Epithelial Ovarian Cancer Tumor Research Articles

MicroRNA let-7g acts as tumor suppressor and predictive biomarker for chemoresistance in human epithelial ovarian cancer

Remarkable deregulation of microRNAs has been demonstrated in epithelial ovarian cancer (EOC). In particular, some of the let-7 miRNA family members have been proposed as tumor suppressors. Here, we explored the functional roles of let-7g in EOC. The ectopic overexpression of let-7g in OVCAR3 and HEY-A8 EOC cells induced i) a down-regulation of c-Myc and cyclin-D2 thus promoting cell cycle arrest, ii) a reduction of Vimentin, Snail and Slug thus counteracting the progression of epithelial to mesenchymal transition, iii) a chemosensitization to cis-platinum treatment. Next, analysis of human EOC tissues revealed that let-7g expression was significantly reduced in tumor tissue specimens of patients with EOC compared to their non-tumor counterparts (p = 0.0002). Notably, low let-7g tissue levels were significantly associated with acquired chemoresistance of patients with late-stage of EOC (n = 17, p = 0.03194). This finding was further validated in the serum samples collected from the same cohort of patients (n = 17, p = 0.003). To conclude, we demonstrate that let-7g acts as tumor suppressor and might be used to disable EOC tumor progression and chemoresistance to cis-platinum-based chemotherapy. Furthermore, we propose that decreased expression of let-7g could serve as a tissue and serum biomarker able to predict the chemo-resistant features of EOC patients.

Increase Paclitaxel Sensitivity to Better Suppress Serous Epithelial Ovarian Cancer via Ablating Androgen Receptor/Aryl Hydrocarbon Receptor-ABCG2 Axis.

Background: Epithelial ovarian cancer (EOC) is one of the most lethal gynecological malignancies and presents chemoresistance after chemotherapy treatment. Androgen receptor (AR) has been known to participate in proliferation. Yet the mechanisms of the resistance of this drug and its linkage to the AR remains unclear. Methods: To elucidate AR-related paclitaxel sensitivity, co-IP, luciferase reporter assay and ChIP assay were performed to identify that AR direct-regulated ABCG2 expression under paclitaxel treatment. IHC staining by AR antibody presented higher AR expression in serous-type patients than other types. AR degradation enhancer (ASC-J9) was used to examine paclitaxel-associated and paclitaxel-resistant cytotoxicity in vitro and in vivo. Results: We found AR/aryl hydrocarbon receptor (AhR)-mediates ABCG2 expression and leads to a change in paclitaxel cytotoxicity/sensitivity in EOC serous subtype cell lines. Molecular mechanism study showed that paclitaxel activated AR transactivity and bound to alternative ARE in the ABCG2 proximal promoter region. To identify AR as a potential therapeutic target, the ASC-J9 was used to re-sensitize paclitaxel-resistant EOC tumors upon paclitaxel treatment in vitro and in vivo. Conclusion: The results demonstrated that activation of AR transactivity beyond the androgen-associated biological effect. This novel AR mechanism explains that degradation of AR is the most effective therapeutic strategy for treating AR-positive EOC serous subtype.

Combining Vascular Normalization with an Oncolytic Virus Enhances Immunotherapy in a Preclinical Model of Advanced-Stage Ovarian Cancer.

Intravenous delivery of oncolytic viruses often leads to tumor vascular shutdown, resulting in decreased tumor perfusion and elevated tumor hypoxia. We hypothesized that using 3TSR to normalize tumor vasculature prior to administration of an oncolytic Newcastle disease virus (NDV) would enhance virus delivery and trafficking of immunologic cell subsets to the tumor core, resulting in systemically enhanced immunotherapy and regression of advanced-stage epithelial ovarian cancer (EOC). Using an orthotopic, syngeneic mouse model of advanced-stage EOC, we pretreated mice with 3TSR (4 mg/kg per day) alone or followed by combination with fusogenic NDV(F3aa) (1.0 × 108 plaque-forming units). Treatment with 3TSR normalized tumor vasculature, enhanced blood perfusion of primary EOC tumors, and induced disease regression. Animals treated with combination therapy had the greatest reduction in primary tumor mass, ascites accumulation, and secondary lesions (50% of mice were completely devoid of peritoneal metastases). Combining 3TSR + NDV(F3aa) led to enhanced trafficking of immunologic cells into the primary tumor core. We have shown, for the first time, that NDV, like other oncolytic viruses, is a potent mediator of acute vascular shutdown and that preventing this through vascular normalization can promote regression in a preclinical model of advanced-stage ovarian cancer. This challenges the current focus on induction of intravascular thrombosis as a requisite for successful oncolytic virotherapy.See related commentary by Bykov and Zamarin, p. 1446.

