Sustained suppression of Fas ligand expression in cisplatin-resistant human ovarian surface epithelial cancer cells

Although cisplatin derivatives are first line chemotherapeutic agents for the treatment of ovarian epithelial cancer, chemoresistance is a major therapeutic problem. Although the cytotoxic effect of these agents are believed to be mediated through the induction of apoptosis, the role of the Fas/FasL system in chemoresistance in human ovarian epithelial cancer is not fully understood. In the present study, we have used cultures of established cell lines of cisplatin-sensitive human ovarian epithelial tumours (OV2008 and A2780-s) and their resistant variants (C13* and A2780-cp, respectively) to assess the role ofFas/FasL system in the chemo-responsiveness of ovarian cancer cells to cisplatin. Cisplatin was effective in inducing the expression of cell-associated Fas and FasL, soluble FasL and apoptosis in concentration and time-dependent fashion in both cisplatin-sensitive cell lines (OV2008 and A2780-s). In contrast, while cisplatin was effective in increasing cell-associated Fas protein content in C13*, it failed to up-regulate FasL (cell-associated and soluble forms) and induce apoptosis, irrespective of concentration and duration of cisplatin treatment. Concentrated spent media from OV2008 cultures after cisplatin treatment were effective in inducing apoptosis in C13* cells which was partly inhibited by the antagonistic Fas monoclonal antibody (mAb) suggesting that the soluble FasL present in the spent media was biologically active. In the resistant A2780-cp cells, neither Fas nor FasL up-regulation were evident in the presence of the chemotherapeutic agent and apoptosis remained low compared to its sensitive counterpart. Activation of the Fas signalling pathway, by addition to the cultures an agonistic Fas mAb, was equally effective in inducing apoptosis in the cisplatin-sensitive (OV2008) and -resistant variant C13*, although these responses were of lower magnitude compared to that observed with cisplatin in the chemosensitive cells. A significant interaction between cisplatin and agonistic Fas mAb was observed in the apoptotic response in OV2008 and C13* when cultured in the presence of both agents. Immunohistochemistry of human ovarian epithelial carcinomas reveals the presence of Fas in low abundance in proliferatively active cells but in high levels in quiescent ones. Although the expression pattern of FasL in the tumour was similar to that of Fas, the protein content was considerably lower. Taken together, these data suggest that the dysregulation of the Fas/FasL system may be an important determinant in cisplatin resistance in ovarian epithelial cancer cells. Our results are also supportive of the notion that combined immuno- and chemo-therapy (i.e., agonistic Fas mAb plus cisplatin) may provide added benefits in the treatment of both chemo-sensitive and -resistant ovarian tumours.     

Overexpression of CXCR4 is significantly associated with cisplatin-based chemotherapy resistance and can be a prognostic factor in epithelial ovarian cancer

The chemokine receptor 4 (CXCR4) plays an important role in the growth, angiogenesis and metastasis of various cancers, including epithelial ovarian cancer (EOC). However, the correlation between CXCR4 and the clinical response of EOC patients to chemotherapy remains unknown. 124 EOC patients were recruited to assess the relationship between CXCR4 and the response to cisplatin-based chemotherapy. The results showed that patients with a higher CXCR4 expression had a significantly lower chemosensitivity, a poorer progression-free survival and a lower overall survival than those with lower CXCR4 expression. In addition, knockdown of CXCR4 by small interfering RNA suppressed cell proliferation and resulted in G1/S arrest, increased apoptosis and chemosensitivity in both cisplatin-sensitive A2780 cells and cisplatin-resistant cell A2780/cis in vitro. Our data suggest that CXCR4 is one of the key molecules in cisplatin-based chemotherapy for EOC patients and that CXCR4 inhibition is a potential strategy to address the chemoresistance of EOC. [BMB Reports 2014; 47(1): 33-38]     

Clinicopathologic predictors of early relapse in advanced epithelial ovarian cancer: development of prediction models using nationwide data

