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PAX8 is a lineage-restricted transcription factor that is expressed in epithelial ovarian cancer (EOC) precursor tissues, and in the major EOC histotypes. Frequent overexpression of PAX8 in primary EOCs suggests this factor functions as an oncogene during tumorigenesis, however, the biological role of PAX8 in EOC development is poorly understood. We found that stable knockdown of PAX8 in EOC models significantly reduced cell proliferation and anchorage dependent growth in vitro, and attenuated tumorigenicity in vivo. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) and transcriptional profiling were used to create genome-wide maps of PAX8 binding and putative target genes. PAX8 binding sites were significantly enriched in promoter regions (p < 0.05) and superenhancers (p < 0.05). MEME-ChIP analysis revealed that PAX8 binding sites overlapping superenhancers or enhancers, but not promoters, were enriched for JUND/B and ARNT/AHR motifs. Integrating PAX8 ChIP-seq and gene expression data identified PAX8 target genes through their associations within shared topological association domains. Across two EOC models we identified 62 direct regulatory targets based on PAX8 binding in promoters and 1,330 putative enhancer regulatory targets. SEPW1, which is involved in oxidation-reduction, was identified as a PAX8 target gene in both cell line models. While the PAX8 cistrome exhibits a high degree of cell-type specificity, analyses of PAX8 target genes and putative cofactors identified common molecular targets and partners as candidate therapeutic targets for EOC.

Mutations or deletions in exons 18–21 in the EGFR) are present in approximately 15% of tumors in patients with non–small cell lung cancer (NSCLC). They lead to activation of the EGFR kinase domain and sensitivity to molecularly targeted therapeutics aimed at this domain (gefitinib or erlotinib). These drugs have demonstrated objective clinical response in many of these patients; however, invariably, all patients acquire resistance. To examine the molecular origins of resistance, we derived a set of gefitinib-resistant cells by exposing lung adenocarcinoma cell line, HCC827, with an activating mutation in the EGFR tyrosine kinase domain, to increasing gefitinib concentrations. Gefitinib-resistant cells acquired an increased expression and activation of JUN, a known oncogene involved in cancer progression. Ectopic overexpression of JUN in HCC827 cells increased gefitinib IC50 from 49 nmol/L to 8 μmol/L (P < 0.001). Downregulation of JUN expression through shRNA resensitized HCC827 cells to gefitinib (IC50 from 49 nmol/L to 2 nmol/L; P < 0.01). Inhibitors targeting JUN were 3-fold more effective in the gefitinib-resistant cells than in the parental cell line (P < 0.01). Analysis of gene expression in patient tumors with EGFR-activating mutations and poor response to erlotinib revealed a similar pattern as the top 260 differentially expressed genes in the gefitinib-resistant cells (Spearman correlation coefficient of 0.78, P < 0.01). These findings suggest that increased JUN expression and activity may contribute to gefitinib resistance in NSCLC and that JUN pathway therapeutics merit investigation as an alternate treatment strategy. Mol Cancer Ther; 16(8); 1645–57. ©2017 AACR.

Summary Long noncoding RNAs (lncRNAs) have emerged as critical regulators of tumorigenesis, and yet their mechanistic roles remain challenging to characterize. Here, we integrate functional proteomics with lncRNA-interactome profiling to characterize Urothelial Cancer Associated 1 (UCA1), a candidate driver of ovarian cancer development. Reverse phase protein array (RPPA) analysis indicates that UCA1 activates transcription coactivator YAP and its target genes. In vivo RNA antisense purification (iRAP) of UCA1 interacting proteins identified angiomotin (AMOT), a known YAP regulator, as a direct binding partner. Loss-of-function experiments show that AMOT mediates YAP activation by UCA1, as UCA1 enhances the AMOT-YAP interaction to promote YAP dephosphorylation and nuclear translocation. Together, we characterize UCA1 as a lncRNA regulator of Hippo-YAP signaling and highlight the UCA1-AMOT-YAP signaling axis in ovarian cancer development., Graphical Abstract, Highlights • A super-enhancer drives the expression of lncRNA UCA1 in EOC • Inactivation of UCA1 impairs tumor growth in vivo • UCA1 activates transcription coactivator YAP and its target genes • UCA1 promotes YAP dephosphorylation and nuclear translocation via AMOTp130, Cancer Systems Biology; Omics; Proteomics; Systems Biology

