Normal Ovarian Surface Epithelium Research Articles

Stearoyl-CoA desaturase 1 inhibition induces ER stress-mediated apoptosis in ovarian cancer cells

Ovarian cancer is a leading cause of death among gynecologic tumors, often detected at advanced stages. Metabolic reprogramming and increased lipid biosynthesis are key factors driving cancer cell growth. Stearoyl-CoA desaturase 1 (SCD1) is a crucial enzyme involved in de novo lipid synthesis, producing mono-unsaturated fatty acids (MUFAs). Here, we aimed to investigate the expression and significance of SCD1 in epithelial ovarian cancer (EOC). Comparative analysis of normal ovarian surface epithelial (NOSE) tissues and cell lines revealed elevated SCD1 expression in EOC tissues and cells. Inhibition of SCD1 significantly reduced the proliferation of EOC cells and patient-derived organoids and induced apoptotic cell death. Interestingly, SCD1 inhibition did not affect the viability of non-cancer cells, indicating selective cytotoxicity against EOC cells. SCD1 inhibition on EOC cells induced endoplasmic reticulum (ER) stress by activating the unfolded protein response (UPR) sensors and resulted in apoptosis. The addition of exogenous oleic acid, a product of SCD1, rescued EOC cells from ER stress-mediated apoptosis induced by SCD1 inhibition, underscoring the importance of lipid desaturation for cancer cell survival. Taken together, our findings suggest that the inhibition of SCD1 is a promising biomarker as well as a novel therapeutic target for ovarian cancer by regulating ER stress and inducing cancer cell apoptosis.

The splicing factor DHX38/PRP16 is required for ovarian clear cell carcinoma tumorigenesis, as revealed by a CRISPR-Cas9 screen.

Certain cancers, such as ovarian clear cell carcinoma (OCCC), display high levels of genetic variation between patients, making it difficult to develop effective therapies. In order to identify novel genes critical to OCCC growth, we carried out a comprehensive CRISPR‐Cas9 knockout screen against cell growth using an OCCC cell line and a normal ovarian surface epithelium cell line. We identified the gene encoding DHX38/PRP16, an ATP‐dependent RNA helicase involved in splicing, as critical for the growth and tumorigenesis of OCCC. DHX38/PRP16 knockdown in OCCC cells, but not normal cells, induces apoptosis and impairs OCCC tumorigenesis in a mouse model. Our results suggest that DHX38/PRP16 may play a role in OCCC tumorigenesis and could potentially be a promising therapeutic target.

Functional analysis of the 1p34.3 risk locus implicates GNL2 in high-grade serous ovarian cancer

The high-grade serous ovarian cancer (HGSOC) risk locus at chromosome 1p34.3 resides within a frequently amplified genomic region signifying the presence of an oncogene. Here, we integrate in silico variant-to-function analysis with functional studies to characterize the oncogenic potential of candidate genes in the 1p34.3 locus. Fine mapping of genome-wide association statistics identified candidate causal SNPs local to H3K27ac-demarcated enhancer regions that exhibit allele-specific binding for CTCF in HGSOC and normal fallopian tube secretory epithelium cells (FTSECs). SNP risk associations colocalized with eQTL for six genes (DNALI1, GNL2, RSPO1, SNIP1, MEAF6, and LINC01137) that are more highly expressed in carriers of the risk allele, and three (DNALI1, GNL2, and RSPO1) were upregulated in HGSOC compared to normal ovarian surface epithelium cells and/or FTSECs. Increased expression of GNL2 and MEAF6 was associated with shorter survival in HGSOC with 1p34.3 amplifications. Despite its activation of β-catenin signaling, RSPO1 overexpression exerted no effects on proliferation or colony formation in our study of ovarian cancer and FTSECs. Instead, GNL2, MEAF6, and SNIP1 silencing impaired in vitro ovarian cancer cell growth. Additionally, GNL2 silencing diminished xenograft tumor formation, whereas overexpression stimulated proliferation and colony formation in FTSECs. GNL2 influences 60S ribosomal subunit maturation and global protein synthesis in ovarian cancer and FTSECs, providing a potential mechanism of how GNL2 upregulation might promote ovarian cancer development and mediate genetic susceptibility of HGSOC.

