Human Ovarian Cancer Tissues Research Articles

Hexokinase 2 promoted cell motility and proliferation by activating Akt1/p-Akt1 in human ovarian cancer cells

BackgroundRecently, increasing evidence has indicated that elevation of Hexokinase 2 (HK2) plays an important role in several cancers on regulating cell motility and growth. However, its role on regulating cell EMT in human ovarian cancer still less to known.MethodsThe transwell and wound-healing assay were used to detect the effective of HK2 on regulating motility of ovarian cancer cells. Real Time PCR and Western Blotting were used to explore the changing of EMT-related proteins in HK2-modified cells. The clonogenic formation, cell growth curves and MTT assays were used to evaluate the effective of HK2 on regulating cell proliferation in HK2-modified cells. The flow cytometry was used to detect the differences in the distribution of cells in the cell cycle between the HK2-modified cells and their control cells. The correlation of HK2 and Akt1/p-Akt1 was explored by using Western Blotting, Akt1 inhibitor (MK2206) and transient transfection of an Akt1 recombinant plasmid. The potential correlation between HK2 and EMT-related proteins in human ovarian cancer tissues and OV (ovarian serous cystadenocarcinoma) was confirmed by using Pearson correlation analysis and TIMER 2.0.ResultsIn ovarian cancer cells, overexpressing of HK2 enhanced cell motility by inducing of EMT-related proteins, such as CDH2, fibronectin, MMP9, ZEB1, ZEB2 and vimentin. Moreover, overexpressing of HK2 promoted cell growth by reducing p21 and p27 expression in ovarian cancer cells. Further studies demonstrated that this promotion of cell motility and growth by HK2 was probably a result of it activating of Akt1 (p-Akt1) in ovarian cancer cells. Additionally, the positive correlation between HK2 and p-Akt1, fibronectin, MMP9 expression in human ovarian cancer samples was verified by using Pearson correlation analysis. The positive correlation between HK2 and CDH2, fibronectin, MMP9, ZEB1, ZEB2 and vimentin in OV (ovarian serous cystadenocarcinoma) was confirmed by using TIMER 2.0.ConclusionThis study demonstrated that HK2 could induce EMT-related proteins and reduce cell cycle inhibitor by activating Akt1 in human ovarian cancer cells, subsequently enhancing cell motility and growth, suggesting that HK2 participate in the malignant process of ovarian cancer by interacting with Akt1.

CpG-binding protein CFP1 promotes ovarian cancer cell proliferation by regulating BST2 transcription

Epigenetic alterations have been functionally linked to ovarian cancer development and occurrence. The CXXC zinc finger protein 1 (CFP1) is an epigenetic regulator involved in DNA methylation and histone modification in mammalian cells. However, its role in ovarian cancer cells is unknown. Here, we show that CFP1 protein is highly expressed in human ovarian cancer tissues. Loss of CFP1 inhibited the growth of human ovarian cancer cells, promoted apoptosis, and increased senescence. CFP1 knockdown resulted in reduced levels of SETD1 (a CFP1 partner) and histone H3 trimethylation at the fourth lysine residue (H3K4me3). RNA-sequencing revealed that deletion of CFP1 resulted in mRNA reduction of bone marrow stromal cell antigen 2 (BST2). Bioinformatics analysis and chromatin immunoprecipitation showed that CFP1 binds to the promoter of BST2 and regulates its transcription directly. Overexpression of BST2 rescued the growth inhibitory effect of CFP1 loss. Furthermore, depletion of cullin-RING ubiquitin ligases 4 (CRL4) components ROC1 or CUL4A had significantly inhibited the expression of CFP1 and BST2 similar to MLN4924 treatment that blocked cullin neddylation and inactivated CRL4s. In conclusion, CFP1 promotes ovarian cancer cell proliferation and apoptosis by regulating the transcription of BST2, and the expression of CFP1 was affected by CRL4 ubiquitin ligase complex.

