EMT Of Ovarian Cancer Cells Research Articles

Abstract 4779: The effects of glutamine deprivation on ovarian cancer initiation and progression

Abstract High-grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer. Most patients with HGSC are diagnosed at advanced stage diseases. Despite the high response rate to platinum/taxane-based chemotherapy, most of these patients will develop recurrent chemoresistant disease. Recent studies showed the cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME) play an important role in modulating the malignant phenotypes of HGSC. CAFs are primarily responsible for producing factors such as cytokines and growth factors to modify the TME niche to promote the malignant phenotypes of cancer cells. Recently, we have identified a previously unrecognized mechanism in which metabolism of reactive CAFs is reprogrammed through an upregulated glutamine anabolic pathway in HGSC. This dysfunctional CAF metabolism confers atypical metabolic flexibility and adaptive mechanisms in CAFs, allowing them to harness carbon and nitrogen from noncanonical sources to synthesize glutamine in nutrient-deprived conditions existing in the TME. Our preliminary data show that tumors developed from syngeneic fallopian epithelial cell-derived cancer cells injected IP into C57BL/6 mice, which started to be fed with normal diet two weeks before tumor cells injection had significantly higher tumor burden than those fed with the glutamine-deprived diet. Moreover, tumor-bearing mice treated with glutaminase inhibitor for 3 weeks demonstrated significantly lower tumor burden than those treated with the control vehicle. These findings suggest that the interplay between CAFs and HGSC cells enriches glutamine in the ovarian TME; and glutamine depletion can not only suppress ovarian tumor growth but also prevent the development of advanced progressive diseases. In addition to the direct promoting effect of glutamine on the glutamine addicted HGSC cells, increased CAF-derived glutamine can also affect the activity of other stromal cell types such as immune cells in the TME, which subsequently modulate HGSC cell growth. Imaging mass cytometry (IMC) and Mass Spectrometry Imaging (MSI) were performed on serial tissue sections of tissue microarrays generated from the abovementioned murine samples. Mice fed with a glutamine-deprived diet or treated with the glutaminase inhibitor not only have a significantly lower tumor burden but also induced dramatic changes in the cellular and molecular composition within the TME. In particular, mice fed with a glutamine-deprived diet had greater B-cell-related immune response, reduced stemness, and EMT of ovarian cancer cells, and more activated CAFs, which may result in increased stiffness of the ECM and subsequently enhance the malignant phenotype of HGSC cells. Further studies to delineate the molecular mechanisms by which spatial cellular and metabolic profiles modulate HGSC pathogenesis and progression are warranted. Citation Format: Sammy Ferri-Borgogno, Kathryn Lu, Basant T. Gamal, Chi Lam Au Yeung, Erin Seeley, Javier A. Gomez, Akshay Basi, Jared K. Burks, Olamide Animasahun, Deepak Nagrath, Samuel C. Mok. The effects of glutamine deprivation on ovarian cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4779.

Tropomyosin1 isoforms underlie epithelial to mesenchymal plasticity, metastatic dissemination, and resistance to chemotherapy in high-grade serous ovarian cancer

Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer. When compared with colon cancer, a distinct spectrum of downstream targets characterizes quasi-mesenchymal ovarian cancer cells, likely to reflect the different modalities of metastasis formation between these two types of malignancy, i.e. hematogenous in colon and transcoelomic in ovarian cancer. Moreover, upstream RNA-binding proteins differentially expressed between epithelial and quasi-mesenchymal subpopulations of ovarian cancer cells were identified that underlie differential regulation of EMT-related isoforms. In particular, the up- and down-regulation of RBM24 and ESRP1, respectively, represent a main regulator of EMT in ovarian cancer cells. To validate the functional and clinical relevance of our approach, we selected and functionally analyzed the Tropomyosin 1 gene (TPM1), encoding for a protein that specifies the functional characteristics of individual actin filaments in contractile cells, among the ovarian-specific downstream AS targets. The low-molecular weight Tpm1.8/9 isoforms are specifically expressed in patient-derived ascites and promote invasion through activation of EMT and Wnt signaling, together with a broad spectrum of inflammation-related pathways. Moreover, Tpm1.8/9 expression confers resistance to taxane- and platinum-based chemotherapy. Small molecule inhibitors that target the Tpm1 isoforms support targeting Tpm1.8/9 as therapeutic targets for the development of future tailor-made clinical interventions.