Natural antisense transcript of hypoxia-inducible factor 1 regulates hypoxic cell apoptosis in epithelial ovarian cancer.

PurposeHypoxia is a key stress that triggers apoptosis in various tumors, including epithelial ovarian cancer (EOC). Previous researches identified a hypoxia-upregulated lncRNA named “a natural antisense transcript of hypoxia-inducible factor 1 (aHIF)” in some tumors. However, the contribution of aHIF to EOC remains unclear. Here, we aimed to investigate the expression, function, and underlying mechanisms of aHIF in EOC progression under hypoxia.Materials and methodsExpression levels of aHIF in EOC tissues were tested. In vitro and in vivo assays were conducted to explore the function and mechanism of aHIF in hypoxia-induced EOC progression.ResultsaHIF levels were increased in EOC tissues and were upregulated by hypoxia in EOC cells. Functional data revealed that aHIF knockdown accelerated cell apoptosis under hypoxia and inhibited EOC tumorigenesis and tumor growth in vivo. Additionally, aHIF overexpression inhibited cell apoptosis and enhanced cell proliferation under hypoxia in EOC. Mechanistically, the dysregulation of certain key mitochondrial apoptosis pathway-related genes, including Bcl-2, Bax, Caspase-7, and Caspase-9, may partially explain aHIF-regulated EOC apoptosis and growth under hypoxia.ConclusionThese data provide the first convincing evidence that aHIF may inhibit EOC apoptosis and thereby promote tumor growth through activation of the mitochondrial apoptosis pathway under hypoxia. Our findings help clarify the role of lncRNA in hypoxia-induced EOC progression.

CD44-Targeting PLGA Nanoparticles Incorporating Paclitaxel and FAK siRNA Overcome Chemoresistance in Epithelial Ovarian Cancer.

Chemotherapy is commonly used in the treatment of ovarian cancer, yet most ovarian cancers harbor inherent resistance or develop acquired resistance. Therefore, novel therapeutic approaches to overcome chemoresistance are required. In this study, we developed a hyaluronic acid-labeled poly(d,l-lactide-co-glycolide) nanoparticle (HA-PLGA-NP) encapsulating both paclitaxel (PTX) and focal adhesion kinase (FAK) siRNA as a selective delivery system against chemoresistant ovarian cancer. The mean size and zeta potential of the HA-PLGA-NP were 220 nm and -7.3 mV, respectively. Incorporation efficiencies for PTX and FAK siRNA in the HA-PLGA-NPs were 77% and 85%, respectively. HA-PLGA-NP showed higher binding efficiency for CD44-positive tumor cells as compared with CD44-negative cells. HA-PLGA (PTX+FAK siRNA)-NP caused increased cytotoxicity and apoptosis in drug-resistant tumor cells. Treatment of human epithelial ovarian cancer tumor models HeyA8-MDR (P < 0.001) and SKOV3-TR (P < 0.001) with HA-PLGA (PTX+FAK siRNA)-NP resulted in significant inhibition of tumor growth. Moreover, in a drug-resistant, patient-derived xenograft (PDX) model, HA-PLGA (PTX+FAK siRNA)-NP significantly inhibited tumor growth compared with PTX alone (P < 0.002). Taken together, HA-PLGA-NP acts as an effective and selective delivery system for both the chemotherapeutic and the siRNA in order to overcome chemoresistance in ovarian carcinoma.Significance: These findings demonstrate the efficacy of a novel, selective, two-in-one delivery system to overcome chemoresistance in epithelial ovarian cancer. Cancer Res; 78(21); 6247-56. ©2018 AACR.