MiR-135b improves proliferation and regulates chemotherapy resistance in ovarian cancer

MicroRNAs act as regulators in ovarian tumorigenesis and progression by involving different molecular pathways. Here, we examined the role of miR-135b on growth, chemotherapy resistance in OVCAR3 and SKOV3 ovarian cancer cells. MTT assay was performed to examine proliferation. Transwell migration and matrigel invasion assays were used to assess migration and invasion. Caspase-Glo3/7 assay was carried out to evaluate apoptosis. The dual-luciferase reporter assay was performed to validate the putative binding site. Meanwhile, the miR-135b levels in human ovarian cancer tissue were detected by qPCR assay. Overexpression of miR-135b increased growth, and improved migration and invasion in ovarian cancer cells. Meanwhile, overexpression of miR-135b decreased the cisplatin treatment sensitivity in OVCAR3 and SKOV3 cells. The cisplatin-induced apoptosis was decreased by miR-135b. Furthermore, miR-135b could alter epithelial to mesenchymal transition (EMT) associated proteins expression including E-cadherin, N-cadherin, snail and Vimentin in ovarian cancer cells. Further study demonstrated aberrant expression of miR-135b regulated PTEN and p-AKT expression in ovarian cancer cells. The expression level of miR-135b was increased in human ovarian cancer tissue, compared with normal ovary tissue. MiR-135b involves in tumorigenesis and progression in ovarian cancer cells, and might serve as a promising biomarker to predict chemotherapy sensitivity and prognosis in ovarian cancer.

MiR-600 promotes ovarian cancer cells stemness, proliferation and metastasis via targeting KLF9

Previous studies have revealed that miRNAs participate in the pathogenesis of ovarian cancer; however, whether miR-600 is also involved remains unclear. In this study, we aimed to investigated the role of miR-600 in ovarian cancer progression. Here, miR-600 expression was significantly upregulated in ovarian cancer tissues and stem cells. Functional studies showed that miR-600 promoted ovarian cancer cell stemness, proliferation and metastasis. Mechanistic studies revealed that Kruppel like factor 9 (KLF9) was indicated as the target of miR-600. The luciferase reporter assay suggested that miR-600 directly bound to the 3′-untranslated region of KLF9. Additionally, miR-600 expression was negatively associated with KLF9 expression in human ovarian cancer tissues. Si-KLF9 partially abolished the discrepancy of self-renewal, growth and metastasis capacity between miR-600 knockdown ovarian cancer cells and control cells. In conclusion, our results suggest that miR-600 promotes ovarian cancer cell stemness, proliferation and metastasis via directly downregulating KLF9, and impairing miR-600 levels may be a new treatment strategy for ovarian cancer in the future.

Online Quaternized Derivatization Mapping and Glycerides Profiling of Cancer Tissues by Laser Ablation Carbon Fiber Ionization Mass Spectrometry.

Mass spectrometry imaging has become a hot research field owing to its ability to reflect the distribution of multiple metabolites in tissue. However, not all kinds of metabolites have great ionization efficiency in mass spectrometry imaging. The mass signals of low polar metabolites like monoglycerides and diglycerides may be seriously suppressed. Many strategies have been proposed to fix the problem, such as on-tissue derivatization and online derivatization. Also, some challenges were encountered when implementing these approaches. Herein, a platform coupled online quaternized derivatization and laser ablation carbon fiber ionization mass spectrometry imaging has been developed. The mass signals of monoglycerides and diglycerides were drastically increased in the platform, and high-quality mass images of these metabolites could be acquired readily. In the platform, metabolites were first desorbed by a laser and then reacted online with a derivatization reagent transmitted by carbon fiber ionization, which also undertook the postionization of derivatization products. Pyridine acted as the main derivatization reagent to target metabolites with hydroxyl groups. Remarkably, the derivatization reaction proceeded rapidly without any catalyst owing to the high energy provided by the laser. The mass images of eight monoglycerides and 21 diglycerides were achieved after applying the platform into human ovarian cancer tissues. Notably, a higher mass intensity of these glycerides was captured in cancerous tissues than in para-cancerous tissues, which might infer aberrations in glyceride metabolisms of cancerous tissues.

The roles of HOXB8 through activating Wnt/β-catenin and STAT3 signaling pathways in the growth, migration and invasion of ovarian cancer cells.