Clerodane Diterpenoids from an Edible Plant Justicia insularis: Discovery, Cytotoxicity, and Apoptosis Induction in Human Ovarian Cancer Cells

Objectives: The toxicity of chemotherapeutic anticancer drugs is a serious issue in clinics. Drug discovery from edible and medicinal plants represents a promising approach towards finding safer anticancer therapeutics. Justicia insularis T. Anderson (Acanthaceae) is an edible and medicinal plant in Nigeria. This study aims to discover cytotoxic compounds from this rarely explored J. insularis and investigate their underlying mechanism of action. Methods: The cytotoxicity of the plant extract was evaluated in human ovarian cancer cell lines and normal human ovarian surface epithelia (HOE) cells using a sulforhodamine B assay. Bioassay-guided isolation was carried out using column chromatography including HPLC, and the isolated natural products were characterized using GC-MS, LC-HRMS, and 1D/2D NMR techniques. Induction of apoptosis was evaluated using Caspase 3/7, 8, and 9, and Annexin V and PI based flow cytometry assays. SwissADME and SwissTargetPrediction web tools were used to predict the molecular properties and possible protein targets of identified active compounds. Key finding: The two cytotoxic compounds were identified as clerodane diterpenoids: 16(α/β)-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (1) and 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (2) from the Acanthaceous plant for the first time. Compound 1 was a very abundant compound (0.7% per dry weight of plant material) and was shown to be more potent than compound 2 with IC50 values in the micromolar range against OVCAR-4 and OVCAR-8 cancer cells. Compounds 1 and 2 were less cytotoxic to HOE cell line. Both compounds induced apoptosis by increasing caspase 3/7 activities in a concentration dependent manner. Compound 1 further increased caspase 8 and 9 activities and apoptosis cell populations. Compounds 1 and 2 are both drug like, and compound 1 may target various proteins including a kinase. Conclusions: Clerodane diterpenoids (1 and 2) in J. insularis were identified as cytotoxic to ovarian cancer cells via the induction of apoptosis, providing an abundant and valuable source of hit compounds for the treatment of ovarian cancer.

Ameliorative Potential of Aqueous Extract of Broccoli Sprouts Against Triazophos Induced Ovarian Toxicity in Wistar Rats

Traditional therapeutic procedures using antioxidant-rich fruits and vegetables have been in vogue for the development of evidence-based biomarkers for assessing reproductive health. Present investigation was designed to study the antioxidative potential of broccoli sprouts aqueous extract (BE), against ovarian toxicity in female rats induced by triazophos (TZ). In the experimental setup, six groups of rats were formed; Control (group 1), BE (group 2), TZ (group 3), and also BE+TZ groups such as BE1 (group 4), BE2 (group 5) and BE3 (group 6) groups. Body weight was weekly recorded of all the rats, while vaginal smear was observed daily during 30 days experiment. After sacrifice, oxidative stress (OS) biomarkers levels viz; catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and lipid peroxidation (LPO) were determined along with histopathological and apoptotic observation. Results revealed differentially modified changes in OS biomarkers as CAT, SOD, GR, GPx, and GST, while LPO levels were significantly improved with broccoli supplementation compared to TZ group rats. Plasma progesterone and estradiol levels were also restored along with improved ovarian histoarchitecture among all BE+TZ treated rats. Reduced apoptotic granulosa cells with reduced atresia and normal ovarian surface epithelium height were also observed with BE treatment. BE exerts multi-mechanistic protective effects against TZ induced ovarian toxicity which is attributable to its antioxidant and protective actions.

Targeting Aurora A Kinase (AAK) in Platinum-Resistant High Grade Serous Ovarian Cancer.

Aurora A kinase (AAK) involved in G2-M transition is functionally involved in centrosome maturation and maintaining an active spindle assembly checkpoint. We tested the hypothesis that in platinum-taxane resistant high grade serous ovarian cancer (HGSOC) inhibition of AAK involved in G2-M transition would enhance the anti-tumor activity of cisplatin (CP) or paclitaxel (PT). Using HGSOC cell lines from platinum-taxane refractory patients that do not harbor BRCA1/2 mutations, we tested the anti-tumor activity of CP, or PT alone or in combination with the AAK inhibitor alisertib (AL). Treatment with CP for 3 h or PT for 6 h followed sequentially by AL for 48 h led to a significant decrease in cell survival (p < 0.001) compared to treatment with either drug alone in HGSOC cells but not in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. The treatment with CP or PT followed by AL also led to a significant increase in reactive oxygen species (p < 0.05), apoptosis (p < 0.001) and accumulation of cells in G2/M that was accompanied by a modest increase in expression of AAK. Downregulation of AAK, but not aurora B kinase, with targeted siRNAs also significantly enhanced apoptosis by CP or PT, suggesting that AL specifically targeted AAK. In summary, in HGSOC without BRCA1/2 mutations, CP, or PT resistance can potentially be circumvented by sequential treatment with AL that inhibits AAK involved in G2-M transition.

