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Background The prognostic value of the expression of estrogen receptor (ER) subtypes ER⍺ and ERβ in ovarian cancer has previously been evaluated by meta-analyses. However, the results are contradictory and controversial. Methods We conducted an updated meta-analysis with stringent inclusion criteria to ensure homogeneous studies to determine the effect of ER subtypes on ovarian cancer prognosis. Articles were retrieved by systematic search of PubMed and Web of Science for articles dated up to June 2021. Only studies with known hazard ratio (HR) and antibody clone for immunochemistry (IHC) were included. Pooled HRs with the corresponding 95% confidence intervals (CIs) were calculated for the effect of ER⍺ and ERβ expression on ovarian cancer patient progression-free survival (PFS) and overall survival (OS). Results A total of 17 studies were included, of which 11 and 13 studies examined the relationships between ER⍺ expression and PFS and OS, respectively, and 5 and 7 studies examined the relationships between ERβ expression and PFS and OS, respectively. Neither ER⍺ expression (random-effects model; HR = 0.99, 95% CI = 0.83–1.18) nor ERβ expression (fixed-effects model; HR = 0.94, 95% CI = 0.69–1.27) was associated with PFS. Random-effects models showed that ER⍺ expression (HR = 0.81, 95% CI = 0.64–1.02) and ERβ expression (HR = 0.75, 95% CI = 0.50–1.13) were only marginally and not significantly associated with better OS. Subgroup analysis revealed that ER⍺ expression determined using antibody clone 1D5 (HR = 0.75, 95% CI = 0.64–0.88) and ERβ expression determined using ERβ1-specific-antibody clone PPG5/10 or EMR02 (HR = 0.65, 95% CI = 0.50–0.86) were associated with significantly better OS, but ER expression determined using other antibodies was not. Conclusions In conclusion, a higher ER⍺ expression and ERβ expression are significantly associated with a better survival of ovarian cancer patients, but the results from previous prognostic studies are significantly dependent on the choice of specific ER antibody clones used in immunohistochemistry analysis.

The AURKA gene, encoding Aurora kinase-A (Aurora-A), is frequently amplified and overexpressed across multiple cancer types correlating with poor prognosis. Although the AURKA gene is frequently amplified in human cancers, underlying mechanism(s) for Aurora-A protein stability through different phases of cell cycle are not well elucidated. Inhibiting the kinase activity and promoting protein degradation are two well-validated conceptual strategies for targeting protein kinases in cancers. Here, we demonstrate that Eukaryotic Elongation Factor 1 Alpha 2 (eEF1A2) facilitates PTEN-GSK3β mediated Aurora-A protein degradation through the SCF complex (SKP1-Cul1-FBXW7) during the S/G2 phase of proliferating cells. In contrast, this mechanism is inactivated in cancer cells accompanying PTEN-GSK3β pathway deficiency. Mechanistically, eEF1A2 interacts with Aurora-A, GSK3β, FBXW7 and Cul1-E3 ligase, as the SCF complex, to facilitate Aurora-A polyubiquitination for 26S proteasomal degradation. eEF1A2 promotes PTEN phosphorylation at T366 and stability, inactivates AKT and activates GSK3β which in turn phosphorylates Aurora-A at S283, S284 and S342. The phosphorylation of Aurora-A at S342 is detected during S/G2 phase of cell mitosis in parallel with eEF1A2-SCF complex formation with active form of GSK3β and neddylated Cul1. Conversely, genetic ablation of EEF1A2 and PTEN, activation of AKT, inhibition of GSK3β, expression of Aurora-A phosphodeficient-mutant attenuates the Aurora-A protein degradation which is corroborated in Aurora-A overexpressing mouse mammary carcinomas and human breast carcinomas. This study identifies a novel mechanism of Aurora-A protein degradation mediated eEF1A2-PTEN-GSK3β pathway and provides a framework for the discovery of Aurora-A therapeutic targets in breast cancer that harbors deficiency of PTEN tumor suppressor pathway. Citation Format: Treekitkarnmongkol W, Solis LM, Kai K, Thompson AM, Tian W, Wistuba II, Sasai K, Jltsumori Y, Sahin AA, Hawke DH, Lee JM, Qin L, Bawa-Khalfe T, Rad R, Wong KK, Abbott CM, Katayama H, Sen S. eEF1A2 facilitates PTEN-GSK3β mediated Aurora-A protein degradation during S-G2 phase inactivated in PTEN-deficient breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-05.

How different KRAS variants impact tumor initiation and progression in vivo has not been thoroughly examined. We hypothesize that the ability of either KRASG12D or KRASG12V mutations to initiate tumor formation is context dependent. Amhr2-Cre mice express Cre recombinase in tissues that develop into the fallopian tubes, uterus, and ovaries. We used these mice to conditionally express either the KRASG12V/+or KRASG12D/+ mutation. Mice with the genotype Amhr2-Cre Pten(fl/fl) KrasG12D/+(G12D mice) had abnormal follicle structures and developed low-grade serous ovarian carcinomas with 100% penetrance within 18 weeks. In contrast, mice with the genotype Amhr2-Cre Pten(fl/fl) KrasG12V/+ (G12V mice) had normal follicle structures, and about 90% of them developed uterine tumors with diverse histological features resembling those of leiomyoma and leiomyosarcoma. Granulosa cell tumors also developed in G12V mice. Differences in cell-signaling pathways in the uterine tissues of G12D and G12V mice were identified using RNA sequencing and reverse-phase protein array analyses. We found that CTNNB1, IL1A, IL1B, TNF, TGFB1, APP, and IL6 had the higher activity in G12V mice than in G12D mice. These mouse models will be useful for studying the differences in signaling pathways driven by KrasG12V/+ or KrasG12D/+ mutations to aid development of targeted therapies for specific KRAS mutant variants. Our leiomyoma model driven by the KrasG12V/+ mutation will also be useful in deciphering the malignant progression from leiomyoma to leiomyosarcoma.

Background: Human paired box 2 (PAX2) plays a key role in cell fate, early patterning and organogenesis. Methods: We investigated the function of PAX2 on the biological behavior of endometrial cancer in vitro and in vivo and to explore the regulation mechanism, stable knocking-down and over-expression PAX2 endometrial cancer cell lines were established. CCK-8 and transwell assays were applied to determine proliferation, invasion and migration ability. Cell cycle distribution was analyzed by flow cytometry. Affymetrix GeneChip® human Exon 1.0 ST arrays was used to screen the downstream target genes of PAX2. Results: PAX2 significantly enhanced proliferation and invasiveness. In addition, PAX2 influenced the expression of cyclin-dependent kinase 1(CDK1), which play pivotal roles in cell cycle pathway. When CDK1 was knocked down, and the cell proliferation promotion role of PAX2 was attenuated dramatically to a level comparable with the control groups. Conclusions: PAX2, though influencing the expression of CDK1, promotes the proliferation, enhances the mobility of endometrial cancer cells, thus exerts an important role in the carcinogenesis of endometrial cancer. PAX2 may be a potential therapeutic target for endometrial cancer.

