Ovarian Cancer Tumorigenesis Research Articles

MALDI-TOF-MS analysis in discovery and identification of serum proteomic patterns of ovarian cancer

BackgroundDue to high mortality and lack of efficient screening, new tools for ovarian cancer (OC) diagnosis are urgently needed. To broaden the knowledge on the pathological processes that occur during ovarian cancer tumorigenesis, protein-peptide profiling was proposed.MethodsSerum proteomic patterns in samples from OC patients were obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Eighty nine serum samples (44 ovarian cancer and 45 healthy controls) were pretreated using solid-phase extraction method. Next, a classification model with the most discriminative factors was identified using chemometric algorithms. Finally, the results were verified by external validation on an independent test set of samples.ResultsMain outcome of this study was an identification of potential OC biomarkers by applying liquid chromatography coupled with tandem mass spectrometry. Application of this novel strategy enabled the identification of four potential OC serum biomarkers (complement C3, kininogen-1, inter-alpha-trypsin inhibitor heavy chain H4, and transthyretin). The role of these proteins was discussed in relation to OC pathomechanism.ConclusionsThe study results may contribute to the development of clinically useful multi-component diagnostic tools in OC. In addition, identifying a novel panel of discriminative proteins could provide a new insight into complex signaling and functional networks associated with this multifactorial disease.

Abstract LB-276: Characterizing tumorigenesis of ovarian cancer across metastatic tumors and circulating, cell-free DNA

Abstract Tumor heterogeneity creates significant challenges for the generation and monitoring of treatment strategies. Circulating, cell-free DNA (cfDNA) can facilitate these processes by providing a noninvasive method to detect and track mutations without the need for multiple biopsies. To study both tumor heterogeneity and its relationship to cfDNA, we used samples taken from multiple positions on two metastatic tumors from a stage 3B ovarian carcinosarcoma patient as well as a cfDNA sample that was collected at the time of surgery. To maximize low frequency variant detection from next-generation sequencing (NGS) data, molecular identifiers (MIDs) were employed, allowing accurate distinction of true variants from PCR-induced errors along with sequencing artifacts. The use of MIDs to uniquely label input DNA generates tagged library molecules for the detection and removal of PCR duplicates while simultaneously preserving fragmentation and strand duplicates. By over-sequencing the MID tagged library, sequencing reads from PCR duplicates can be grouped based on their shared MID tag, generating a consensus sequence to identify and subsequently remove errors. To validate this technology, we performed low frequency spike-in experiments to 0.5% and 1% by combining Coriell NA12878 and HG005 genomic DNA as well as different cfDNA samples. Libraries were sequenced to 8000x coverage and a consensus sequence was generated with BMFtools (ARUP labs). All known variants present at 1% and 0.5% allele frequencies were maintained in the resulting data set. Further, true variants were preserved while sequencing and PCR-induced errors were removed, demonstrating improved sensitivity but also improved specificity using MIDs. After validation of the technology, libraries with MIDs were prepared from the ovarian carcinosarcoma FFPE and cfDNA samples. Libraries were sequenced on an Illumina® HiSeq® to a minimum of 13,000x coverage, and we determined data retention after de-duplication with and without the use of MIDs. We observed an increase in data retention for both Covaris-sheared FFPE and cfDNA libraries that led to a 2 to 3-fold increase in coverage using MIDs. Variant calling depicted significant inter-tumor heterogeneity in these samples as well as low frequency variants that represent intra-tumor heterogeneity. Specifically, we identified a pathogenic TP53 mutation present in one tumor sample and the cfDNA but absent from the second tumor. Additionally, we observed a significant loss of heterozygosity for only one of the tumors. We also identified low frequency, pathogenic mutations (as low as 1%) that are either unique to one tumor or unique to one sample from a tumor. This study highlights the use of MID technology to enable low frequency variant calling during disease diagnosis and tracking to facilitate precise, personalized treatment of complex cancers. Citation Format: Ashley Wood, Sukhinder Sandhu, Matthew Dashkoff, Olga Camacho-Vanegas, Laurie Kurihara, Timothy Harkins, John Martignetti, Vladimir Makarov, Peter Dottino. Characterizing tumorigenesis of ovarian cancer across metastatic tumors and circulating, cell-free DNA [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 LB-276. doi:10.1158/1538-7445.AM2017-LB-276

High expressions of BCL6 and Lewis y antigen are correlated with high tumor burden and poor prognosis in epithelial ovarian cancer.