Summary: Fallopian tube secretory epithelial cells (FTSECs) are likely the main precursor cell type of high-grade serous ovarian cancers (HGSOCs), but these tumors may also arise from ovarian surface epithelial cells (OSECs). We profiled global landscapes of gene expression and active chromatin to characterize molecular similarities between OSECs (n = 114), FTSECs (n = 74), and HGSOCs (n = 394). A one-class machine learning algorithm predicts that most HGSOCs derive from FTSECs, with particularly high FTSEC scores in mesenchymal-type HGSOCs (padj < 8 × 10−4). However, a subset of HGSOCs likely derive from OSECs, particularly HGSOCs of the proliferative type (padj < 2 × 10−4), suggesting a dualistic model for HGSOC origins. Super-enhancer (SE) landscapes were also more similar between FTSECs and HGSOCs than between OSECs and HGSOCs (p < 2.2 × 10−16). The SOX18 transcription factor (TF) coincided with a HGSOC-specific SE, and ectopic overexpression of SOX18 in FTSECs caused epithelial-to-mesenchymal transition, indicating that SOX18 plays a role in establishing the mesenchymal signature of fallopian-derived HGSOCs. : Lawrenson et al. profile gene expression and active chromatin in ∼200 ovarian and fallopian epithelial isolates and implement machine learning to demonstrate that most high-grade serous ovarian cancers (HGSOCs) derive from fallopian tube epithelial cells, but a subset may originate from ovarian epithelia. SOX18 induces mesenchymal features to drive early neoplasia in fallopian tube precursors. Keywords: high-grade serous ovarian cancer, ovarian surface epithelial cell, fallopian tube secretory epithelial cell, super enhancers, transcription factors, SOX18, single-cell RNA-seq, RNA-seq, machine learning, one-class logistic regression models, dual origins

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of tumorigenesis and yet their mechanistic role remains challenging to characterize. Here, we present LncRNA Interpreter, a strategy integrating functional proteomics with interacting proteins to characterize lncRNAs. Its robustness is exemplified by lncRNA Urothelial Cancer Associated 1 (UCA1), a driver of ovarian cancer development. Reverse phase protein array (RPPA) analysis indicates that UCA1 inhibits Hippo signaling pathway and in vivo RNA antisense purification (iRAP) of UCA1 interacting proteins identified angiomotin (AMOT), a known YAP regulator, as direct binding partner. Loss-of-function experiments show that AMOT mediates Hippo signaling pathway inhibition by UCA1. UCA1 enhances the AMOT-YAP interaction to prevent YAP phosphorylation and facilitate its nuclear translocation. Together, our LncRNA Interpreter pipeline identified UCA1 as an lncRNA regulator of the Hippo signaling pathway and highlighted the UCA1-AMOT-YAP signaling axis in ovarian cancer development. LncRNA Interpreter can readily be applied to other lncRNAs implicated in complex diseases. Citation Format: Xianzhi Lin, Tassja J. Spindler, Marcos A de Souza Fonseca, Rosario I. Corona, Ji-Heui Seo, Felipe S Dezem, Lewyn Li, Janet M. Lee, Henry W. Long, Thomas A. Sellers, Beth Y. Karlan, Houtan Noushmehr, Matthew L. Freedman, Simon A. Gayther, Kate Lawrenson. Super-enhancer-associated long noncoding RNA UCA1 interacts directly with AMOT to inhibit Hippo signaling pathway in epithelial ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A30.

Historically, high-grade serous ovarian cancers (HGSOCs) were thought to arise from ovarian surface epithelial cells (OSECs) but recent data implicate fallopian tube secretory epithelial cells (FTSECs) as the major precursor. We performed transcriptomic and epigenomic profiling to characterize molecular similarities between OSECs, FTSECs and HGSOCs. Transcriptomic signatures of FTSECs were preserved in most HGSOCs reinforcing FTSECs as the predominant cell-of-origin; though an OSEC-like signature was associated with increased chemosensitivity (Padj= 0.03) and was enriched in proliferative-type tumors, suggesting a dualistic model for HGSOC origins. More super-enhancers (SEs) were shared between FTSECs and HGSOCs than between OSECS and HGSOCs (P< 2.2 × 10−16). SOX18, ELF3 and EHF transcription factors (TFs) coincided with HGSOC SEs and represent putative novel drivers of tumor development. Our integrative analyses support a predominantly fallopian origin for HGSOCs and indicate tumorigenesis may be driven by different TFs according to cell-of-origin.

We sought to identify susceptibility genes for high-grade serous ovarian cancer (HGSOC) by performing a transcriptome-wide association study of gene expression and splice junction usage in HGSOC-relevant tissue types (N = 2,169) and the largest genome-wide association study available for HGSOC (N = 13,037 cases and 40,941 controls). We identified 25 transcriptome-wide association study significant genes, 7 at the junction level only, including LRRC46 at 19q21.32, (P = 1 × 10−9), CHMP4C at 8q21 (P = 2 × 10−11) and a PRC1 junction at 15q26 (P = 7 × 10−9). In vitro assays for CHMP4C showed that the associated variant induces allele-specific exon inclusion (P = 0.0024). Functional screens in HGSOC cell lines found evidence of essentiality for three of the new genes we identified: HAUS6, KANSL1 and PRC1, with the latter comparable to MYC. Our study implicates at least one target gene for 6 out of 13 distinct genome-wide association study regions, identifying 23 new candidate susceptibility genes for HGSOC. A multi-tissue transcriptome-wide association study based on genetic predictors of expression level and alternative splicing in relevant tissues identifies 25 candidate genes associated with high-grade serous ovarian cancer.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of tumorigenesis and yet their mechanistic role remains challenging to characterize. Here, we present LncRNA Interpreter, a pipeline integrating functional proteomics with interacting proteins to characterize lncRNAs. Its robustness is exemplified by lncRNA Urothelial Cancer Associated 1 (UCA1), a driver of ovarian cancer development. Reverse phase protein array (RPPA) analysis indicates that UCA1 activates Hippo-YAP signaling and in vivo RNA antisense purification (iRAP) of UCA1 interacting proteins identified angiomotin (AMOT), a known YAP regulator, as direct binding partner. Loss-of-function experiments show that AMOT mediates Hippo-YAP signaling activation by UCA1. UCA1 enhances the AMOT-YAP interaction to prevent YAP phosphorylation and facilitate its nuclear translocation. Together, our LncRNA Interpreter pipeline identified UCA1 as an lncRNA regulator of the Hippo-YAP signaling and highlighted the UCA1-AMOT-YAP signaling axis in ovarian cancer development. LncRNA Interpreter can readily be applied to other lncRNAs implicated in complex diseases.