The tumor microenvironment (TME), composed primarily of fibroblasts, endothelial cells, lymphocytic infiltrates and extracellular matrix proteins, can directly affect cancer cell growth, migration, differentiation as well as immune cell landscapes, thereby presenting a unique aspect of diagnosing and treating cancer. Cancer-associated fibroblasts (CAF) and immune cells are the main orchestrators of the ovarian cancer TME. We hypothesized that spatial transcriptomics (ST), which can provide a rich spatial context to gene expression, will identify crosstalk signaling networks among various cell types in the ovarian TME. Advanced stage high-grade serous ovarian cancer (HGSC) tissue frozen sections from two treatment naïve long-term survivors (LTS, overall survival ≥ 10 years) and two short-term survivors (STS, overall survival ≤ 2 years) were placed onto a ST expression slide pre-spotted with 1000 unique barcoded RNA-capturing probes. The ST slides were then stained with H&E and imaged. Tissue permeabilization and cDNA synthesis were then performed directly on the tissue section. The derived barcoded cDNA libraries from each sample were sequenced using Illumina NextSeq500 flow cells. After RNA-Seq analysis, supervised analysis was then performed by selecting different regions (based on tissue histology) in the tumor, tumor/stroma mixture, stroma close or far away from tumor areas to identify differentially expressed genes between STS and LTS. Stromal cells from different spatial locations had unique differentially expressed genes based on their proximity (11 genes in LTS; 11 genes in STS) or remoteness (18 genes in LTS; 10 genes in STS) to cancer cell compartments. Among the major modulated gene networks, higher levels of antigen-presenting molecules were found in the stroma in close proximity to the tumor cell nest than in stroma located far away in LTS tumor samples but not in the STS samples, suggesting an increased number of activated antigen-presenting cells in LTS tumors. In addition, higher levels of adhesion molecules and angiogenetic factors were found in the stroma in close proximity to the tumor cell nest than in stroma located far away in STS tumor samples but not in LTS samples, suggesting that adhesion molecules and angiogenetic factors produced by stromal cells may facilitate the invasiveness of the tumor cells, which subsequently leads to short term survival in patients with the disease. Our findings demonstrate for the first time that spatially resolved transcriptomics allows the identification of prognostic biomarkers associated with overall survival in HGSC patients. Further studies using deconvoluted ST data to further delineate the specific stromal cell subtypes in close proximity to tumors, and imaging mass cytometry (IMC) or multiplexed immunohistochemistry to validate the protein expression of the potential biomarkers are ongoing. Citation Format: Sammy Ferri-Borgogno, Jianting Sheng, Ying Zhu, Kwong K. Wong, Stephen T. Wong, Samuel C. Mok. Spatially resolved transcriptomics identified distinct tumor-stroma crosstalk networks in long term ovarian cancer survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 103.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that is known to facilitate vaccine efficacy by promoting the development and prolongation of both humoral and cellular immunity. Here, we investigated a novel vaccine approach using a human papillomavirus (HPV)-16 E6/E7-transformed cell line, TC-1, that ectopically expresses a codon-optimized 26-11-2015 murine GM-CSF (cGM-CSF). Ectopically expressing cGM-CSF in TC-1 (TC-1/cGM) cells significantly increased expression of a GM-CSF that was functionally identical to wt GM-CSF by 9-fold compared with ectopically expressed wild type GM-CSF in TC-1 cells (TC-1/wt). Mice vaccinated with irradiated TC-1/cGM cells exhibited enhanced survival compared with mice vaccinated with TC-1/wt cells when both groups were subsequently injected with live TC-1. Consistently, mice vaccinated with irradiated TC-1/cGM cells exhibited stronger IFN-γ production in HPV E7-specific CD8(+) T cells. More dendritic cells were recruited to the draining lymph nodes (dLNs) of mice vaccinated with TC-1/cGM cells than C-1/wt cells. Regarding dLN cell recall responses, both proliferation and IFN-γ production in the HPV E7-specific CD8(+) T cells were enhanced in mice that were vaccinated with TC-1/cGM cells. Our results demonstrate that a novel practical molecular strategy utilizing a codon-optimized GM-CSF gene overcomes the limitation and improves the efficacy of tumor cell-based vaccines.

Simple Summary Patients with wild-type p53 ovarian cancer appear to have a poorer survival rate than those with mutant p53 due to resistance to chemotherapy. The mechanism underlying this observation is not clearly understood. The aim of this study was to identify potential biomarkers regulated by p53 that conferred resistance using in vitro and in vivo studies. Growth differentiation factor 15 (GDF15) expression was demonstrated to be controlled by p53 in both ovarian cancer cell lines and orthotopic mouse models. The histological and RNAseq studies of the GDF15-knocked down, A2780 cell line-induced tumor revealed that the ratio and canonical pathways of stromal/tumor were modified by secretory GDF15. Abstract Background: The standard treatment of ovarian cancer is surgery followed by a chemotherapeutic combination consisting of a platinum agent, such as cisplatin and a taxane-like paclitaxel. We previously observed that patients with ovarian cancer wild-type for p53 had a poorer survival rate than did those with p53 mutations. Thus, a better understanding of the molecular changes of epithelial ovarian cancer cells with wild-type p53 in response to treatment with cisplatin could reveal novel mechanisms of chemoresistance. Methods: Gene expression profiling was performed on an ovarian cancer cell line A2780 with wild-type p53 treated with cisplatin. A gene encoding a secretory protein growth differentiation factor 15 (GDF15) was identified to be highly induced by cisplatin treatment in vitro. This was further validated in a panel of wild-type and mutant p53 ovarian cancer cell lines, as well as in mouse orthotopic models. The mouse tumor tissues were further analyzed by histology and RNA-seq. Results: GDF15 was identified as one of the highly induced genes by cisplatin or carboplatin in ovarian cancer cell lines with wild-type p53. The wild-type p53-induced expression of GDF15 and GDF15-confered chemotherapy resistance was further demonstrated in vitro and in vivo. This study also discovered that GDF15-knockdown (GDF15-KD) tumors had less stromal component and had different repertoires of activated and inhibited canonical pathways in the stromal cell and cancer cell components from that of the control tumors after cisplatin treatment. Conclusions: GDF15 expression from the wild-type p53 cancer cells can modulate the canonical pathways in the tumor microenvironment in response to cisplatin, which is a possible mechanism of chemoresistance.

Background The standard treatment for ovarian cancer is surgery, followed by chemotherapy with a platinum agent, such as cisplatin or carboplatin, and a taxane, such as paclitaxel. We previously observed that patients with ovarian cancer that was wild-type for p53 had a lower survival rate than did those with p53 mutations. A better understanding of the molecular changes that occur in epithelial ovarian cancer cells with wild-type p53 in response to cisplatin treatment could reveal a novel mechanism of chemoresistance. Therefore, we might be able to develop novel strategy to overcome chemoresistance. Methods Gene expression profiling was performed on the wild-type p53 ovarian cancer cell line A2780 to identify genes that are highly induced by cisplatin. Growth differentiation factor 15 (GDF15) was identified as being highly induced by cisplatin or carboplatin. This finding was validated in a panel of wild-type and mutant p53 ovarian cancer cell lines. Mouse orthotopic models were used to further investigate the induction of GDF15 by cisplatin in vivo and the effect of GDF15 knockdown (GDF15-KD) on tumor growth and cisplatin response. A novel analysis was performed of RNAseq data generated from tumors formed in cisplatin-treated orthotopic mouse models to understand the tumor and stromal cell signaling changes. Results The induction of GDF15 was demonstrated in vivo, and the serum level of GDF15 after cisplatin treatment was also found to be significantly up-regulated in an A2780 ovarian cancer orthotopic mouse model. More importantly, cisplatin treatment was more effective in shrinking the mouse tumors developed from GDF15-KD A2780 cell line than did control tumors. Histopathological and novel RNAseq analyses of mouse tumors revealed that GDF15-KD tumors had a lower stromal component than did control tumors after cisplatin treatment. GDF15-KD tumors also had a different repertoire of activated and inhibited canonical pathways in their stromal cell and cancer cell components than did control tumors in response to cisplatin treatment. Conclusions GDF15 expression in wild-type p53 ovarian cancer cells can modulate the canonical pathways in the tumor microenvironment in response to cisplatin and may be a mechanism of chemoresistance. Targeting GDF15 signaling could be a novel strategy to enhance the effectiveness of chemotherapy. Citation Format: Daisy I. Izaguirre, Yvonne TM Tsang, Eucharist H. Kun, David M. Gershenson, Kwong-Kwok Wong. GDF15 expression level impacts the canonical pathways of the tumor microenvironment and ovarian tumors with wild-type p53 in response to chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3985.

Uterine serous carcinoma (USC) is the most aggressive form of endometrial cancer, with poor survival rates and high recurrence risk. Therefore, the purpose of this study was to identify therapeutic targets that could aid in the management of USC. By analyzing endometrial cancer samples from The Cancer Genome Atlas (TCGA), we found Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) to be highly expressed in USC and to correlate with poorer overall survival. UCHL1 silencing reduced cell proliferation in vitro and in vivo, cyclin B1 protein levels and cell cycle progression. Further studies showed that UCHL1 interacts with cyclin B1 and increases cyclin B1 protein stability by deubiquitination. Treatment of USC-bearing mice with the UCHL1-specific inhibitor reduced tumor growth and improved overall survival. Our findings suggest that cyclin B1 is a novel target of UCHL1 and targeting UCHL1 is a potential therapeutic strategy for USC.

Background: Low-grade serous carcinoma of the ovary or peritoneum is a distinct, well- recognized histologic subtype characterized by young age at diagnosis, relative chemoresistance, and prolonged overall survival. Common mutations reported to be found within this subtype include KRAS and BRAF. Methods: Using clinical information of patients from our IRB-approved registry and tissue from a subset of these patients, we performed mutational analysis for KRAS and BRAF using the direct Sanger sequencing technique and correlated findings with the clinical outcome, overall survival (OS). Results: In 79 cases, patients with KRAS or BRAF mutations (n=21) had a significantly better OS than those with wild-type KRAS or BRAF (n=58) (106.7 months (95% CI, 50.6, 162.9) vs 66.8 months (95% CI, 43.6, 90.0)), respectively (P=0.018). Conclusions: Mutational status appears to be a potential prognostic factor in low-grade serous carcinoma of the ovary or peritoneum.

We have previously found that Sema5A, a member of the semaphorin gene family, is up-regulated in pediatric pilocytic astrocytomas (PA) at the mRNA level by microarray analysis and real-time RT-PCR. By further analysis of the expression level of all 17 semaphorin genes in the microarray dataset, we found that Sema5A and Sema3E are the only two semaphorin genes that are highly up-regulated in pilocytic astrocytomas. In this study, the up-regulation of Sema3E was further confirmed by real-time RT-PCR. Furthermore, the over-expression of both SEMA3E and SEMA5A proteins were also confirmed by Western blot analysis and immunohistochemistry. Since pilocytic astrocytoma is characterized by extensive vasculature, co-immunofluorescent staining of both CD31 and SEMA3E (or SEMA5A) was performed. The result showed that a higher expression of SEMA3E was around the CD31 positive endothelial cells. Based on semaphorin’s function in angiogenesis and a higher expression of SEMA3E and SEMA5A around endothelial cells in these PA samples, these genes could be potential biological markers and anti-angiogenesis therapeutic targets for pilocytic astrocytomas.