TNFAIP8 promotes cell growth by regulating the Hippo pathway in epithelial ovarian cancer.

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is an independent prognostic factor for cancer-specific and disease-free survival in patients with epithelial ovarian cancer (EOC). However, the exact mechanism of the biological role of TNFAIP8 in EOC remains unclear. In the present study, a siRNA specifically targeting TNFAIP8 was prepared to knock down TNFAIP8 in EOC cells. Cell growth, colony formation, apoptosis, and cell cycle distribution in TNFAIP8-deficient EOC cells were determined. In addition, the underlying molecular mechanisms were investigated by western blot analysis and reverse transcription quantitative polymerase chain reaction assays. It was demonstrated that the knockdown of TNFAIP8 inhibited EOC cell growth and colony formation, along with increased levels of apoptosis and cell cycle arrest. The results of the western blot analysis suggested that TNFAIP8 inhibited the expression of phosphorylated yes-associated protein 1 (YAP) while promoting total and nuclear YAP expression, followed by the regulation of apoptosis and cell cycle checkpoint protein expression in EOC. Overexpression of YAP in EOC cells efficiently attenuated cell growth inhibition in TNFAIP8-deficient EOC cells. In addition, knockdown of TNFAIP8 significantly impaired EOC tumor growth in vivo. Collectively, the data from the present study suggested that TNFAIP8 is an oncogene and a novel therapeutic target for EOC.

Clinical associations between ASCT2 and p‑mTOR in the pathogenesis and prognosis of epithelial ovarian cancer

Alanine serine cysteine‑preferring transporter2 (ASCT2; also known as SLC1A5) is an important glutamine transporter, and it serves a crucial role in tumor growth and progression. ASCT2 is highly expressed in numerous types of cancer, but the pathological significance of its expression in epithelial ovarian cancer (EOC) remains unclear. The mechanistic target of rapamycin (mTOR) level is hyperelevated in a number of tumor types, including ovarian cancer. The aim of the present study was to elucidate the prognostic role of ASCT2 and phosphorylated (p)‑mTOR in EOC. The levels of ASCT2 and p‑mTOR/mTOR were detected in normal ovarian tissues, benign ovarian tumors, borderline ovarian tumors and EOC tissues by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot assays. The protein levels of ASCT2 and p‑mTOR in EOC patients were then detected by immunohistochemistry (IHC). Furthermore, EOC tumor sections were stained for Ki‑67 and cluster of differentiation34 (CD34) to assess proliferation and microvessel density by IHC. The results of RT‑qPCR and western blot analysis demonstrated that ASCT2 and p‑mTOR protein levels were significantly higher in EOC tissues compared with those in other groups. IHC analysis of 104 EOC tissues suggested that ASCT2 expression was associated with clinicopathological parameters, including International Federation of Gynecology and Obstetrics stage, pathological grade, serum cancer antigen 125 level, Ki‑67 status and CD34 status. Kaplan‑Meier survival curve analysis indicated that high expression of ASCT2 and p‑mTOR were important factors predicting a poor prognosis for patients with EOC. The expression levels of ASCT2 and p‑mTOR in EOC were positively correlated (r=0.385, P<0.001). This positive correlation between ASCT2 and p‑mTOR indicates that they have a synergistic effect on the growth and development of early EOC. The combined detection of ASCT2 and p‑mTOR may serve as a potential marker to inform diagnosis, postoperative follow‑up requirements and targeted therapy options for patients with early‑stage EOC, but not for terminal‑stage patients.

The down-regulation of long non-coding RNA LINC01088 is associated with the poor prognosis of epithelial ovarian cancer patients.