Genome-wide association studies have identified several risk associations for ovarian carcinomas (OC) but not for mucinous ovarian carcinomas (MOC). Genotypes from OC cases and controls were imputed into the 1000 Genomes Project reference panel. Analysis of 1,644 MOC cases and 21,693 controls identified three novel risk associations: rs752590 at 2q13 (P = 3.3 × 10−8), rs711830 at 2q31.1 (P = 7.5 × 10−12) and rs688187 at 19q13.2 (P = 6.8 × 10−13). Expression Quantitative Trait Locus (eQTL) analysis in ovarian and colorectal tumors (which are histologically similar to MOC) identified significant eQTL associations for HOXD9 at 2q31.1 in ovarian (P = 4.95 × 10−4, FDR = 0.003) and colorectal (P = 0.01, FDR = 0.09) tumors, and for PAX8 at 2q13 in colorectal tumors (P = 0.03, FDR = 0.09). Chromosome conformation capture analysis identified interactions between the HOXD9 promoter and risk SNPs at 2q31.1. Overexpressing HOXD9 in MOC cells augmented the neoplastic phenotype. These findings provide the first evidence for MOC susceptibility variants and insights into the underlying biology of the disease.

The OCAC OncoArray genotyping project: et al., To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC., The OCAC OncoArray genotyping project was funded through grants from the US National Institutes of Health (CA1X01HG007491-01 (C.I.A.), U19-CA148112 (T.A.S.), R01-CA149429 (C.M.P.) and R01-CA058598 (M.T.G.)); Canadian Institutes of Health Research (MOP-86727 (L.E.K.)); and the Ovarian Cancer Research Fund (A.B.). Funding for the CIMBA OncoArray genotyping was provided by the Government of Canada through Genome Canada and the Canadian Institutes of Health Research, the Ministère de l’Économie, de la Science et de l’Innovation du Québec through Génome Québec, the Quebec Breast Cancer Foundation for the PERSPECTIVE project, the US National Institutes of Health (CA1X01HG007491-01 (C.I.A.)), the Odense University Hospital Research Foundation (M.T.), the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (1420190 (S.K.P.)), the Italian Association for Cancer Research (IG16933 (L.O.)) and German Cancer Aid (110837 (R.K.S.).

For many cancers, there is a real need for more effective therapies. Although many drugs show promising results in vitro, most fail to translate into an in vivo model system, and only ∼5% show anti-tumor activity in clinical trials. It remains a significant challenge to accurately replicate in vitro the complex in vivo microenvironment in which cancers thrive, but this will be key to increasing the success of translating novel therapies into clinical practice. Three-dimensional (3D) cell culture models may better mimic primary tumors in vivo than traditional two-dimensional (2D) cultures. Therefore, we established and characterized 3D in vitro models of 31 epithelial ovarian cancer (EOC) cell lines, compared their biological and molecular features with 2D cultures and primary tumors, and tested their efficacy as models for evaluating chemoresponse. When cultured in 3D using polyhydroxoethylamethacrylate-coated plastics, EOC lines formed multicellular aggregates that could be classified as 'large dense', 'large loose', and 'small', based on size, light permeability, and proportion of cells incorporated into the complex structures. Features of histological differentiation characteristic of primary tumors that were not present in 2D cultures were restored in 3D. For many cell lines, the transition from a 2D to 3D microenvironment induced changes in the expression of several biomarkers relevant to disease. Generally, EOC cell lines proliferated more slowly and were more chemoresistant in 3D compared with 2D culture. In summary, 3D models of EOCs better reflect the histological, biological, and molecular features of primary tumors than the same cells cultured using traditional 2D techniques; 3D in vitro models also exhibit different sensitivities to chemotherapeutic agents compared with 2D models, which may have a significant impact on the success of drug testing pipelines for EOC. These findings could also impact in vitro modeling approaches and drug development strategies for other solid tumor types.

