Human Epithelial Ovarian Cancer Tissues Research Articles

Abstract B33: Genomic analysis of immunosuppressive and proangiogenic genes in recombinant HE4 treated immune cells and implications for T-cell cytotoxicity in ovarian cancer cell co-culture

Abstract Objective: To determine the effect of HE4 on expression of immune-related genes in CD8+ T cells and explore the role of HE4 in immunosuppression and angiogenesis in epithelial ovarian cancer (EOC). Methods: Tissue HE4 levels and T cell counts were determined by immunohistochemistry (IHC). Recombinant HE4 (rHE4)-mediated gene expression in CD8+ T cells was measured by qPCR array. Cytokine levels were determined using a human cytokine array. Cell viability of ovarian cancer cells co-cultured with pooled peripheral blood mononuclear cells (PBMC) in the presence or absence of rHE4 was determined using MTS cell viability reagent. P-values were determined by unpaired 2-tailed student t test. Activation of STAT3 was evaluated by Western blot, and the interaction of STAT3 and HE4 was queried by immunoprecipitation of STAT3. Finally, HE4 and IL8 levels were measured by IHC in a microarray of human EOC and normal adjacent tissue (NAT), and levels were correlated using Spearman Rank test. Results: Tissue HE4 levels correlated with CD8+ T cell counts in EOC. rHE4 treatment of CD8+ T cells upregulated expression of a panel of immunosuppressive and proangiogenic genes, including VEGF, HIF1A, STAT3, IDO1, FOXP3, and IL8. IL8 was most robustly upregulated (99-fold vs control). HE4 also resulted in alterations in cytokine levels in ovarian cancer cells co-cultured with PBMCs. OVCAR8 and SKOV3 ovarian cancer cell lines were treated with rHE4, and levels of phospho-STAT3—a known regulator of IL8—were measured. HE4 was found to activate STAT3 in a time-dependent manner, which occurred in the absence of a physical interaction between HE4 and STAT3. rHE4 treatment of OVCAR8 and SKOV3 cells co-cultured with PBMCs suppressed PBMC-mediated cytotoxicity. Finally, IL8 and HE4 mean levels were elevated in EOC tissue compared to NAT and significantly correlated (R=0.49332; p=0.00142). Conclusions: HE4 upregulates expression of genes involved in promoting a proangiogenic and immunosuppressive microenvironment in EOC, which may play a role in reduced cytotoxic ability of immune cells. Upregulation of IL8 may occur via HE4 regulation of STAT3 signaling. Future directions include testing the effect of HE4 on endothelial cell angiogenesis and determining if STAT3 and VEGF inhibitors modulate HE4 suppression of immune cell cytotoxicity. Citation Format: Nicole James, Jenna Emerson, Ashley Borgstadt, Lindsey Beffa, Anze Urh, Kyukwang Kim, Rakesh Singh, Richard Moore, Paul DiSilvestro, Jennifer Ribeiro. Genomic analysis of immunosuppressive and proangiogenic genes in recombinant HE4 treated immune cells and implications for T-cell cytotoxicity in ovarian cancer cell co-culture [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B33.

Abstract 4754: CHTOP is a novel therapeutic target for chemoresistant epithelial ovarian cancer therapy