Long intergenic non-coding RNA 1088 (LINC01088) has been suggested to act as a tumor suppressor in epithelial ovarian cancer (EOC); however, the prognostic role of LINC01088 has not been evaluated in cancer patients. This study aimed to investigate the expression of LINC01088 in EOC, along with evaluating its clinical-pathological and prognostic importance. A bioinformatics tool (GEPIA) was used to screen the dysregulated lncRNAs. Quantitative Real-time PCR (qRT-PCR) was used to measure expression level of LINC01088 in EOC tumor samples and adjacent non-tumor tissues. Then, the association between LINC01088 expression and pathological parameters were further evaluated. Overall survival (OS) was estimated using the Kaplan-Meier method, and the differences in survival were compared using the log-rank test. Univariate and multivariate analysis were performed using the Cox proportional hazard analysis. We found that LINC01088 expression was significantly down-regulated in EOC tissues via "GEPIA". Then, the results of RT-PCT confirmed that the expression levels of LINC01088 were significantly lower in EOC tissues compared to adjacent noncancerous tissues (p < 0.01). Interestingly, lower LINC01088 expression levels were associated with FIGO stage (p = 0.000), grade (p = 0.003) and distant metastasis (p = 0.006). Moreover, Kaplan-Meier analysis indicated that patients with low LINC01088 expression had a poor overall survival (p = 0.0013). Finally, univariate and multivariate analysis show that LINC01088 expression is an independent predictor for overall survival. Low LINC01088 expression was associated with the progression of EOC and could serve as a potential independent prognostic biomarker for patients with EOC.

Epigenetic Crosstalk between the Tumor Microenvironment and Ovarian Cancer Cells: A Therapeutic Road Less Traveled.

Metastatic dissemination of epithelial ovarian cancer (EOC) predominantly occurs through direct cell shedding from the primary tumor into the intra-abdominal cavity that is filled with malignant ascitic effusions. Facilitated by the fluid flow, cells distribute throughout the cavity, broadly seed and invade through peritoneal lining, and resume secondary tumor growth in abdominal and pelvic organs. At all steps of this unique metastatic process, cancer cells exist within a multidimensional tumor microenvironment consisting of intraperitoneally residing cancer-reprogramed fibroblasts, adipose, immune, mesenchymal stem, mesothelial, and vascular cells that exert miscellaneous bioactive molecules into malignant ascites and contribute to EOC progression and metastasis via distinct molecular mechanisms and epigenetic dysregulation. This review outlines basic epigenetic mechanisms, including DNA methylation, histone modifications, chromatin remodeling, and non-coding RNA regulators, and summarizes current knowledge on reciprocal interactions between each participant of the EOC cellular milieu and tumor cells in the context of aberrant epigenetic crosstalk. Promising research directions and potential therapeutic strategies that may encompass epigenetic tailoring as a component of complex EOC treatment are discussed.

Abstract B64: The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer

Abstract The role and regulation of ZIP4, a zinc (Zn) transporter, have been studied in the context of extracellular Zn transporting. Factors other than Zn with ZIP4 regulatory effects are essentially unknown. In addition, its role in cancer stem cell (CSC) has not been reported in any type of cancer. RNA-seq analyses were conducted to compare genes differentially expressed in less and more aggressive epithelial ovarian cancer (EOC) cells. ZIP4 expression and its regulation was assessed by immunoblotting, quantitative PCR, or immunofluorescence staining in human ovarian tissues. CSC-related activities were examined to evaluate the role of ZIP4 in human high-grade serous ovarian cancer (HGSOC) cells. RNAi and CRISPR techniques were used to knock down or knock out ZIP4 and related genes. EOC tumors overexpressed ZIP4 when compared with normal and benign tissues. ZIP4 knockout significantly reduced several CSC-related activities in EOC cells, including proliferation, anoikis resistance, colony formation, spheroid formation, drug resistance, and side-population (SP). Only ZIP4-expressing SP highly expressed known CSC markers ALDH1 and OCT4. ZIP4 knockout dramatically reduced tumorigenesis and ZIP4 overexpression increased tumorigenesis in vivo. In addition, the ZIP4-expressing SP population had the tumor initiating activity. Moreover, the oncolipid lysophosphatic acid (LPA) effectively upregulated ZIP4 expression via the nuclear receptor peroxisome proliferator-activated receptor gamma, and LPA’s promoting effects in CSC-related activities in HGSOC cells was at least partially mediated by ZIP4 in an extracellular Zn-independent manner. Our critical data imply that ZIP4 is a new and important cancer stem cell regulator in EOC. Our data also provide an innovative interpretation for the apparent disconnection between low levels of zinc and upregulation of ZIP4 in EOC tissues. Citation Format: Yan Xu, Qipeng Fan, Qingchun Cai, Kenneth P. Nephew. The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B64.