Ovarian cancer is one of the gynecological malignancies ranked third in incidence and first in mortality in the world. Homoboxb8 (HOXB8) has been demonstrated to play crucial roles in various tumors. However, the function of HOXB8 in ovarian cancer remains to be addressed. Quantitative real-time polymerase chain reaction, immunohistochemistry staining and western blot assays demonstrated that HOXB8 expression was up-regulated in human ovarian cancer tissues and cells. The results of CCK-8 and colony formation assays indicated that HOXB8 promoted the proliferation of ovarian cancer cells. Transwell and immunofluorescence (IF) staining assay demonstrated that HOXB8 promoted the migration and invasion of ovarian cancer cells. Importantly, mechanism analysis implied that HOXB8 increased the expression of β-catenin and phosphorylation of STAT3, and the downstream target molecules of Cyclin D1, c-Myc, TWIST1, MMP7 and MMP9, indicating that HOXB8 could promote the activation of Wnt/β-catenin and STAT3 pathways. Moreover, HOXB8 knockdown suppressed xenograft tumor growth, and inhibited the levels of HOXB8 and Ki-67, while increasing the level of E-cadherin in mice. In conclusion, HOXB8 promotes cell proliferation, migration and invasion through modulating Wnt/β-catenin and STAT3 signaling pathways in ovarian cancer, suggesting that HOXB8 may provide a promising target for the therapy of ovarian cancer.

Role of alternatively spliced, pro-survival Protein Kinase C delta VIII (PKCδVIII) in ovarian cancer.

Ovarian cancer is the deadliest malignant disease in women. Protein Kinase C delta (PRKCD; PKCδ) is serine/threonine kinase extensively linked to various cancers. In humans, PKCδ is alternatively spliced to PKCδI and PKCδVIII. However, the specific function of PKCδ splice variants in ovarian cancer has not been elucidated yet. Hence, we evaluated their expression in human ovarian cancer cell lines (OCC): SKOV3 and TOV112D, along with the normal T80 ovarian cells. Our results demonstrate a marked increase in PKCδVIII in OCC compared to normal ovarian cells. Therefore, we elucidated the role of PKCδVIII and the underlying mechanism of its expression in OCC. Using overexpression and knockdown studies, we demonstrate that PKCδVIII increases cellular survival and migration in OCC. Further, overexpression of PKCδVIII in T80 cells resulted in increased expression of Bcl2 and knockdown of PKCδVIII in OCC decreased Bcl2 expression. Using co‐immunoprecipitations and immunocytochemistry, we demonstrate nuclear localization of PKCδVIII in OCC and further show increased association of PKCδVIII with Bcl2 and Bcl‐xL in OCC. Using PKCδ splicing minigene, mutagenesis, siRNA and antisense oligonucleotides, we demonstrate that increased levels of alternatively spliced PKCδVIII in OCC is regulated by splice factor SRSF2. Finally, we verified that PKCδVIII levels are elevated in samples of human ovarian cancer tissue. The data presented here demonstrate that the alternatively spliced, signaling kinase PKCδVIII is a viable target to develop therapeutics to combat progression of ovarian cancer.

Dual mTORC1/2 inhibitor AZD2014 diminishes myeloid-derived suppressor cells accumulation in ovarian cancer and delays tumor growth.

Mechanistic target of rapamycin (mTOR) forms two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2. Here we investigated the antitumor effect of dual mTORC1/2 inhibitor AZD2014 on epithelial ovarian cancer (EOC) and its potential effect on immunosuppressive myeloid-derived suppressor cells (MDSCs). Immunohistochemical analysis of mTORC1 and mTORC2 was performed on a human ovarian cancer tissue microarray. High mTORC2 expression level was associated with shorter survival in EOC, whereas mTORC1 was not correlate with patients' prognosis. AZD2014 suppressed mTOR signaling pathway in ovarian cancer cells, inhibited proliferation and induced G1-phase cell cycle arrest and apoptosis. In tumor-bearing mice, AZD2014 treatment limited tumor growth, reduced peritoneal ascites, and prolonged survival. AZD2014 specifically reduced MDSCs migration and accumulation in EOC peritoneal fluid but not in the spleen. Moreover, subsequent AZD2014 treatment after cisplatin chemotherapy delayed EOC recurrence. Collectively, we observed that high mTORC2 expression level in EOC indicated a poor prognosis. Remarkably, in tumor-bearing mice, AZD2014 diminished MDSC accumulation and delayed tumor growth and recurrence.