Mfn2-mediated mitochondrial fusion promotes autophagy and suppresses ovarian cancer progression by reducing ROS through AMPK/mTOR/ERK signaling.

Mitochondrial dynamics are balanced fission and fusion events that regulate mitochondrial morphology, and alteration in these events results in mitochondrial dysfunction and contributes to many diseases, including tumorigenesis. Ovarian cancer (OC) cells exhibit fragmented mitochondria, but the mechanism by which mitochondrial dynamics regulators contribute to OC is considerably less clear. Here, we elucidated the potential role of Mfn2-mediated mitochondrial fusion in OC and present evidence that genetic or pharmacological activation of Mfn2 leads to mitochondrial fusion and reduces ROS generation, which correlates with reduced cell proliferation, invasion, migration, and EMT in OC cells. Also, increased mitochondrial fusion promotes the F-actin remodeling, reduces lamellipodia formation, and thus reduces EMT. Increased expression of Mfn2 triggers AMPK, promotes autophagy, reduces ROS,andsuppresses OC progression by downregulating the p-mTOR (2481 and 2448) and p-ERK axis. OC patients with higher Mfn2 expression have better survival than those with lower Mfn2 levels. Our findings demonstrate that restoration of Mfn2-mediated mitochondrial fusion suppressed OC progression and suggest that this process could be a potential strategy in OC treatment.

Rad50 promotes ovarian cancer progression through NF-κB activation.

Rad50 is a component of MRN (Mre11‐Rad50‐Nbs1), which participates in DNA double‐strand break repair and DNA‐damage checkpoint activation. Here, we sought to investigate the clinical and functional significance of Rad50 in high‐grade serous ovarian cancer (HGSOC). We found that Rad50 was frequently upregulated in HGSOCs and enhanced Rad50 expression inversely correlated with patient survival. In addition, ectopic expression of Rad50 promoted proliferation/invasion and induced EMT of ovarian cancer cells, whereas knockdown of Rad50 led to decreased aggressive behaviors. Mechanistic investigations revealed that Rad50 induced aggressiveness in HGSOC via activation of NF‐κB signaling pathway. Moreover, we identified CARD9 as an interacting protein of Rad50 in ovarian cancer cells and the activation of NF‐κB pathway by Rad50 is CARD9 dependent. Our findings provide evidence that Rad50 exhibits oncogenic property via NF‐κB activation in HGSOC.

A Newly Identified lncBCAS1-4_1 Associated With Vitamin D Signaling and EMT in Ovarian Cancer Cells.

Long noncoding RNAs (lncRNAs) were identified rapidly due to their important role in many biological processes and human diseases including cancer. 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and its analogues are widely applied as preventative and therapeutic anticancer agents. However, the expression profile of lncRNAs regulated by 1α,25(OH)2D3 in ovarian cancer remains to be clarified. In the present study, we found 606 lncRNAs and 102 mRNAs that showed differential expression (DE) based on microarray data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the DE genes were mainly enriched in TGF-β, MAPK, Ras, PI3K-Akt, and Hippo signaling pathways, as well as the vitamin D-related pathway. We further assessed the potential lncRNAs that linked vitamin D signaling with EMT, and lncBCAS1-4_1 was identified in the first time. Moreover, we found that the most upregulated lncBCAS1-4_1 showed 75% same transcripts with CYP24A1 (metabolic enzyme of 1α,25(OH)2D3). Finally, the lncBCAS1-4_1 gain-of-function cell model was established, which demonstrated that the knockdown of lncBCAS1-4_1 inhibited the proliferation and migration of ovarian cancer cells. Furthermore, lncBCAS1-4_1 could resist the antitumor effect of 1α,25(OH)2D3, which was associated with upregulated ZEB1. These data provide new evidences that lncRNAs served as a target for the antitumor effect of 1α,25(OH)2D3.