Super-enhancers demarcate regions of master regulators involved in cellular differentiation and tumorigenesis. While many trait-associated variants discovered through genome wide association studies are enriched in regulatory elements, the role of super-enhancers in disease susceptibility is less well understood. We evaluated the role of super-enhancers, defined by H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) analysis of epithelial ovarian cancers (EOC) and precursor cells, in mediating the genetic risk of EOC. We characterized super-enhancer-risk SNP intersections to identify a series of risk-associated loci where multiple candidate causal alleles overlap with ovarian super-enhancers (approximately 33% of candidate causal loci). In particular, a risk locus at 3q25 marked by SNP rs7651446, (odds ratio = 1.59, p = 1.5x10-34), is tagged by 79 candidate causal risk SNPs, more than half of which coincide with super-enhancers detected in EOC precursor cells (ovarian and fallopian tube epithelia) but not in ovarian cancer cell lines or primary tumors suggesting that loss of activity of this super-enhancer is associated with EOC development. We hypothesized that SNPs at the 3q25 locus modulate activity of super-enhancers that regulate in cis the expression of genes involved in the identity of normal tissue precursors, and that these genes are downregulated during tumorigenesis. We tested the effect of decreasing the binding affinity of BRD4, a protein that binds super-enhancer regions, using a small molecule inhibitor (JQ1). We measured the expression of genes at this locus following JQ1 treatment in EOC precursor cells, identifying three candidate genes, LEKR1, SSR3, and TiPARP, that show decreased expression due to BRD4 binding inhibition. For SSR3 and TIPARP, these expression level changes mirrored the differential expression observed between normal precursor cells and primary high grade serous ovarian cancer. We then established stable shRNA-mediated knockdown models for SSR3 and TIPARP in normal EOC precursor cell lines to establish a role for these genes in ovarian cancer development. In summary, we have identified a potential role for super-enhancers at a subset of EOC risk loci, and present a functional pipeline for identification of the target gene/genes and evaluation of their role in neoplastic transformation. Citation Format: Kevin C. Vavra, Rosario I. Corona, Ji-Heui Seo, Simon G. Coetzee, Janet M. Lee, Matthew L. Freedman, Paul D. Pharoah, Dennis J. Hazelett, Simon A. Gayther, Kate Lawrenson. The role of tissues specific super-enhancers in mediating the genetic risk of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1498.

Contains fulltext : 152042.pdf (Publisher’s version ) (Closed access) Genome-wide association studies have identified 20 genomic regions associated with risk of epithelial ovarian cancer (EOC), but many additional risk variants may exist. Here, we evaluated associations between common genetic variants [single nucleotide polymorphisms (SNPs) and indels] in DNA repair genes and EOC risk. We genotyped 2896 common variants at 143 gene loci in DNA samples from 15 397 patients with invasive EOC and controls. We found evidence of associations with EOC risk for variants at FANCA, EXO1, E2F4, E2F2, CREB5 and CHEK2 genes (P

Genome-wide association studies have reported 11 regions conferring risk of high-grade serous epithelial ovarian cancer (HGSOC). Expression quantitative trait locus (eQTL) analyses can identify candidate susceptibility genes at risk loci. Here we evaluate cis-eQTL associations at 47 regions associated with HGSOC risk (P≤10−5). For three cis-eQTL associations (P, Genome-wide association studies have identified regions which confer risk of high-grade serous epithelial ovarian cancer. Here the authors use expression quantitative train locus analysis to identify candidate genes and functionally characterise them, identifying a role for HOXD9 in ovarian cancer.

The management of patients with intradural spinal tumors differs in many respects from approaches taken for patients with intracranial tumors. Intramedullary lesions are often completely surrounded by normal spinal cord, displacing vital functional tracts eccentrically. Extramedullary lesions can drastically compress the spinal cord and nerve roots, reducing normal tissue to a ribbon-like consistency. The small amount of normal tissue relative to tumor has implications for surgery and postoperative adjuvant therapy. In addition, operative intervention must take spinal stability into consideration. In this report, we describe the current best care practices and strategies for patients with a diagnosis of spinal astrocytoma, ependymoma, hemangioblastoma, schwannoma, and meningioma. Treatment of patients with intradural tumors of the spinal cord and adjoining structures has changed over the past 20 years. Advances in many disciplines including neuroradiology, neurosurgery, neurooncology, and neuropathology have contributed to expediting diagnosis and improving outcomes.