ObjectiveTo identify clinicopathologic factors predictive of early relapse (platinum-free interval (PFI) of ≤6 months) in advanced epithelial ovarian cancer (EOC) in first-line treatment, and to develop and internally validate risk prediction models for early relapse.MethodsAll consecutive patients diagnosed with advanced stage EOC between 01-01-2008 and 31-12-2015 were identified from the Netherlands Cancer Registry. Patients who underwent cytoreductive surgery and platinum-based chemotherapy as initial EOC treatment were selected. Two prediction models, i.e. pretreatment and postoperative, were developed. Candidate predictors of early relapse were fitted into multivariable logistic regression models. Model performance was assessed on calibration and discrimination. Internal validation was performed through bootstrapping to correct for model optimism.ResultsA total of 4,557 advanced EOC patients were identified, including 1,302 early relapsers and 3,171 late or non-relapsers. Early relapsers were more likely to have FIGO stage IV, mucinous or clear cell type EOC, ascites, >1 cm residual disease, and to have undergone NACT-ICS. The final pretreatment model demonstrated subpar model performance (AUC = 0.64 [95 %-CI 0.62−0.66]). The final postoperative model based on age, FIGO stage, pretreatment CA-125 level, histologic subtype, presence of ascites, treatment approach, and residual disease after debulking, demonstrated adequate model performance (AUC = 0.72 [95 %-CI 0.71−0.74]). Bootstrap validation revealed minimal optimism of the final postoperative model.ConclusionA (postoperative) discriminative model has been developed and presented online that predicts the risk of early relapse in advanced EOC patients. Although external validation is still required, this prediction model can support patient counselling in daily clinical practice.     

Targeted therapies in epithelial ovarian cancer: Molecular mechanisms of action

Ovarian cancer is the leading cause of death in women with gynecological cancer. Most patients are diagnosed at an advanced stage and have a poor prognosis. Currently, surgical tumor debulking, followed by platinum- and taxane-based chemotherapy is the standard treatment for advanced ovarian cancer. However, these patients are at great risk of recurrence and emerging drug resistance. Therefore, novel treatment strategies are required to improve outcomes for women with advanced ovarian cancer. A variety of molecular targeted agents, the majority of which are monoclonal antibodies and small-molecule protein-kinase inhibitors, have been explored in the management of ovarian cancer. The targets of these agents include angiogenesis, the human epidermal growth factor receptor family, ubiquitin-proteasome pathway, epigenetic modulators, poly(ADP-ribose) polymerase (PARP), and mammalian target of rapamycin (mTOR) signaling pathway, which are aberrant in tumor tissue. The antiangiogenic agent, bevacizumab, has been reported as the most effective targeted agent and should be included in the standard chemotherapeutic regimen for advanced ovarian cancer. PARP inhibitors, which are mainly used in breast and ovarian cancer susceptibility gene-mutated patients, and mTOR inhibitors are also attractive treatment strategies, either alone or combination with chemotherapy, for ovarian cancer. Understanding the tumor molecular biology and identification of predictive biomarkers are essential steps for selection of the best treatment strategies. This article reviews the molecular mechanisms of the most promising targeted agents that are under early phase clinical evaluation for ovarian cancer.     

miRNA-21 inhibition suppresses the human epithelial ovarian cancer by targeting PTEN signal pathway

ObjectTo explore the role of microRNA-21 in human epithelial ovarian cancer (EOC).MethodsWe used RT-PCR to test the expressions of miRNA-21 in EOC cells and normal ovarian epithelial cells, as well as the tumor samples and the tumor-adjacent normal tissues. The vector of LV3 pGLV-H1-GFP-miR-21 was used to decrease the level expression of endogenous miR-21 in cells. Further, we investigated how miR-21 affected the biological events of EOC through determining the changes in proliferation, cycle and invasion of EOC cells, and measured the tumorigenesis in xenograft models. The association between phosphatase and tensin homolog deleted on chromosome ten (PTEN) and miR-21 were tested by RT-PCR. Next, siRNA was used to knockdown PTEN gene which help us to assess the functional association between miR-21 and PTEN in vivo and in vitro.ResultsIn EOC cell lines and human epithelial ovarian tumor cells, we found that miR-21 altered the biological features of EOC cells, including suppression of proliferation and invasion and arrest of cell cycle, and also resulted in a decrease in tumorigenesis in the in vitro xenograft models. The association between PTEN and miR-21 was confirmed in previous research. From our results, the down-regulation of PTEN gave rise to the miR-21 decrease, regardless of the cells or tissues.ConclusionThe suppression of microRNA-21 inhibits the progression of EOC profoundly. In EOC, miR-21 is negatively correlated with the expression of PTEN gene.     

A short peptide derived from the antisense homology box of Fas ligand induces apoptosis in anti-Fas antibody-insensitive human ovarian cancer cells

We found that a short synthetic peptide corresponding to the antisense homology box of Fas ligand induced apoptotic cell death of Fas-expressing human ovarian cancer cell lines. The peptide was deduced from residues 256–265 of human Fas ligand, based on the hypothesis that it should contain a specific binding site to the corresponding Fas. Interestingly, the ovarian cancer cell line NOS4, which was sensitive to anti-Fas antibody induced apoptosis, was not affected by the peptide, whereas another cell line, SKOV-3, which was insensitive to anti-Fas antibody, was killed by the peptide. Thus, this short peptide was shown to have a unique activity to induce apoptosis in human ovarian cancer cells in a manner different from anti-Fas antibody.     