ALDH1A1+ ovarian cancer stem cells co-expressing surface markers CD24, EPHA1 and CD9 form tumours in vivo

One of the reasons for recurrence following treatment of high grade serous ovarian carcinoma (HGSOC) is the persistence of residual cancer stem cells (CSCs). There has been variability between laboratories in the identification of CSC markers for HGSOC. We have identified new surface markers (CD24, CD9 and EPHA1) in addition to those previously known (CD44, CD117 and CD133) using a bioinformatics approach. The expression of these surface markers was evaluated in ovarian cancer cell lines, primary malignant cells (PMCs), normal ovary and HGSOC. There was no preferential expression of any of the markers or a combination. All the markers were expressed at variable levels in ovarian cancer cell lines and PMCs. Only CD117 and CD9 were expressed in the normal ovarian surface epithelium and fallopian tube. Both ALDEFLUOR (ALDH1A1) and side population assays identified a small proportion of cells (<3%) separately that did not overlap with little variability in cell lines and PMCs. All surface markers were co-expressed in ALDH1A1+ cells without preference for one combination. The cell cycle analysis of ALDH1A1+ cells alone revealed that majority of them reside in G0/G1 phase of cell cycle. Further separation of G0 and G1 phases showed that ALDH1A1+ cells reside in G1 phase of the cell cycle. Xenograft assays showed that the combinations of ALDH1A1 + cells co-expressing CD9, CD24 or EPHA1 were more tumorigenic and aggressive with respect to ALDH1A1-cells. These data suggest that a combined approach could be more useful in identifying CSCs in HGSOC.

ROR2 induces cell apoptosis via activating IRE1\u03b1/JNK/CHOP pathway in high-grade serous ovarian carcinoma in vitro and in vivo

BackgroundEpithelial ovarian cancer (EOC) is the most lethal cancer in female genital tumors. New disease markers and novel therapeutic strategies are urgent to identify considering the current status of treatment. Receptor tyrosine kinases family plays critical roles in embryo development and disease progression. However, ambivalent research conclusions of ROR2 make its role in tumor confused and the underlying mechanism is far from being understood. In this study, we sought to clarify the effects of ROR2 on high-grade serous ovarian carcinoma (HGSOC) cells and reveal the mechanism.MethodsImmunohistochemistry assay and western-blot assay were used to detect proteins expression. ROR2 overexpression adenovirus and Lentivirus were used to create ROR2 overexpression model in vitro and in vivo, respectively. MTT assay, colony formation assay and transwell assay were used to measure the proliferation, invasion and migration ability of cancer cells. Flow cytometry assay was used to detect cell apoptosis rate. Whole transcriptome analysis was used to explore the differentially expressed genes between ROR2 overexpression group and negative control group. SiRNA targeted IRE1α was used to knockdown IRE1α. Kira6 was used to inhibit phosphorylation of IRE1α.ResultsExpression of ROR2 was significantly lower in HGSOC tissues compared to normal fallopian tube epithelium or ovarian surface epithelium tissues. In HGSOC cohort, patients with advanced stages or positive lymph nodes were prone to express lower ROR2. Overexpression of ROR2 could repress the proliferation of HGSOC cells and induce cell apoptosis. RNA sequencing analysis indicated that ROR2 overexpression could induce unfold protein response. The results were also confirmed by upregulation of BIP and phosphorylated IRE1α. Furthermore, pro-death factors like CHOP, phosphorylated JNK and phosphorylated c-Jun were also upregulated. IRE1α knockdown or Kira6 treatment could reverse the apoptosis induced by ROR2 overexpression. Finally, tumor xenograft experiment showed ROR2 overexpression could significantly repress the growth rate and volume of transplanted tumors.ConclusionsTaken together, ROR2 downregulation was associated with HGSOC development and progression. ROR2 overexpression could repress cell proliferation and induce cell apoptosis in HGSOC cells. And the underlying mechanism might be the activation of IRE1α/JNK/CHOP pathway induced by ROR2.

Integrated Analysis To Identify Molecular Biomarkers Of High-Grade Serous Ovarian Cancer.