Abstract Background: Chemotherapy is the mainstay treatment for ovarian cancer (OC). Chemoresistance is a major challenge in epithelial ovarian cancer (EOC) therapy. CHTOP was identified as a potential chemoresistant biomarker in chemoresistant EOC cell lines using label-free LC-MS/MS proteomic technique. However, the role of CHTOP in EOC chemoresistance is still unclear. Aim: In this study, we aimed to investigate whether CHTOP can be used as a therapeutic target in chemoresistant EOC cells and to reveal the mechanism underlying chemosensitization. Methods: The expression difference of CHTOP was detected in chemoresistant and metastatic EOC cell lines by immunofluorescence (IF) and Western blot (WB). The expression of CHTOP in human EOC tissues was examined using immunohistochemistry (IHC). The effect of CHTOP knockdown (KD) on metastasis was examined using the Transwell® matrigel invasion and wound healing assays. Flow cytometry and TUNEL assay were employed to determine the association of CHTOP with apoptosis, while mammary sphere formation assay and IF were used to evaluate its regulation on EOC-cis cell stemness. Results: The higher expression of CHTOP was found in EOC-cis (A2780-cis and IGROV-1-cis) and metastatic EOC (SKOV-3 and OV-90) cells as compared to normal epithelial ovarian cells (HOSE) by IF and WB. Also, high expression of CHTOP was found in human EOC tissues and associated with poor prognosis in patients. In contrast, CHTOP KD significantly reduced the metastatic potential of EOC-cis cells and increased their apoptosis at the presence of cisplatin. Furthermore, CHTOP KD decreased the stemness of EOC-cis cells. Conclusion: Our findings suggest that CHTOP is associated with metastasis, apoptosis, and stemness in chemoresistant EOC cells, the survival in EOC patients, and might be a promising therapeutic target to overcome chemoresistance in EOC treatment. Keywords: ovarian cancer, chemoresistance, CHTOP, metastasis, apoptosis, stemness Citation Format: Xupeng Bai, Xiaojie Feng, Jie Ni, Julia Beretov, Junli Deng, Ying Zhu, Peter Graham, Yong Li. CHTOP is a novel therapeutic target for chemoresistant epithelial ovarian cancer therapy [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 4754.

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.

Increased expression of long noncoding RNA HMMR-AS1 in epithelial ovarian cancer: an independent prognostic factor.

Evidence has indicated that long noncoding RNA (lncRNAs) may have significant roles in cancer. In this study, we aimed to investigate the expression pattern and prognostic value of a noncoding RNA named as HMMR antisense RNA 1 (HMMR-AS1) in epithelial ovarian cancer (EOC). Differences in the expression of HMMR-AS1 between EOC and matched normal tissues were analyzed using RT-PCR. The correlation between HMMR-AS1 levels and the clinicopathological factors of the EOC patients was analyzed by x2-test. Kaplan-Meier analysis and Cox proportional hazards regression models were explored to reveal the correlations of HMMR-AS1 expression with survival of patients. HMMR-AS1 was significantly upregulated in human EOC tissues compared with adjacent normal tissues (p < 0.01). Clinicopathologic analysis revealed that high expression of HMMR-AS1 was associated with advanced FIGO stage (p = 0.013) and positive lymphatic metastasis (p = 0.010). Moreover, patients with higher HMMR-AS1 expression displayed shorter overall survival time (p = 0.0075) and progression-free survival time (p = 0.0013) than those with lower HMMR-AS1 expression. More importantly, multivariate analysis suggested that high expression of HMMR-AS1 was an independent prognostic indicator for EOC patients. Our data suggested that HMMR-AS1 may be considered a novel prognostic factor in EOC and a specific diagnostic indicator for patients with EOC.

Keratinocyte growth factor binding to fibroblast growth factor receptor 2-IIIb promotes epithelial ovarian cancer cell proliferation and invasion.

To analyze the function of keratinocyte growth factor (KGF) and it ligand fibroblast growth factor receptor 2-IIIb (FGFR2-IIIb) in the epithelial ovarian cancer (EOC) progression. In this study, the protein KGF and corresponding ligand FGFR2-IIIb expression were detected in both normal epithelial ovarian tissues and in EOC tissues. Seventy-one ovarian tumor tissues were examined for KGF and FGFR2-IIIb expression by immunohistochemistry; seven normal epithelial ovarian tissues as control were examined. By using a monoclonal antibody to inhibit KGF activation, we tested KGF-induced EOC cells invasion ability. By means of Western blot, we tested extracellular signal-regulated kinase (ERK), phosphorylation ERK, myosin light chain (MLC), and phosphorylation MLC with or without KGF protein. We found that the expression FGFR2-IIIb increased in EOC cells and tissues comparing with its normal counterpart, and the expression of KGF protein decreased or undetectable in human EOC cells and tissues comparing with its normal part. The effect of KGF in promoting EOC cell invasion was blocked by an FGFR2-IIIb antibody. We further discovered that KGF upregulated ERK and MLC phosphorylation in the highly invasive ovarian cancer cell line HO8910PM. Therefore, regarding the highly invasive ovarian cancer cells, we speculated that KGF might promote proliferation and invasion through the ERK-MLC pathway. These results suggest that KGF might play an important role in the progression of ovarian cancer and could be an attractive target for ovarian cancer therapy.

