA1 mutation were significantly associated with amplification both at 8q24 (frequencies: BRCA1 tumors 50%, BRCA2 tumors 32%, and wild-type tumors 9%) and regions of the X-chromosome specifically dysregulated in basal-like breast cancer (BLBC; BRCA1 62%, BRCA2 34%, and wild-type 35%). Tumors associated with BRCA1/BRCA2 mutations shared a negative association with amplification at 19p13 (BRCA1 0%, BRCA2 3%, and wild-type 20%) and 19q12 (BRCA1 6%, BRCA2 3%, and wild-type 29%). CONCLUSION: The molecular differences between tumors associated with BRCA1 compared with BRCA2 mutations are in accord with emerging clinical and pathologic data and support a growing appreciation of the relationship between HGSC and BLBC.
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ItemNo Preview AvailableSynthetic lethality between CCNE1 amplification and loss of BRCA1(NATL ACAD SCIENCES, 2013-11-26)High-grade serous ovarian cancers (HGSCs) are characterized by a high frequency of TP53 mutations, BRCA1/2 inactivation, homologous recombination dysfunction, and widespread copy number changes. Cyclin E1 (CCNE1) gene amplification has been reported to occur independently of BRCA1/2 mutation, and it is associated with primary treatment failure and reduced patient survival. Insensitivity of CCNE1-amplified tumors to platinum cross-linking agents may be partly because of an intact BRCA1/2 pathway. Both BRCA1/2 dysfunction and CCNE1 amplification are known to promote genomic instability and tumor progression. These events may be mutually exclusive, because either change provides a path to tumor development, with no selective advantage to having both mutations. Using data from a genome-wide shRNA synthetic lethal screen, we show that BRCA1 and members of the ubiquitin pathway are selectively required in cancers that harbor CCNE1 amplification. Furthermore, we show specific sensitivity of CCNE1-amplified tumor cells to the proteasome inhibitor bortezomib. These findings provide an explanation for the observed mutual exclusivity of CCNE1 amplification and BRCA1/2 loss in HGSC and suggest a unique therapeutic approach for treatment-resistant CCNE1-amplified tumors.
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ItemNo Preview AvailableLRP1B Deletion in High-Grade Serous Ovarian Cancers Is Associated with Acquired Chemotherapy Resistance to Liposomal Doxorubicin(AMER ASSOC CANCER RESEARCH, 2012-08-15)High-grade serous cancer (HGSC), the most common subtype of ovarian cancer, often becomes resistant to chemotherapy, leading to poor patient outcomes. Intratumoral heterogeneity occurs in nearly all solid cancers, including ovarian cancer, contributing to the development of resistance mechanisms. In this study, we examined the spatial and temporal genomic variation in HGSC using high-resolution single-nucleotide polymorphism arrays. Multiple metastatic lesions from individual patients were analyzed along with 22 paired pretreatment and posttreatment samples. We documented regions of differential DNA copy number between multiple tumor biopsies that correlated with altered expression of genes involved in cell polarity and adhesion. In the paired primary and relapse cohort, we observed a greater degree of genomic change in tumors from patients that were initially sensitive to chemotherapy and had longer progression-free interval compared with tumors from patients that were resistant to primary chemotherapy. Notably, deletion or downregulation of the lipid transporter LRP1B emerged as a significant correlate of acquired resistance in our analysis. Functional studies showed that reducing LRP1B expression was sufficient to reduce the sensitivity of HGSC cell lines to liposomal doxorubicin, but not to doxorubicin, whereas LRP1B overexpression was sufficient to increase sensitivity to liposomal doxorubicin. Together, our findings underscore the large degree of variation in DNA copy number in spatially and temporally separated tumors in HGSC patients, and they define LRP1B as a potential contributor to the emergence of chemotherapy resistance in these patients.
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ItemNo Preview AvailableBRCA Mutation Frequency and Patterns of Treatment Response in BRCA Mutation-Positive Women With Ovarian Cancer: A Report From the Australian Ovarian Cancer Study Group(AMER SOC CLINICAL ONCOLOGY, 2012-07-20)PURPOSE: The frequency of BRCA1 and BRCA2 germ-line mutations in women with ovarian cancer is unclear; reports vary from 3% to 27%. The impact of germ-line mutation on response requires further investigation to understand its impact on treatment planning and clinical trial design. PATIENTS AND METHODS: Women with nonmucinous ovarian carcinoma (n = 1,001) enrolled onto a population-based, case-control study were screened for point mutations and large deletions in both genes. Survival outcomes and responses to multiple lines of chemotherapy were assessed. RESULTS: Germ-line mutations were found in 14.1% of patients overall, including 16.6% of serous cancer patients (high-gradeserous, 17.1%); [corrected] 44% had no reported family history of breast orovarian cancer.Patients carrying germ-line mutations had improved rates of progression-free and overall survival. In the relapse setting, patients carrying mutations more frequently responded to both platin- and nonplatin-based regimens than mutation-negative patients, even in patients with early relapse after primary treatment. Mutation-negative patients who responded to multiple cycles of platin-based treatment were more likely to carry somatic BRCA1/2 mutations. CONCLUSION: BRCA mutation status has a major influence on survival in ovarian cancer patients and should be an additional stratification factor in clinical trials. Treatment outcomes in BRCA1/2 carriers challenge conventional definitions of platin resistance, and mutation status may be able to contribute to decision making and systemic therapy selection in the relapse setting. Our data, together with the advent of poly(ADP-ribose) polymerase inhibitor trials, supports the recommendation that germ-line BRCA1/2 testing should be offered to all women diagnosed with nonmucinous, ovarian carcinoma, regardless of family history.