PurposeOvarian cancer is the leading cause of gynecologic cancer-related death worldwide. Early diagnosis of ovarian cancer can significantly improve patient prognosis. Hence, there is an urgent need to identify key diagnostic and prognostic biomarkers specific for ovarian cancer. Because high-grade serous ovarian cancer (HGSOC) is the most common type of ovarian cancer and accounts for the majority of deaths, we identified potential biomarkers for the early diagnosis and prognosis of HGSOC.MethodsSix datasets (GSE14001, GSE18520, GSE26712, GSE27651, GSE40595, and GSE54388) were downloaded from the Gene Expression Omnibus database for analysis. Differentially expressed genes (DEGs) between HGSOC and normal ovarian surface epithelium samples were screened via integrated analysis. Hub genes were identified by analyzing protein–protein interaction (PPI) network data. The online Kaplan-Meier plotter was utilized to evaluate the prognostic roles of these hub genes. The expression of these hub genes was confirmed with Oncomine datasets and validated by quantitative real-time PCR and Western blotting.ResultsA total of 103 DEGs in patients with HGSOC—28 upregulated genes and 75 downregulated genes—were successfully screened. Enrichment analyses revealed that the upregulated genes were enriched in cell division and cell proliferation and that the downregulated genes mainly participated in the Wnt signaling pathway and various metabolic processes. Ten hub genes were associated with HGSOC pathogenesis. Seven overexpressed hub genes were partitioned into module 1 of the PPI network, which was enriched in the cell cycle and DNA replication pathways. Survival analysis revealed that MELK, CEP55 and KDR expression levels were significantly correlated with the overall survival of HGSOC patients (P < 0.05). The RNA and protein expression levels of these hub genes were validated experimentally.ConclusionBased on an integrated analysis, we propose the further investigation of MELK, CEP55 and KDR as promising diagnostic and prognostic biomarkers of HGSOC.

Defining the molecular evolution of extrauterine high grade serous carcinoma

ObjectiveHigh grade serous carcinoma (HGSC) is the most common and most aggressive, subtype of epithelial ovarian cancer. It presents as advanced stage disease with poor prognosis. Recent pathological evidence strongly suggests HGSC arises from the fallopian tube via the precursor lesion; serous tubal intraepithelial carcinoma (STIC). However, further definition of the molecular evolution of HGSC has major implications for both clinical management and research. This study aims to more clearly define the molecular pathogenesis of HGSC. MethodsSix cases of HGSC were identified at the Northern Ireland Gynaecological Cancer Centre (NIGCC) that each contained ovarian HGSC (HGSC), omental HGSC (OMT), STIC, normal fallopian tube epithelium (FTE) and normal ovarian surface epithelium (OSE). The relevant formalin-fixed paraffin embedded (FFPE) tissue samples were retrieved from the pathology archive via the Northern Ireland Biobank following attaining ethical approval (NIB11:005).Full microarray-based gene expression profiling was performed on the cohort. The resulting data was analysed bioinformatically and the results were validated in a HGSC-specific in-vitro model. ResultsThe carcinogenesis of HGSC was investigated and showed the molecular profile of HGSC to be more closely related to normal FTE than OSE. STIC lesions also clustered closely with HGSC, indicating a common molecular origin. ConclusionThis study provides strong evidence suggesting that extrauterine HGSC arises from the fimbria of the distal fallopian tube. Furthermore, several potential pathways were identified which could be targeted by novel therapies for HGSC. These findings have significant translational relevance for both primary prevention and clinical management of the disease.

Abstract 1774: Targeting aurora A kinase (AAK) in platinum-resistant high grade serous ovarian cancer (HGSOC)