S100A7 Regulates Ovarian Cancer Cell Metastasis and Chemoresistance Through MAPK Signaling and Is Targeted by miR-330-5p.

Epithelial ovarian cancer (EOC) is the most common cause of gynecological cancer-associated death. The high mortality rate is largely due to early stage metastasis and posttreatment recurrence. Identifying crucial regulators of EOC cells as well as ways to target them promises to improve the disease's prognosis. The S100 calcium-binding protein A7 (S100A7) has been shown to promote cell proliferation, migration, invasion, and tumor metastasis. In this study, we have investigated the role that S100A7 plays in regulating EOC cells. We have also identified the microRNA protein miR-330-5p, a known suppressor of oncogenesis and chemoresistance, as an inhibitor of S100A7 activity. We employed both fresh EOC tissue specimens as well as EOC cell lines Caov3 and SKOV3. S100A7 levels were analyzed using quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry. siRNA was used to knockdown S100A7. Cell proliferation, colony formation, and Transwell migration and invasion assays were performed on EOC cell lines with and without cisplatin treatment. TargetScan was used to identify miR-330-5p as a regulator of S100A7. Luciferase reporter plasmids were constructed with either the wild-type or mutant S100A7 3'UTR to determine the effect of miR-330-5p on S100A7. S100A7 was significantly overexpressed in human EOC tissue specimens and EOC cell lines Caov3 and SKOV3. Knocking down S100A7 reduced EOC cell proliferation, migration, and invasion. Furthermore, S100A7 knockdown cells demonstrated increased sensitivity to the chemotherapeutic cisplatin. Finally, miR-330-5p was shown to target the S100A7 3'UTR and to reduce EOC cell growth by inhibiting S100A7 expression. As a regulator of EOC cell proliferation, metastasis, and chemoresistance, S100A7 represents a potential prognostic biomarker for EOC as well as a treatment target. Because miR-330-5p functions as an inhibitor of EOC cell growth and S100A7 expression, it promises to improve EOC outcomes. Further research into the clinical significance of both S100A7 and miR-300-5p is warranted.

LncRNA myocardial infarction-associated transcript promotes cell proliferation and inhibits cell apoptosis by targeting miR-330-5p in epithelial ovarian cancer cells.

IntroductionLong non-coding RNAs (lncRNAs) have been shown to have great importance in cancer development and progression. However, the mechanism of lncRNAs in epithelial ovarian cancer remains unclear. In the present study, we aimed to explore the role of the lncRNA myocardial infarction-associated transcript (MIAT) in epithelial ovarian cancer tumorigenesis.Material and methodsQuantitative real-time PCR (qRT-PCR) was used to determine MIAT expression in human epithelial ovarian cancer tissues and cell lines, and the effects of MIAT on cell proliferation and cell apoptosis were determined by CCK-8 assay or flow cytometry analysis. Dual-Luciferase Reporter assay and Western blot assay were used to explore the molecular mechanisms of MIAT in epithelial ovarian cancer cells progression.ResultsOur data showed that the expression of lncRNA MIAT was remarkably increased in human epithelial ovarian cancer tissues and cell lines (p < 0.05). High MIAT expression was associated with poor overall survival of epithelial ovarian cancer patients (p < 0.05). Function assays showed that knockdown of MIAT expression significantly inhibited epithelial ovarian cancer cell proliferation and promoted cell apoptosis in vitro (p < 0.05). Moreover, we revealed that MIAT might function as an endogenous miR-330-5p sponge to regulate the target gene of miR-330-5p in epithelial ovarian cancer progression.ConclusionsLncRNA MIAT was found to be a tumor oncogenic lncRNA in epithelial ovarian cancer tumorigenesis. LncRNA MIAT promoted cell proliferation and inhibited cell apoptosis by negative regulation of miR-330-5p in epithelial ovarian cancer cells. Our findings suggested that MIAT might act as a candidate prognostic biomarker and new therapeutic target for treating epithelial ovarian cancer patients.

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.