The functional status of the tumor repressor protein (TP53 or TRP53) is a defining feature of ovarian cancer. Mutant or null alleles of TP53 are expressed in greater than 90% of all high-grade serous adenocarcinomas. Wild-type TP53 is elevated in low-grade serous adenocarcinomas in women and in our Pten;Kras;Amhr2-Cre mutant mouse model. Disruption of the Trp53 gene in this mouse model did not lead to high-grade ovarian cancer but did increase expression of estrogen receptor α (ESR1) and markedly enhanced the responsiveness of these cells to estrogen. Specifically, when Trp53-positive and Trp53 null mutant mice were treated with estradiol or vehicle, only the Trp53 null and Esr1-positive tumors respond vigorously to estradiol in vivo and exhibit features characteristic of high-grade type ovarian cancer: invasive growth into the ovarian stroma, rampant metastases to the peritoneal cavity, and nuclear atypia. Estrogen promoted and progesterone suppressed the growth of Trp53 null ovarian tumors and tumor cells injected ip, sc, or when grown in matrigel. Exposure of the Trp53 depleted cells to estrogen also has a profound impact on the tumor microenvironment and immune-related events. These results led to the new paradigm that TRP53 status is related to the susceptibility of transformed ovarian surface epithelial cells to estradiol-induced metastases and nuclear atypia via increased levels of estradiol receptor α.

BRAF and KRAS mutations in ovarian serous borderline tumours (OSBTs) and ovarian low-grade serous carcinomas (LGSCs) have been previously described. However, whether those OSBTs would progress to LGSCs or whether those LGSCs were developed from OSBT precursors in previous studies is unknown. Therefore, we assessed KRAS and BRAF mutations in tumour samples from 23 recurrent LGSC patients with a known initial diagnosis of OSBT. Paraffin blocks from both OSBT and LGSC samples were available for five patients, and either OSBTs or LGSCs were available for another 18 patients. Tumour cells from paraffin-embedded tissues were dissected out for mutation analysis by conventional polymerase chain reaction (PCR) and Sanger sequencing. Tumours that appeared to have wild-type KRAS by conventional PCR–Sanger sequencing were further analysed by full COLD (co-amplification at lower denaturation temperature)-PCR and deep sequencing. Full COLD-PCR was able to enrich the amplification of mutated alleles. Deep sequencing was performed with the Ion Torrent personal genome machine (PGM). By conventional PCR–Sanger sequencing, BRAF mutation was detected only in one patient and KRAS mutations were detected in ten patients. Full COLD-PCR deep sequencing detected low-abundance KRAS mutations in eight additional patients. Three of the five patients with both OSBT and LGSC samples available had the same KRAS mutations detected in both OSBT and LGSC samples. The remaining two patients had only KRAS mutations detected in their LGSC samples. For patients with either OSBT or LGSC samples available, KRAS mutations were detected in seven OSBT samples and six LGSC samples. Surprisingly, patients with the KRAS G12V mutation have shorter survival times. In summary, KRAS mutations are very common in recurrent LGSC, while BRAF mutations are rare. The findings indicate that recurrent LGSC can arise from proliferation of OSBT tumour cells with or without detectable KRAS mutations. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

TGF-β has limited effects on ovarian cancer cells, but its contributions to ovarian tumor growth might be mediated through elements of the tumor microenvironment. In the present study, we tested the hypothesis that TGF modulates ovarian cancer progression by modulating the contribution of cancer-associated fibroblasts (CAF) that are present in the microenvironment. Transcriptome profiling of microdissected stromal and epithelial components of high-grade serous ovarian tumors and TGF-β–treated normal ovarian fibroblasts identified versican (VCAN) as a key upregulated target gene in CAFs. Functional evaluations in coculture experiments showed that TGF-β enhanced the aggressiveness of ovarian cancer cells by upregulating VCAN in CAFs. VCAN expression was regulated in CAFs through TGF-β receptor type II and SMAD signaling. Upregulated VCAN promoted the motility and invasion of ovarian cancer cells by activating the NF-κB signaling pathway and by upregulating expression of CD44, matrix metalloproteinase-9, and the hyaluronan-mediated motility receptor. Our work identified a TGF-β–inducible gene signature specific to CAFs in advanced high-grade serous ovarian tumors, and showed how TGF-β stimulates ovarian cancer cell motility and invasion by upregulating the CAF-specific gene VCAN. These findings suggest insights to develop or refine strategies for TGF-β–targeted therapy of ovarian cancer. Cancer Res; 73(16); 5016–28. ©2013 AACR.

Purpose: To evaluate the prognostic value of fibroblast growth factor receptor 4 (FGFR4) protein expression in patients with advanced-stage, high-grade serous ovarian cancer, delineate the functional role of FGFR4 in ovarian cancer progression, and evaluate the feasibility of targeting FGFR4 in serous ovarian cancer treatment. Experimental Design: Immunolocalization of FGFR4 was conducted on 183 ovarian tumor samples. The collected FGFR4 expression data were correlated with overall survival using Kaplan–Meier and Cox regression analyses. The effects of FGFR4 silencing on ovarian cancer cell growth, survival, invasiveness, apoptosis, and FGF1-mediated signaling pathway activation were evaluated by transfecting cells with FGFR4-specific siRNAs. An orthotopic mouse model was used to evaluate the effect of injection of FGFR4-specific siRNAs and FGFR4 trap protein encapsulated in nanoliposomes on ovarian tumor growth in vivo. Results: Overexpression of FGFR4 protein was significantly associated with decreased overall survival durations. FGFR4 silencing significantly decreased the proliferation, survival, and invasiveness and increased apoptosis of ovarian cancer cells. Also, downregulation of FGFR4 significantly abrogated the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and WNT signaling pathways, which are activated by FGF1. Targeting FGFR4 with the FGFR4-specific siRNAs and FGFR4 trap protein significantly decreased ovarian tumor growth in vivo. Conclusions: FGFR4 is a prognostic marker for advanced-stage, high-grade serous ovarian carcinoma. Silencing FGFR4 and inhibiting ligand-receptor binding significantly decrease ovarian tumor growth both in vitro and in vivo, suggesting that targeting ovarian cancer cells with high levels of FGFR4 protein expression is a new therapeutic modality for this disease and will improve survival of it. Clin Cancer Res; 19(4); 809–20. ©2012 AACR.

The oncogene RAS is known to induce genomic instability, leading to cancer development; the underlying mechanism, however, remains poorly understood. To better understand how RAS functions, we measured the activity of the functionally related genes Aurora-A and BRCA2 in ovarian cancer cell lines and tumor samples containing RAS mutations. We found that Aurora-A and BRCA2 inversely controlled RAS-associated genomic instability and ovarian tumorigenesis through regulation of cytokinesis and polyploidization. Overexpression of mutated RAS ablated BRCA2 expression but induced Aurora-A accumulation at the midbody, leading to abnormal cytokinesis and ultimately chromosomal instability via polyploidy in cancer cells. RAS regulates the expression of Aurora-A and BRCA2 through dysregulated protein expression of farnesyl protein transferase β and insulin-like growth factor binding protein 3. Our results suggest that the imbalance in expression of Aurora-A and BRCA2 regulates RAS-induced genomic instability and tumorigenesis.

Mutations in the tumor protein p53 (TP53) are the most frequently occurring genetic events in high-grade ovarian cancers, especially the prevalence of the Trp53R172H-mutant allele. In this study, we investigated the impact of the Trp53R172H-mutant allele on epithelial ovarian cancer (EOC) in vivo. We used the Pten/KrasG12D–mutant mouse strain that develops serous EOC with 100% penetrance to introduce the mutant Trp53R172H allele (homolog for human Trp53R172H). We demonstrate that the Trp53R172H mutation promoted EOC but had differential effects on disease features and progression depending on the presence or absence of the wild-type (WT) TP53 allele. Heterozygous WT/Trp53R172H alleles facilitated invasion into the ovarian stroma, accelerated intraperitoneal metastasis, and reduced TP53 transactivation activity but retained responsiveness to nutlin-3a, an activator of WT TP53. Moreover, high levels of estrogen receptor α in these tumors enhanced the growth of both primary and metastatic tumors in response to estradiol. Ovarian tumors homozygous for Trp53R172H mutation were undifferentiated and highly metastatic, exhibited minimal TP53 transactivation activity, and expressed genes with potential regulatory functions in EOC development. Notably, heterozygous WT/Trp53R172H mice also presented mucinous cystadenocarcinomas at 12 weeks of age, recapitulating human mucinous ovarian tumors, which also exhibit heterozygous TP53 mutations (∼50%–60%) and KRAS mutations. Therefore, we present the first mouse model of mucinous tumor formation from ovarian cells and supporting evidence that mutant TP53 is a key regulator of EOC progression, differentiation, and responsiveness to steroid hormones. Cancer Res; 76(8); 2206–18. ©2016 AACR.

