Tumorigenicity Of Ovarian Cancer Cells Research Articles

Hsa_circ_0000129 targets miR-383-5p/tropomyosin 3 axis to facilitate ovarian cancer progression.

Ovarian cancer is one of the most prevalent malignancies among women. CircRNAs play key roles in the progression of ovarian cancer. This study aimed to investigate the mechanism of action of hsa_circ_0000129 and its effects on ovarian cancer. Expression of hsa_circ_0000129, tropomyosin 3 (TPM3), and miR-383-5p in ovarian cancer cell lines and tissue specimens was detected using qRT-PCR or western blotting. Cell counting kit-8 (CCK-8), colony formation, and transwell assays were performed to assess viability, proliferation, and migration of ovarian cancer cells. A xenograft model was used to study tumorigenicity of ovarian cancer cells in vivo. Luciferase and RNA immunoprecipitation assays were performed to determine binding between miR-383-5p and hsa_circ_0000129 or TPM3. Upregulation of hsa_circ_0000129 and TPM3 and downregulation of miR-383-5p were observed in ovarian cancer. Low hsa_circ_0000129 and TPM3 expression repressed viability, migration, and proliferation of ovarian cancer cells. Inhibition of miR-383-5p had a contrary effect. Furthermore, knockdown of hsa_circ_0000129 restricted the tumorigenicity of ovarian cancer cells. Mechanistically, hsa_circ_0000129 has a sponging effect on miR-383-5p, which targets TPM3. Hsa_circ_0000129 stimulated development of the malignant ovarian cancer phenotype by sponging miR-383-5p and releasing TPM3.

Circular RNA circMAN1A2 promotes ovarian cancer progression through the microRNA-135a-3p/IL1RAP/TAK1 pathway.

Ovarian cancer (OC) is the most lethal malignancy in women owing to its diagnosis only at the advanced stage. Elucidation of its molecular pathogenesis may help identify new tumor markers and targets for therapy. Circular RNAs (circRNAs) are stable, conserved, and functional biomolecules that can be used as effective biomarkers for various cancers. In this study, a potential circRNA related to early diagnosis of OC, circMAN1A2, was analyzed. Overexpression/knockdown of circMAN1A2 in OC cells was used to decipher its effects on cell proliferation with a Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine (EdU), cell cycle, clone formation, and wound healing assay. RNA pull-down and Dual luciferase assay were used to explain the underlying mechanism by which circMAN1A2 regulates OC cell proliferation. In vivo, the effect of circMAN1A2 in OC was evaluated using nude mouse xenograft experiments. CircMAN1A2 was highly expressed in OC and promoted proliferation, clone formation, and tumorigenicity of OC cells. In addition, we found that circMAN1A2 acted as a sponge for microRNA (miR)-135a-3p; miR-135a-3p directly targeted the 3' untranslated region of interleukin 1 receptor accessory protein (IL1RAP) in OC cells, thereby regulating the phosphorylation of transforming growth factor-beta activated kinase 1 (TAK1), which resulted in promotion of OC cell growth. CircMAN1A2 promotes OC cell proliferation by inhibiting the miR-135a-3p/IL1RAP/TAK1 axis. In conclusion, circMAN1A2 may be a biomarker for early detection of OC and a target for subsequent therapy.

TRAF4 promotes the malignant progression of high-grade serous ovarian cancer by activating YAP pathway