PAX8 is a lineage-restricted transcription factor expressed in a large proportion of epithelial ovarian cancers (EOCs). PAX8 is commonly upregulated in EOCs relative to precursor tissues, suggesting it functions as an oncogene. However, the biological role of PAX8 during cancer initiation and development is poorly understood, and the genome-wide transcriptional targets have yet to be comprehensively catalogued. Using stable models of PAX8 knockdown in HEYA8 and IGROV1 EOC cell lines we show that PAX8 knockdown reduces cell proliferation in vitro and tumor growth in vivo. To understand how PAX8 regulates neoplastic phenotypes in cancer cells we performed RNA expression profiling by microarray in HEYA8 and IGROV1 before and after PAX8 knockdown. We also performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) for PAX8 and a marker of active chromatin (H3K27ac) in both cell lines. De novo motif discovery in the ChIP-seq profiles identified a PAX-like binding motif and several potential PAX8 cofactors in IGROV1 that are not enriched in HEYA8. These cofactors include members of the TEAD and Sp/KLF families of transcription factors. We divided the genes from our differential expression (DE) analysis in PAX8 knockdown into three non-overlapping categories: (a) direct regulatory targets (RTs), i.e. DE genes with PAX8 binding sites in their promoter regions; (b) putative enhancer RTs, where DE genes and PAX8 binding sites reside within the same topological association domain (TAD) defined from embryonic stem cells; and (c) indirect RTs. Our findings reveal that PAX8 can function as either an activator or a repressor, and that PAX8 regulates a very different set of genes in each cell line, as only a small fraction RTs (~50) is shared between HEYA8 and IGROV1. Nonetheless, independent pathway enrichment analysis reveals four pathways downstream of PAX8 that are common to both cell lines: DNA replication, cellular response to lipopolysaccharides, TNFA signaling via NFKB and anatomical structure morphogenesis. Individually, we discovered enrichment for G2M checkpoint and EMT signaling in HEYA8, and tissue morphogenesis and mesenchymal cell development in IGROV1. Collectively, our results suggest that PAX8 regulates cell proliferation and cancer promoting processes via highly tissue-specific regulation of common oncogenic pathways. Citation Format: Rosario I. Corona, Emily Adler, Janet M. Lee, Norma Rodriguez-Malave, Paulette Mhawech-Fauceglia, Heidi Sowter, Dennis J. Hazelett, Kate Lawrenson, Simon A. Gather. Characterization of the PAX8 regulatory network in epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2418. doi:10.1158/1538-7445.AM2017-2418

Superenhancers are master regulators of genes involved in cellular differentiation and tumorigenesis. While it is now well established that typical enhancers are key mediators of alleles associated with a mild risk of polygenic diseases, including cancer, the role of superenhancers is less understood. We evaluated the role of superenhancers in mediating risk of epithelial ovarian cancer (EOC). Using FunciVar software and superenhancers defined using H3K27ac chromatin immunoprecipitation followed by next generation sequencing, performed in cell lines and tissues, we characterized the superenhancer-risk SNP intersect and identified a series of risk-associated loci where multiple candidate causal alleles overlap with ovarian superenhancers. In particular, locus 3q25, the locus most associated with epithelial ovarian cancer (EOC) risk from GWAS studies, has >70 candidate causal risk SNPs, all of which coincide with superenhancers detected in ovarian cancer precursor cells (ovarian and fallopian tube epithelia) but not in ovarian cancers. We are proposing that the presence of one or more SNPs occurring at this locus leads to a higher expression of one or more genes by modulating activation of the superenhancer region. We tested the effect of decreasing the binding affinity of BRD4, a protein that binds superenhancer regions, using a small molecule inhibitor on the expression of genes in this locus. We identified three candidate genes—LEKR1, SSR3, and TiPARP—that demonstrated a decrease expression level as a result of BRD4 binding inhibition. Preliminary results show that partial knockdown of either SSR3 or TiPARP decreases normal ovarian and fallopian cell proliferation, and knocking down both SSR3 and TiPARP simultaneously shows an enhanced proliferation decrease compared with knockdown of a single gene. We then created stable knockout (KO) models using the CRISPR-Cas9 system to fully KO one, two or all three of our candidate causal genes: LEKR1, SSR3, and TiPARP. Functional evaluation of these KO models is currently underway to test the hypothesis that risk SNPs may regulate more than one gene at this locus. In all we identify a potential role for superenhancers at a subset of EOC risk loci, and present a functional pipeline for identification of the target gene/genes and evaluation of their role in neoplastic transformation. Citation Format: Kevin C. Vavra, Simon Coetzee, Janet M. Lee, Paul Pharoah, Dennis J. Hazelett, Kate Lawrenson, Simon A. Gayther. Functional evaluation of superenhancers as mediators of epithelial ovarian cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5502. doi:10.1158/1538-7445.AM2017-5502

High-grade serous ovarian cancers (HGSOCs) are the most common subtype of epithelial ovarian cancer. Pathways driving HGSOC development are poorly understood, and the cellular origins are debated in the literature. Previously, ovarian surface epithelial cells (OSECs) were thought to be cellular precursors of HGSOC, but there is now strong evidence that at least half of all HGSOCs arise from fallopian tube secretory epithelial cells (FTSECs). We took a large-scale integrated transcriptomic-epigenomic profiling approach to defining the molecular hallmarks of HGSOC and to explore the disease cell-of-origin. RNA sequencing was performed on 121 OSEC cultures and 84 FTSEC cultures and integrated with 394 HGSOC transcriptomes profiled by TCGA. We identified around 100 genes that differentiate OSECs and FTSECs, and pathway analysis of this gene list identified ovarian cancer genes as highly enriched (p=2.2x10-8), with ZEB1 and estrogen the most significant predicted upstream regulators (P Citation Format: Kate Lawrenson, Marcos Abraao, Felipe Segato, Janet M. Lee, Simon Coetzee, Ji-Heui Seo, Matthew L. Freedman, Dennis Hazelett, Simon Gayther, Houtan Noushmehr. Large scale integrated transcriptomic and epigenetic profiling defines the molecular hallmarks of HGSOC and disease origins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2417. doi:10.1158/1538-7445.AM2017-2417