Aberrant regulation of BCL6 plays crucial oncogenic roles in various malignant tumors; howbeit, the function of BCL6 in tumorigenesis of ovarian cancer remains unclear. The aim of this study is to investigate the role of BCL6 in ovarian cancer. The methods of immunohistochemical staining, quantitative real-time polymerase chain reaction, immunocytochemical staining, and gene expression profile enrichment analysis were performed to identify the possible role of BCL6 in ovarian cancer. We observed that the expression of BCL6 was significantly higher in ovarian cancer tissues and correlated with higher tumor burden including advanced International Federation of Gynecology and Obstetrics stages, poor differentiation, Type II ovarian cancer, the presence of >1 cm residual tumor size, and appearance of recurrence or death (all p < 0.05). The expression patterns of Lewis y were similar to these of BCL6. Multivariate Cox analysis demonstrated that advanced International Federation of Gynecology and Obstetrics stage, lymph node metastasis, residual tumor size >1 cm, as well as high expressions of BCL6 and Lewis y antigen were independent factors of worse progression-free survival and overall survival (all p < 0.05). There was a positive correlation of the expressions of BCL6 and Lewis y antigen. The associated genes with BCL6 in response to Lewis y antigen were identified, including four upregulated genes ( SOCS3, STAT1, PPARG, and GADD45A) and three downregulated genes ( ACAN, E2F3, and ZBTB7B). In conclusion, the high expressions of BCL6 and Lewis y antigen are associated with development, high tumor burden, and worse prognosis of ovarian cancer and targeting BCL6 could be a novel therapeutic strategy for ovarian cancer treatment.

Abstract 1988: Exosome-mediated ovarian cancer tumorigenesis mediated by miR1246/Rb/Cav1 axis

Abstract Exosomes are secreted from many cell types and play an important role in the tumor microenvironment. The most impressive breakthrough in exosomes research was that they contain the genetic material of the host cell. However, whether cancer cells use their exosomes to transfer their oncogenic material to recipient cells, or to get rid of their tumor suppressor material is not well understood. We previously identified that miR-1246 was hundreds of folds higher expressed in six different ovarian cancer exosomes compared to their originating cells. Here, we showed that miR-1246 co-localized in the exosomes in ovarian cancer cells. miR-1246 act as an oncogenic miRNA and the levels were elevated in ovarian cancer patients compared to health donors. We also demonstrated that miR-1246 inhibitor treatment in combination with paclitaxel was significantly inhibited tumor burden in SKOV3-ip1 orthotopic ovarian cancer model. Our results suggest that miR-1246 inhibited RB tumor suppressor protein and regulate Cav-1 and platelet-derived growth factor receptor beta precursor signaling in ovarian cancer. In addition Inhibiting miR-1246 led a significant decrease in exosome release. Together, our findings provide strong evidence that oncogenic miR-1246 can be targeted as a potential novel therapeutic approach in the treatment of ovarian cancer. . Note: This abstract was not presented at the meeting. Citation Format: Pinar Kanlikilicer, Recep Bayraktar, Mohammed Rashed, Burcu Aslan, George A. Calin, Anil K. Sood, Gabriel Lopez-Berestein. Exosome-mediated ovarian cancer tumorigenesis mediated by miR1246/Rb/Cav1 axis [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 1988. doi:10.1158/1538-7445.AM2017-1988

Microarray expression profiling of long non-coding RNAs in epithelial ovarian cancer.

Although numerous long non-coding RNAs (lncRNAs) have been identified to be important in human cancer, their potential regulatory roles in epithelial tumorigenesis and tumor progression in ovarian cancer remain unclear. The purpose of the present study was to investigate lncRNAs that were differentially expressed (DE) in epithelial ovarian cancer and to explore their potential functions. The lncRNA profiles in five pairs of human epithelial ovarian cancer tissues and their adjacent normal tissues were described using microarrays. The results of the microarray analysis revealed that 672 upregulated and 549 downregulated (fold-change ≥2.0) lncRNAs were DE between the cancerous and normal tissues. Reverse transcription-quantitative polymerase chain reaction was used to validate the microarray results using four upregulated (RP11-1C1.7, XLOC_003286, growth arrest-specific 5 and ZNF295-AS1) and four downregulated (protein tyrosine kinase 7, maternally expressed gene 3, AC079776.2 and ribosomal protein lateral stalk subunit P0 pseudogene 2) lncRNAs. Furthermore, gene ontology and pathway analyses were used to carry out functional analyses of the candidate genes of DE lncRNAs. The results identified lncRNAs with significantly altered expression profiles in human epithelial ovarian cancer cells compared with those in adjacent normal cells. These data offer new insights into the occurrence and development of epithelial ovarian cancer, and these lncRNAs may provide novel molecular biomarkers for further research on epithelial ovarian cancer.