The insulin-like growth factor (IGF) family and the IGF-1 receptor (IGF-1R) play an important role in cancer. This intricate and complex signaling pathway provides many opportunities for therapeutic intervention, and several novel therapeutics aimed at the IGF-1R, particularly monoclonal antibodies and small molecule tyrosine kinase inhibitors, are under clinical investigation. This article provides a patent overview of the IGF signaling pathway and its complexity, addresses the justification for the use of IGF-1R-targeted therapy, and reviews the results of in vivo and in vitro novel therapeutics. Over the past year, the completion of several phase I, II, and III trials have provided interesting new information about the clinical activity of these novel compounds, particularly CP-751,871, IMC-A12, R1507, AMG-479, AVE-1642, MK-0646, XL-228, OSI-906, and BMS-754807. We review the important preliminary results from clinical trials with these compounds and conclude with a discussion about future therapeutic efforts.

To validate the overexpression of insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) in low-grade serous ovarian carcinoma (SOC), and to investigate whether the IGF-1 pathway is a potential therapeutic target for low-grade SOC.Gene expression profiling was performed on serous borderline ovarian tumors (SBOTs) and low-grade SOC, and overexpression of IGF-1 in low-grade SOC was validated by RT-PCR and immunohistochemistry. The effect of exogenous IGF-1 on cell proliferation was determined in cell lines by cell proliferation assays, cell migration assays, and Western blot. Signaling pathways downstream of IGF-1 and the effects of the AKT inhibitor MK-2206 were investigated by Western blot analysis and by generating IGF-1R short hairpin RNA stable knockdown cell lines. Low- and high-grade cell lines were treated with the dual IGF-1R- and insulin receptor-directed tyrosine kinase inhibitor OSI-906, and cellular proliferation was measured.mRNA analysis and immunostaining revealed significantly higher IGF-1 expression in low-grade SOCs than in SBOTs or high-grade SOCs. In response to exogenous treatment with IGF-1, low-grade cell lines exhibited more intense upregulation of phosphorylated AKT than did high-grade cell lines, an effect that was diminished with IGF-1R knockdown and MK-2206 treatment. Low-grade SOC cell lines were more sensitive to growth inhibition with OSI-906 than were high-grade cell lines.IGF-1 is overexpressed in low-grade SOCs compared with SBOTs and high-grade SOCs. Additionally, low-grade SOC cell lines were more responsive to IGF-1 stimulation and IGF-1R inhibition than were high-grade lines. The IGF-1 pathway is therefore a potential therapeutic target in low-grade SOC.

Low-grade serous ovarian carcinoma is believed to arise from serous borderline ovarian tumors, yet the progression from serous borderline tumors to low-grade serous ovarian carcinoma remains poorly understood. The purpose of this study was to identify differentially expressed genes between the two groups. Expression profiles were generated from 6 human ovarian surface epithelia (HOSE), 8 serous borderline ovarian tumors (SBOT), 13 low-grade serous ovarian carcinomas (LG), and 24 high-grade serous ovarian carcinomas (HG). The anterior gradient homolog 3 (AGR3) gene was found to be highly upregulated in serous borderline ovarian tumors; this finding was validated by real-time quantitative RT-PCR, Western blotting, and immunohistochemistry. Anti-AGR3 immunohistochemistry was performed on an additional 56 LG and 103 HG tissues and the results were correlated with clinical data. Expression profiling determined that 1254 genes were differentially expressed (P < 0.005) between SBOT, LG and HG tumors. Serous borderline ovarian tumors exhibited robust positive staining for AGR3, with a lower percentage of tumor cells stained in LG and HG. Immunofluorescence staining indicated that AGR3 expression was limited to ciliated cells. Tumor samples with a high percentage (>10%) of AGR3 positively stained tumor cells were associated with improved longer median survival in both the LG (P = 0.013) and HG (P = 0.008) serous ovarian carcinoma groups. The progression of serous borderline ovarian tumors to low-grade serous ovarian carcinoma may involve the de-differentiation of ciliated cells. AGR3 could serve as a prognostic marker for survival in patients with low-grade and high-grade serous ovarian carcinomas.

Ovarian cancer is a complex and deadly disease that remains difficult to detect at an early curable stage. Furthermore, although some oncogenic (Kras, Pten/PI3K and Trp53) pathways that are frequently mutated, deleted or amplified in ovarian cancer are known, how these pathways initiate and drive specific morphological phenotypes and tumor outcomes remain unclear. We recently generated Pten(fl/fl); Kras(G12D); Amhr2-Cre mice to disrupt the Pten gene and express a stable mutant form of Kras(G12D) in ovarian surface epithelial (OSE) cells. On the basis of histopathologic criteria, the mutant mice developed low-grade ovarian serous papillary adenocarcinomas at an early age and with 100% penetrance. This highly reproducible phenotype provides the first mouse model in which to study this ovarian cancer subtype. OSE cells isolated from ovaries of mutant mice at 5 and 10 weeks of age exhibit temporal changes in the expression of specific Mullerian epithelial marker genes, grow in soft agar and develop ectopic invasive tumors in recipient mice, indicating that the cells are transformed. Gene profiling identified specific mRNAs and microRNAs differentially expressed in purified OSE cells derived from tumors of the mutant mice compared with wild-type OSE cells. Mapping of transcripts or genes between the mouse OSE mutant data sets, the Kras signature from human cancer cell lines and the human ovarian tumor array data sets, documented significant overlap, indicating that KRAS is a key driver of OSE transformation in this context. Two key hallmarks of the mutant OSE cells in these mice are the elevated expression of the tumor-suppressor Trp53 (p53) and its microRNA target, miR-34a-c. We propose that elevated TRP53 and miR-34a-c may exert negatively regulatory effects that reduce the proliferative potential of OSE cells leading to the low-grade serous adenocarcinoma phenotype.

Low-grade ovarian serous carcinomas are believed to arise via an adenoma-serous borderline tumor-serous carcinoma sequence. In this study, we found that advanced-stage, low-grade ovarian serous carcinomas both with and without adjacent serous borderline tumor shared similar regions of loss of heterozygosity. We then analyzed 91 ovarian tumor samples for mutations in TP53, BRAF, and KRAS. TP53 mutations were not detected in any serous borderline tumors (n = 30) or low-grade serous carcinomas (n = 43) but were found in 73% of high-grade serous carcinomas (n = 18). BRAF (n = 9) or KRAS (n = 5) mutation was detected in 47% of serous borderline tumors, but among the low-grade serous carcinomas (39 stage III, 2 stage II, and 2 stage I), only one (2%) had a BRAF mutation and eight (19%) had a KRAS mutation. The low frequency of BRAF mutations in advanced-stage, low-grade serous carcinomas, which contrasts with previous findings, suggests that aggressive, low-grade serous carcinomas are more likely derived from serous borderline tumors without BRAF mutation. In addition, advanced-stage, low-grade carcinoma patients with BRAF or KRAS mutation have a better apparent clinical outcome. However, further investigation is needed.

Low-grade serous ovarian carcinoma (LGSOC) accounts for a relatively large percentage of epithelial ovarian cancers in young women. Although LGSOC is considered to be an indolent cancer, with an estimated median overall survival of 80 months, an updated analysis of 350 women with LGSOC from our Low-Grade Serous Tumor Database indicated that over 85% developed recurrent disease, and almost all ultimately died of the disease. The overall response rate of recurrent LGSOC to various chemotherapy drugs is dismal - less than 4%. To explore the potential of immunotherapy in low-grade serous ovarian carcinoma, we investigate the immune profiles in low-grade serous ovarian carcinoma. In this study, we have generated gene expression profiles from microdissected epithelial tumor cells and the corresponding LGSOC bulk tumors. Such a novel comparison revealed an immune active tumor stromal microenvironment with highly expressed genes involved in immune response, antigen processing and presentation. The down-regulation of MHC-I gene expression or antigen-processing components of the MHC-I pathway in the tumor cells may lead to an escape from immune surveillance. A recent study has indicated that activation of the MAPK pathway in melanoma cancer cells could lead to internalization of MHC-I and subsequent sequestration within endolysosomal compartments. This sequestration may diminish antigen presentation and CD8+ T-cell recognition of tumor cells. This is further supported by our immunostaining analysis of CD8+ cells in LGSOC. The data indicated that the majority of LGSOC has less than five intratumoral CD8+ T-cells per high-power field, which is much less than the number of CD8+ T-cells observed in high-grade ovarian serous carcinoma. Thus, this low-number of intratumoral CD8+ cells and low level of MHC-I complex in LGSOC tumor cells would suggest a compromised antigen processing and presentation system. Furthermore, we also compared the number of CD45+ cells in LGSOC with or without BRAF/KRAS mutations. CD45 antigen (leukocyte common antigen) is a unique and ubiquitous membrane glycoprotein expressed on almost all hematopoietic cells, from which CD4+ helper T-lymphocyte, CD8+ cytotoxic T-lymphocyte and other lymphocytes are derived. Our data indicate that LGSOC with BRAF or KRAS mutant proteins had a high number of infiltrating CD45+ hematopoietic cells, which would suggest BRAF or KRAS mutant proteins are immunogenic and would partly explain our observation that patients with LGSOC carrying BRAF or KRAS mutation had longer overall survival. However, there were only a few or no CD8+ T-lymphocytes in the corresponding samples stained with CD45+ cells. In summary, our data suggest that an immune suppressive environment related to MAPK activation exists in LOSOC, which may prevent antigen presentation, maturation and activation of infiltrating cytotoxic T-cells. Citation Format: Kwong-Kwok Wong, David M. Gershenson. Immunosuppressive microenvironment in low-grade serous ovarian carcinoma [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 135.