MiR-590-3p Promotes Ovarian Cancer Growth and Metastasis via a Novel FOXA2-Versican Pathway.

miRNAs play important roles in gene regulation, and their dysregulation is associated with many diseases, including epithelial ovarian cancer (EOC). In this study, we determined the expression and function of miR-590-3p in EOC. miR-590-3p levels were higher in high-grade carcinoma when compared with low-grade or tumors with low malignant potential. Interestingly, plasma levels of miR-590-3p were significantly higher in patients with EOC than in subjects with benign gynecologic disorders. Transient transfection of miR-590-3p mimics or stable transfection of mir-590 increased cell proliferation, migration, and invasion. In vivo studies revealed that mir-590 accelerated tumor growth and metastasis. Using a cDNA microarray, we identified forkhead box A2 (FOXA2) and versican (VCAN) as top downregulated and upregulated genes by mir-590, respectively. miR-590-3p targeted FOXA2 3' UTR to suppress its expression. In addition, knockdown or knockout of FOXA2 enhanced cell proliferation, migration, and invasion. Overexpression of FOXA2 decreased, whereas knockout of FOXA2 increased VCAN mRNA and protein levels, which was due to direct binding and regulation of the VCAN gene by FOXA2. Interrogation of the TCGA ovarian cancer database revealed a negative relationship between FOXA2 and VCAN mRNA levels in EOC tumors, and high FOXA2/low VCAN mRNA levels in tumors positively correlated with patient survival. Finally, overexpression of FOXA2 or silencing of VCAN reversed the effects of mir-590. These findings demonstrate that miR-590-3p promotes EOC development via a novel FOXA2-VCAN pathway.Significance: Low FOXA2/high VCAN levels mediate the tumor-promoting effects of miR-590-3p and negatively correlate with ovarian cancer survival. Cancer Res; 78(15); 4175-90. ©2018 AACR.

Upregulation of miR-874-3p and miR-874-5p inhibits epithelial ovarian cancer malignancy via SIK2.

Based on miR-874 expression levels in the GSE47841 microarray, we hypothesized that the mature products of miR-874, miR-874-3p, or miR-874-5p, would inhibit epithelial ovarian cancer (EOC) cell proliferation, metastasis, and chemoresistance. We first examined miR-874-3p and miR-874-5p expression levels in primary EOC tumor tissue samples and found that they were significantly decreased. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation and transwell assays revealed that miR-874-3p and miR-874-5p significantly inhibit EOC cell proliferation, migration, and invasion. Then, using MTT and soft agar assays of paclitaxel-treated Caov3 and SKOV3 cells transfected with miR-874-3p and miR-874-5p, we found that miR-874-3p and miR-874-5p enhance EOC cell chemosensitivity. We then confirmed that serine/threonine-protein kinase 2 (SIK2) was a target gene of miR-874-3p and miR-874-5p. Overall, the results of this study indicate that SIK2 expression can serve as a prognostic biomarker for EOC and that miR-874-3p and miR-874-5p have the potential to enhance clinical treatment of EOC.

Lysophospholipid Signaling in the Epithelial Ovarian Cancer Tumor Microenvironment.

As one of the important cancer hallmarks, metabolism reprogramming, including lipid metabolism alterations, occurs in tumor cells and the tumor microenvironment (TME). It plays an important role in tumorigenesis, progression, and metastasis. Lipids, and several lysophospholipids in particular, are elevated in the blood, ascites, and/or epithelial ovarian cancer (EOC) tissues, making them not only useful biomarkers, but also potential therapeutic targets. While the roles and signaling of these lipids in tumor cells are extensively studied, there is a significant gap in our understanding of their regulations and functions in the context of the microenvironment. This review focuses on the recent study development in several oncolipids, including lysophosphatidic acid and sphingosine-1-phosphate, with emphasis on TME in ovarian cancer.