Increased expression of LncRNA BANCR and its prognostic significance in human epithelial ovarian cancer.

Long non-coding RNAs (lncRNAs) have been proved to play important roles in the tumorigenesis and development of human epithelial ovarian cancer (EOC). The aim of the present study was to investigate the expression and clinical value of BRAF-activated non-coding RINA (BANCR) in EOC patients. BANCR expression was detected in 84 EOC and 36 normal ovarian epithelial tissue samples. Association between BANCR levels and clinicopathological factors and patient prognosis was also analyzed. BANCR expression was increased in EOC compared with normal ovarian epithelial tissues. Moreover, high expression of BANCR was closely correlated with advanced FIGO stage, higher serum, CAI125 expression level, and lymph node metastasis. Multivariate regression analysis identified BANCR overexpression as an independent unfavorable prognostic factor in EOC patients. These findings suggested that BANCR may act as a tumor promoter in EOC and would be a novel diagnostic and prognostic marker for this disease.

Histone demethylase KDM2B upregulates histone methyltransferase EZH2 expression and contributes to the progression of ovarian cancer in vitro and in vivo.

Aberrant histone methylation contributes to the progression and development of many tumors. Histone methylation is a dynamic process regulated by both histone demethylase and histone methyltransferase, which ultimately alters the levels of gene transcription. However, the relationship between histone demethylase and histone methyltransferase, as well as their regulatory mechanisms in ovarian cancer development, is still unclear. Lysine-specific demethylase 2B (KDM2B) is a key demethylase of H3K36me3 and H3K4me3 that regulates gene expression and plays a role in tumorigenesis via epigenetic mechanisms. To determine the expression pattern of KDM2B in ovarian neoplasms, we analyzed the mRNA and protein levels of KDM2B and the histone methyltransferase enhancer of zester homolog 2 (EZH2) in normal, benign, borderline, and malignant ovarian tissue samples. We found that KDM2B expression was gradually increased in ovarian tumors, with the highest expression found in the malignant ovarian tissues, and the differences in KDM2B expression among the different International Federation of Gynecology and Obstetrics stages and pathological grades/types were statistically significant. Moreover, KDM2B expression was positively correlated with EZH2 expression in ovarian tissues. To determine the role of KDM2B in tumorigenesis in vitro and in vivo, we silenced KDM2B expression in ovarian cancer cells using the KDM2B short hairpin RNA expression lentivirus and established a nude mouse xenograft model. Downregulation of endogenous KDM2B decreased the expression of EZH2 and reduced the proliferation and migration of ovarian cancer cells. Loss of KDM2B suppressed ovarian tumor formation in vivo. Our results suggest that KDM2B plays an important role in the tumorigenesis of ovarian cancer, with a possible mechanism of increasing the expression of the oncogene EZH2; this indicates that certain histone methyltransferase may be positively regulated by certain histone demethylase in the epigenetic regulation of ovarian tumors. KDM2B may be a novel therapeutic target for the clinical treatment of ovarian cancer.

Abstract TMEM-030: INTERLEUKIN–6 (IL6) AND LEUKEMIA INHIBITORY FACTOR (LIF) ARE FUNCTIONALLY REDUNDANT IN PROMOTING OVARIAN CANCER GROWTH AND REQUIRE DUAL BLOCKADE FOR THERAPEUTIC EFFECT