Rhizoctonia bataticola lectin induces apoptosis and inhibits metastasis in ovarian cancer cells by interacting with CA 125 antigen differentially expressed on ovarian cells.

A N-glycan specific lectin from Rhizoctonia bataticola [RBL] was shown to induce growth inhibitory and apoptotic effect in human ovarian, colon and leukemic cells but mitogenic effect on normal PBMCs as reported earlier, revealing its clinical potential. RBL has unique specificity for high mannose tri and tetra antennary N-glycans, expressed in ovarian cancer and also recognizes glycans which are part of CA 125 antigen, a well known ovarian cancer marker. Hence, in the present study diagnostic and therapeutic potential of RBL was investigated using human ovarian epithelial cancer SKOV3 and OVCAR3 cells known for differentially expressing CA 125. RBL binds differentially to human ovarian normal, cyst and cancer tissues. Flow cytometry, western blot analysis of membrane proteins showed the competitive binding of RBL and CA 125 antibody for the same binding sites on SKOV3 and OVCAR3 cells. RBL has strong binding to both SKOV3 and OVCAR3 cells with MFI of 173 and 155 respectively. RBL shows dose and time dependent growth inhibitory effect with IC50 of 2.5 and 8μg/mL respectively for SKOV3 and OVCAR3 cells. RBL induces reproductive cell death, morphological changes, nuclear degradation and increased release of ROS in SKOV3 and OVCAR3 cells leading to cell death. This is also supported by increase in hypodiploid population, altered MMP leading to apoptosis possibly involving intrinsic pathway. Adhesion, wound healing, invasion and migration assays demonstrated anti-metastasis effect of RBL apart from its growth inhibitory effect. These results show the promising potential of RBL both as a diagnostic and therapeutic agent.

Selection and Characterization of Vimentin-Binding Aptamer Motifs for Ovarian Cancer.

The application of aptamers in biomedicine is emerging as an essential technology in the field of cancer research. As small single-stranded DNA or RNA ligands with high specificity and low immunogenicity for their targets, aptamers provide many advantages in cancer therapeutics over protein-based molecules, such as antibodies. Vimentin is an intermediate filament protein that is overexpressed in endothelial cells of cancerous tissue. High expression levels of vimentin have been associated with increased capacity for migration and invasion of the tumor cells. We have selected and identified thioated aptamers with high specificity for vimentin using human ovarian cancer tissues. Tentative binding motifs were chosen for two vimentin aptamers based on predicted secondary structures. Each of these shorter, tentative binding motifs was synthesized, purified, and characterized via cell binding assays. Two vimentin binding motifs with high fidelity binding were selected and further characterized via cell and tissue binding assays, as well as flow cytometric analysis. The equilibrium binding constants of these small thioated aptamer constructs were also determined. Future applications for the vimentin binding aptamer motifs include conjugation of the aptamers to synthetic dyes for use in targeted imaging and therapy, and ultimately more detailed and precise monitoring of treatment response and tumor progression in ovarian pathology.

Niraparib exhibits a synergistic anti-tumor effect with PD-L1 blockade by inducing an immune response in ovarian cancer

BackgroundImmune checkpoint blockades (ICBs) therapy showed limited efficacy in ovarian cancer management. Increasing evidence indicated that conventional and targeted therapies could affect tumor-associated immune responses and increase the effectiveness of immunotherapy. However, the effects of Niraparib, one of the poly (ADP) ribose polymerase (PARP) inhibitors, on the immune response remains unclear. Delineating the crosstalk between cytotoxic anticancer agents and cancer-associated immunity may lead to more efficient combinatorial strategies.MethodsProgrammed death ligand 1 (PD-L1) expression in human ovarian cancer cells after PARP inhibitors treatment was examined by western blotting (WB) and flow cytometry. The expression of poly ADP-ribose polymerase (PARP1), PD-L1, and CD8 in human ovarian cancer tissues was detected by immunohistochemistry(IHC). The effect of Niraparib and PD-L1 blockade in ovarian cancer progression was investigated in vivo. The changes of immune cells and cytokines in vitro and in vivo were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Changes of cGAS/STING signal pathway after Niraparib treatment were determined by WB, ELISA.ResultsNiraparib upregulated membrane PD-L1 and total PD-L1 expression in ovarian cancer cells and had a synergistic effect with PD-L1 blockade in vivo. In clinical patient samples, Niraparib augmented cytotoxic CD8+T cell proportion and function. In vivo and vitro, Niraparib can also increase the proportion of T cells and combined with PD-L1 blockade could further enhance the effect. Besides, Niraparib activated the cGAS-STING pathway, increasing the levels of cytokines such as CCL5 and CXCL10, which played a vital role in augmenting the infiltration and activation of cytotoxic T cells.ConclusionsNiraparib could modulate the immune response via the activation of the cGAS/STING pathway, and combination with PD-L1 blockade could further enhance the effect. These results provide a sound theoretical basis for clinical treatment.