Abstract Resistance to platinum-based chemotherapy is a major problem in the clinical management of recurrent HGSOC. While the use of poly ADP-ribose polymerase inhibitors (PARPi) have made an impact in BRCA1/2 mutated and homologous recombinant-deficient HGSOC, resistance and limited response-rates to PARPi persist. Alisertib (AL) an inhibitor of AAK can inhibit entry and progression of cells through mitosis as well as the interaction with N-myc and its degradation (Front. Oncol. doi:10.3389/fonc.2015.00189). In models of HGSOC from patients with recurrent platinum-resistant disease, cisplatin (CP) treatment does not lead to cell cycle traverse perturbations and significant accumulation of cells in G2-M. Since a defect in the G2-M DNA damage checkpoint is a potential mechanism of resistance to cisplatin, we sought to test the hypothesis that inhibition of AAK that is involved in centrosome maturation and entry into mitosis would enhance the anti-tumor activity of cisplatin in platinum-resistant HGSOC. To test this hypothesis, we treated HGSOC cell lines from patients with recurrent platinum-refractory disease with increasing doses of CP (1- 2.5 μM), of AL (0.05 μM) or the combination and evaluated effects on cell proliferation, apoptosis and generation of reactive oxygen species (ROS). The sequential treatment of four different HGSOC cell lines with CP for 3 hours followed by AL for 48 hours and recovery in drug-free medium for 96-120 hours led to a synergistic increase in apoptosis (p&amp;lt;0.01) and a synergistic decrease in cell survival (p&amp;lt;0.001) compared to treatment with either drug alone. The enhanced apoptosis and decreased cell survival with (CP→AL) treatment was accompanied by a significant increase (p&amp;lt;0.05) in ROS. In contrast, sequential treatment with AL followed by CP (AL→CP) led to an antagonistic response. The synergistic effect of (CP→AL) was accompanied by a modest increase in mRNA and protein levels of AAK. However, protein levels of N-myc protein, which interacts with AAK and is degraded, were not altered. Interestingly, (CP→AL) treatment did not elicit the synergistic cytotoxic response or increased ROS in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. In platinum-refractory HGSOC cells that are wild-type for BRCA1/2 and resistant to olaparib, treatment with the combination of 0.01 μM AL and 1 μM olaparib for 48 hours resulted in an additive cytotoxic response. Based on the mutation landscape of the HGSOC cell panel evaluated, the synergistic cytotoxicity with the CP→AL treatment was independent of BRCA1/2 or TP53 mutation status. In summary, CP resistance in HGSOC due to a defective G2-M DNA damage response, can be potentially circumvented by sequential treatment with CP followed by AL. Citation Format: Ram N. Ganapathi, Eric J. Norris, Ashley P. Sutker, Hala Soliman, Mahrukh K. Ganapathi. Targeting aurora A kinase (AAK) in platinum-resistant high grade serous ovarian cancer (HGSOC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1774.

Common and specific genes in ovarian clear cell carcinoma and serous carcinoma by gene expression analysis

Background: Gene expression profiles differences of clear cell carcinoma (CCC) and serous carcinoma (SC) have been documented, however, these studies usually miss the specific genes changed only in SC or CCC. This study analyzes gene expression profiles of the two histological types and normal ovarian surface epithelium to determine the common and specific genes in these tumors.

Overexpression of the recently identified oncogene REDD1 correlates with tumor progression and is an independent unfavorable prognostic factor for ovarian carcinoma

BackgroundRegulated in development and DNA damage response (REDD1), a gene responding to hypoxia or multiple DNA damage events, was recently implicated in cancer development and progression. Previously, in vivo and in vitro experiments indicated that REDD1 functions as an oncogene in ovarian cancer cells. However, the role of REDD1 in cancer cell migration and invasion and in clinical significance of prognostic values is not examined in detail.MethodsWe detected the REDD1 protein expression by immunohistochemistry in 18 normal ovarian surface epithelium or fallopian tube epithelium specimens, 24 ovarian borderline tumors, and 229 ovarian cancers. Fisher’s exact test, logistic regression analysis, the Kaplan–Meier method, and the log-rank test were used to evaluate the association of REDD1 with clinical factors, overall survival and disease-free survival. The prognostic predictive value of REDD1 for ovarian cancer patients was evaluated using multivariate Cox proportional hazard regression models. REDD1 expression in HEY, HEY A8, SKOV3, SKOV3 ip1, OVCA429, OVCA433 and A2780 human ovarian epithelial cancer cell lines was detected by western blotting. The role of REDD1 in cell invasion and migration was assessed by transwell migration and invasion assays using SKOV3, A2780, HEY, HEYA8, and SKOV3-REDD1 with parental A2780-REDD1 HEY-REDD1i and HEY A8-REDD1i.ResultsHigh expression of REDD1 was observed in 35.4% of primary ovarian carcinoma samples. Overexpression of cytoplasmic REDD1 in ovarian cancer was significantly associated with serous carcinoma (P < 0.001), late-stage disease (P < 0.001), ascites (P < 0.001), and partial or non-response to chemotherapy (P < 0.001). High cytoplasmic expression of REDD1 was correlated with poorer overall survival (P < 0.001) and disease-free survival (P < 0.001). The multivariate Cox proportional hazards regression analysis indicated that patients with high cytoplasmic REDD1 expression had a high risk of death (P < 0.001) and high risk of an event (i.e., recurrence, progression, or death) (P < 0.001). REDD1 was first reported as an independent prognostic factor in ovarian cancer patients. In addition, REDD1 overexpression enhanced ovarian cancer cell migration and invasion.ConclusionREDD1 is an independent unfavorable prognostic factor in ovarian carcinoma and may promote ovarian cancer metastasis.