The LINC00922 aggravates ovarian cancer progression via sponging miR-361-3p

BackgroundLong noncoding RNA (lncRNA) LINC00922 has been reported to promote tumorigenesis of lung and breast cancer. However, the functions and mechanisms of LINC00922 in ovarian cancer (OC) remain unclarified. The current study aims to clarify the detailed functions and underlying mechanisms of LINC00922 in the progression of OC.MethodsLINC00922 expression in OC tissues and cells was identified by a comprehensive strategy of data miming, computational biology and quantitative real-time polymerase chain reaction (RT-qPCR) experiment. In vitro CCK-8, wound healing, transwell invasion, western blotting and in vivo tumorigenesis assays LINC00922 were conducted to evaluate the functions of LINC00992. Subsequently, bioinformatics technology and dual luciferase reporter assay were performed to confirm the between miR-361-3p and LINC00922 or CLDN1. Finally, rescue experiments were performed to confirm whether LINC00922 effect functions of OC cells through regulation of miR-361-3p.ResultsLINC00922 was significantly upregulated in OC tissues and cell lines, which is significantly positively corelated with the poor prognosis of patients with OC. LINC00922 knockdown inhibited proliferation and tumorigenesis of OC cells in vitro and vivo. In addition, LINC00922 knockdown suppressed migration, invasion, and EMT of OC cells in vitro. Mechanically, LINC00922 could competitively bind with miR-361-3p to relieve the repressive effect of miR-361-3p on its target gene CLDN1 in OC cells. In addition, silencing miR-361-3p promoted OC cell proliferation, migration, invasion, EMT and Wnt/β-catenin signaling, while LINC00922 knockdown inhibited Wnt/β-catenin signaling by upregulating miR-361-3p. Rescue experiments revealed that LINC00922 knockdown inhibited OC cell proliferation, migration, invasion and EMT by regulating miR-361-3p.ConclusionThis study suggested that LINC00922 could competitively bind with miR-361-3p to promote the CLDN1 expression and activate Wnt/β-catenin signaling in OC progression, which providing a promising therapeutically target for OC.

CCAAT enhancer binding protein α suppresses proliferation, metastasis, and epithelial-mesenchymal transition of ovarian cancer cells via suppressing the Wnt/β-catenin signaling.

CCAAT enhancer-binding protein alpha (CEBPA, also known as C/EBPα) is a transcription factor that plays an essential role in regulating terminal differentiation and cell proliferation of many tissues. The objective of this study was to explore the potential function of CEBPA in ovarian cancer. The expression of CEBPA in ovarian cancer samples and adjacent normal tissues was evaluated by qRT-PCR. The putative role of CEBPA in ovarian cancer cells was evaluated by immunohistochemistry, western blot, cell viability assay, BrdU incorporation assay, soft agar colony formation assay, Transwell cell migration and invasion assay, tumor xenograft formation, and lung metastasis model. We found that CEBPA was downregulated in ovarian cancer samples and predicted a poor prognosis. CRISPR/Cas9-mediated CEBPA knockout promoted proliferation, anchorage-independent growth, migration, invasion, and EMT of ovarian cancer cells, while enforced CEPBA expression suppressed proliferation, anchorage-independent growth, migration, invasion, EMT, tumor xenograft growth, and lung metastasis of ovarian cancer cells. Furthermore, we found that the knockout of CEBPA activated Wnt/β-catenin signaling in ovarian cancer cells, while CEBPA overexpression suppressed Wnt/β-catenin activation. Our data indicated that CEBPA acted as a tumor suppressor in ovarian cancer, and might be a potential prognostic marker for ovarian cancer treatment.

Long-Chain Non-Coding SOX21-AS1 Promotes Proliferation and Migration of Breast Cancer Cells Through the PI3K/AKT Signaling Pathway.