Long noncoding RNA LINC00858 promotes the progression of ovarian cancer via regulating the miR-134-5p/TRIM44 axis

Recent studies have shown that many long noncoding RNAs (lncRNAs) are abnormally expressed in ovarian cancer and involved in the pathological progress of ovarian cancer. In the present study, we aimed to investigate the role of lncRNA LINC00858 and the potential mechanism in ovarian cancer. The qRT-PCR was used to measure the expression levels of LINC00858 and miR-134-5p in ovarian cancer tissue specimens and cell lines. Loss-of-function assays were performed to investigate the role of LINC00858 in ovarian cancer. MTT assay was carried out to measure cell proliferation. Transwell assays were performed to determine cell migration and invasion. Biological information analysis and luciferase report gene assay were used to verify potential downstream genes of LINC00858. The xenograft mouse model was established to analyze tumor growth in vivo. Our results showed that LINC00858 was highly expressed in human ovarian cancer tissues and cell lines. Knockdown of LINC00858 inhibited cell proliferation, migration and invasion of SKOV3 cells, and suppressed tumor growth in mouse xenograft models. Mechanistic studies revealed that LINC00858 acted as a sponge of miR-134-5p and then regulated TRIM44 expression in SKOV3 cells. Furthermore, rescue experiments illustrated that inhibition of miR-134-5p restored the inhibitory effects of LINC00858 knockdown on cell proliferation, migration and invasion. TRIM44 overexpression could counteract the inhibitory effects of miR-134-5p mimics on ovarian cancer cells. In conclusion, these findings demonstrated that LINC00858 exerted oncogenic role in ovarian cancer, which was mediated by miR-134-5p/TRIM44 axis. Thus, LINC00858 might be a therapeutic target for the treatment of ovarian cancer.

Abstract 1534: Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors

Abstract Mesothelin (MSLN) is a tumor-associated antigen over-expressed on the cell surface of various malignant tumor cells, including majority of mesothelioma and ovarian cancer. MSLN-directed therapy has been intensively studied in preclinical and clinical settings. However, the clinical efficacy and safety of mesothelin-targeted therapy remains to be demonstrated. To identify a safe and effective target for CAR-T cell therapy in solid tumors, we used artificial intelligent platform developed with public and private RNA transcription database to identify cancer-associated antigens caused by alternative spicing. One of interesting targets is an alternative spicing isoform from MSLN. Human MSLN transcript has at least three isoforms. The isoform 1 is the predominant transcript detected in normal and tumor tissues and has been a promising target for cancer immunotherapy. The isoform 2 is the minor transcript using alternatively spliced exons producing 8 additional amino acids insertion compared to isoform 1. The isoform 3 produces a truncated and soluble MSLN. Our data demonstrated that MSLN isoform 2 is specifically expressed in ovarian cancers but not normal tissues, confirmed by Q-PCR studies. Furthermore, we generated mouse hybridoma antibodies specifically targeting MSLN isoform 2. The antibody specificity was screened and confirmed by ELISA-based reaction to MSLN isoform 2-specific peptide and flow cytometry-based binding to 293T cells overexpressing MSLN isoform 2 but not isoform 1 proteins. We further detected the endogenous MSLN isoform 2 expression in human mesothelioma cell line NCI H226 and further confirmed MSLN isoform 2 expression on human primary mesothelioma and ovarian cancer tissue but not normal tissues by immunohistochemistry staining. To study if targeting MSLN isoform 2 with chimeric antigen receptor (CAR) can control the tumor growth, we made CAR retrovirus construct and showed that genetically modified allogenic gamma-delta T cells expressing CAR for MSLN isoform 2 can kill human mesothelioma cells in vitro and in vivo. In summary, we have demonstrated that MSLN isoform 2 is a tumor-specific antigen, which can be targeted for CAR-T cell therapy. Citation Format: Xiaohong Wang, Yibin Chen, Anupama Gopisetty, Leonardo Mirandola, Lucia Piccotti, Quynh Nguyen, Maurizio Chiriva-Internati. Mesothelin isoform 2 is a novel target for allogenic CARγδT cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1534.

