Summary The Cancer Genome Atlas (TCGA) project has analyzed mRNA expression, miRNA expression, promoter methylation, and DNA copy number in 489 high-grade serous ovarian adenocarcinomas (HGS-OvCa) and the DNA sequences of exons from coding genes in 316 of these tumors. These results show that HGS-OvCa is characterized by TP53 mutations in almost all tumors (96%); low prevalence but statistically recurrent somatic mutations in 9 additional genes including NF1, BRCA1, BRCA2, RB1, and CDK12; 113 significant focal DNA copy number aberrations; and promoter methylation events involving 168 genes. Analyses delineated four ovarian cancer transcriptional subtypes, three miRNA subtypes, four promoter methylation subtypes, a transcriptional signature associated with survival duration and shed new light on the impact on survival of tumors with BRCA1/2 and CCNE1 aberrations. Pathway analyses suggested that homologous recombination is defective in about half of tumors, and that Notch and FOXM1 signaling are involved in serous ovarian cancer pathophysiology.

Some stimulatory receptors of the innate immune system, such as the NKG2D receptor expressed by NK cells and activated CD8 + T cells, recognize self-molecules that are upregulated in diseased cells by poorly understood mechanisms 1 . Here we show that mouse and human NKG2D ligands are upregulated in non-tumor cell lines by genotoxic stress and stalled DNA replication, conditions known to activate a major DNA damage checkpoint pathway initiated by ATM (Ataxia telangiectasia, mutated) or ATR (ATM-and Rad3-related) protein kinases 2 . Ligand upregulation was prevented by pharmacological or genetic inhibition of ATR, ATM or Chk1, the latter a downstream transducer kinase in the pathway. Furthermore, constitutive ligand expression by a tumor cell line was inhibited by ATM siRNA, suggesting that ligand expression in established tumor cells, which often harbor genomic irregularities, may be due to chronic activation of the DNA damage response pathway. Thus, the DNA damage response, previously shown to arrest the cell cycle and enhance DNA repair functions or to trigger apoptosis, may also participate in alerting the immune system to the presence potentially dangerous cells.To investigate mechanisms leading to NKG2D ligand upregulation, we examined two transformed ovarian epithelial cell lines from p53 −/− mice 3 . The C1 cell line had been transduced with K-ras and c-myc, whereas the C2 cell line had been transduced with AKT and c-myc (Fig. 1A). Both transformed cell lines grew well in cell culture but did not express appreciable levels of mouse NKG2D ligands as detected by staining with a tetrameric NKG2D reagent that binds to all mouse NKG2D ligands (Rae1, MULT1 and H60 1 ) (Fig. 1B). Ligand upregulation failed to occur when C1 and C2 cells were transfected or super-transduced with numerous other oncogenes, including E6, E7, E1A, or Ras V12 (data not shown), some of which interfere with expression of the retinoblastoma tumor suppressor gene. When injected into the ovaries of nude mice, both cell lines generated ovarian epithelial tumors, which were established as cell lines T1 and T2 3 . Both T1 and T2 exhibited significant upregulation of NKG2D ligands (Fig. 1B) including Rae1 (see below). These findings suggested that ligand upregulation was not associated with transformation per se.Ligand expression by C1 or C2 cells was not upregulated by numerous cell stress conditions, including heat shock, hyperoxia, hypoxia, inhibition of the cell cycle (by roscovitine), exposure to inflammatory cytokines such as TNF, interferon or IL-6, or incubation in medium of pH6 or pH8.5 ( Fig. 2A, S1, data not shown). In contrast, NKG2D ligands were upregulated in C1 or C2 cells exposed to high doses of ionizing radiation, inhibitors of DNA replication such as mitomycin C, hydroxyurea (HU), 5-fluorouracil (5-FU) and the DNA polymerase inhibitor aphidicolin, or chromatin modifying treatments such as trichostatin A, chloroquine and

We analyzed molecular data on 2,579 tumors from The Cancer Genome Atlas (TCGA) of four gynecological types plus breast. Our aims were to identify shared and unique molecular features, clinically significant subtypes, and potential therapeutic targets. We found 61 somatic copy-number alterations (SCNAs) and 46 significantly mutated genes (SMGs). Eleven SCNAs and 11 SMGs had not been identified in previous TCGA studies of the individual tumor types. We found functionally significant estrogen receptor-regulated long non-coding RNAs (lncRNAs) and gene/lncRNA interaction networks. Pathway analysis identified subtypes with high leukocyte infiltration, raising potential implications for immunotherapy. Using 16 key molecular features, we identified five prognostic subtypes and developed a decision tree that classified patients into the subtypes based on just six features that are assessable in clinical laboratories.