Ovarian cancer is the most lethal gynecologic cancer, and this is largely related to its late diagnosis. High grade serous cancers often initially respond to chemotherapy, resulting in a better survival rate, compared to other ovarian carcinoma subtypes. We review recent work identifying a survival-associated gene expression profile for advanced serous ovarian cancer. Within this signature, the authors identified MAGP2, also known as microfibrillar associated protein 5 (MFAP5), as a highly significant indicator of survival and chemosensitivity. MAGP2 is a multifunctional secreted protein—important for elastic microfibril assembly and modulating endothelial cell behavior—with a newly identified role in cell survival. Through αVβ3 integrin-mediated signaling, MAGP2 promotes tumor and endothelial cell survival and endothelial cell motility, providing a potential mechanistic link between MAGP2 and angiogenesis as well as patient survival.

Gene copy number change is an essential characteristic of many types of cancer. However, it is important to distinguish copy number variation (CNV) in the human genome of normal individuals from bona fide abnormal copy number changes of genes specific to cancers. Based on Affymetrix 50K single nucleotide polymorphism (SNP) array data, we identified genome-wide copy number variations among 104 normal subjects from three ethnic groups that were used in the HapMap project. Our analysis revealed 155 CNV regions, of which 37% were gains and 63% were losses. About 21% (30) of the CNV regions are concordant with earlier reports. These 155 CNV regions are located on more than 100 cytobands across all 23 chromosomes. The CNVs range from 68bp to 18 Mb in length, with a median length of 86 Kb. Eight CNV regions were selected for validation by quantitative PCR. Analysis of genomic sequences within and adjacent to CNVs suggests that repetitive sequences such as long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) may play a role in the origin of CNVs by facilitating non-allelic homologous recombination. Thirty-two percent of the CNVs identified in this study are associated with segmental duplications. CNVs were not preferentially enriched in gene-encoding regions. Among the 364 genes that are completely encompassed by these 155 CNVs, genes related to olfactory sensory, chemical stimulus, and other physiological responses are significantly enriched. A statistical analysis of CNVs by ethnic group revealed distinct patterns regarding the CNV location and gain-to-loss ratio. The CNVs reported here will help build a more comprehensive map of genomic variations in the human genome and facilitate the differentiation between copy number variation and somatic changes in cancers. The potential roles of certain repeat elements in CNV formation, as corroborated by other studies, shed light on the origin of CNVs and will improve our understanding of the mechanisms of genomic rearrangements in the human genome.

Ovarian tumors of low-malignant potential and low-grade ovarian serous carcinomas are thought to represent different stages on a tumorigenic continuum and to develop along pathways distinct from high-grade ovarian serous carcinoma. We performed gene expression profiling on 3 normal human ovarian surface epithelia samples, and 10 low-grade and 10 high-grade ovarian serous carcinomas. Analysis of gene expression profiles of these samples has identified 80 genes up-regulated and 232 genes down-regulated in low-grade ovarian serous carcinomas. PAX2 was found to be one of the most up-regulated genes in low-grade ovarian serous carcinoma. The up-regulation of PAX2 was validated by real-time quantitative RT-PCR, Western blot and immunohistochemical analyses. Real-time RT-PCR demonstrated a statistically significant difference in PAX2 mRNA expression (expressed as fold change in comparison to normal human ovarian surface epithelia) among ovarian tumors of low-malignant potential (1837.38, N=8), low-grade (183.12, N=17), and high-grade (3.72, N=23) carcinoma samples (p=0.015). Western blot analysis revealed strong PAX2 expression in ovarian tumors of low-malignant potential (67%, N=3) and low-grade carcinoma samples (50%, N=10) but no PAX2 protein expression in high-grade carcinomas (0%, N=10). Using immunohistochemistry, tumors of low-malignant potential (59%, N=17) and low-grade carcinoma (63%, N=16) samples expressed significantly stronger nuclear staining than high-grade ovarian carcinoma samples (9.1%, N=263). Furthermore, consistent with previous immunohistochemical findings, PAX2 expression was found to be expressed in the epithelial cells of fallopian tubes but not in normal ovarian surface epithelial cells. Our findings further support the two-tiered hypothesis that tumors of low-malignant potential and low-grade ovarian serous carcinoma are on a continuum and are distinct from high-grade ovarian carcinomas. Additionally, the absence of PAX2 expression in normal ovarian epithelia but expression in fallopian tube fimbria and ciliated epithelial inclusions would suggest the potential development of tumors of low-malignant potential and of low-grade ovarian serous carcinomas from secondary Müllerian structures.

Hepatoblastoma is the most common malignant tumor of the liver of children worldwide. Histologically, hepatoblastomas show marked variation in the type and proportion of epithelial (fetal, embryonal, or small cell) and mesenchymal components with differing prognosis and response to therapy. The pure fetal-type hepatoblastoma, presenting as stage 1 and resectable, has the best prognosis, whereas the small cell histology has been associated with unfavorable outcome. Using gene expression profiling, we demonstrate that in addition to Wnt pathway deregulation, cell growth and survival pathways are also globally deregulated in hepatoblastomas. Furthermore, the different histologic subtypes are characterized by specific gene expression and pathway signatures that give insight into the degree of molecular heterogeneity that is present among these tumors. Although Wnt signaling pathway upregulation is common to all histologic types of hepatoblastoma, this pathway is even more significantly deregulated in aggressive hepatoblastomas. In addition, deregulation of MAPK signaling pathway and antiapoptotic signaling is preferentially upregulated in aggressive epithelial hepatoblastomas with a small cell component. The gene expression signatures reported here provide possible prognostic and diagnostic markers as well as therapeutic targets for this disease.

Purpose: The Aurora kinase family plays pivotal roles in mitotic integrity and cell cycle. We sought to determine the effects of inhibiting Aurora kinase on ovarian cancer growth in an orthotopic mouse model using a small molecule pan-Aurora kinase inhibitor, MK-0457.Experimental Design: We examined cell cycle regulatory effects and ascertained the therapeutic efficacy of Aurora kinase inhibition both alone and combined with docetaxel using both in vitro and in vivo ovarian cancer models.Results: In vitro cytotoxicity assays with HeyA8 and SKOV3ip1 cells revealed >10-fold greater docetaxel cytotoxicity in combination with MK-0457. After in vivo dose kinetics were determined using phospho-histone H3 status, therapy experiments with the chemosensitive HeyA8 and SKOV3ip1 as well as the chemoresistant HeyA8-MDR and A2780-CP20 models showed that Aurora kinase inhibition alone significantly reduced tumor burden compared with controls (P values < 0.01). Combination treatment with docetaxel resulted in significantly improved reduction in tumor growth beyond that afforded by docetaxel alone (P ≤ 0.03). Proliferating cell nuclear antigen immunohistochemistry revealed that MK-0457 alone and in combination with docetaxel significantly reduced cellular proliferation (P values < 0.001). Compared with controls, treatment with MK-0457 alone and in combination with docetaxel also significantly increased tumor cell apoptosis by ∼3-fold (P < 0.01). Remarkably, compared with docetaxel monotherapy, MK-0457 combined with docetaxel resulted in significantly increased tumor cell apoptosis.Conclusions: Aurora kinase inhibition significantly reduces tumor burden and cell proliferation and increases tumor cell apoptosis in this preclinical orthotopic model of ovarian cancer. The role of Aurora kinase inhibition in ovarian cancer merits further investigation in clinical trials.