AimThis study aimed to investigate the effect of long-chain non-coding SOX21-AS1 on the proliferation and migration of breast cancer (BC) cells through the PI3K/AKT signaling pathway.MethodsEighty-eight BC and adjacent tissues were collected, and BC cells and normal breast epithelial cells were purchased. LncRNA SOX21-AS1 expression in tissues and cells was detected by RT-PCR. miR-NC, si-SOX21-AS1, and Sh-SOX21-AS1 were transfected into BC cells. The PI3K/AKT signaling pathway was interfered with L740Y-P (activator of the PI3K/AKT pathway) and LY294002 (inhibitor of the PI3K/AKT pathway) in BC cells. The SOX21-AS1 expression in BC tissues and cells was tested by qRT-PCR, and the expression levels of p-PI3K, p-AKT, N-cadherin, E-cadherin, and vimentin were detected by WB.ResultsSOX21-AS1 was highly expressed in BC, and the p-PI3K and p-AKT levels were also high. Cell experiments showed that inhibiting SOX21-AS1 expression could inhibit the proliferation, invasion, migration, and EMT of BC cells, and up-regulating its expression could promote the proliferation, invasion, migration, and EMT of ovarian cancer cells. The tumor-forming experiment in nude mice was consistent with the results in vitro. 740Y-P intervention could reverse the inhibition effect of Si-SOX21-AS1 on BC cell proliferation, invasion, migration, and EMT, while LY294002 intervention could reverse the promotion effect of Sh-SOX21-AS1 on BC cell proliferation, invasion, migration, and EMT.ConclusionSOX21-AS1 is highly expressed in BC tissues. Silencing BC expression can inhibit the proliferation, invasion, migration, and EMT of cells by inhibiting the PI3K/AKT signaling pathway, which may be a new target for diagnosis and treatment.

MiR-22 suppresses cell viability and EMT of ovarian cancer cells via NLRP3 and inhibits PI3K/AKT signaling pathway.

miR-22 plays a great role in inhibiting cell growth, metastasis and enhanced cell apoptosis in several cancers. The purpose of this study was to investigate the functions of miR-22 in ovarian cancer. The proliferative ability was measured using CCK-8 assay. The protein expression associated with EMT and PI3K/AKT signaling biomarkers were measured by western blot. Luciferase assay applied to measure the luciferase activity. Kaplan-Meier method was performed to evaluate the overall survival rate of ovarian cancers. miR-22 was low expressed and NLRP3 was overexpressed in ovarian cancer tissues and cells, and downregulation of miR-22 was associated with poor prognosis. The expression of NLRP3 had a negative correlation with miR-22 expression in ovarian cancer. miR-22promoted cell viability and EMT through directly binding to the 3'-UTR of NLRP3 mRNA and inhibited PI3K/AKT signaling pathway. NLRP3 partially restored functions of miR-22 on cell proliferation and EMT in ovarian cancer. miR-22 impaired cell viability and EMT by NLRP3 and inhibited PI3K/AKT signaling pathway in ovarian cancer. The newly identified miR-22/NLRP3/PI3K/AKT axis provides novel insight into the pathogenesis of ovarian cancer.

Lidocaine Inhibits Ovarian Cancer Cell Proliferation and Induces Apoptosis: An In Vitro Study

Objective: The present study aimed to explore the effects and related mechanism of lidocaine on human ovarian cancer cell lines. Methods: Human ovarian cancer cell lines (SKOV3 and ES-2) were treated with different concentrations of lidocaine for different time. We treated SKOV3 and ES-2 cells using lidocaine then used MTT assay and flow cytometry to detect the cell proliferation and cell apoptosis. In addition, we used western blot analysis to explore the protein expression of Bax and Bcl-2 in SKOV3 and ES-2 cells. Western blot analysis and qRT-PCR were performed for the detection of EMT markers (E-cadherin, N-cadherin). The protein expression levels of TRAF3 and p-p65 in SKOV3 and ES-2 cells were determined by Western blot analysis. Results: Compared to the control group, 0.5, 1, 5, and 10 mM of lidocaine significantly inhibited ovarian cancer cell proliferation at different time points, while 0.1 mM of lidocaine had no significant effect. 1, 5 mM of lidocaine induced the cell apoptosis, and observably reduced expression of Bcl-2 protein, but improved Bax expression markedly compared with the control group. Treatment of lidocaine increased E-cadherin expression, but decreased N-cadherin expression when compared with control group. Treatment of lidocaine increased TRAF3 protein expression, but decreased p-p65 protein expression in ES-2 and SKOV3 cells. Conclusion: We demonstrated that lidocaine inhibited cell proliferation, induced apoptosis, and inhibited EMT in ovarian cancer cells via regulating TRAF3/NF-κB pathway.