Abstract PURPOSE: We have previously isolated mesenchymal stem cells from the microenvironment of tumors from ovarian cancer patients (cancer-associated MSC, CA-MSC). We found that CA-MSC promote tumorigenesis in vitro and in vivo by increasing cancer stem cells (CSC) in the epithelial tumor cells. Microarray analysis demonstrates the upregulation of the cytokines IL6 and LIF in CA-MSC versus control MSC. The purpose of this work is to characterize the role of cytokines interleukin-6 (IL6) and leukemia inhibitory factor (LIF) secreted by CA-MSC in ovarian cancer tumorigenesis. EXPERIMENTAL PROCEDURES: We have isolated and established cultures of CA-MSC. Healthy donor adipose-derived MSC were used as controls (Invitrogen). Ovarian cancer cells were treated with MSC-conditioned media or recombinant IL6 and/or LIF (EBioscience). Cytokine function was inhibited with IL6 blocking antibody, LIF blocking antibody, both antibodies or the JAK2 inhibitor ruxolitinib for dual blockade. Phosphorylated STAT3 and total STAT3 immunoblotting was performed by standard methods. Sphere assays were performed using low-adherence plates and X-Vivo20 serum free media (Lonza). Flow cytometry was carried out using the Aldefluor assay for aldehyde dehydrogenase activity (Stem Cell Technologies). For in vivo studies, NSG mice were injected with OVCAR3 ovarian cancer cells without or with CA-MSC, and then either mock treated or treated with IL6 blocking antibody (tocilizumab, Genentech), LIF blocking antibody or ruxolitinib. Tumor size was monitored both during treatment and at sacrifice, and tumors were analyzed. RESULTS: Treatment of ovarian cancer cells with either recombinant IL6 or LIF alone leads to STAT3 phosphorylation, suggesting functional redundancy in stimulation of this pathway. Neither LIF nor IL6 blocking antibodies alone can prevent upregulation of phospho-STAT3, but combined therapy with both LIF and IL6 blocking antibodies prevents STAT3 phosphorylation. The JAK2 inhibitor ruxolitinib inhibits cytokine-mediated upregulation of phospho-STAT3. Either LIF or IL6 treatment results in an increase the percentage of cancer stem cells in ovarian cancer cell lines, as measured using both sphere assays and aldehyde dehydrogenase activity. Ruxolitinib blocks both LIF and IL6 mediated increase in cancer stem cells. In vivo, the pro-tumorigenic effect of CA-MSC is abrogated by dual blockade with ruxolitinib but not by treatment with anti-IL6 antibody alone or anti-LIF antibody alone. Ruxolitinib treated tumors demonstrate decreased phospho-STAT3, indicating on-target activity. CONCLUSIONS: IL6 and LIF are secreted by CA-MSC in the ovarian cancer tumor microenvironment. Both cytokines upregulate phospho-STAT3 and increase the percentage of cancer stem cells in epithelial tumor cells. Functional inhibition of these effects cannot be achieved in vitro or in vivo with blockade of either cytokine individually; dual blockade with compounds such as the JAK2 inhibitor ruxolitinib is necessary. Thus the shared downstream signaling molecules of the JAK/STAT pathway may be preferred targets for novel anticancer therapeutics for ovarian cancer. Citation Format: Karen McLean, Lijun Tan and Ronald J. Buckanovich. INTERLEUKIN–6 (IL6) AND LEUKEMIA INHIBITORY FACTOR (LIF) ARE FUNCTIONALLY REDUNDANT IN PROMOTING OVARIAN CANCER GROWTH AND REQUIRE DUAL BLOCKADE FOR THERAPEUTIC EFFECT [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 TMEM-030.

Abstract NTOC-083: TARGETED APPROACH WITH MOMELOTINIB TREATMENT ATTENUATES PACLITAXEL–INDUCED ENRICHMENT OF CANCER STEM CELLS (CSC)–LIKE AND SUPPRESSES OVARIAN CANCER TUMORIGENESIS AND RECURRENCE

Abstract INTRODUCTION: In the era of personalised medicine, chemotherapy treatment alone is inadequate to treat advanced-stage ovarian cancer patients, given that a steady 75% of patients relapse within five years and die of this disease. We have shown that chemotherapy-treated ovarian cancer cell lines express significantly elevated JAK2/STAT3 activation and CSC-like compared to untreated cells in vitro and in vivo mouse models. We aim to investigate the in vivo significance of targeting paclitaxel-induced ovarian CSC-like and the associated JAK2/STAT3 activation using the JAK2-specific inhibitor, Momelotinib. METHODS: Female Balbc nu/nu mice were injected ip with HEY cells and treated at day 19 with paclitaxel (15mg/kg bodyweight) weekly by ip injection and/or Momelotinib (25mg/kg bodyweight) daily by oral gavage. Study 1: All treatment groups (n=3/group) were sacrificed at the same time as the untreated control group (n=4). Study 2: All treatments were terminated after the endpoint of untreated group groups and were sacrificed at their respective endpoints (n=4). Study 3: Paclitaxel+Momelotinib-treated group was further divided into two groups (n=9/group) after the endpoint of the untreated group: (i) termination of paclitaxel and Momelotinib treatments, and (ii) termination of paclitaxel treatment alone for 13 weeks. One-way ANOVA was used to compare the tumour burden (tumour wt/mice wt), survival period, and immunohistochemistry analysis of the tumour xenograft for the activated JAK2/STAT3 and CSC-like markers (Oct3/4 and c-Kit) expressions between multiple groups. Statistical significance was defined as a p value &amp;lt; 0.05. RESULTS: In Study 1, paclitaxel+Momelotinib-treated group produced the smallest tumour burden compared to all groups, and had tumours that showed a reduction in the activated JAK2/STAT3 pathway and CSC-like markers expressions when compared to the paclitaxel-treated group. In Study 2, paclitaxel+Momelotinib-treated group survived the longest compared to all groups. In Study 3, paclitaxel+(ongoing)Momelotinib-treated group survived substantially longer and produced a significantly smaller tumour burden compared to the untreated and paclitaxel+(terminated) Momelotinib-treated groups. Expressions of the activated JAK2/STAT3 and CSC-like markers were significantly upregulated in the tumours isolated from paclitaxel+Momelotinib-treated group post-Momelotinib termination compared to pre-Momelotinib termination. CONCLUSION: Targeting the paclitaxel-induced JAK2/STAT3 activation and CSC-like using Momelotinib not only enhanced paclitaxel cytotoxicity but also prolonged the survival of HEY-bearing mice. This strongly supports the idea for future clinical testing using Momelotinib (currently in Phase 3 clinical trials for treating myelofibrosis) in combination with paclitaxel for advanced-stage ovarian cancer patients. These results also support the use of Momelotinib as a maintenance treatment for the ongoing suppression of the activated JAK2/STAT3 pathway in an attempt to prevent a relapse in ovarian cancer patients. Citation Format: Chan. E, Findlay. JK, Luwor. R, Hickey. M, Ahmed. N. TARGETED APPROACH WITH MOMELOTINIB TREATMENT ATTENUATES PACLITAXEL–INDUCED ENRICHMENT OF CANCER STEM CELLS (CSC)–LIKE AND SUPPRESSES OVARIAN CANCER TUMORIGENESIS AND RECURRENCE [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-083.