Abstract 5435: Role of the WT1 transcription factor in high-grade serous ovarian cancer

Abstract The Wilms Tumor protein 1 (WT1) transcription factor (TF) is a ubiquitous biomarker of high-grade serous ovarian carcinoma (HGSOC), which is the most common and aggressive subtype of epithelial ovarian cancer (EOC). This TF interacts with a host of cellular factors including the tumor suppressor P53, which is mutated in virtually all HGSOC cases. Studies have suggested a role for WT1 in HGSOC development, but little is known about the underlying mechanism. We have generated unique catalogues of epigenomic landscapes (specifically H3K27ac) in primary HGSOC using chromatin immunoprecipitation sequencing (ChIP-seq). Using Bayesian analyses of H3K27ac profiling in HGSOCs, we identified significant tissue specific enrichment of active chromatin at the WT1 locus in HGSOC when compared to all other EOC tumor histotypes. Furthermore, this locus contains a superenhancer in HGSOCs and in normal fallopian tube secretory epithelial cells (FTSECs). This locus features a bidirectional promoter that is shared by WT1-AS lncRNA and that contains a WT1 binding site. LncRNAs are essential in many cellular processes, and have been shown to play pivotal roles in tumorigenicity. The WT1-AS lncRNA is implicated in other diseases but little is known of its function in EOC. We have investigated the mechanisms of interaction between WT1 and WT1-AS. We observed that both protein and transcript expression for WT1 and WT1-AS are positively correlated in HGSOC cell lines. Analysis of data for &amp;gt;400 primary HGSOCs from TCGA further supported this correlation. We hypothesized that WT1 is a critical TF in the development of HGSOC, and that WT1-AS plays a key role in its regulatory pathway. We evaluated the regulatory role of WT1 first by analyzing cellular phenotypes and target gene expression changes in HGSOC cell lines after shRNA mediated knockdown (KD) of WT1. HGSOC cells with stable WT1 knockdown had impaired proliferation and concomitant reduction of WT1-AS expression. We also identified a decrease in expression of SHMT1 in WT1 KD cells, which is a known WT1 target gene. Integrated genome-wide analysis of the WT1 transcriptome by RNA-seq and the WT1 cistrome by ChIP-seq, in parallel with ChIP-MS, is being used to establish the architecture of the WT1 regulome, specifically to establish the regulatory mechanisms and relationships between WT1 and WT1-AS. Ultimately, these studies aim to establish the mechanisms of action for the WT1 TF in neoplastic transformation of HGSOC. Citation Format: Norma I. Rodriguez-Malave, Justyna E. Kanska, Kevin C. Vavra, Dennis Hazelett, Rosario I. Corona, Ji-Heui Seo, Matthew Freedman, Simon R. Knott, Sandra Orsulic, Beth Y. Karlan, Kate Lawrenson, Simon A. Gayther. Role of the WT1 transcription factor in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5435.

CircHIPK3 is upregulated and predicts a poor prognosis in epithelial ovarian cancer.

CircRNAs have been recently identified as important regulators in tumors biological functions. However, the clinical significance of circHIPK3 in epithelial ovarian cancer (EOC) remains unknown. The expression of circHIPK3 in EOC tumor tissues and adjacent noncancerous tissues was analyzed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The association between circHIPK3 expression and clinicopathological factors was analyzed by using Chi-square test. Kaplan-Meier method and log-rank test were used to analyze the association of circHIPK3 expression with disease-free survival (DFS) and overall survival (OS) time of EOC patients. Univariate and multivariate Cox analysis was also performed. We found that circHIPK3 was higher expressed in EOC tissues and cells compared to adjacent normal tissue and ovarian epithelium cell line, respectively. Higher circHIPK3 expression associated with lymph node invasion, FIGO stage, and worse DFS and OS of patients. Moreover, multivariate Cox analysis showed that higher circHIPK3 was an independent predictor of DFS and OS in EOC patients. Thus, circHIPK3 may be a novel biomarker for predicting EOC prognosis.

Expression of multiple immune checkpoint molecules on T cells in malignant ascites from epithelial ovarian carcinoma.