Role of BRCA1 and BRCA2 gene mutations in epithelial ovarian cancer in Indian population: a pilot study

Ovarian cancer is a silent killer as most patients have non-specific symptoms and usually present in advanced stage of the disease. It occurs due to certain genetic alterations and mutations namely founder mutations, 187delAG and 5385insC in BRCA1 and 6174delT in BRCA2 which are associated with specific family histories. These highly penetrant susceptibility genes responsible for approximately half of families containing 2 or more ovarian cancer cases account for less than 40% of the familial excess malignancy risk. The remaining risk may be due to single nucleotide polymorphisms (SNPs) which are single base change in a DNA sequence with usual alternatives of two possible nucleotides at a given position. Preliminary study involving 30 women with histologically proven epithelial ovarian cancer was conducted and their detailed genetic analysis was carried out. Regions of founder mutations on BRCA1 and BRCA2 were amplified and sequenced using primers designed based on 200 bp upstream and downstream regions of the mutation sites. Five sequence variants in BRCA1 were identified of which three novel sequence variants were found in 23 patients while in BRCA2, one novel sequence variant was found. The three founder mutations 187delAG, 5385insC in BRCA1 and 6174delT in BRCA2 were not seen in any of the subjects.     

Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients

Early detection of resistance to platinum-based therapy is critical for improving the treatment of ovarian cancers. We have previously found that increased expression of annexin A3 is a mechanism for platinum resistance in ovarian cancer cells. Here we demonstrate that annexin A3 can be detected in the culture medium of ovarian cancer cells, particularly these cells that express high levels of annexin A3. Levels of annexin A3 were then determined in sera from ovarian cancer patients using an enzyme-linked immunosorbent assay. Compared with those from normal donors, sera from ovarian cancer patients contain significantly higher levels of annexin A3. Furthermore, serum levels of annexin A3 were significantly higher in platinum-resistant patients than in platinum-sensitive patients. To gain insight into the mechanism of secretion, the ovarian cancer cell lines were examined using both transmission electron microscopy and immunoelectron microscopy. Compared with parent cells, there are significantly more vesicles in the cytoplasm of ovarian cancer cells that express high levels of annexin A3, and at least some vesicles are annexin A3-positive. Moreover, some vesicles appear to be fused with the cell membrane, suggesting that annexin A3 secretion may be associated with exocytosis and the release of exosomes. This is supported by our observation that ovarian cancer cells expressing higher levels of annexin A3 released increased numbers of exosomes. Furthermore, annexin A3 can be detected in exosomes released from cisplatin-resistant cells (SKOV3/Cis) by immunoblotting and immunoelectron microscopy.     

Molecular pathogenesis and therapeutic targets in epithelial ovarian cancer

Ovarian cancer, the most aggressive gynecologic cancer, is the foremost cause of death from gynecologic malignancies in the developed world. Two primary reasons explain its aggressive behavior: most patients present with advanced disease at diagnosis, and die of recurrences from disease that has become resistant to conventional chemotherapies. In this paper on epithelial ovarian cancer (EOC), we will review molecular alterations associated with the few precursor lesions identified to date, followed by the more commonly recognized processes of de novo carcinogenesis, metastasis, and the development of chemoresistance. We will propose a unifying model of ovarian epithelial tumorigenesis that takes into account various hypotheses. We will also review novel approaches to overcome the major problem of chemoresistance in ovarian cancer. Finally, we will discuss advances and new challenges in the development of mouse model systems to investigate EOC precursor lesions, progression, metastasis, and chemoresistance.     

Loss of 4.1N in epithelial ovarian cancer results in EMT and matrix-detached cell death resistance