NF-\u03baB-activated SPRY4-IT1 promotes cancer cell metastasis by downregulating TCEB1 mRNA via Staufen1-mediated mRNA decay

Previous study demonstrated that most long non-coding RNAs (lncRNAs) function as competing endogenous RNAs or molecular sponges to negatively modulate miRNA and regulate tumor development. However, the molecular mechanisms of lncRNAs in cancer are not fully understood. Our study describes the role of the lncRNA SPRY4 intronic transcript 1 (SPRY4-IT1) in cancer metastasis by mechanisms related to Staufen1 (STAU1)-mediated mRNA decay (SMD). Briefly, we found that, high SPRY4-IT1 expression was associated with aggressiveness and poor outcome in human colorectal, breast and ovarian cancer tissues. In addition, functional assays revealed that SPRY4-IT1 significantly promoted colorectal, breast and ovarian cancer metastasis in vitro and in vivo. Mechanistically, microarray analyses identified several differentially-expressed genes upon SPRY4-IT1 overexpression in HCT 116 colorectal cancer cells. Among them, the 3′-UTR of transcription elongation factor B subunit 1 (TCEB1) mRNA can base-pair with the Alu element in the 3′-UTR of SPRY4-IT1. Moreover, SPRY4-IT1 was found to bind STAU1, promote STAU1 recruitment to the 3′-UTR of TCEB1 mRNA, and affect TCEB1 mRNA stability and expression, resulting in hypoxia-inducible factor 1α (HIF-1α) upregulation, and thereby affecting cancer cell metastasis. In addition, STAU1 depletion abrogated TCEB1 SMD and alleviated the pro-metastatic effect of SPRY4-IT1 overexpression. Significantly, we revealed that SPRY4-IT1 is also transactivated by NF-κB/p65, which activates SPRY4-IT1 to inhibit TCEB1 expression, and subsequently upregulate HIF-1α. In conclusion, our results highlight a novel mechanism of cytoplasmic lncRNA SPRY4-IT1 in which SPRY4-IT1 affecting TCEB1 mRNA stability via STAU1-mediated degradation during cancer metastasis.

Tagln Mediated Stiffness-Regulated Ovarian Cancer Progression Via RhoA/ROCK Pathway

Background: Ovarian cancer (OC) progression is an unmet medical challenge. Since omental metastases were palpated harder than their primary counterparts during cytoreductive surgery of patients with epithelial ovarian cancer, we were inspired to investigate OC progression from the perspective of biomechanics. Methods: Atomic Force Microscope (AFM) was used to measure the young’s moduli of tissues. The collagen-coated polyacrylamide hydrogel (PA gel) system was prepared to mimic the soft and stiff substrates in vitro. The effect of TAGLN were evaluated both in vitro and in vivo using transwell, immunofluorescence, western blot analysis and immunohistochemistry. Findings: We quantitatively confirmed that omental metastases were stiffer and more abundant in desmoplasia compared with paired primary tumors, and further demonstrated that matrix stiffness could notably regulate OC progression. Remarkably, TAGLN, encoding an actin cross-linking/gelling protein, was identified as a potent mechanosensitive gene that could form a regulation loop with Src activation reacting to environmental stiffness, thus mediating stiffness-regulated OC progression through regulating RhoA/ROCK pathway. Interpretation: These data demonstrate that targeting extra-cellular matrix stiffness could probably hamper OC progression, and of note, targeting TAGLN might provide promising clinical therapeutic value for OC therapy. Funding: This work was supported by the “973” Program of China (No. 2015CB553903 to Ding Ma and Junbo Hu), Technical Innovation Special Project of Hubei Province (2018ACA138), the Fundamental Research Funds for the Central Universities (2019kfyXMBZ024), National Science and Technology Major Sub- Project (2018ZX10301402-002), and the National Science Foundation of China (81902661, 81772787, 82072889). Declaration of Interest: None to declare. Ethical Approval: Human ovarian cancer tissues were all obtained from patients undergoing surgery at the Department of Obstetrics and Gynecology, Tongji Hospital, Huazhong University of Science and Technology after obtaining written informed consent of the patients and the authorization of the Ethics Committee of Tongji Hospital (TJ-iRB20181103). Fresh tissues were prepared for the Atomic Force Microscope (AFM) measurement, frozen or formalin fixed.