Expression of immune checkpoint molecules, including programmed cell death protein-1 (PD-1), has been reported on T cells in various types of cancer. However, the expression status of these molecules in the tumor microenvironment of epithelial ovarian cancer (EOC) has not yet been studied. A total of 54 cases of malignant ascites from patients with EOC were analyzed in the present study. The expression of PD-1, lymphocyte-activation gene-3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and B and T lymphocyte attenuator (BTLA) on cluster of differentiation (CD)4+ and CD8+ T cells in malignant EOC ascites were investigated using multicolor flow cytometric analysis. The expression of PD-L1 in tumor cells, PD-L2 in HLA-DR-positive cells and galectin-9 in ascitic fluid was also analyzed. In addition, cytokine profiling of ascitic fluid was performed to understand the immune microenvironment of EOC. PD-1, LAG-3 TIM-3, and BTLA were expressed on 65.8, 10.6, 4.3 and 37.6% of CD4+ T cells, and on 57.7, 5.0, 4.9 and 15.7% of CD8+ T cells, respectively. Programmed cell death protein-1 (PD-1), LAG-3 and BTLA were more frequently expressed on CD4+ compared with CD8+ T cells. The co-expression of immune checkpoints was further investigated and results indicated that 39 (72.2%) and 37 patients (68.5%) expressed multiple immune checkpoints on CD4+ T cells and CD8+ T cells, respectively. In addition, lower levels of TNF-α and interleukin-6 in ascitic fluid were significantly associated with multiple immune checkpoint expression on CD8+ T cells. The present findings indicated that multiple immune checkpoint molecules were expressed on T cells in the EOC tumor microenvironment and the results may suggest the significance of simultaneous blockade of immune checkpoints to control EOC.

Dysregulation of fibulin-5 and matrix metalloproteases in epithelial ovarian cancer.

Fibulin 5 (FBLN5) is an extracellular matrix glycoprotein that suppresses matrix metalloprotease 9 (MMP-9), angiogenesis and epithelial cell motility. Here, we investigated the regulation and function of FBLN5 in epithelial ovarian cancer (EOC). FBLN5 mRNA was down-regulated 5-fold in EOC relative to benign ovary. Not surprisingly, MMP9 mRNA and enzyme activity were increased significantly, and inversely correlated with FBLN5 gene expression. FBLN5 degradation products of 52.8 and 41.3 kDa were increased substantially in EOC. We identified two candidate proteases (serine elastase and MMP-7, but not MMP-9) that cleave FBLN5. MMP-7, but not neutrophil elastase, gene expression was increased dramatically in EOC. Recombinant FBLN5 significantly inhibited adhesion of EOC cells to both laminin and collagen I. Finally, using immunohistochemistry, we found immunoreactive FBLN5 within tumor macrophages throughout human EOC tumors. This work indicates that FBLN5 is degraded in EOC most likely by proteases enriched in macrophages of the tumor microenvironment. Proteolysis of FBLN5 serves as a mechanism to promote cell adhesion and local metastasis of ovarian cancer cells. Promotion of a stable ECM with intact FBLN5 in the tumor matrix may serve as a novel therapeutic adjunct to prevent spread of ovarian cancer.

Ovarian carcinoma glyco-antigen targeted by human IgM antibody.

Epithelial Ovarian Cancer (EOC) cells expression of a novel carbohydrate antigen was defined using a human VH4-34 encoded IgM monoclonal antibody (mAb216). MAb216 binds to a poly N-acetyllactosamine epitope expressed on B cells and kills normal and malignant B cells in vitro and in vivo. EOC patient ascites and EOC cell lines were used to study the anti tumor effect of mAb216. Various assays were used to characterize the epitope and demonstrate antibody-mediated binding and cytotoxicity in EOC. Drug and antibody combination effects were determined by calculating the combination index values using the Chou and Talalay method. MAb216 displays direct antibody mediated cytotoxicity on a population of human EOC tumor and ascites samples and EOC cell lines, which express high amounts of poly N-acetyllactosamine epitope, carried by CD147/CD98. Eighty four percent of patient samples, including platin resistant, had a tumor population that bound the monoclonal antibody. The binding pattern of mAb216 and mechanism of cytotoxicity was similar to that seen on normal and malignant B cells with unique general membrane disruption and “pore” formation. In vitro incubation with mAb216 and cisplatin enhanced killing of OVCAR3 cell line. In EOC cell lines percent cytotoxicity correlated with percent expression of epitope. Although in vitro data shows specific EOC cytotoxicity, for possible treatment of EOC MAb216 would need to be evaluated in a clinical trial with or without chemotherapy.