Downregulation of lncRNA TUBA4B is Associated with Poor Prognosis for Epithelial Ovarian Cancer

A host of studies have revealed that long non-coding RNAs (lncRNAs) are critically involved in the development and progression of epithelial ovarian cancer. LncRNA TUBA4B is recently identified to be a critical mediator in non-small cell lung cancer. However, the clinical roles and biological functions of lncRNA TUBA4B in epithelial ovarian cancer have yet to be fully clarified. The present study was conducted to explore the expression of lncRNA TUBA4B in human epithelial ovarian cancer tissues and potential roles of lncRNA TUBA4B in ovarian cancer cells. The matched epithelial ovarian cancer specimens and adjacent normal tissues were employed to detect the expression of lncRNA TUBA4B. The prognostic value of lncRNA TUBA4B for tumor progression and survival rate was investigated. The effects of lncRNA TUBA4B on ovarian cancer cell proliferation and migration were also explored. The expression of lncRNA TUBA4B was significantly decreased in epithelial ovarian cancer tissue specimens. The low lncRNA TUBA4B level was closely related with pathological grade, FIGO stage and lymph node metastases, and serum CA125 level. Enforced expression of lncRNA TUBA4B obviously reduced the proliferation of SKOV3 cells, and attenuated the activation of ERK and Akt signaling pathways. Our data demonstrate for the first time that lower lncRNA TUBA4B may be a novel independent prognostic biomarker for overall survival of epithelial ovarian cancer. Overexpression of lncRNA TUBA4B inhibits the proliferation of ovarian cancer cells. LncRNA TUBA4B may be an important target for therapeutic intervention in ovarian cancer.

Targeting HSF1 leads to an antitumor effect in human epithelial ovarian cancer.

Late diagnosis and lack of specific therapeutic targets contribute to the low survival rate of patients with epithelial ovarian cancer(EOC), the most lethal gynecologic malignancy. Therefore, the screening of diagnostic markers and the identification of therapeutic targets are urgently required. Heat shock factor1 (HSF1) has been demonstrated to be overexpressed in certain malignancies and to be involved in tumor initiation, development, transformation and metastasis. It is believed that HSF1 is a promising candidate for antitumor therapy. However, its expression pattern and function in ovarian cancer are far from being fully elucidated. Therefore, we examined the HSF1 expression in human EOC tissues, and evaluated its carcinogenesis-promoting activity in a xenograft tumor model. Examination of HSF1 expression in human EOC tissues was performed by immunohistochemical assay using ovarian tissue blots. Specific short hairpin RNA (shRNA) against HSF1 was employed to knockdown HSF1 in SKOV3 cells. Cell proliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay; cell cycle distribution and apoptosis were determined by flow cytometric analysis. In normal ovarian tissues, HSF1 was barely detected, whereas, high expression of HSF1 was found in malignant EOC tissues, including serous, mucinous, endometrioid, and clear cell EOC tissues. Suppressed proliferative activity and intensified apoptosis were observed in HSF1-knockdown SKOV3 cells. In nude mouse xenografts, downregulation of HSF1 was found to cause reduced carinogenesis, indicating the antitumor effect induced by modulation of HSF1 against EOC. Our findings suggest that HSF1 may be considered as a potential candidate diagnostic marker of human EOC, and that modulation of HSF1 could be a promising therapeutic strategy against human EOC.

Let-7e sensitizes epithelial ovarian cancer to cisplatin through repressing DNA double strand break repair

BackgroundResistance to platinum-based chemotherapy remains a great challenge for ovarian cancer treatment. The human let-7 family contains 13 members located on nine different chromosomes, and most members have been implicated in the modulation of drug sensitivity in cancers. Our previous study showed that deregulation of let-7e in epithelial ovarian cancer (EOC) promoted the development of resistance to cisplatin. In the present study, we aimed to investigate the underlying mechanism and further evaluate the clinical value of let-7e in predicting chemo-response and prognosis in EOC.ResultsIn situ hybridization assays revealed a significantly decreased expression of let-7e in chemo-resistant EOC tissues compared with chemo-sensitive cases. Transfection with let-7e agomir sensitized EOC cells to cisplatin, down-regulated BRCA1 and Rad51 expression, and repressed the repair of cisplatin-induced DNA double strand break, while let-7e inhibitor exerted the opposite effects. In human EOC tissues, BRCA1 and Rad51 levels were increased in the chemo-resistant group compared with the sensitive group and were negatively correlated with let-7e. Low let-7e and high Rad51 were significantly associated with poor progression-free survival and overall survival and multivariate regression analyses showed that let-7e was an independent predictor for overall survival and chemotherapy response in EOC. Receiver operating characteristic analysis indicated that let-7e level was highly predictive of resistance to platinum-taxane chemotherapy with an area under the curve of 0.826.ConclusionsIn EOC, low let-7e leads to activation of BRCA1 and Rad51 expression and subsequent enhancement of DSB repair, which in turn results in cisplatin-resistance. Let-7e is a potential predictor for survival and chemo-response in EOC and re-expression of let-7e might be an effective strategy for overcoming chemo-resistance.