Epithelial ovarian cancer(EOC)is one of the leading causes of death from gynecologic cancers and peritoneal dissemination is the major cause of death in patients with EOC.Although the loss of 4.1N is associated with increased risk of malignancy,its association with EOC remains unclear.To explore the underlying mechanism of the loss of 4.1N in constitutive activation of epithelial-mesenchymal transition(EMT)and matrixdetached cell death resistance,we investigated samples from 268 formalin-fixed EOC tissues and performed various in vitro and in vivo assays.We report that the loss of 4.1N correlated with progress in clinical stage,as well as poor survival in EOC patients.The loss of 4.1N induces EMT in adherent EOC cells and its expression inhibits anoikis resistance and EMT by directly binding and accelerating the degradation of 14-3-3 in suspension EOC cells.Furthermore,the loss of 4.1N could increase the rate of entosis,which aggravates cell death resistance in suspension EOC cells.Moreover,xenograft tumors in nude mice also show that the loss of 4.1N can aggravate peritoneal dissemination of EOC cells.Single-agent and combination therapy with a ROCK inhibitor and a 14-3-3 antagonist can reduce tumor spread to varying degrees.Our results not only define the vital role of 4.1N loss in inducing EMT,anoikis resistance,and entosis-induced cell death resistance in EOC,but also suggest that individual or combined application of 4.1N,14-3-3 antagonists,and entosis inhibitors may be a promising therapeutic approach for the treatment of EOC.     

Vasohibin-1基因在上皮性卵巢癌中的表达和临床病理意义

目的:探讨卵巢癌中Vasohibin-1的表达情况及临床病理学意义。方法:采用实时定量PCR和免疫组化方法检测在60例上皮性卵巢癌和12例正常卵巢组织中Vasohibin-1的表达情况,ELISA法检测卵巢癌组织中VEGF蛋白表达,分析vasohibin-1表达与VEGF之间的关系及其与卵巢癌分期,分级和预后之间的关系。结果:卵巢癌中vasohibin-1表达明显高于正常卵巢(P0.05)。Vasohibin-1表达水平与卵巢癌分期相关(P0.05),而与卵巢癌分级和淋巴转移无关。Vasohibin-1表达与VEGF蛋白水平呈正相关(P0.01)。高vasohibin-1表达卵巢癌患者三年生存率(50%)低于低vasohibin-1表达的患者(83%)。结论:vasohibin-1可以反映卵巢癌的血管生成潜能,是卵巢癌的不良预后因素。     

Common miRNAs,candidate genes and their interaction network across four subtypes of epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is categorized into four major histological subtypes such as clear cell carcinoma (CCC), endometrioid carcinoma (EC), mucinous carcinoma (MC), and serous carcinoma (SC). Heterogeneity of the EOC leads to different clinical outcomes of the disease, although all the subtypes are originated from the same layer of tissue. Therefore, it is of interest to identify the common candidate genes, miRNA and their interaction network in four the subtypes of EOC. A comparative gene expression analysis identified 248 common differentially expressed genes (DEGs) in the four subtypes of EOC. Identified common DEGs were found to be enriched in cancer specific pathways. A protein-protein interaction (PPI) network of the common DEGs were constructed, and subsequent module and survival analyses identified seven key candidate genes (CCNB1, CENPM, CEP55, RACGAP1, TPX2, UBE2C, and ZWINT). We also documented 10 key candidate miRNAs (hsa-mir-16-5p, hsa-mir-23b-3p, hsa-mir-34a-5p, hsa-mir-103a-3p, hsa-mir-107, hsa-mir-124-3p, hsa-mir-129-2-3p, hsa-mir-147a, hsa-mir-205-5p, and hsa-mir-195-5p) linked to the candidate genes. These derived data find application in the understanding of EOC.     

Galectin-1 overexpression promotes progression and chemoresistance to cisplatin in epithelial ovarian cancer

This study was performed to investigate the role of galectin-1 (Gal-1) in epithelial ovarian cancer (EOC) progression and chemoresistance. Tissue samples from patients with EOC were used to examine the correlation between Gal-1 expression and clinical stage of EOC. The role of Gal-1 in EOC progression and chemoresistance was evaluated in vitro by siRNA-mediated knockdown of Gal-1 or lentivirus-mediated overexpression of Gal-1 in EOC cell lines. To elucidate the molecular mechanisms underlying Gal-1-mediated tumor progression and chemoresistance, the expression and activities of some signaling molecules associated with Gal-1 were analyzed. We found overexpression of Gal-1 in advanced stages of EOC. Knockdown of endogenous Gal-1 in EOC cells resulted in the reduction in cell growth, migration, and invasion in vitro, which may be caused by Gal-1''s interaction with H-Ras and activation of the Raf/extracellular signal-regulated kinase (ERK) pathway. Additionally, matrix metalloproteinase-9 (MMP-9) and c-Jun were downregulated in Gal-1-knockdown cells. Notably, Gal-1 overexpression could significantly decrease the sensitivities of EOC cells to cisplatin, which might be ascribed to Gal-1-induced activation of the H-Ras/Raf/ERK pathway and upregulation of p21 and Bcl-2. Taken together, the results suggest that Gal-1 contributes to both tumorigenesis and cisplatin resistance in EOC. Thus, Gal-1 is a potential therapeutic target for EOC.     