Epithelial ovarian cancer (EOC) is still considered the most lethal gynecological malignancy and improved early detection of ovarian cancer is crucial to improving patient prognoses. To address this need, we tested whether candidate EOC biomarkers can be identified using three-dimensional (3D) in vitro models. We quantified changes in the abundance of secreted proteins in a 3D genetic model of early-stage EOC, generated by expressing CMYC and KRAS(G) (12) (V) in TERT-immortalized normal ovarian epithelial cells. Cellular proteins were labeled in live cells using stable isotopic amino acid analogues, and secreted proteins identified and quantified using liquid chromatography-tandem mass spectrometry. Thirty-seven and 55 proteins were differentially expressed by CMYC and CMYC+KRAS(G) (12) (V) expressing cells respectively (p 0.05;2-fold). We evaluated expression of the top candidate biomarkers in ∼210 primary EOCs: CHI3L1 and FKBP4 are both expressed by96% of primary EOCs, and FASN and API5 are expressed by 86 and 75% of cases. High expression of CHI3L1 and FKBP4 was associated with worse patient survival (p = 0.042 and p = 0.002, respectively). Expression of LGALS3BP was positively associated with recurrence (p = 0.0001) and suboptimal debulking (p = 0.018) suggesting that these proteins may be novel prognostic biomarkers. Furthermore, within early stage tumours (I/II), high expression of API5, CHI3L1 and FASN was associated with high tumour grade (p = 3 × 10(-4) , p = 0.016, p = 0.010, respectively). We show in vitro cell biology models of early-stage cancer development can be used to identify novel candidate biomarkers for disease, and report the identification of proteins that represent novel potential candidate diagnostic and prognostic biomarkers for this highly lethal disease.

Background Endometriosis is a common condition that is associated with an increased risk of developing ovarian carcinoma. Improved in vitro models of this disease are needed to better understand how endometriosis, a benign disease, can undergo neoplastic transformation, and for the development of novel treatment strategies to prevent this progression. Methods We describe the generation and in vitro characterization of novel TERT immortalized ovarian endometriosis epithelial cell lines (EEC16-TERT). Results Expression of TERT alone was sufficient to immortalize endometriosis epithelial cells. TERT immortalization induces an epithelial-to-mesenchymal transition and perturbation in the expression of genes involved in the development of ovarian cancer. EEC16-TERT was non-tumorigenic when xenografted into immunocompromised mice but grew in anchorage-independent growth assays in an epidermal growth factor and hydrocortisone dependent manner. Colony formation in agar was abolished by inhibition of Src, and the Src pathway was found to be activated in human endometriosis lesions. Conclusions This new in vitro model system mimics endometriosis and the early stages of neoplastic transformation in the development of endometriosis associated ovarian cancer. We demonstrate the potential clinical relevance of this model by identifying Src activation as a novel pathway in endometriosis that could be targeted therapeutically, perhaps as a novel strategy to manage endometriosis clinically, or to prevent the development of endometriosis-associated ovarian cancer.

Contains fulltext : 118576.pdf (Publisher’s version ) (Open Access) Epithelial ovarian cancer (EOC) has a heritable component that remains to be fully characterized. Most identified common susceptibility variants lie in non-protein-coding sequences. We hypothesized that variants in the 3' untranslated region at putative microRNA (miRNA)-binding sites represent functional targets that influence EOC susceptibility. Here, we evaluate the association between 767 miRNA-related single-nucleotide polymorphisms (miRSNPs) and EOC risk in 18,174 EOC cases and 26,134 controls from 43 studies genotyped through the Collaborative Oncological Gene-environment Study. We identify several miRSNPs associated with invasive serous EOC risk (odds ratio=1.12, P=10(-8)) mapping to an inversion polymorphism at 17q21.31. Additional genotyping of non-miRSNPs at 17q21.31 reveals stronger signals outside the inversion (P=10(-10)). Variation at 17q21.31 is associated with neurological diseases, and our collaboration is the first to report an association with EOC susceptibility. An integrated molecular analysis in this region provides evidence for ARHGAP27 and PLEKHM1 as candidate EOC susceptibility genes.

Contains fulltext : 118459.pdf (Publisher’s version ) (Closed access) Genome-wide association studies (GWAS) have identified four susceptibility loci for epithelial ovarian cancer (EOC), with another two suggestive loci reaching near genome-wide significance. We pooled data from a GWAS conducted in North America with another GWAS from the UK. We selected the top 24,551 SNPs for inclusion on the iCOGS custom genotyping array. We performed follow-up genotyping in 18,174 individuals with EOC (cases) and 26,134 controls from 43 studies from the Ovarian Cancer Association Consortium. We validated the two loci at 3q25 and 17q21 that were previously found to have associations close to genome-wide significance and identified three loci newly associated with risk: two loci associated with all EOC subtypes at 8q21 (rs11782652, P = 5.5 x 10(-9)) and 10p12 (rs1243180, P = 1.8 x 10(-8)) and another locus specific to the serous subtype at 17q12 (rs757210, P = 8.1 x 10(-10)). An integrated molecular analysis of genes and regulatory regions at these loci provided evidence for functional mechanisms underlying susceptibility and implicated CHMP4C in the pathogenesis of ovarian cancer.