MicroRNA-152 inhibits ovarian cancer cell proliferation and migration and may infer improved outcomes in ovarian cancer through targeting FOXP1.

microRNA (miR) are a class of endogenous small non-coding RNA that are aberrantly expressed and are critical in tumorigenesis. Amongst them, miR-152 was reported to be dysregulated in epithelial ovarian cancer (EOC). However, the function and mechanism of miR-152 is not well understood. In the present study, total RNA was extracted from 58 ovarian epithelial carcinoma tissue samples and adjacent non-tumor tissues and measured by reverse transcription-quantitative polymerase chain reaction. The observations of the present study revealed that the expression of miR-152 was significantly downregulated in EOC specimens, as well as three ovarian cancer (OC) cell lines. The higher expression of miR-152 indicated a better overall survival rate in patients with EOC. Following miR-152 mimic transfection into SKOV3 or OVCAR3 cells, MTT assay revealed that cell proliferation was significantly inhibited (P<0.05). Although miR-152 had no effect on SKOV3 cell migration, miR-152 inhibited OVCAR3 cell migration. Bioinformatics analyses and luciferase reporter assays demonstrated that miR-152 targeted the 3'-untranslated region (3'-UTR) of the forkhead box protein 1 (FOXP1). Furthermore, introducing FOXP1 without the 3'-UTR abrogated the effect of miR-152-induced proliferation and migration alteration, respectively. In addition, the expression level of FOXP1 was higher in the EOC tumor tissues and cell lines. Additionally, the level of miR-152 and FOXP1 was inversely correlated in grade 3 and 4 ovarian tumor tissues. Altogether, these observations indicated that miR-152 may be involved in the inhibition of OC through repression of FOXP1. In the future, miR-152 and FOXP1 may act as novel biomarkers for early detection of EOC or therapeutic targets.

Altered expression of different GalNAc‑transferases is associated with disease progression and poor prognosis in women with high-grade serous ovarian cancer.

Protein glycosylation perturbations are implicated in a variety of diseases, including cancer. Aberrant glycosylation in cancer is frequently attributed to altered expression of polypeptide GalNAc transferases (GalNAc‑Ts) - enzymes initiating mucin-type O-glycosylation. A previous study from our group demonstrated that one member of this family (GALNT3) is overexpressed in epithelial ovarian cancer (EOC), and GALNT3 expression correlated with shorter progression-free survival (PFS) in EOC patients with advanced disease. As considerable degree of redundancy between members of the GalNAc‑Ts gene family has been frequently observed, we decided to investigate whether other members of this family are essential in EOC progression. Insilico analysis based on publically available data was indicative for altered expression of five GalNAc‑Ts (GALNT2, T4, T6, T9 and T14) in ovarian high-grade serous carcinoma (HGSC) samples compared to non-tumoral (control) ovarian tissue. We analyzed protein expression of these GalNAc‑Ts in EOC cells and tumors by western blotting, followed by immunohistochemical (IHC) evaluation of their expression in EOC tumor and control samples using tissue microarrays (TMAs). Western blot analyses were indicative for low expression of GALNT2 and strong expression of GALNT6, T9 and T14 in both EOC cells and tumors. These observations were confirmed by IHC. GALNT2 displayed significantly lower expression, while GALNT6, GALNT9 and GALNT14 showed significantly higher expression in HGSC tumors compared to control tissue. Importantly, GALNT6 and GALNT14 expression correlated with poor prognosis of serous EOC patients. Moreover, our results suggest for overlapping functions of some GalNAc‑Ts, more specifically GALNT3 and GALNT6, in directing EOC progression. Our results are indicative for a possible implication of different members of the GalNAc‑T gene family in modulating EOC progression, and the potential use of GALNT6 and GALNT14 as novel prognostic EOC biomarkers. These data warrant future studies on the role of members of the GalNAc‑Ts gene family in ovarian tumorigenesis.