Activation of caspase-8 is critical for sensitivity to cytotoxic anti-Fas antibody-induced apoptosis in human ovarian cancer cells

Two ovarian cancer cell lines named NOS4 and SKOV-3 have been shown to have different sensitivities to a cytotoxic anti-Fas antibody, CH-11. Although both cell lines express Fas molecules on the cell surfaces at the same intensities, apoptosis is induced by CH-11 in NOS4 cells but not in SKOV-3 cells. In this study, the different apoptosis-sensitivities of these cells were assessed. Both cell lines express almost the same levels of FADD, RIP, c-FLIP, FAP-1, Bax, Bcl-2 and Bcl-XL. Evidence of caspase-8, caspase-9 and caspase-3 activation and of cleavage of PARP and Bid was obtained in NOS4 cells but not in SKOV-3 cells. When triggered by FasL protein, DNA fragmentation and caspase-8 activation were observed in SKOV-3 cells, though they were not as clear as in NOS4 cells. All the anti-Fas antibody-mediated signals for apoptosis induction in NOS4 cells were completely blocked by a caspase-8-specific inhibitor, Z-IETD-FMK. These results indicate that the different sensitivities to the anti-Fas antibody are solely dependent on the activation of caspase-8, which could be influenced by yet unknown qualitative or quantitative abnormalities in molecules involved in DISC formation.     

In-depth proteomic analyses of ovarian cancer cell line exosomes reveals differential enrichment of functional categories compared to the NCI 60 proteome

Molecular communication between cancer cells and its stromal microenvironment is a key factor for cancer progression. Alongside classic secretory pathways, it has recently been proposed that small membranous vesicles are alternative mediators of intercellular communication. Exosomes carry an effector-rich proteome with the ability to modulate various functional properties of the recipient cell. In this study, exosomes isolated from four epithelial ovarian cancer cell lines (OVCAR3, OVCAR433, OVCAR5 and SKOV3) were characterized using mass spectrometry-based proteomics. Using an optimized workflow consisting of efficient exosome solubilization and the latest generation of proteomic instrumentation, we demonstrate improved detection depth. Systematic comparison of our cancer cell line exosome proteome against public data (Exocarta) and the recently published NCI 60 proteome revealed enrichment of functional categories related to signaling biology and biomarker discovery.     

Knockdown of TMEM14A expression by RNAi inhibits the proliferation and invasion of human ovarian cancer cells

Transmembrane protein 14A (TMEM14A) is a member of TMEMs. Alterations in TMEMs expression have been identified in several types of cancer, but the expression and function of TMEM14A in ovarian cancer is still unclear. Here, analysis on the expression data of the Cancer Genome Atlas (TCGA) ovarian serous cystadenocarcinoma (OV) dataset demonstrated the overexpression of TMEM14A in ovarian cancer tissues compared with normal tissues, which was consistent with our real-time PCR analysis on ovarian cancer and normal tissues collected from 30 patients. In addition, TMEM14A knockdown in two ovarian cancer cell lines, A2780 and HO-8910, reduced cell proliferation, causes cell cycle arrest and suppressed cell invasion. Moreover, silencing of TMEM14A notably repressed G1/S cell cycle transition and cell invasion via down-regulating the expression of cell cycle related proteins (Cyclin D1, Cyclin E and PCNA) and metastasis-related proteins (MMP-2 and MMP-9), respectively. TMEM14A knockdown significantly reduced the phosphorylation status of Smad2 and Smad3, downstream effectors of TGF-β signalling. In summary, these results indicate that TMEM14A has a pro-tumorigenic effect in ovarian cancer cells, suggesting an important role of this protein in ovarian cancer oncogenesis and metastasis.     

Roles of metformin-mediated girdin expression in metastasis of epithelial ovarian cancer

Antimetastatic effect of Metformin has been documented in epithelial ovarian cancer (EOC). Presently, we investigated the regulatory mechanism of Metformin in EOC metastasis. First, Girdin was significantly enhanced in EOC tumorous tissues and cell lines. Seconded, knockdown of Girdin significantly suppressed EOC cell viability, migration, and invasion, while upregulation of Girdin produced the opposite effects in vitro and facilitated lung metastasis in EOC cell xenograft in vivo. In addition, we confirmed that the inhibitory effect of Metformin on Girdin expression. Mechanistically, the oncogenic effects of Girdin could be reversed by LY294002 (an AKT pathway inhibitor) and Metformin. These results suggested that Metformin attenuated EOC metastasis through Girdin and targeting Girdin may be a promising therapeutic strategy for EOC in the future.