Background The molecular biology and cellular origins of mixed type endometrial carcinomas (MT-ECs) are poorly understood, and a Type II component of 10 percent or less may confer poorer prognoses. Methodology/Principal Findings We studied 10 cases of MT-EC (containing endometrioid and serous differentiation), 5 pure low-grade endometrioid adenocarcinoma (EAC) and 5 pure uterine serous carcinoma (USC). Endometrioid and serous components of the MT-ECs were macrodissected and the expression of 60 candidate genes compared between MT-EC, pure USC and pure EAC. We found that four genes were differentially expressed when MT-ECs were compared to pure low-grade EAC: CDKN2A (P = 0.006), H19 (P = 0.010), HOMER2 (P = 0.009) and TNNT1 (P = 0.006). Also while we found that even though MT-ECs closely resembled the molecular profiles of pure USCs, they also exhibit lower expression of PAX8 compared to all pure cases combined (P = 0.035). Conclusion Our data suggest that MT-EC exhibits the closest molecular and epidemiological similarities to pure USC and supports clinical observations that suggest patients with MT-EC should receive the same treatment as patients with pure serous carcinoma. Novel specific markers of MT-EC could be of diagnostic utility and could represent novel therapeutic targets in the future.

To identify factors important for the regulation of microtubule stability in yeast, dosage suppressors of the hyperstable microtubule phenotype of the budding yeast tub2-150 β-tubulin mutation were isolated. Of the two suppressors reported here, one (JSN2) encodes a tRNAVal, and the other (JSN3) is an antimorphic allele of the methionine biosynthesis transcription factor Met4p. Furthermore, growth of tub2-150 mutants and suppression of tub2-150 mutants by JSN3 are sensitive to levels of methionine in the growth medium. We explore several possible explanations for these findings, including the potential involvement of the general amino acid control and the involvement of Cbflp, a component of yeast kinetochores that is also necessary for Met4p-mediated transcription.

To better understand the dynamic regulation of microtubule structures in yeast, we studied a conditional-lethal beta-tubulin mutation tub2-150. This mutation is unique among the hundreds of tubulin mutations isolated in Saccharomyces cerevisiae in that it appears to cause an increase in the stability of microtubules. We report here that this allele is a mutation of threonine 238 to alanine, and that tub2-150 prevents the spindle from elongating during anaphase, suggesting a nuclear microtubule defect. To identify regulators of microtubule stability and/or anaphase, yeast genes were selected that, when overexpressed, could suppress the tub2-150 temperature-sensitive phenotype. One of these genes, JSN1, encodes a protein of 125 kDa that has limited similarity to a number of proteins of unknown function. Overexpression of the JSN1 gene in a TUB2 strain causes that strain to become more sensitive to benomyl, a microtubule-destabilizing drug. Of a representative group of microtubule mutants, only one other mutation, tub2-404, could be suppressed by JSN1 overexpression, showing that JSN1 is an allele-specific suppressor. As tub2-404 mutants are also defective for spindle elongation, this provides additional support for a role for JSN1 during anaphase.