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, 9q31.1) and one for endometrioid EOC (5q12.3). We then meta-analysed 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 an additional three 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 novel susceptibility gene for low grade/borderline serous EOC.

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However, our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore, we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types, n = 792) by immunohistochemical staining. Using the ALDEFUOR assay, ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition, an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct, and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers, we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439, p = 0.0036). Finally, ALDHbr tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker, ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast, lung, ovarian or colon cancer.

Purpose To elucidate molecular pathways contributing to metastatic cancer progression and poor clinical outcome in serous ovarian cancer. Experimental Design Poor survival signatures from three different serous ovarian cancer datasets were compared and a common set of genes was identified. The predictive value of this gene signature was validated in independent datasets. The expression of the signature genes was evaluated in primary, metastatic, and/or recurrent cancers using qPCR and in situ hybridization. Alterations in gene expression by TGFβ1 and functional consequences of loss of COL11A1 were evaluated using pharmacologic and knockdown approaches, respectively. Results We identified and validated a 10-gene signature (AEBP1, COL11A1, COL5A1, COL6A2, LOX, POSTN, SNAI2, THBS2, TIMP3, VCAN) that is associated with poor overall survival in patients with high-grade serous ovarian cancer. The signature genes encode extracellular matrix proteins involved in collagen remodeling. Expression of the signature genes is regulated by TGFβ1 signaling and is enriched in metastases in comparison to primary ovarian tumors. We demonstrate that levels of COL11A1, one of the signature genes, continuously increase during ovarian cancer disease progression, with the highest expression in recurrent metastases. Knockdown of COL11A1 decreases in vitro cell migration and invasion and tumor progression in mice. Conclusion Our findings suggest that collagen-remodeling genes regulated by TGFβ1 signaling promote metastasis and contribute to poor overall survival in patients with serous ovarian cancer. Our 10-gene signature has both predictive value and biological relevance and thus may be useful as a therapeutic target.

We have developed a mouse model for ovarian carcinoma by using an avian retroviral gene delivery technique for the introduction of multiple genes into somatic ovarian cells of adult mice. Ovarian cells from transgenic mice engineered to express the gene encoding the avian receptor TVA were efficiently infected in vitro with multiple vectors carrying coding sequences for oncogenes and marker genes. When target cells were derived from TVA transgenic mice deficient for p53, the addition of any two of the oncogenes c-myc, K-ras, and Akt were sufficient to induce ovarian tumor formation when infected cells were injected at subcutaneous, intraperitoneal, or ovarian sites. We demonstrated that the ovarian surface epithelium is the precursor tissue for these ovarian carcinomas, and that introduction of oncogenes causes phenotypic changes in the ovarian surface epithelial cells. The induced ovarian tumors in mice resembled human ovarian carcinomas in their rapid progression and intraperitoneal metastatic spread.

Abstract. Armadillo, the Drosophila homologue of vertebrate 13-catenin, plays a pivotal role both in Wingless signaling and in assembly of adherens junctions. We performed the first in vivo structure-function study of an adherens junction protein, by generating and examining a series of Armadillo mutants in the context of the entire animal. We tested each mutant by assaying its biological function, its ability to bind proteins that normally associate with Armadillo in adherens junctions, its cellular localization, and its pattern of phosphorylation. We mapped the binding sites for DE-cadherin and o~-catenin, Although these bind to Armadillo independently of each other, binding of each is required for the function of adherens junctions. We identified two separate regions of Armadillo critical for Wingless signaling. We demonstrated that endogenous Armadillo accumulates in the nucleus and provide evidence that it may act there in transducing Wingless signal. We found that the Arm repeats, which make up the central two-thirds of Armadillo, differ among themselves in their functional importance in different processes. Finally, we demonstrated that Armadillo's roles in adherens junctions and Wingless signaling are independent. We discuss the potential biochemical role of Armadillo in each process.