MiR-29c-3p, a target miRNA of LINC01296, accelerates tumor malignancy: therapeutic potential of a LINC01296/miR-29c-3p axis in ovarian cancer

As one of the main gynecological cancers, ovarian cancer (OC) has an unfavourable outcomes owing to its high recurrence and metastasis rate. Our previous studies have revealed that LINC01296 functions as an oncogene in OC, but the underlying mechanism has not been explored. The aim of this paper was to further investigate that how LINC01296 plays a role in OC. Through online software prediction, miR-29c-3p has been discriminated as the target miRNA of LINC01296 for further research, and subsequent luciferase assay confirmed bioinformatics prediction. Then the data obtained from the two databases (GSE119055 and GSE83693) were analyzed by GEO2R for differential gene analysis. The results indicated that the miR-29c-3p was lowly expressed in OC tissues than that in normal ovarian tissues, and its expression in recurrent OC tissues was lower than that in primary OC tissues. Simultaneously, Kaplan-Meier survival analysis illustrated that the lower expression of miR-29c-3p was interrelated to unfavourable outcomes of OC. Further, the qRT-PCR data revealed that the miR-29c-3p expression in OC cell lines (SKOV-3 and OVCAR-3) was markedly declined than that in normal control cells (IOSE80). Subsequently, the functional experiments, such as CCK8, colony formation and Transwell assays, prompted that inhibition of miR-29c-3p can obviously increase the proliferation, invasion and migration of OVCAR3 and SKOV3 cells compared with control group, while downregulation of LINC01296 showed an opposite result. It is worth noting that downregulation of LINC01296 can reverse the effect of miR-29c-3p suppression on OC cells. Finally, we detected the changes of EMT-related proteins by western blot experiment, and reached a similar conclusion that knockdown of LINC01296 reversed the EMT caused by miR-29c-3p inhibition. In sum up, the cancer-promoting function of LINC01296 was achieved by regulating the expression of miR-29c-3p, and LINC01296/miR-29c-3p axis mediates the mechanical regulation of EMT in OC cells, hoping to provide the novel biomarkers and possibilities for OC therapy.

Regulatory mechanism of lncRNA NORAD on proliferation and invasion of ovarian cancer cells through miR-199a-3p.

To explore the regulatory mechanism of lncRNA NORAD on proliferation and invasion of ovarian cancer cells through miR-199a-3p. Eighty-six ovarian cancer tissues and 86 tissues adjacent to cancer, human ovarian cancer cell lines SKOV3, HO-8910, A2780, OVCAR-3, and human normal ovarian epithelial cell line IOSE80 were collected. MiR-199a-3p-mimics, miR-199a-3p-inhibitor, miR-NC, si-NORAD, Sh-NORAD, and NC were transfected into HO-8910 and A2780 cells, the expression levels of lncRNA NORAD and miR-199a-3p in ovarian cancer tissues and cells were detected by qRT-PCR, and the expression levels of N-cadherin, E-cadherin, and vimentin in cells were detected by WB. Cell Counting Kit-8 (CCK-8), transwell, and cell scratch tests were used to detect proliferation, invasion, and migration of cells, and the relationship between lncRNA NORAD and miR-199a-3p was confirmed by the Dual-Luciferase reporter assay. LncRNA NORAD was highly expressed and miR-199a-3p was lowly expressed in ovarian cancer, and the expression levels of LNCRNARAD and miR-199a-3p were negatively correlated. Cell experiments showed that inhibiting the expression of lncRNA NORAD or up-regulating the expression of miR-199a-3p could inhibit the proliferation, invasion, migration, and EMT of ovarian cancer cells, while up-regulating the expression of lncRNA NORAD or inhibiting the expression of miR-199a-3p could promote their proliferation, invasion, migration, and EMT. Dual-Luciferase reporter assay confirmed that there was a regulatory relationship between lncRNA NORAD and miR-199a-3p. LncRNA NORAD was highly expressed in ovarian cancer tissues, while silencing lncRNA NORAD expression could inhibit the proliferation, invasion, migration, and EMT of ovarian cancer cells by regulating miR-199a-3p, which might be a new target for the diagnosis and treatment of ovarian cancer.