Recent identification of cancer stem cells in medulloblastoma (MB) and high-grade glioma has stimulated an urgent need for animal models that will not only replicate the biology of these tumors, but also preserve their cancer stem cell pool. We hypothesize that direct injection of fresh surgical specimen of MB and high-grade glioma tissues into anatomically equivalent locations in immune-deficient mouse brains will facilitate the formation of clinically accurate xenograft tumors by allowing brain tumor stem cells, together with their non-stem tumor and stromal cells, to grow in a microenvironment that is the closest to human brains. Eight of the 14 MBs (57.1%) and two of the three high-grade gliomas (66.7%) in this study developed transplantable (up to 12 passages) xenografts in mouse cerebellum and cerebrum, respectively. These xenografts are patient specific, replicating the histopathologic, immunophenotypic, invasive/metastatic, and major genetic (analyzed with 10K single nucleotide polymorphism array) abnormalities of the original tumors. The xenograft tumor cells have also been successfully cryopreserved for long-term preservation of tumorigenicity, ensuring a sustained supply of the animal models. More importantly, the CD133+ tumor cells, ranging from 0.2%–10.4%, were preserved in all the xenograft models following repeated orthotopic subtransplantations in vivo. The isolated CD133+ tumor cells formed neurospheres and displayed multi-lineage differentiation capabilities in vitro. In summary, our study demonstrates that direct orthotopic transplantation of fresh primary tumor cells is a powerful approach in developing novel clinical relevant animal models that can reliably preserve CD133+ tumor cell pools even during serial in vivo subtransplantations.Disclosure of potential conflicts of interest is found at the end of this article.

The role of serous tumors of low malignant potential (LMP) in the development of invasive epithelial cancer of the ovary is debatable. This review summarizes the current clinical, genetic, and genomic evidence for the existence of a continuum comprising both LMP serous tumors and low-grade serous ovarian carcinomas.

The growth pattern of pilocytic astrocytoma (PAs) is unpredictable. Gene expression profiling has recently demonstrated an inverse relationship between myelin basic protein (MBP) expression and progression free survival (PFS) in PAs. We present here the pattern of expression of oligodendroglial differentiation markers (ODMs) in PAs by immunohistochemistry and their correlation with PI and PFS. Sixty-four cases of PA were reviewed and representative sections were stained for Ki-67 and ODMs, including MBP, platelet-derived growth factor receptor-α (PDGFR-α), Olig-1, and Olig-2. Sections were graded semi-quantitatively for intensity (I: 0–3+) and extent (E: 0–4+) of staining. PI was expressed as a percentage of Ki-67 positive cells. Immunoreactivity of MBP, PDGFR-α, Olig-1, and Olig-2 was observed in 84, 56, 97, and 75% of cases, respectively. There was a statistically significant inverse correlation between MBP expression and PI (r 2 = .696, p = .014). A positive correlation was observed between PDGFR-α and PI (r 2 = .727, p = .011). Further analysis showed a significant difference in PFS between low expressors [I + E score ≤ 3] and high expressors (I + E score ≥ 4) for PDGFR-α with p

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a--a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis--as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC50 values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.

Suppression of ovarian tumor growth by chromosome 3p was demonstrated in a previous study. Deleted in Lung and Esophageal Cancer 1 (DLEC1) on 3p22.3 is a candidate tumor suppressor in lung, esophageal, and renal cancers. The potential involvement of DLEC1 in epithelial ovarian cancer remains unknown. In the present study, DLEC1 downregulation was found in ovarian cancer cell lines and primary ovarian tumors. Focus-expressed DLEC1 in two ovarian cancer cell lines resulted in 41% to 52% inhibition of colony formation. No chromosomal loss of chromosome 3p22.3 in any ovarian cancer cell line or tissue was found. Promoter hypermethylation of DLEC1 was detected in ovarian cancer cell lines with reduced DLEC1 transcripts, whereas methylation was not detected in normal ovarian epithelium and DLEC1-expressing ovarian cancer cell lines. Treatment with demethylating agent enhanced DLEC1 expression in 90% (9 of 10) of ovarian cancer cell lines. DLEC1 promoter methylation was examined in 13 high-grade ovarian tumor tissues with DLEC1 downregulation, in which 54% of the tumors showed DLEC1 methylation. In addition, 80% of ovarian cancer cell lines significantly upregulated DLEC1 transcripts after histone deacetylase inhibitor treatment. Therefore, our results suggested that DLEC1 suppressed the growth of ovarian cancer cells and that its downregulation was closely associated with promoter hypermethylation and histone hypoacetylation.

Purpose: Mucinous adenocarcinoma of the ovary is one of the common histologic types of ovarian cancer. Its pathogenesis is largely unknown. In addition, the differential diagnosis of metastatic mucinous carcinomas to the ovaries, particularly those originating from the appendix, remains challenging. The purpose of this study is to identify molecular biomarkers for mucinous ovarian adenocarcinoma and compare them with those of appendiceal origin. Experimental Design: Genome-wide loss-of-heterozygosity (LOH) analysis was done on DNA isolated from 28 microdissected primary mucinous ovarian carcinomas and five appendiceal adenocarcinomas. Markers from high-loss regions were selected for further analysis on a total of 32 ovarian and 14 appendiceal cancers. Results: High levels of LOH rates (>40%) were detected on chromosome arms 9p, 17p, and 21q in mucinous ovarian carcinoma cases. The frequency of allelic loss was similar between high-grade and low-grade mucinous ovarian carcinoma cases but was significantly higher in ovarian versus appendiceal cases. In addition, LOH rates on five chromosomal loci were statistically different between ovarian and appendiceal carcinomas. Conclusion: A high frequency of LOH can be found in mucinous ovarian adenocarcinomas independent of grade. Despite histologic similarities between mucinous ovarian carcinomas and metastatic appendiceal carcinomas, they have distinct LOH profiles, which may be used for distinguishing the two diseases.

Deleted in liver cancer (DLC1) is a candidate tumor suppressor gene recently isolated from human hepatocellular carcinoma. Structurally, DLC1 protein contains a conserved GTPase-activating protein for Rho family protein (RhoGAP) domain, which has been thought to regulate the activity of Rho family proteins. Previous studies indicated that DLC1 was frequently inactivated in cancer cells. In the present study, we aimed to characterize the tumor suppressor roles of DLC1 in hepatocellular carcinoma. We showed that DLC1 significantly inhibited cell proliferation, anchorage-independent growth, and in vivo tumorigenicity when stably expressed in hepatocellular carcinoma cells. Moreover, DLC1 expression greatly reduced the motility and invasiveness of hepatocellular carcinoma cells. With RhoGAP-deficient DLC1 mutant (DLC1-K714E), we showed that the RhoGAP activity was essential for DLC1-mediated tumor suppressor function. Furthermore, the 292– to 648–amino acid region and the steroidogenic acute regulatory related lipid transfer domain played an auxiliary role to RhoGAP and tumor suppressor function of DLC1. Taken together, our findings showed that DLC1 functions as a tumor suppressor in hepatocellular carcinoma and provide the first evidence to support the hypothesis that DLC1 suppresses cancer cell growth by negatively regulating the activity of Rho proteins.

We have established a method for using a cDNA array platform in combination with degenerate oligonucleotide primer polymerase chain reaction (DOP-PCR) and taramide signal amplification (TSA) to identify DNA copy number abnormalities (CNA) in cancer cell lines and cancer cells procured with laser-based microdissection. To determine the sensitivity and specificity for detecting single-copy gain and loss, receiver-operator curve analysis was performed on hybridization signal ratios generated from non-DOP and DOP amplified female and male DNA using a 10,816-element cDNA microarray. A cutoff value of 1.12 and 1.07 average signal ratio for X-chromosomal genes versus autosomal genes provided a sensitivity and specificity of 50 and 79%, respectively, for non-DOP amplified DNA and a sensitivity and specificity of 50 and 72%, respectively, for DOP amplified DNA. We used this approach to identify DNA copy number abnormalities in the ovarian cancer cell line OVCA633, which has previously been shown to have 12p amplification. Transcription profiling of OVCA633 was also performed. Two amplified and overexpressed genes located on 12p11, KRAS2 and LRMP, were identified; these were validated with quantitative real-time PCR. Subsequently, the same approach was used to identify CNAs and gene expression alterations in 11 microdissected serous ovarian adenocarcinoma cases. Validated data revealed amplification and overexpression of ERBB3 and FOS and deletion and underexpression of KRT6 and APXL in more than 50% of the tissue samples. These results show the feasibility of using the cDNA array platform to identify changes in DNA and mRNA copy number simultaneously in microdissected tumor tissues.

Gene amplifications have been observed in many different tumor cells, and many of these changes are related to tumor pathogenesis. Comparative genomic hybridization (CGH) using metaphase chromosomes can detect changes in chromosome copy number with a resolution of 10–20 Mb. Current advances in CGH analysis in a microarray format allow us to refine such changes down to the gene level. We applied microarray technology to detect novel gene amplification in a malignant mixed tumor of salivary gland. Besides detecting previously known gene amplifications ( MDM2 and MYC ), we identified four other highly amplified genes located at 8q11.2∼q13: MGC2177 , PLAG1 , PSMC6P , and LYN . The amplification was further validated with real-time quantitative polymerase chain reaction.