Abstract MIP-060: EPIGENETIC DYSREGULATION OF A HISTONE MARK REVEALS A METABOLIC VULNERABILITY IN OVARIAN CANCER

Abstract High-grade serous ovarian cancer (HGSOC) is frequently complicated by the inevitable development of therapeutic resistance, and thus remains an incurable disease. Recent insights supporting the fallopian tube epithelium (FTE) as the tissue of origin and serous tubal intraepithelial carcinomas (STICs) as the precursor lesion for the majority of HGSOCs provide a necessary context to study the mechanisms that drive the development and progression of HGSOC. Understanding these key molecular processes is essential to inform the development of next generation therapies. In this study we investigated the role of the ubiquitin ligase RNF20 and histone H2B monoubiquitylation (H2Bub1) in serous tumorigenesis. H2Bub1, catalyzed largely by RNF20, is an epigenetic mark with tumor suppressor properties. Conversely, the loss of RNF20/H2Bub1 has been linked with cancer progression. By analyzing the TCGA data we found that heterozygous loss of RNF20 defines the majority of HGSOC tumors. Our IHC results show that H2Bub1 is lost or downregulated in a large proportion of STICs and invasive HGSOC tumors, implicating RNF20/H2Bub1 loss as an early event in serous ovarian cancer tumorigenesis. Functionally, we demonstrated that shRNA-mediated RNF20 knockdown, with concomitant H2Bub1 downregulation, was sufficient to increase cell migration and clonogenic growth of immortalized tubal epithelial cells as well as cells derived from invasive HGSOC. Subsequent proteomic profiling (RPPA), of RNF20/H2Bub1-depleted cells revealed pronounced changes in the steady-state levels of a number of metabolic and signaling pathways. In particular, two of the top differentially expressed proteins were glutaminase (GLS) and glutamate dehydrogenase (GLUD1/2), key enzymes in the glutamine metabolism pathway. These changes are predicted to result in elevated levels of glutamate that can fuel the TCA cycle, amino acid biogenesis, and/or glutathione production. We hypothesize that loss of RNF20/H2Bub1 rewires glutamine metabolism in cancer cells and leads to an increase in transamination and glutathione production which fuels the progression of HGSOC. In conclusion, our study identifies that loss of H2Bub1 is an early epigenetic event in HGSOC progression, which rewires glutamine metabolism and may represent a unique therapeutic vulnerability. Citation Format: Jagmohan Hood, Marián Novak, Emily MacDuffie, Jenny Lester, Vinita Parkash, Gordon B. Mills, Beth Y. Karlan, Moshe Oren, and Ronny Drapkin. EPIGENETIC DYSREGULATION OF A HISTONE MARK REVEALS A METABOLIC VULNERABILITY IN OVARIAN CANCER [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 MIP-060.

Impact of beta blocker medication on survival outcome of ovarian cancer: A nationwide population-based cohort study.