α2,3-sialyltransferase type I regulates migration and peritoneal dissemination of ovarian cancer cells.

Epithelial ovarian cancer (EOC) has the highest mortality rate among gynecologic cancers due to advanced stage presentation, peritoneal dissemination, and refractory ascites at diagnosis. We investigated the role of α2,3-sialyltransferase type I (ST3GalI) by analyzing human ovarian cancer datasets and human EOC tissue arrays. We found that high expression of ST3GalI was associated with advanced stage EOC. Transwell migration and cell invasion assays showed that high ST3GalI expression enhanced migration of EOC cells. We also observed that there was a linear relation between ST3GalI expression and epidermal growth factor receptor (EGFR) signaling in EOC patients, and that high ST3GalI expression blocked the effect of EGFR inhibitors. Co-Immunoprecipitation experiments demonstrated that ST3GalI and EGFR were present in the same protein complex. Inhibition of ST3GalI using a competitive inhibitor, Soyasaponin I (SsaI), inhibited tumor cell migration and dissemination in the in vivo mouse model with transplanted MOSEC cells. Further, SsaI synergistically enhanced the anti-tumor effects of EGFR inhibitor on EOC cells. Our study demonstrates that ST3GalI regulates ovarian cancer cell migration and peritoneal dissemination via EGFR signaling. This suggests α2,3-linked sialylation inhibitors in combination with EGFR inhibitors could be effective agents for the treatment of EOC.

Higher Numbers of T-Bet+ Tumor-Infiltrating Lymphocytes Associate with Better Survival in Human Epithelial Ovarian Cancer

Background/Aims: T-bet, a member of the T-box family of transcription factors, is a key marker of type I immune response within the tumor microenvironment, and has been previously reported by us to serve as an important prognostic indicator for human gastric cancer patients and a potential biomarker for immunotherapy. In the present study, we aimed to assess the clinical significance and prognostic value of T-bet⁺ tumor-infiltrating lymphocytes in human epithelial ovarian cancer. Methods: The immunohistochemistry was used to analyze the infiltration density of T-bet⁺ lymphoid cells in human epithelial ovarian cancer tissues, and the flow cytometry analysis was used to further analyze the presence of T-bet⁺ tumor-infiltrating lymphocytes subgroups in cancer tissues. Results: Our immunohistochemistry analysis showed increased number of T-bet⁺ lymphoid cells in the human epithelial ovarian cancer tissues, and the flow cytometry analysis further demonstrated the presence of T-bet⁺ tumor-infiltrating lymphocytes subgroups including CD4⁺ , CD8⁺ T cells and NK cells. In addition, we also observed a significant association of T-bet⁺ tumor-infiltrating lymphocytes density in the tumor nest of cancer with not only serum CA125 levels but also with distant metastasis. However no association was observed with other characteristics like patients' age, pathological type, FIGO stage, tumor site and tumor size. Furthermore, the survival analysis showed that higher density of T-bet⁺ tumor-infiltrating lymphocytes both in tumor nest and tumor stroma of cancer tissues was significantly associated with better patient survival. In addition, the density of T-bet⁺ tumor-infiltrating lymphocytes in tumor nest appeared to be an independent risk factor for predicting patients’ postoperative prognoses. Conclusions: Our data indicated that the key transcription factor T-bet might play an important role in the type I immune cells mediated antitumor response, and the density of T-bet⁺ lymphocytes in human epithelial ovarian cancer tissues could serve as a prognostic predictor for ovarian cancer patients.