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organism's dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans . This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Ovarian cancer is the fifth leading cause for cancer-associated deaths among women in the US. High-grade serous ovarian carcinoma is the most common histological subtype and is characterized by frequent (>90%) mutations in the TP53 (p53) gene. Gain-of-function (GOF) p53 mutant proteins form highly stable aggregates that accumulate in ovarian cancer cells and have been shown to have oncogenic activity. Increasing evidence indicates the stabilization of GOF p53 mutants to be important for tumor progression and drug resistance. Therefore, selective degradation of the GOF p53 mutant proteins is a highly attractive therapeutic strategy to specifically target ovarian cancer cells. We identified that a small molecule named MCB-613, originally discovered as a stimulator of steroid receptor coactivator-3 (SRC-3), causes rapid degradation of the usually stable p53R175H conformational mutant aggregates leading to catastrophic cell death in human ovarian cancer cell lines. Our results indicate this effect is independent of SRC-3 mediated gene expression, but rather by post-translational modification of p53 mutant protein. Upon MCB-613 treatment, the p53R175H mutant is rapidly exported from the nucleus, ubiquitinated and degraded through a lysosomal pathway. Interestingly, in contrast to its effect on the p53R175H mutant, MCB-613 causes a slight stabilization of wild type (WT) p53 but had less of an effect on the turnover of other frequently observed p53 mutants (R248Q and R273H) in human ovarian cancer cell lines. MCB-613 also causes a rapid decrease in MDM2 protein but not mRNA. Further, the effect of MCB-613 on p53R175H is phenocopied by multiple deubiquitinase (DUB) inhibitors, confirming the role of ubiquitination in the selective turnover of p53R175H mutant aggregates. Taken together, these results show that different pathways regulate the turnover of WT p53 and the specific GOF p53 mutant proteins. We are currently investigating the precise mechanism by which MCB-613 and the different DUB inhibitors induce selective turnover of p53R175H mutant protein. We anticipate the proposed work to significantly advance our understanding of pathways important in ovarian cancer progression and pave way for the development of new targeted therapies for ovarian cancer patients with specific p53 mutations. This work is funded by NIH-CA-181808, NIH-HD-07857 and NIH-HD-076596. Citation Format: Achuth Padmanabhan, Nicholes Candelaria, Kwong-Kwok Wong, Bryan C. Nikolai, David Lonard, Bert W. O'Malley, JoAnne S. Richards. Identifying pathways that regulate the turnover of gain-of-function p53 mutant proteins in ovarian cancer cells [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 2579. doi:10.1158/1538-7445.AM2017-2579

Inactivating mutations in AT-rich interaction domain 1A (ARID1A) are most frequently found in ovarian clear cell carcinomas (OCCC) and ovarian endometrioid carcinomas (OEC). The functional roles of ARID1A are not completely understood and there are limited therapeutic strategies that specifically target ARID1A-mutant cancers. Given that ARID1A expression is lost in cancer, ARID1A mutations cannot be targeted directly and novel therapeutic strategies are required to target ARID1A-mutant cancers. In this study, drug responses between ARID1A-wildtype and ARID1A-mutant cell lines were compared using the ‘Genomics of Drug Sensitivities in Cancer’ database. From this analysis, we found that ARID1A-mutant cell lines are more sensitive to elesclomol, which is a reactive oxygen species (ROS)-inducing agent. This finding was validated using a panel of well-characterized ovarian and endometrial cancer cell lines. ARID1A-mutant OCCC cell lines exhibited lower IC50 values and higher apoptotic rates when treated with elesclomol. Knockdown and re-expression of ARID1A in ovarian cancer cells showed that ARID1A is required to protect cancer cells from oxidative stress. In the absence of ARID1A, intracellular ROS levels were increased as determined by flow cytometry. Immunostaining of 4-hydroxy-2-nonenals (4-HNE), one of the products from lipid peroxidation induced by ROS, in OCCC patient samples indicated an up-regulation of oxidative stress in the tumor. We also found that ARID1A negatively regulates the expression of NRF2 which is the major regulator of the antioxidant response in the cell. Knockdown of NRF2 in ovarian cancer cell lines revealed that NRF2 expression may be preferentially required for protection from oxidative stress and cell proliferation in ARID1A-mutant cells. Analysis using The Cancer Genome Atlas (TCGA) UCEC dataset revealed that ARID1A-mutant tumor samples have higher expression of NRF2 and NRF2-target genes. In summary, this study revealed novel roles of ARID1A in protecting ovarian cancer cells against oxidative stress. In the absence of ARID1A, NRF2 is up-regulated and may be required to compensate for ARID1A deficiency. These findings suggest that ARID1A-mutant ovarian cancer cells may be targeted for treatment with ROS-inducing agents and NRF2 inhibitors. Citation Format: Suet-Yan Kwan, Xuanjin Cheng, Suet-Ying Kwan, Daisy I. Izaguirre, Yvonne T.M. Tsang, Jong-Sun Choi, Hoi-Shan Kwan, David M. Gershenson and Kwong-Kwok Wong. ARID1A DEFICIENCY IN OVARIAN CLEAR CELL CARCINOMAS UP–REGULATES OXIDATIVE STRESS AND SENSITIZES CELLS TO ROS–INDUCING AGENTS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-114.

Ovarian cancer is the most lethal gynaecologic malignancy in the United States, and advanced serous ovarian adenocarcinoma is responsible for most ovarian cancer deaths. However, the stroma-derived molecular determinants that modulate patient survival are yet to be characterized. Here we identify a stromal gene signature for advanced high-grade serous ovarian cancer using microdissected stromal ovarian tumour samples and find that stromal microfibrillar-associated protein 5 (MFAP5) is a prognostic marker for poor survival. Further functional studies reveal that FAK/CREB/TNNC1 signalling pathways mediate the effect of MFAP5 on ovarian cancer cell motility and invasion potential. Targeting stromal MFAP5 using MFAP5-specific siRNA encapsulated in chitosan nanoparticles significantly decreases ovarian tumour growth and metastasis in vivo, suggesting that it may be a new modality of ovarian cancer treatment.

BACKGROUND Screening biomarkers for ovarian cancer are needed because of its late stage at diagnosis and poor survival. We used microarray technology to identify overexpressed genes for secretory proteins as potential serum biomarkers and selected prostasin, a serine protease normally secreted by the prostate gland, for further study. METHODS RNA was isolated and pooled from three ovarian cancer cell lines and from three normal human ovarian surface epithelial (HOSE) cell lines. Complementary DNA generated from these pools was hybridized to a microarray slide, and genes overexpressed in the cancer cells were identified. Real-time quantitative polymerase chain reaction was used to examine prostasin gene expression in ovarian cancer and HOSE cell lines. Anti-prostasin antibodies were used to examine prostasin expression and to measure serum prostasin by an enzyme-linked immunosorbent assay in 64 case patients with ovarian cancer and in 137 control subjects. Previously determined levels of CA 125, an ovarian cancer marker, were available from about 70% of all subjects. All statistical tests were two-sided. RESULTS Prostasin was detected by immunostaining more strongly in cancerous ovarian epithelial cells and stroma than in normal ovarian tissue. The mean level of serum prostasin was 13.7 microg/mL (95% confidence interval [CI] = 10.5 to 16.9 microg/mL) in 64 case patients with ovarian cancer and 7.5 microg/mL (95% CI = 6.6 to 8.3 microg/mL) in 137 control subjects (P

Capillary isoelectric focusing (CIEF) can provide high-resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400-1000 putative proteins in the mass range of 2-100 kDa from total injections of approximately 300 ng protein in single CIEF-FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome-wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.

Using a genomic approach, we have identified a new Salmonella pathogenicity island, SPI-4, which is the fourth Salmonella pathogenicity island to be identified. SPI-4 was located at 92 min on the chromosome map and was flanked by the ssb and soxSR loci. The DNA sequence covering the entire SPI-4 and both boundaries was determined. The size of SPI-4 was about 25 kb and it contains 18 putative open reading frames (ORFs). Three of these ORFs encode proteins that have significant homology with proteins involved in toxin secretion. Another five ORFs encode proteins that have significant homology with hypothetical proteins from Synechocystis sp. strain PCC6803 or Acinetobacter calcoaceticus . The rest of the ORFs encode novel proteins, one of which has five membrane-spanning domains. SPI-4 is likely to carry a type I secretion system involved in toxin secretion. Furthermore, a previously identified locus ( ims98 ), which is required for intramacrophage survival, was also mapped within the SPI-4 region. These findings suggested that SPI-4 is needed for intramacrophage survival.