5578 Background: In experimental studies, adrenergic hormones are involved in tumorigenesis of ovarian cancer and its progression. We investigated the impact of beta adrenergic blocker on survival outcome of ovarian cancer since few studies have investigated its relevance. Methods: Data of Korean National Health Insurance Service was analyzed (n = 866). We analyzed the impact of beta blocker on survival outcome of ovarian cancer according to the duration on medication and age groups of patients. Cox proportional hazards regression was used to analyze hazard ratios (HR) for all-cause mortality with 95% confidence intervals (CI) adjusting for confounding factors. Results: Median years of follow-up was 5.98 and 6.71 for non-users and users, respectively. Among the 866 patients, 206 (23.8%) were users and 660 (76.2%) were non-users. In total, there was no survival difference between the 2 groups. But, when patietns were grouped according to the duration of medication, patients with longer duration of medication (≥1 year) showed better survival outcome (adjusted HR 0.305 [95% CI: 0.187-0.500], P &lt; 0.001). Also, beta blocker use in patients with &gt; 60 years showed better survival compared to younger patients (adjusted HR 0.579 [95% CI: 0.408-0.822], P = 0.002). In patients with &gt; 60 years, medication longer than 720 days was associated with better survival outcome (adjusted HR 0.267 [95% CI: 0.140-0.511], P &lt; 0.001). Both selective and non-selective beta blocker showed identical survival benefit in these settings without difference between each other. Conclusions: Beta blocker medication was associated with favorable survival outcome in ovarian cancer, especially when used in older patients and in long term duration.

MicroRNA-205 promotes cell invasion by repressing TCF21 in human ovarian cancer

BackgroundOvarian cancer is the leading lethal, gynecological malignancy in the United States. No doubt, the continued morbidity and mortality of ovarian cancer reflects a poor understanding of invasive mechanisms. Recent studies reveal that ovarian cancers express aberrant microRNAs (miRNAs or miRs), some of which have oncogenic or tumor suppressor properties. Several studies suggested that miR-205 is involved in tumorigenesis. Presently, we investigate the molecular mechanisms and target of miR-205 in ovarian cancer.MethodsQuantitative real-time polymerase chain reaction and western blot were performed to assess miR-205 and transcription factor 21 (TCF21) expression in ovarian cancer and normal ovary samples. The effect of miR-205 on TCF21 was determined by luciferase reporter assay and western blot. The effect of miR-205 and TCF21 on cell invasion was quantitated using transwell invasion assay.ResultmiR-205 expression was increased in ovarian cancer and it promoted the invasive behavior of ovarian cancer cell lines (OVCAR-5, OVCAR-8 and SKOV-3). miR-205 directly targeted TCF21, which was significantly decreased in ovarian cancer tissue. miR-205 inhibited TCF21 expression and as a consequence blunted the inhibitory effect of TCF21 on cell invasion. Matrix Metalloproteinases (MMPs) play an important role in cancer invasion and metastasis. TCF21 inhibited MMP-2 and MMP-10 and decreased ovarian cancer cell invasion. Co-transfection of TCF21 expression plasmid with miR-205 mimic diminished the inhibitory effect of TCF21 on MMP-2 and MMP-10 in ovarian cancer cells.ConclusionmiR-205 appears to have an important role in the spread of ovarian cancer by targeting TCF21. These findings offer a new mechanism of ovarian cancer tumorigenesis, which could be useful for the development of new therapeutic approaches to ovarian cancer treatment.

High WAVE3 expression correlates with proliferation, migration and invasion in human ovarian cancer.

BackgroundWiskott-Aldrich syndrome verprolin-homologous (WAVE) 3, a member of the WASP/WAVE family of proteins, plays a critical role in cell motility and acts as an oncogene in some human cancers, but no sufficient information available to illustrate its involvement in ovarian cancer tumorigenesis and progression.MethodsThe expression of WAVE3 in human ovarian cancer and normal tissue was analyzed by immunohistochemistry. WAVE3 gene and protein expression in different human ovarian cancer cell lines was tested by RT-PCR and western blotting. Stable cells of WAVE3-knockdown in SKOV3 cells or transfected high expression in A2780 cells were constructed. The WAVE3 expression and its correlation with MMPs, p38 MAPK and other factors were studied. The relationship between WAVE3 and oncogenicity in vivo was also evaluated by nude mice xenograft model.ResultsImmunohistochemistry staining showed the highest WAVE3 expression in ovarian cancer metastases, high in ovarian cancer and weak in normal. In different cell lines, SKOV3 cells showed the highest WAVE3 expression, A2780 cells expressed the lowest. Elevated WAVE3 expression in A2780 cells promoted proliferation and decreased apoptosis, increased the cell number in G2/M phase and promoted migration significantly. Correspondingly, knockdown of WAVE3 in SKOV3 cells showed opposite effects. The WAVE3 expression showed positive correlation with MMPs, NF-κB, COX-2, VEGF and phospho-p38 MAPK, but not p38. The high expression of WAVE3 promoted tumorigenesis in vivo.ConclusionsOur results suggested that WAVE3 may be pivotal in ovarian cancer cell motility, invasion and oncogenesis, which might be related with MMPs production and p38 MAPK pathway.