Abstract 2006: Targeting AXL kinase with TP-0903 successfully reverses the mesenchymal phenotype and extends survival in preclinical models of advanced ovarian cancer

Abstract Epithelial-to-mesenchymal transition (EMT) is a critical step in the progression of ovarian cancer (OVCA), which drives local invasion, metastasis and drug resistance leading to poor survival. AXL, a receptor tyrosine kinase, functions as a molecular driver of EMT in multiple cancers, including OVCA. The high mortality of this tumor is explained by the fact that the majority (75%) of patients present at an advanced stage, with widely metastatic disease within the peritoneal cavity often associated with ascites. The extent of ascites is a prognostic factor for survival, impacts quality of life, and correlates with the EMT phenotype. Approaches to block or reverse EMT are needed to improve the prognosis of OVCA patients, particularly those with refractory or early relapse (<6 months) disease following debulking surgery and conventional adjuvant treatments. TP-0903 is a potent, small molecule AXL inhibitor that has activity in reversing EMT in multiple models of disease and embryonic development. The objective of this study was to explore the anti-tumor activity in preclinical mesenchymal OVCA-derived models, which may provide rationale to evaluate TP-0903 in treatment-resistant or refractory OVCA patients. TP-0903 showed potent effects on cell viability in multiple OVCA-derived cell lines. The IC50 values in cell viability studies ranged from 33 nM (OVTOKO cells) to 840 nM (OVMANA cells). Kuramochi cells represent the most common ovarian cancer subtype, high-grade serous ovarian cancer and was particularly platinum-resistant (>5 μM), yet sensitive to TP-0903 (210 nM). Baseline phospho-AXL (Y702) was exceptionally high in Kuramochi cells and TP-0903 inhibited this phosphorylation at concentrations as low as 100 nM. TP-0903 reversed EMT in OVCA cells as evident by the down-regulation of mesenchymal markers including Snail and Slug. Consistent with the reversal of the EMT, TP-0903 inhibited the migration of ES-2 OVCA cells in a scratch assay. TP-0903 suppressed ascites development in preclinical models of OVCA and inhibited metastatic intraperitoneal tumor dissemination. Treatment suppressed ascites development (ascites volume, vehicle: 3.34 mL, TP-0903: 0.28 mL; abdominal circumference, vehicle: 7.96 cm (day 15), TP-0903: 6.82 cm (day 15), p<0.05) in an orthotopic ES-2 model and extended overall survival compared to vehicle or cisplatin alone. Phospho-AXL modulation and changes in other mechanism-validating biomarkers were observed in the ES-2 model. Similar preclinical activities, including decreased ascites volume, were observed in A2780cis and OVCAR-3 xenograft models. These results support further clinical evaluation of TP-0903 in either single-agent or combination regimens treating recurrent or refractory OVCA. TP-0903 is investigated in a Phase I trial that includes an expansion cohort of this patient population (NCT02729298). Citation Format: Nozomi Tomimatsu, Ken Fujimura, Yuta Matsumura, Hiroki Umehara, Lars Mouritsen, Steven L. Warner, David J. Bearss. Targeting AXL kinase with TP-0903 successfully reverses the mesenchymal phenotype and extends survival in preclinical models of advanced ovarian cancer [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 2006.

Platelets promote invasion and induce epithelial to mesenchymal transition in ovarian cancer cells by TGF-β signaling pathway

ObjectiveTo test whether platelets could increase invasion potential and initiate EMT in ovarian cancer cells via a TGF-β signaling pathway. MethodsBlood samples were collected in 69 patients with ovarian cancer, 16 patients with benign ovarian tumor and 64 healthy donors. SK-OV-3 and OVCAR-3 ovarian cancer cells were treated with platelets. Transwell assays were used to analyze the invasive capacity, and EMT was assessed by microarray analysis, quantitative real-time PCR (qPCR) and Western blotting. Activation of TGF-β pathway was examined by ELISA and Western blotting. TGF-β type I receptor (TβR I) inhibitor A83-01 was used to confirm the role of TGF-β pathway in vitro and in vivo. ResultsClinical data showed ovarian cancer patients with elevated platelet counts had a higher incidence of advanced stages. Treatment with platelets increased the invasive properties of both cell lines. Mesenchymal markers (snail family transcriptional repressor-1, vimentin, neural cadherin, fibronectin-1 and matrix metalloproteinase-2) were up-regulated in platelet-treated cells, while the epithelial marker (epithelial cadherin) was down-regulated. Higher TGF-β level was observed in patients with elevated platelet counts when compared to the subjects. Higher levels of TGF-β were also found in culture medium treated with platelets, and cells treated with platelets also showed increased phosphorylation of Smad2. TβR I inhibitor A83–01 reversed the EMT-like alterations and inhibited platelet-induced invasion in vitro and in vivo. ConclusionPlatelet increased invasion potential and induced EMT in ovarian cancer cells in a TGF-β dependent pathway. Platelet-derived TGF-β may be useful as a new target treatment for ovarian cancer.