Using RNA fingerprinting (RAP) strategy and Northern blot analysis, we identified a differentially expressed sequence DOC-2 which is detectable in all normal human ovarian surface epithelial (HOSE) cell cultures but not in ovarian cancer cell lines and tissues. Subsequent cloning of DOC-2 from a cDNA library generated from the HOSE cells was carried out using the 3' and 5' RACE approach. A 3268 base pair full length cDNA of DOC-2 was isolated and sequenced. The predicted protein has a length of 770 amino acids. Homology search of all NCBI sequences indicated that the amino acid sequence of DOC-2 shares 93% homology with the mouse p96/mDab2 phosphoprotein and has a phosphotyrosine interacting domain (PID) and multiple SH3 binding motifs. Chromosomal localization by FISH showed that the DOC-2 gene is located on 5p13. Western blot analysis showed that the 105 kDa DOC-2 protein was down-regulated in all the carcinoma cell lines. In-situ immunohistochemistry performed on normal ovaries, and benign, borderline and invasive ovarian tumor tissues showed down regulation of DOC-2 protein particularly in serous ovarian tumor tissues. When DOC-2 was transfected into the ovarian carcinoma cell line SKOV3, the stable transfectants showed significantly reduced growth rate and ability to form tumors in nude mice. These data suggest that down-regulation of DOC-2 may play an important role in ovarian carcinogenesis.

PAX2 is one of nine PAX genes that regulate tissue development and cellular differentiation in embryos. However, the functional role of PAX2 in ovarian cancer is not known. Twenty-six ovarian cancer cell lines with different histology origins were screened for PAX2 expression. Two ovarian cancer cell lines: RMUGL (mucinous) and TOV21G (clear cell), with high PAX2 expression were chosen for further study. Knockdown PAX2 expression in these cell lines was achieved by lentiviral shRNAs targeting the PAX2 gene. PAX2 stable knockdown cells were characterized for cell proliferation, migration, apoptosis, protein profiles, and gene expression profiles. The result indicated that these stable PAX2 knockdown cells had reduced cell proliferation and migration. Microarray analysis indicated that several genes involved in growth inhibition and motility, such as G0S2, GREM1, and WFDC1, were up-regulated in PAX2 knockdown cells. On the other hand, over-expressing PAX2 in PAX2-negative ovarian cell lines suppressed their cell proliferation. In summary, PAX2 could have both oncogenic and tumor suppression functions, which might depend on the genetic content of the ovarian cancer cells. Further investigation of PAX2 in tumor suppression and mortality is warranted.

Human paired box 2 (PAX2) plays a key role in cell fate, early patterning and organogenesis. To investigate the function of PAX2 on the biological behavior of endometrial cancer and the regulation mechanism, stable knocking-down and over-expression PAX2 endometrial cancer cell lines were established. CCK-8 and transwell assays were applied to determine proliferation, invasion and migration ability. Cell cycle distribution was analyzed by flow cytometry. Nude mice were used for in vivo experiments and Affymetrix GeneChip® human Exon 1.0 ST arrays was used to screen the downstream target genes of PAX2. The upstream regulation mechanism of PAX2 was explored by dual luciferase reporter gene assay and bioinformatics analysis.Results showed that,no matter in vivo or in vitro,PAX2 significantly enhanced tumorigenicity and invasiveness of endometrial cancer cells, and promoted liver metastasis in vivo. In addition, PAX2 influenced the expression of cyclin-dependent kinase 1(CDK1) and YWHAZ, which play pivotal roles in cell cycle pathway. Knocking down CDK1 in PAX2 over-expressing cells attenuated cell viability.A myeloid zinc finger gene-1(MZF-1)binding site was found in a negative regulation region of PAX2 upstream and down-regulation of MZF-1 reduced the transcriptional activity of PAX2, as well as its protein level.Together, PAX2, through influencing the activity of CDK1 and YWHAZ, promotes proliferation,invasion and progression of endometrial cancer and can be regulated by MZF-1. PAX2 may be a target therapeutic site for endometrial cancer. Citation Format: Jieyu Wang, Weiwei Feng, Yinhua Yu, Kwong-Kwok Wong, Nan Jia, Tianjiao Lyu. Paired box 2 promotes progression of endometrial cancer via cell cycle pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2016.

Most ovarian cancers are diagnosed at an advanced stage when the tumor is widely metastatic. The 5-year survival drops to 50% for the cancer cases that spread beyond the pelvis to the omentum. However, the mechanisms underlying the effect of omental adipose tissue on ovarian cancer progression are poorly understood. Recent studies showed that exosomes also contain non-coding RNAs such as microRNAs (miRNAs). Thus, we hypothesize that the transfer of microRNAs and their variants from ovarian cancer-associated omental adipose tissues to ovarian cancer cells via exosomes may contribute to the nearby microenvironment for ovarian cancer metastasis and cancer progression. Ion Torrent next generation sequencing was performed on miRNAs isolated and enriched from exosomes and cell lysates of ovarian cancer cell lines (OVCA), the epithelial component of microdissected omental ovarian cancer tissues (CT), normal omental adipose tissues (OMN) and ovarian cancer-associated omental adipose tissues (OMT). By integrating the miRNA expression profiles, 65 miRNAs were expressed at significant higher levels in OMT-derived exosomes compared with those in OMN-derived exosomes and OVCA-derived exosomes. A set of miRNAs (miR-32a, miR-221 and miR320a), which had been implicated in controlling cell growth and chemoresistance, was identified. Also, the Ion Torrent results were validated and exosomal transfer of OMT-derived miRNAs was confirmed in vitro. The exosomal communication between adipose tissues and ovarian cancer cells in the omental tumor microenvironment is verified. The transferable miRNAs and their variants may remain functional in the recipient ovarian cancer cells and confer more aggressive phenotypes in these cells. Citation Format: Chi Lam Au Yeung, Tetsushi Tsuruga, Ngai Na Co, Tsz-Lun Yeung, Cecilia S. Leung, Kwong K. Wong, Samuel C. Mok. Adipose tissues derived exosomal microRNAs and their variants in ovarian cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 715.

Ovarian cancer is the most lethal gynecologic malignancy in the US. Our group and others has shown that CD8+ lymphocyte infiltration in the ovarian tumor epithelium is associated with prolonged survival in patients with high-grade serous ovarian cancer (HGSOC). Despite the increasing evidence on stromal involvement in tumor progression, the underlying genetic composition of the stromal cells that could regulate the infiltration and activation of CD8+ cytotoxic T lymphocytes (CTLs) in ovarian cancer is not fully understood. How tumor heterogeneity contributes to a different in immune response remains a challenge question that needs to be addressed. The present study seeks to evaluate the roles and to delineate the underlying mechanisms by which stromal cancer associated fibroblasts (CAFs) modulates immune response in ovarian cancer, particularly immune suppression by CAF-derived protein factors. By laser microdissection of tumor tissue samples from HGSOC patients, we generated cell type specific expression profiles and identified a CAF-specific gene signature for ovarian cancer. Genes expressed exclusively by CAFs that are associated with differential immune response were identified by comparing CAF expression profiles from HGSOC patients with high and low tumor-infiltrated CD8+ T cell densities. Among the differentially expressed genes identified, immunostaining results showed a significant inverse correlation between stromal MFAP5 expression and intratumoral CD8+ T cell density in HGSOC tissue samples (p = 0.006). The results were further confirmed by correlating stromal MFAP5 protein expression and intratumoral CD8+ T cell density. Our recent studies showed that increased stromal MFAP5 expression is associated with poorer survival in HGSOC patients and MFAP5 modulates ovarian caner invasion and motility potential. Together with our preliminary data showing that MFAP5 modulates the expression of immune-related genes, we hypothesize that stromal MFAP5 may generate an immunosuppressive microenvironment through suppressing CD8+ T cell activation and trafficking in the ovarian tumor tissue. Cell culture experiment showed that recombinant MFAP5 protein treatment induced expression of CD47, an immune checkpoint protein, in cancer cells and downregulated CXCL13, a chemokine essential for immune cell adhesion, in endothelial cells. Using animal models, these findings were further validated. Based on our data, animal studies will be performed to further evaluate the efficacy of immune activation by targeting stromal MFAP5 in ovarian cancer treatment. Delineating the molecular mechanism by which MFAP5 modulates the immune responses in ovarian cancer will help to design novel treatment modalities based on stromal MFAP5 blockade, which will promote activation and trafficking of cytotoxic CD8+ T cells and improve patient survival rates. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Stromal heterogeneity and immune response in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4145.

A DNA-fingerprinting approach has been adapted to detect differentially expressed genes in human ovarian carcinoma. This method is based on the use of arbitrary primers to generate fingerprints from total RNA isolated from normal ovarian epithelial cells and ovarian carcinoma cells by polymerase chain reaction (PCR). Using this method, we cloned two cDNA fragments (DOC-1 and DOC-2) which were present in normal ovarian surface epithelial cells but consistently absent in all of the ovarian cancer cell lines from the differential display. In addition, we also identified a cDNA fragment (LF4.0) which is overexpressed in most of the tumor cell lines and tumor tissues in comparison to the normal ovarian surface epithelial cells. The differential expression of the genes in the tumor cell lines as well as in the tumor tissues was also confirmed by Northern analysis. The clone DOC-2, which is a 800-bp cDNA fragment, has one open reading frame suggesting that the gene may be translated. Assuming that this frame is the sense strand, we generated both sense and antisense riboprobe for in situ mRNA hybridization. Only the antisense DOC-2 riboprobe revealed a hybridization signal which was restricted to the human surface ovarian epithelium. The potential functional roles of these genes is now under investigation.