Knockdown of Nucleostemin in an ovarian cancer SKOV-3 cell line and its effects on cell malignancy

Nucleostemin plays an essential role in the proliferation of some stem cells and cancer cells, but no research has been conducted to assess the link between Nucleostemin and ovarian cancer. To investigate the role of Nucleostemin in ovarian cancer tumorigenesis, we generated short hairpin (sh)RNA to knockdown the expression of the Nucleostemin gene in an ovarian cancer SKOV-3 cell line. We found that knockdown of this gene led to cell-cycle arrest, as well as to an increase in apoptosis. In addition, migration and invasion demonstrated significant inhibition in Nucleostemin-deficient cells. Furthermore, the knockdown of Nucleostemin dramatically suppressed xenograft progression in BALB/c nude mice. Our findings suggest that Nucleostemin is associated with malignancy in an ovarian cancer SKOV-3 cell line.

Knockdown of astrocyte elevated gene-1 inhibited cell growth and induced apoptosis and suppressed invasion in ovarian cancer cells

Emerging evidence has demonstrated that AEG-1 (astrocyte elevated gene-1) plays a pivotal oncogenic role in tumorigenesis. However, the molecular mechanism by which AEG-1 exerts its oncogenic function is elusive in ovarian cancer. To explore the role and molecular insight on AEG-1-mediated tumorigenesis in ovarian cancer, multiple approaches are performed including MTT assay, flow cytometry for apoptosis and cell cycle assay, gene transfection, real-time RT-PCR, Western blotting, and Transwell assay. Our MTT assay showed that knockdown of AEG-1 by its siRNA significantly inhibited cell growth in ovarian cancer cells. Moreover, AEG-1 siRNA treatment induced G0/G1 cell cycle arrest and triggered cell apoptosis in ovarian cancer cells. Notably, inhibition of AEG-1 suppressed cell migration and invasion in ovarian cancer cells. Intriguingly, we identified that knockdown of AEG-1 remarkably inhibited the activation of Akt pathway. Our results also validated that knockdown of AEG-1 inhibited the expression of MMP-2 and VEGF, which could lead to inhibition of cell migration and invasion. These data suggest that AEG-1 could be a potential therapeutic target for the treatment of ovarian cancer.

Upregulation of the long non-coding RNA SPRY4-IT1 indicates a poor prognosis and promotes tumorigenesis in ovarian cancer.

Long non-coding RNAs (lncRNAs) have been identified to be critical mediators in various tumors associated with cancer progression. LncRNA SPRY4-IT1 serves as a novel prognostic biomarker for hepatocellular carcinoma. However, the biological role and clinical significance of lncRNA SPRY4-IT1 in human ovarian cancer (OC) need to be completely elucidated. The aim of the present study was to explore the lncRNA SPRY4-IT1 expression in human OC patients and its role in OC cells. We show that lncRNA SPRY4-IT1 expression is significantly upregulated in ovarian tumor tissues and OC cell lines in comparison with adjacent non-tumor control tissues and the human ovarian immortalized nontumorigenic ovarian surface epithelial (IOSE), respectively. Further analysis by Kaplan-Meier survival analysis and multivariate analysis indicated that high lncRNA SPRY4-IT1 expression may be an independent prognostic factor for progression-free survival (PFS) and overall survival (OS) in OC patients. Furthermore, the area under the receiver operating characteristic (ROC) curve of lncRNA SPRY4-IT1 was up to 0.8512, indicating lncRNA SPRY4-IT1 has diagnostic values to discriminate tumor tissues from nontumorous tissues. Also, knockdown of lncRNA SPRY4-IT1 inhibited the proliferation of OC cells by CCK-8 assay and clonogenic assay and arrested cell cycle at a G0/G1 stage in OC cells. In conclusion, these results suggest that lncRNA SPRY4-IT1 may be considered as a new predictor in the clinical prognosis of OC patients.

VSV based virotherapy in ovarian cancer: the past, the present and …future?