In natural populations of musk thistle (Carduus nutans L.) germination occurs over several months in the fall and spring. To better understand the physical environmental mechanisms that influence this pattern of germination, a greenhouse experiment was established consisting of ten treatments in which soil surface topography and the amount and consistency of litter cover was varied. Significant differences occurred among the treatments for germination percentage, levels of mortality and individual growth rates. Optimum levels ofgermination, survival and growth occurred in treatments that provided microhabitats with reduced evaporation. Treatments with cracks or moderately irregular surface topographies provided the best conditions. A light covering of litter benefited establishment and growth but thick layers of litter prevented seeds from reaching the soil surface and reduced germination and establishment. These results were consistent with what is known about the germination ecology of musk thistle in natural populations. IN MANY PLANT species the highest levels of mortality occur between the seed dispersal and the seedling establishment phases of the life cycle (Harper and White, 1974; Harper, 1977). Unfortunately, this is often the most poorly understood portion of the life cycle. Usual monitoring procedures are capable of quantifying seed production and seedling establishment but are generally incapable of describing interactions between seeds and the soil surface environment. Yet many biologists (e.g., Grubb, 1977; Grime, 1979) feel that these interactions are critical to the reproduction of populations and the composition of plant communities. Successful germination involves complex interactions between the seed's physical position, its physiological state, and its microenvironment. Some species have genetically determined, innate dormancy requirements (Harper, 1977) that prevent germination until after specific environmental keys have been experienced (e.g., Betula pubescens, Black and I Received for publication 13 February 1986; revision accepted 19 August 1986. We wish to express our gratitude to Karen J. Hamrick and Jenna R. Hamrick for their technical assistance. Steve Stewart provided valuable assistance during the data analyses. The comments of two anonymous reviewers greatly improved this paper. This work was supported by funds supplied by University of Kansas General Research Fund Grant #3675 and Environmental Protection Agency Grant R-805455030. 2 Current address: Departments of Botany and Genetics, University of Georgia, Athens, GA 30602. 3Current address: Department of Botany and Plant Sciences, University of Califomia, Riverside, CA 92521. Wareing, 1955; Avenafatua, Marshall and Jain, 1970; Naylor and Jana 1976; Ambrosia artemisifolia, Willemsen 1975a, b; Avena barbata, Marshall and Jain, 1970). Other species have less rigid dormancy (enforced, Harper, 1977) and can germinate whenever minimum environmental conditions are met (usually heat, light, moisture, and proper aeration). Adverse conditions such as drought, cold, or deep burial in soil or litter prevent or retard germination in these species (Werner 1975; Harper, 1977). Several investigators (Harper and Sager, 1953; Harper, Williams and Sager, 1965; Sheldon, 1974) have demonstrated that subtle features of the soil surface topography and the position of the seed in relation to this topography can have dramatic effects on germination and seedling establishment. Studies of the population ecology of musk thistle (Carduus nutans L.) indicate that germination occurs over periods of several months in both fall and spring (Lee and Hamrick, 1983). Subsequent survival, growth and fecundity of monitored individuals was influenced by the their time of germination. Generally, individuals germinating in the early fall had lower mortality, attained larger vegetative sizes and produced more seeds than those that germinated later in the fall or in the spring. Later studies (J. L. Hamrick, unpubl. data) indicate, however, that germination early in the fall can be followed by a period of high mortality if a period of drought immediately follows germination. It is clear, therefore, that the dynamics of musk thistle populations may be

Detailed interpretation of triose phosphate isomerase (TPI) isozymes in seed plants has been restricted to only a few species. Three sets of TPI bands are regularly observed in the cherimoya (Annona cherimola), a primitive angiosperm. The slowest, set I, is expressed as one or three bands; the second-slowest set II, as one or two bands; and the fastest, set IV, as one or three bands. A faint set III, just cathodal to set IV, is detected rarely with overstaining. Set IV bands are expressed in macerated extracted pollen but not in pollen leachate. Dissociation-reassociation experiments reveal that the set II bands are heterodimers involving, in part, the enzymes involved in the set I bands. These data combined with those from full-sib progeny analysis lead us to propose a three-locus model to explain the TPI isozyme banding patterns in cherimoya. Sets I and IV consist of the allelic products of individual, single loci. Sets I and II occur in the cytoplasm. Set IV occurs in organelles. Set II isozymes are the intergenic heterodimers of the locus coding for set I and the locus coding for set III. Our results reported here are contrasted with the TPI isozyme patterns known for other vascular plants and suggest that the locus coding for set III may be a duplication of very ancient origin.

Two sets of alcohol dehydrogenase (ADH) bands are regularly observed in grain sorghum (Sorghum bicolor): set I is a permanent triplet; set II is variable, as either two or three bands. A faint set III is detected only when extracts from seeds subjected to anaerobiosis are run in neutral pH gels. Dissociation-reassociation experiments reveal that the central band of the set I triplet is a heterodimer of the other two. Full-sib progeny analysis from self-fed plants shows that the set II bands are doublets, with heterozygotes having only three apparent bands instead of four because of the similar mobilities of the fast-migrating isozyme specified by the slow allele and the slow isozyme specified by the fast allele. We propose a three-locus model as the best explanation of these patterns. Set I consists of the products of two loci and their intergenic heterodimer. Set III is specified by a third locus. Set II isozymes are the intergenic heterodimers of the two set I loci and the set III locus. This explanation is similar to that of Schwartz and Freeling for maize but suggests that the evolution of Sorghum includes a gene duplication of the homologue of the Adh-1 locus in Zea.

(1) Mortality and fecundity were determined for two populations of musk thistle (Carduus nutans) for the period June 1975 to May 1977. The populations occurred in old-fields but differed in density and competing vegetation. (2) Three life-cycle patterns were observed: winter annual; spring biennial; and autumn biennial. Greatest mortality occurred in summer and late spring. There were significant differences between the populations in the time of germination, mortality, rosette growth rates, time to flowering, and seed production. (3) Generally those cohorts that germinated after autumn 1975 were slower growing, had higher rates of mortality, took longer to flower and produced fewer seeds per individual. (4) The autumn 1975 cohort was divided into September, October and November germination cohorts. The October and November cohorts had lower rosette growth rates, lower survivorship, flowered later and produced fewer seeds per individual. (5) Survivorship, flowering and seed production were all closely associated with rosette size. Larger plants were more likely to survive and to flower, and they produced more seed. (6) Early autumn germination with competition-free growth favours high survivorship and early flowering. Variation in the demography of musk thistle can best be explained by phenotypic plasticity rather than genetic causes.