Alteration of BRCA-1 tumor suppressor gene expression in serous and mucinous ovarian neoplasms in the benign-borderline-malignant pathway

Alteration of expression of the tumor suppressor gene BRCA-1 has been widely studied in breast and ovarian carcinoma. However, pattern of this alteration in the benign-borderline-carcinoma sequence in serous and mucinous ovarian neoplasms have not yet fully described. Tissue sections from 214 formalin-fixed paraffin-embedded ovarian specimens were stained immunohistochemically with BRCA-1 antibody. Specimens were 10 normal ovarian surface epithelium, 10 fallopian tube epithelium, 70 benign adenoma (50 serous and 20 mucinous), 28 borderline (13 serous and 15 mucinous), 78 carcinoma (58 serous and 20 mucinous), and 18 metastatic deposit (13 serous and 5 mucinous). Expression was evaluated into 0, +1, +2, and +3. Score +3 staining similar to normal tissues was considered normal and other scores were considered altered expression. Strong expression was seen in all normal epithelium specimens. Altered expression was seen in 34 serous neoplasms; 17 of 50 (34%) of benign cystadenomas, 6 of 13 (46%) of borderline tumors, 43 of 58 (74%) of primary carcinoma, and in 8 of 13 (62%) of metastatic carcinoma. This alteration was significantly associated with higher histopathologic grade (P = 0.049), presence of necrosis (P = 0.0001), and higher proliferation rate (P = 0.001). In mucinous neoplasms; altered BRCA-1 was detected in 25 specimens; 7 of 20 (41%) of benign cystadenomas, 5 of 15 (33%) of borderline neoplasms, 9 of 20 (45%) of primary carcinoma, and 4 of 5 (80%) of the metastatic deposits. This alteration was not associated with any of the clinicopathologic tumor characteristics. In conclusion, alteration of BRCA-1 expression is more frequent in serous than in mucinous carcinomas and is associated with tumors of higher grades and high proliferation rate.

Integrated transcriptomic and epigenomic analysis of ovarian cancer reveals epigenetically silenced GULP1

Many epigenetically inactivated genes involved in ovarian cancer (OC) development and progression remain to be identified. In this study we undertook an integrated approach that consisted of identification of genome-wide expression patterns of primary OC samples and normal ovarian surface epithelium along with a pharmacologic unmasking strategy using 3 OC and 3 immortalized normal ovarian epithelial cell lines. Our filtering scheme identified 43 OC specific methylated genes and among the 5 top candidates (GULP1, CLIP4, BAMBI, NT5E, TGFβ2), we performed extended studies of GULP1. In a training set, we identified GULP1 methylation in 21/61 (34%) of cases with 100% specificity. In an independent cohort, the observed methylation was 40% (146/365) in OC, 12.5% (2/16) in borderline tumors, 11% (2/18) in cystadenoma and 0% (0/13) in normal ovarian epithelium samples. GULP1 methylation was associated with clinicopathological parameters such as stage III/IV (p = 0.001), poorly differentiated grade (p = 0.033), residual disease (p < 0.0003), worse overall (p = 0.02) and disease specific survival (p = 0.01). Depletion of GULP1 in OC cells led to increased pro-survival signaling, inducing survival and colony formation, whereas reconstitution of GULP1 negated these effects, suggesting that GULP1 is required for maintaining cellular growth control.

CTD-2020K17.1, a Novel Long Non-Coding RNA, Promotes Migration, Invasion, and Proliferation of Serous Ovarian Cancer Cells In Vitro.