Galectin-1 promotes tumor progression via NF-κB signaling pathway in epithelial ovarian cancer.

Purpose: We previously reported that Galectin-1 (Gal-1) played a role in epithelial ovarian cancer (EOC) progression. In this study, we aimed to further investigate the association between Gal-1 expression and prognosis in EOC patients and tried to reveal some novel potential mechanisms of Gal-1 in EOC invasion and migration.Materials and Methods: Gal-1 and nucleus NF-κBp65 expression in 109 human epithelial ovarian cancer tissue specimens were evaluated by immunohistochemistry. The Cox model and survival curves were used to investigate the effect of Gal-1 on EOC prognosis. Correlation between Gal-1 expression and NF-κB activation in EOC patients was also analyzed. In vitro experiments were further performed to reveal the function and mechanisms of Gal-1 in invasion and migration of EOC cells.Results: Expression level of Gal-1 in EOC tissue was an independent prognostic factor on overall survival (p<0.05) and progression-free survival (p<0.05). Patients with high Galectin-1 expression had shorter overall survival (OS, p<0.05)) and progression-free survival (PFS, p<0.05). Immunohistochemistry revealed that expression of Gal-1 was positively associated with activation of NF-κBp65 in EOC tissues (Kappa coefficient=0.458, p<0.001). Patients with tumors concomitantly co-over-expressing Gal-1 and NF-κBp65 had the worse OS (p<0.001) and PFS (p<0.001). The abilities of migration and invasion for EOC cells were significantly reduced after Gal-1 knocked-down in human EOC cell line HO8910, which was accompanied with the suppression of NF-κb pathway activation and with the matrix metalloproteinase-2 and matrix metalloproteinase-9 down-regulation.Conclusions: Our results suggest that Gal-1 is associated with poor outcome in EOC and Galectin-1 promotes tumor progression via NF-κB pathway activation in EOC.

MicroRNA-298 inhibits malignant phenotypes of epithelial ovarian cancer by regulating the expression of EZH2.

MicroRNA (miRNA or miR)-298 has been reported to be downregulated and to modify the expression of the polycomb protein enhancer of zeste 2 (EZH2) in recurrent epithelial ovarian cancer (EOC). To date, no functional evidence of a miR-298-EZH2 axis in EOC has been documented. The present study aimed to investigate the associations of miR-298 and/or EZH2 expression with clinicopathological features of EOC patients, and revealed their roles in cell motility based on EOC cell lines. Reverse transcription-quantitative polymerase chain reaction was performed to detect the expression levels of miR-298 and EZH2 messenger RNA in human EOC tissues and cell lines. Wound healing and transwell assays were performed to determine the function of the miR-298-EZH2 axis on cell migration and invasion, respectively. Compared with normal tissues, miR-298 expression was significantly downregulated, while EZH2 expression was significantly upregulated, in human EOC tissues (both P=0.001). In addition, miR-298 downregulation and EZH2 upregulation were significantly associated with high clinical stage (both P=0.01) and pathological grade (both P=0.02) of EOC patients. Furthermore, the ectopic expression of miR-298 could efficiently inhibit cell migration and invasion. Notably, the overexpression of EZH2 could restore the cell migration and invasion abilities suppressed by miR-298. Our data offer convincing evidence that the dysregulation of the miR-298-EZH2 axis may be important in tumor progression of EOC patients. The present study also confirmed a tumor-suppressive role of miR-298 in modulating EOC cell motility by regulating the expression of EZH2, implying its potential as a novel miRNA-based therapeutic target for the treatment of human EOC.

FOXM1 promotes reprogramming of glucose metabolism in epithelial ovarian cancer cells via activation of GLUT1 and HK2 transcription.