The lncRNA CCAT1 upregulates TGF\u03b2R1 via sponging miR-490-3p to promote TGF\u03b21-induced EMT of ovarian cancer cells

BackgroundOvarian cancer is the fifth leading cause of cancer deaths in women worldwide. LncRNACCAT1 was reported to play a critical role in cell metastasis of ovarian cancer. However, little is known about the detailed mechanism of how CCAT1 enhances TGFβ1-induced EMT of ovarian cancer cells.MethodsWe used RT-qPCR to examine the level of miR-490-3p and CCAT1 and western blot to detect the protein level of TGFβR1 and EMT-associated markers. We utilized luciferase reporter assay to confirm the direct interaction of CCAT1 or TGFβ1 with miR-490-3p. Wound healing and invasion assay were employed to investigate the role of CCAT1 and miR-490-3p in the TGFβ1-induced migration and cell invasion of ovarian cancer cells, respectively.ResultsTGFβ1 stimulated the expression of CCAT1. And CCAT1 knockdown decreased cell migration, invasion and EMT-associated markers expression of ovarian cancer cells treated with TGFβ1. CCAT1 directly targeted and downregulated miR-490-3p, then increasing TGFβR1 level. miR-490-3p was shown to regulate cell invasion, migration and EMT markers expression via TGFβR1. In addition, we also observed that miR-490-3p was essential for TGFβ1-induced tumor cell invasion and migration influenced by CCAT1. CCAT1 level was significantly higher in tumors than adjacent normal tissue, in contrast, miR-490-3p level was lower in ovarian tumors.ConclusionHere, we reveal that CCAT1 contributes to TGFβ1-induced EMT of ovarian tumor cells through miR-490-3p/TGFR1 axis. These findings will provide deep insights into the mechanism by which CCAT1 exerts its oncogenic role in ovarian cancer progression and facilitate developing novel therapeutical therapies for treating ovarian cancer.

MicroRNA-340 Induces Apoptosis and Inhibits Metastasis of Ovarian Cancer Cells by Inactivation of NF-κB1

Aims: Aberrant expression of microRNA-340 (miR-340) has been frequently reported in some cancers excluding ovarian cancer (OC). The role and its molecular mechanism of miR-340 in OC have not been reported. Methods: Real-time PCR was performed to detect the expression of miR-340 in OC cell lines. MiR-340 mimic and negative control were transfected into OC cells and the effects of miR-340 on the cell proliferation, cell cycle, apoptosis and metastasis were investigated by Brdu-ELISA assay, flow cytometry, qRT-PCR, Transwell and ELISA assays. Furthermore, protein level of NF-κB1 was measured by Western blotting. Meanwhile, luciferase assays were performed to validate NF-κB1 as miR-340 target in OC cells. Results: In this study, we explored the effects of miR-340 overexpression on apoptosis, invasion and EMT in OC cells. The mRNA level of miR-340 in OC cell lines and tissues was evidently reduced. The miR-340 mimic was transiently transfected into OC cells using Lipofectamine™ 2000 reagent. Subsequently, the Brdu-ELISA results showed that introduction of miR-340 inhibited cell proliferation. Our data also demonstrated that miR-340 mimic arrested cell cycle progression and promoted apoptosis of OC cells. In addition, miR-340 overexpression could also inhibit invasion and EMT of OC cells. qRT-PCR were used to determined the expressions of matrix metalloproteinase-2 and -9 (MMP-2 and -9) in OC cells. Next, we found that NF-κB1 expression was evidently reduced by up-regulation of miR-340. Bioinformatics analysis predicted that the NF-κB1 was a potential target gene of miR-340. Luciferase reporter assay further confirmed that miR-340 could directly target the 3' UTR of NF-κB1. Moreover, overexpression of NF-κB1 in OC cells transfected with miR-340 mimic partially reversed the inhibitory of miR-340 mimic. Conclusion: miR-340 induced cell apoptosis and inhibited metastasis in OC cells by down-regulation of NF-κB1.