BackgroundOvarian cancer is the most lethal malignant tumor of the female reproductive system, and the metastasis is one of the major factors that contribute to the poor outcome of patients with OC. Accumulating evidence indicates that lncRNAs are expressed and play important regulatory roles in ovarian cancer.Material/MethodsAberrant lncRNAs in primary ovarian cancer tissues (POCTs) and paired omental metastasis tissues (OMTs) of patients with HGSOC were studied via lncRNA microarray. Real-time PCR was performed to examine CTD-2020K17.1 expression in HGSOC tissues from 38 patients, a normal ovarian surface epithelium cell line, and 4 ovarian cancer cell lines. Additionally, Transwell assays, wound healing assays, CCK-8 proliferation assays, and flow cytometry were used to explore the biological function of CTD-2020K17.1 in ovarian cancer cells. Finally, Western blot analysis was used to verify the potential target gene of CTD-2020K17.1.ResultsA novel lncRNA named CTD-2020K17.1 was identified via microarray analysis. Expression of CTD-2020K17.1 was significantly increased in OMTs and in 4 ovarian cancer cell lines compared with POCTs (P<0.05) or normal ovarian surface epithelial cell line (P<0.05). Moreover, CTD-2020K17.1 overexpression promoted migration, invasion, and proliferation of ovarian cancer cells, and CTD-2020K17.1 regulated the expression of CARD11.ConclusionsCTD-2020K17.1 is significantly upregulated in OMTs and ovarian cancer cell lines. It can promote the migration, invasion, and proliferation of ovarian cancer cells, and CARD11 is regulated by CTD-2020K17.1.

Increased RHAMM expression relates to ovarian cancer progression

BackgroundElevated hyaluronan-mediated motility receptor (RHAMM) has been reported to contribute to disease progression, aggressive phenotype and poor prognosis in multiple cancer types, however, RHAMM’s role in ovarian cancer (OC) has not been elucidated. Therefore, we sought to evaluate the role for RHAMM in epithelial OC.ResultsDespite little to no expression in normal ovarian surface epithelium, western immunoblotting, immunohistochemical staining and enzyme linked immunosorbent assay showed elevated RHAMM levels in clinical tissue sections, omental metastasis and urine specimens of serous OC patients, as well as in cell lysates. We also found that RHAMM levels increase with increasing grade and stage in serous OC tissues and that RHAMM localizes to the apical cell surface and inclusion cysts. Apical localization of RHAMM suggested protein secretion which was validated by detection of significantly elevated urinary RHAMM levels (p < 0.0001) in OC patients (116.66 pg/mL) compared with normal controls (8.16 pg/mL). Likewise, urinary RHAMM levels decreased following cytoreductive surgery in OC patients suggesting the source of urinary RHAMM from tumor tissue. Lastly, we validated RHAMM levels in OC cell lysate and found at least 12× greater levels compared to normal ovarian surface epithelial cells.ConclusionThis pilot study shows, for the first time, that RHAMM may contribute to OC disease and could potentially be used as a prognostic marker.

Abstract 1991: Enhanced role of the extracellular matrix in ovarian cancer recurrence

Abstract Purpose: Despite improvements in care and advancements in the understanding of ovarian cancer (OC) pathobiology and genetic alterations, the survival rate is disappointingly low when compared to that of other types of cancer. One factor contributing to the poor survival rate is the development of chemotherapeutic resistance and a high rate of recurrent tumor growth in the months and years following primary OC treatment. Our objective was to identify potential new therapeutic targets for prevention of recurrence through examination of changes in gene regulation that occur in recurrent versus matched primary tumors. Procedures: We used 16 primary-recurrent tumor pairs from patients with stage III/IV serous epithelial OC from the Duke Gynecologic Oncology Tissue Bank. Illumina Infinium HumanMethylation450 BeadChips and Affymetrix Human Genome U133A arrays were used to quantify methylation and gene expression, respectively. Expression and methylation of POSTN and COL11A1 were analyzed using an independent OC dataset comprising 38 women with &amp;lt;3 years survival and 26 women with &amp;gt;7 years survival, 3 normal ovarian surface epithelium (OSE) and 2 fallopian tube fimbriae epithelium (FTFE) samples. siRNA knockdown of POSTN and COL11A1 in OC cells was followed by expression and proliferation assays. Results: Paired student t tests showed a &amp;gt;2-fold difference for 13 of the 1,569 significantly differentially expressed genes, seven with increased expression in recurrent OC (POSTN, COL11A1, MMP1, MMP13, TNC, ASPN, and EPYC), and six with decreased expression (GATA6, PEG3, SST, MAOB, TSPAN8, and C7). Interestingly, all seven up-regulated genes are involved in extracellular matrix (ECM) functions, an important component of the tumor microenvironment. With an independent OC microarray dataset, we found elevated COL11A1 mRNA in OCs as compared to OSE and FTFE tissues (p&amp;lt;0.05). There are inverse correlations between COL11A1 expression and methylation at five promoter CG sites (R=-0.58 to -0.65, p&amp;lt;0.05). Immunohistochemistry showed higher expression of POSTN and COL11A1 in recurrent OC. Knockdown of POSTN and COL11A1 in OC cell lines led to significantly reduced proliferation (p&amp;lt;0.05). Conclusion: Our findings indicate that the genes involved in extracellular matrix function that contribute to the composition and regulation of the tumor microenvironment play critical roles in OC recurrence, and may offer new therapeutic opportunities. Citation Format: Zhiqing Huang, Brad Foster, Carole Grenier, Gregory Sfakianos, Regina Whitaker, Andrew Berchuck, Susan K. Murphy. Enhanced role of the extracellular matrix in ovarian cancer recurrence [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 1991. doi:10.1158/1538-7445.AM2017-1991