Cancer cells exhibit the reprogrammed metabolism mainly via aerobic glycolysis, a phenomenon known historically as the Warburg effect; however, the underlying mechanisms remain largely unknown. In this study, we characterized the critical role of transcription factor Forkhead box protein M1 (FOXM1) in aerobic glycolysis of human epithelial ovarian cancer (EOC) and its molecular mechanisms. Our data showed that aberrant expression of FOXM1 significantly contributed to the reprogramming of glucose metabolism in EOC cells. Aerobic glycolysis and cell proliferation were down-regulated in EOC cells when FOXM1 gene expression was suppressed by RNA interference. Moreover, knockdown of FOXM1 in EOC cells significantly reduced glucose transporter 1 (GLUT1) and hexokinase 2 (HK2) expression. FOXM1 bound directly to the GLUT1 and HK2 promoter regions and regulated the promoter activities and the expression of the genes at the transcriptional level. This reveals a novel mechanism by which glucose metabolism is regulated by FOXM1. Importantly, we further demonstrated that the expression levels of FOXM1, GLUT1 and HK2 were significantly increased in human EOC tissues relative to normal ovarian tissues, and that FOXM1 expression was positively correlated with GLUT1 and HK2 expression. Taken together, our results show that FOXM1 promotes reprogramming of glucose metabolism in EOC cells via activation of GLUT1 and HK2 transcription, suggesting that FOXM1 may be an important target in aerobic glycolysis pathway for developing novel anticancer agents.

Genetic determinants of FOXM1 overexpression in epithelial ovarian cancer and functional contribution to cell cycle progression.

The FOXM1 transcription factor network is frequently activated in high-grade serous ovarian cancer (HGSOC), the most common and lethal subtype of epithelial ovarian cancer (EOC). We used primary human EOC tissues, HGSOC cell lines, mouse and human ovarian surface epithelial (OSE) cells, and a murine transgenic ovarian cancer model to investigate genetic determinants of FOXM1 overexpression in EOC, and to begin to define its functional contribution to disease pathology. The Cancer Genome Atlas (TCGA) data indicated that the FOXM1 locus is amplified in ~12% of HGSOC, greater than any other tumor type examined, and that FOXM1 amplification correlates with increased expression and poor survival. In an independent set of primary EOC tissues, FOXM1 expression correlated with advanced stage and grade. Of the three known FOXM1 isoforms, FOXM1c showed highest expression in EOC. In murine OSE cells, combined knockout of Rb1 and Trp53 synergistically induced FOXM1. Consistently, human OSE cells immortalized with SV40 Large T antigen (IOSE-SV) had significantly higher FOXM1 expression than OSE immortalized with hTERT (IOSE-T). FOXM1 was overexpressed in murine ovarian tumors driven by combined Rb1/Trp53 disruption. FOXM1 induction in IOSE-SV cells was partially dependent on E2F1, and FOXM1 expression correlated with E2F1 expression in human EOC tissues. Finally, FOXM1 functionally contributed to cell cycle progression and relevant target gene expression in human OSE and HGSOC cell models. In summary, gene amplification, p53 and Rb disruption, and E2F1 activation drive FOXM1 expression in EOC, and FOXM1 promotes cell cycle progression in EOC cell models.

MiR-373 targeting of the Rab22a oncogene suppresses tumor invasion and metastasis in ovarian cancer.

Metastasis is major cause of mortality in patients with ovarian cancer. MiR-373 has been shown to play pivotal roles in tumorigenesis and metastasis; however, a role for miR-373 in ovarian cancer has not been investigated. In this study, we show that the miR-373 expression is down-regulated in human epithelial ovarian cancer (EOC) and inversely correlated with clinical stage and histological grade. Ectopic overexpression of miR-373 in human EOC cells suppressed cell invasion in vitro and metastasis in vivo, and the epithelial-mesenchymal transition process. Silencing the expression of miR-373 resulted in an increased migration and invasion of EOC cells. Using integrated bioinformatics analysis, gene expression arrays, and luciferase assay, we identified Rab22a as a direct and functional target of miR-373 in EOC cells. Expression levels of miR-373 were inversely correlated with Rab22a protein levels in human EOC tissues. Rab22a knockdown inhibited invasion and migration of EOC cells, increased E-cadherin expression, and suppressed the expression of N-cadherin. Moreover, overexpression of Rab22a abrogated miR-373-induced invasion and migration of EOC cells. Taken together, these results demonstrate that miR-373 suppresses EOC invasion and metastasis by directly targeting Rab22a gene, a new potential therapeutic target in EOC.