The standard approach to treating patients with advanced epithelial ovarian cancer (EOC) after primary debulking surgery remains taxane and platinum-based chemotherapy. Despite treatment with this strategy, the vast majority of patients relapse and develop drug-resistant metastatic disease that may be driven by cancer stem cells (CSCs) or cancer initiating cells (CICs). Oncolytic viruses circumvent typical drug-resistance mechanisms, therefore they may provide a safe and effective alternative treatment for chemotherapy-resistant CSCs/CICs. Among oncolytic viruses vesicular stomatitis virus (VSV) has demonstrated oncolysis and preferential replication in cancer cells.In this review, we summarize the recent findings regarding existing knowledge on biology of the ovarian cancer and the role of ovarian CSCs (OCSCs) in tumor dissemination and chemoresistance. In addition we also present an overview of recent advances in ovarian cancer therapies with oncolytic viruses (OV). We focus particularly on key genetic or immune response pathways involved in tumorigenesis in ovarian cancer which facilitate oncolytic activity of vesicular stomatitis virus (VSV). We highlight the prospects of targeting OCSCs with VSV. The importance of testing an emerging ovarian cancer animal models and ovarian cancer cell culture conditions influencing oncolytic efficacy of VSV is also addressed.

Rap1A promotes ovarian cancer metastasis via activation of ERK/p38 and notch signaling.

As one of the Ras‐associated proteins, Rap1A has been linked to cancer initiation and development. However, the precise function of Rap1A in ovarian cancer is still not understood. Here, we show that Rap1A promotes ovarian cancer tumorigenesis and metastasis via stimulating cell proliferation, migration and invasion both in vivo and in vitro. Mechanistic study showed that Rap1A activates extracellular signal‐regulated kinase (ERK), p38 mitogen‐activated protein kinase (MAPK) and Notch pathways, leading to the enhanced expression of several epithelial‐mesenchymal transition (EMT) markers such as slug, zeb1, vimentin, fibronectin, and MMP9. However, the pretreatment of Rap1A‐overexpressing cells with the Notch inhibitor DAPT or ERK inhibitor (U0126) inhibited the up‐regulated expression of those molecules. These findings provide the first evidence linking Rap1A with ovarian cancer development through the ERK/p38 and Notch signaling pathways, indicating that Rap1A may be used as a novel diagnostic marker or a therapeutic target for ovarian cancer.

CUL4A functions as an oncogene in ovarian cancer and is directly regulated by miR-494

Cullin 4A (CUL4A), as a well-defined oncogene, has been reported to be upregulated in ovarian cancer clinically. However, the biological functions of CUL4A and the molecular mechanism underlying its upregulation in ovarian cancer remains unknown throughly. Here, we show that expression of CUL4A is significantly higher in ovarian cancer tissues compared to corresponding non-cancerous tissues. Moreover, silencing of CUL4A by siRNA markedly inhibits cell proliferation, invasion and epithelial-mesenchymal transition (EMT). We identified CUL4A as a novel target gene of miR-494. Further investigations showed that miR-494 was remarkably downregulated and correlated with poor prognosis in ovarian cancer. Overexpression of miR-494 inhibited proliferation, migration, invasion and EMT of ovarian cancer cells by directly suppressing CUL4A expression. Therefore, our findings indicate that miR-494/CUL4A axis is important in the control of ovarian cancer tumorigenesis.

MiR-203 promotes the growth and migration of ovarian cancer cells by enhancing glycolytic pathway.

MicroRNAs (miRNAs) play an important role in the tumorigenesis of ovarian cancer. Previously, we have reported the dysregulation of miR-203 in the ovarian cancer tissues. However, the biological functions and molecular mechanisms of miR-203 in ovarian cancer remain unknown. Here, we showed that the expression of miR-203 was increased in ovarian cancer tissues compared with the adjacent non-cancerous tissues and the transcription of miR-203 was inhibited by P53. Forced expression of miR-203 in ovarian cancer promoted cell growth and migration, while depletion of miR-203 inhibited the growth and migration of ovarian cancer cells. In addition, miR-203 promoted the metastasis of ovarian cancer cells in vivo and shorted the survival of the nude mice. Mechanically, miR-203 targeted the 3'-UTR of pyruvate dehydrogenase B (PDHB) and increased the consumption of glucose and the production of lactate. Overexpression of PDHB abolished the oncogenic effects of miR-203 on the growth of ovarian cancer cells. Together, our data suggested the oncogenic roles of miR-203 in ovarian cancer by promoting glycolysis, and miR-203 might be a therapeutic target for ovarian cancer.