Characterization of MicroRNA-200 pathway in ovarian cancer and serous intraepithelial carcinoma of fallopian tube

BackgroundOvarian cancer is the leading cause of death among gynecologic diseases in Western countries. We have previously identified a miR-200-E-cadherin axis that plays an important role in ovarian inclusion cyst formation and tumor invasion. The purpose of this study was to determine if the miR-200 pathway is involved in the early stages of ovarian cancer pathogenesis by studying the expression levels of the pathway components in a panel of clinical ovarian tissues, and fallopian tube tissues harboring serous tubal intraepithelial carcinomas (STICs), a suggested precursor lesion for high-grade serous tumors.MethodsRNA prepared from ovarian and fallopian tube epithelial and stromal fibroblasts was subjected to quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) to determine the expression of miR-200 families, target and effector genes and analyzed for clinical association. The effects of exogenous miR-200 on marker expression in normal cells were determined by qRT-PCR and fluorescence imaging after transfection of miR-200 precursors.ResultsOvarian epithelial tumor cells showed concurrent up-regulation of miR-200, down-regulation of the four target genes (ZEB1, ZEB2, TGFβ1 and TGFβ2), and up-regulation of effector genes that were negatively regulated by the target genes. STIC tumor cells showed a similar trend of expression patterns, although the effects did not reach significance because of small sample sizes. Transfection of synthetic miR-200 precursors into normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cells confirmed reduced expression of the target genes and elevated levels of the effector genes CDH1, CRB3 and EpCAM in both normal OSE and FTE cells. However, only FTE cells had a specific induction of CA125 after miR-200 precursor transfection.ConclusionsThe activation of the miR-200 pathway may be an early event that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian carcinomas (HGSOC) usually express high levels of CA125, the induction of CA125 expression in FTE cells by miR-200 precursor transfection is consistent with the notion that HGSOC has an origin in the distal fallopian tube.

ElncRNA1, a long non-coding RNA that is transcriptionally induced by oestrogen, promotes epithelial ovarian cancer cell proliferation.

Previously, the novel oestrogen (E2)-upregulated lncRNA TC0101441, was identified by us, via microarray analysis. However, the detailed mechanism by which E2 upregulates TC0101441 and the role of TC0101441 in epithelial ovarian cancer (EOC) progression have not been elucidated. In the present study, we further analysed TC0101441, which we designated oestrogen-induced long non-coding RNA-1 (ElncRNA1). We showed that E2 transcriptionally upregulates ElncRNA1 through the oestrogen receptor α (ERα)-oestrogen response element (ERE) pathway using RNA stability assays, bioinformatics-based searches for ERE binding sites, chromatin immunoprecipitation (ChIP) assays and dual luciferase reporter assays. Clinically, ElncRNA1 levels are significantly higher in EOC tissues than in normal ovarian surface epithelium. Invitro and invivo loss-of-function assays revealed that ElncRNA1 promotes EOC cell proliferation. This pro-proliferation effect of ElncRNA1 was partially mediated by the regulation of CDK4, CDK6 and cyclinD1. These findings provide the first evidence that E2 upregulates ElncRNA1 at the transcriptional level through the ERα-ERE pathway and that this novel E2-upregulated lncRNA has an oncogenic role in EOC growth. The placement of ElncRNA1 in the E2-ERα-ERE signalling pathway may provide greater insight into the effects of oestrogen on EOC progression from the perspective of lncRNA.