Abstract 3144: PDZ-RhoGEF/β-arrestin-1 interaction mediates endothelin A receptor-induced RhoA activation and cell motility in ovarian tumor cells

Abstract β-arrestins (β-arrs), consisting of β-arr1 and β-arr2, are involved in G-protein-coupled receptor signaling, and guide the receptor signals in chemotaxis and cytoskeletal reorganization. Among them, receptor A for endothelin-1 (ET-1), namely ETAR, is overexpressed in epithelial ovarian cancer (EOC) and promote metastatic spread. We have recently reported that β-arr1 regulates ETAR-mediated β-catenin signaling to promote EOC cell invasion. Here we evaluated the effects of ET-1 on polarized cell migration correlating with the regulation of Rho-GTPase activity and its subsequent organization of the actin structures supporting migration and the involvement of β-arr1 in these effects. We show that, in EOC cells cultured on collagen type I, ET-1 promotes actin-based dynamic protrusion resembling to pseudopodia, containing β-arr-1. Furthermore, in a β-arr1/ETAR-dependent manner, ET-1 activates RhoA GTPase and downstream the dephosphorylation/activation of cofilin, as shown by pull down assay and immunoblotting analysis. The inhibitory effect of the Rho pathway inhibitor I, Y-27632, demontrates that RhoA GTPase is involved in ET-1-induced cell migration. By screening members of a Rho-guanine nucleotide exchange factor (Rho-GEF) family involved in RhoA activation, we found that PDZ-RhoGEF mRNA levels are highly expressed in EOC cells. Moreover, PDZ-RhoGEF is responsible for ET-1-induced RhoA activation through interaction with β-arr1, that in turn, promotes PDZ-RhoGEF tyrosine phosphorylation and activation. Depletion of PDZ-RhoGEF expression significantly impaired ET-1-stimulated migration of EOC cells, demonstrating that PDZ-RhoGEF/β-arrestin-1 interaction mediates ETAR-driven ovarian tumor cell motility through regulation of RhoA. Analysis of EOC human tissues revealed that PDZ-RhoGEF is overexpressed in human EOC tissues. Collectively, our data establish a novel role for β-arr1 and PDZ-RhoGEF as regulator of ET-1-induced EOC cell migration, unraveling new components required for tumor progression. Supported by AIRC. Citation Format: Piera Tocci, Roberta Cianfrocca, Elisa Semprucci, Valeriana Di Castro, Anna Bagnato, Laura Rosanò. PDZ-RhoGEF/β-arrestin-1 interaction mediates endothelin A receptor-induced RhoA activation and cell motility in ovarian tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3144. doi:10.1158/1538-7445.AM2014-3144

MicroRNA-150 predicts a favorable prognosis in patients with epithelial ovarian cancer, and inhibits cell invasion and metastasis by suppressing transcriptional repressor ZEB1.

MicroRNA (miR)-150 has been reported to be dramatically downregulated in human epithelial ovarian cancer (EOC) tissues and patients’ serum compared to normal controls. This study aimed to investigate clinical significance and molecular mechanisms of miR-150 in EOC. In the current study, quantitative real-time PCR analysis showed that miR-150 was significantly downregulated in human EOC tissues compared to normal tissue samples. Then, we demonstrated the significant associations of miR-150 downregulation with aggressive clinicopathological features of EOC patients, including high clinical stage and pathological grade, and shorter overall and progression-free survivals. More importantly, the multivariate analysis identified miR-150 expression as an independent prognostic biomarker in EOC. After that, luciferase reporter assays demonstrated that Zinc Finger E-Box Binding Homeobox 1 (ZEB1), a crucial regulator of epithelial-to-mesenchymal transition (EMT), was a direct target of miR-150 in EOC cells. Moreover, we found that the ectopic expression of miR-150 could efficiently inhibit cell proliferation, invasion and metastasis by suppressing the expression of ZEB1. Furthermore, we also observed a significantly negative correlation between miR-150 and ZEB1 mRNA expression in EOC tissues (rs = –0.45, P<0.001). In conclusion, these findings offer the convincing evidence that aberrant expression of miR-150 may play a role in tumor progression and prognosis in patients with EOC. Moreover, our data reveal that miR-150 may function as a tumor suppressor and modulate EOC cell proliferation, and invasion by directly and negatively regulating ZEB1, implying the re-expression of miR-150 might be a potential therapeutic strategy for EOC.