MiR-4461 Regulates the Proliferation and Metastasis of Ovarian Cancer Cells and Cisplatin Resistance.

microRNAs (miRNAs) are of great significance in cancer treatment, which may have a desirable result on the regulation of tumorigenesis, progression, recurrence, and chemo-resistance of ovarian cancer. However, the research on the further potential application of miR-4461 in ovarian cancer is little and limited. Therefore, the study in this paper focus on the investigation of the of miR-4461 in ovarian cancer progression and chemo-resistance. The phenomenon that the proliferation and metastasis of ovarian cancer cells can be promoted by miR4461 is revealed in functional assays. Through the bioinformatics and luciferase reporter analysis, the PTEN is validated to be the direct target of miR-4461 in ovarian. The association between the expression of miR-4461 and PTEN is negative in in human ovarian cancer tissues. The distinction of growth and metastasis capacity between miR-4461 knockdown ovarian cancer cells and control cells is partially abolished by si-PTEN. Moreover, it was found that cisplatin treatment has obvious effect on the miR-4461 knockdown ovarian cancer cells. In summary, the data given in this paper indicate that the miR-4461 can be regarded as a potential onco-miRNA in ovarian cancer by targeting PTEN.

Bromodomain-containing protein 4 silencing by microRNA-765 produces anti-ovarian cancer cell activity.

Bromodomain-containing protein 4 (BRD4) overexpression promotes ovarian cancer progression, and represents an important therapeutic oncotarget. This current study identified microRNA-765 (miR-765) as a novel BRD4-targeting miRNA. We showed that miR-765 directly associated with and silenced BRD4. In primary ovarian cancer cells and established cell lines (SKOV3 and CaOV3), ectopic overexpression of miR-765 inhibited cancer cell proliferation, migration and invasion, and induced apoptosis activation. In contrast, miR-765 inhibition by its anti-sense induced BRD4 upregulation to promote ovarian cancer cell proliferation, migration and invasion. Significantly, miR-765 overexpression-induced anti-ovarian cancer cell activity was largely attenuated by restoring BRD4 expression through an UTR-null BRD4 construct. Moreover, CRISPR/Cas9-induced BRD4 knockout (KO)inhibited proliferation and activated apoptosis in ovarian cancer cells. BRD4 KO in ovarian cancer cells abolished the functional impact of miR-765. miR-765 expression levels were downregulated in human ovarian cancer tissues and cells, correlating with the upregulation of BRD4 mRNA. Collectively, BRD4 silencing by miR-765produces significant anti-ovarian cancer cell activity. miR-765 could be further tested for its anti-ovarian cancer potential.

Interleukin‑8 released by cancer‑associated fibroblasts attenuates the autophagy and promotes the migration of ovarian cancer cells.

The tumor microenvironment composed of a mixture of stromal cells and their secretions has a marked impact on cancer progression. In particular, soluble factors and metabolites contribute to malignancy through the dysregulation of autophagy in cancer cells. The present study investigated the effects of ovarian cancer-associated fibroblasts (OVCAFs) with their secretory substances on the autophagy and migration of ovarian cancer cells. The conditioned-medium (CM) of OVCAFs isolated from fresh human ovarian cancer tissues was analyzed for the levels of 27 common cytokines/chemokines using a cytokine array. Autophagy in cancer cells was assessed by determining the expression of the vacuolar form of LC3 by western blot analysis and immunofluorescence. Cancer cell migration was assessed by Transwell migration assay. Interleukin (IL)-8 was found to be the most highly upregulated cytokine among the cytokines/chemokines found in the OVCAF-CM. The role of IL-8 in ovarian cancer cell migration and its mechanistic link with autophagy was investigated. Recombinant human IL-8 (rhIL-8) stimulated the migration of SKOV3 and Kuramochi ovarian cancer cells, and concurrently downregulated basal autophagy, in concentration-dependent manner. Compared to the CM of control counterpart normal fibroblasts isolated from benign ovaries (OVNF-CM), the CM from 3 OVCAF isolates (namely, OVCAF-9, -20 and -43) exerted effects similar to rhIL-8 on both cancer cell lines. The pharmacological induction of autophagy with rapamycin or metformin attenuated the pro-migratory effects of IL-8. Neutralizing anti-IL-8 antibody counteracted the inhibitory effect of OVCAF-CM on basal autophagy. On the whole, the present study highlights the involvement of IL-8 released by CAFs in the ovarian tumor microenvironment in promoting cancer cell migration through the suppression of autophagy.

CircATRNL1 activates Smad4 signaling to inhibit angiogenesis and ovarian cancer metastasis via miR-378.

Ovarian cancer is one of the most frequent carcinomas in females, and the occurrence rate is still rising despite many advances made. The pathogenesis of ovarian cancer remains greatly unclear. Here, we investigated the mechanisms of ovarian cancer, with the focus on circATRNL1. Human ovarian cancer tissues and cell lines were used to examine levels of circATRNL1, miR‐378, Smad4, AKT, and other proliferation‐related and migration‐related proteins. Cellular assays were used to determine cancer cell proliferation, invasion, migration, apoptosis, and angiogenesis. We validated the interactions of circATRNL1/miR‐378 and miR‐378/Smad4, and a mouse tumor xenograft model was employed to assess the effect of circATRNL1 on tumor growth and metastasis in vivo. We found that circATRNL1 was decreased while miR‐378 was increased in human ovarian cancer tissues and cells. circATRNL1 bound to miR‐378 while miR‐378 directly targeted Smad4. Overexpression of circATRNL1 or knockdown of miR‐378 suppressed angiogenesis and ovarian cancer cell proliferation, invasion, and migration via decreasing proliferation‐ and migration‐related proteins via miR‐378 or Smad4, respectively. Overexpression of circATRNL1 restrained ovarian cancer growth and abdominal metastasis in vivo. Our findings indicate that circATRNL1 acts as a miR‐378 sponge to active Smad4 signaling and suppresses angiogenesis and ovarian cancer metastasis.

PAR2 induces ovarian cancer cell motility by merging three signalling pathways to transactivate EGFR.

Specific cellular functions mediated by GPCRs are often associated with signalling through a particular G protein or β-arrestin. Here, we examine signalling through a GPCR, protease-activated receptor 2 (PAR2), in a high-grade serous ovarian cancer cell line (OV90). Human ovarian cancer tissues (n = 1,200) and nine human ovarian cancer cell lines were assessed for PAR2 expression. PAR2 signalling mechanisms leading to cell migration and invasion were dissected using cellular assays, western blots, CRISPR-Cas9 gene knockouts, pharmacological inhibitors of PAR2 and downstream signalling proteins in OV90 cancer cells. PAR2 was significantly overexpressed in clinical ovarian cancer tissues and in OV90 ovarian cancer cells. PAR2 agonists, an endogenous protease (trypsin) and a synthetic peptide (2f-LIGRL-NH2 ), induced migration and invasion of OV90 ovarian cancer cells through activating a combination of Gαq/11 , Gα12/13 and β-arrestin1/2, but not Gαs or Gαi . This novel cooperative rather than parallel signalling resulted in downstream serial activation of Src kinases, then transactivation of epidermal growth factor receptor (EGFR), followed by downstream MEK-ERK1/2-FOS/MYC/STAT3-COX2 signalling. Either a PAR2 antagonist (I-191), CRISPR-Cas9 gene knockouts (PAR2 or Gα proteins or β-arrestin1/2), or inhibitors of each downstream protein attenuated human ovarian cancer cell motility. This study highlights a novel shared signalling cascade, requiring each of Gαq/11 , Gα12/13 and β-arrestin1/2 for PAR2-induced ovarian cancer cell migration and invasion. This mechanism controlling a cellular function is unusual in not being linked to a specific individual G protein or β-arrestin-mediated signalling pathway.