High-grade serous ovarian cancer (HGSOC) accounts for the majority of deaths caused by epithelial ovarian cancer. The specific molecular changes attributable to the pathogenesis of HGSOC are still largely unknown. TRAF4 has been identified to be up-regulated in certain cancers. However, the role and mechanism of TRAF4 in HGSOC remain unclear. In this study, we aim to explore the prognostic value and function of TRAF4 in HGSOC. Immunohistochemical staining and prognostic analysis were used to estimate the prognosis value of TRAF4 in HGSOC. Cell counting assays, colony formation assays, sphere formation assays and tumorigenic assays were used to explore the function of TRAF4 in ovarian cancer cells. Furthermore, RNA-seq, qPCR and western blotting were performed to investigate the molecular mechanism of TRAF4 in ovarian cancer cells. The results showed that TRAF4 was significantly higher expressed in ovarian cancer than normal ovarian epithelium. Moreover, high expression of TRAF4 was significantly associated with shorter overall survival and recurrence-free survival in HGSOC. Knockdown of TRAF4 significantly inhibited the proliferation and tumorigenicity of ovarian cancer cells, whereas overexpression of TRAF4 promoted the proliferation and tumorigenicity of ovarian cancer cells both in vitro and in vivo. Mechanistically, our study demonstrated that TRAF4 expression was positively correlated with the YAP pathway gene signatures, and the malignant progression induced by TRAF4 was inhibited after silencing YAP signaling by its selective inhibitor. In conclusion, our findings suggested that TRAF4 promoted the malignant progression of ovarian cancer cells by activating YAP pathway and might serve as a prognostic biomarker for HGSOC.

CircASH2L Promotes Ovarian Cancer Tumorigenesis, Angiogenesis, and Lymphangiogenesis by Regulating the miR-665/VEGFA Axis as a Competing Endogenous RNA.

Ovarian cancer is the leading cause of gynecologic cancer-related deaths. Emerging research has revealed a close relationship between circular RNAs (circRNAs) and ovarian cancer development, metastasis, and prognosis. The objective of our research was to further explore the relationship between circASH2L and ovarian cancer. Quantitative real-time polymerase chain reaction was used to detect the differential expression of circRNAs between normal ovaries and ovarian cancer tissues. The impact of circASH2L on the proliferation, invasion, and tumorigenicity of ovarian cancer cells was evaluated using gain- and loss-of-function experiments. The molecular mechanisms of circASH2L function were investigated using bioinformatics analysis, RNA fluorescence in situ hybridization, western blots, and dual-luciferase reporter assays. The results showed that circASH2L was remarkably upregulated in ovarian cancer. The invasion and growth of ovarian cancer cells were suppressed by circASH2L knockdown in vitro, and downregulation of circASH2L restrained both angiogenesis and lymphangiogenesis of tumor xenografts in vivo. Furthermore, circASH2L was mostly distributed in the cytoplasm, where it competes with vascular endothelial growth factor A (VEGFA) for binding to miR-665. These findings indicate that circASH2L has an oncogenic function in ovarian cancer. In conclusion, circASH2L plays a critical role in regulating ovarian cancer cell tumorigenesis, angiogenesis, and lymphangiogenesis through the miR-665/VEGFA axis and, therefore, is a possible candidate target for ovarian cancer treatment.

CD83, a Novel MAPK Signaling Pathway Interactor, Determines Ovarian Cancer Cell Fate.

Ovarian cancer is a leading cause of death from gynecologic malignancies worldwide. Although CD83 is widely described as a solid marker for mature dendritic cells, emerging pieces of evidence indicate the expression of membrane protein CD83 by various tumor cells, including ovarian cancer cells. However, the potential role of CD83 in ovarian cancer cell properties and development remains absolutely unknown. By using human CD83 stable overexpression and knockdown sublines of several ovarian cancer cells, we observed that CD83 advanced the growth proliferation, colony formation ability, spheroid formation, and in vivo tumorigenicity of ovarian cancer cells; surprisingly, CD83 limited their migration and invasion potentials. Positive regulation of proliferation/stemness factors (e.g., cyclin-CDKs and KIT/CD44) but negative regulation of matrix metallopeptidases (e.g., MMP1 and 7) by CD83 were revealed by the integrated analysis of transcriptome and proteome. Furthermore, immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP) first identified the association of CD83 with MAP3K7 (also known as TAK1) and MAP3K7-binding protein TAB1 on the cell membrane. Moreover, CD83 functions through the activation of MAP3K7-MEK1/2-ERK1/2 cascades to further regulate downstream FOXO1/p21/CDK2/CCNB1 and STAT3/DKK1 signaling pathways, thus activating proliferation and spheroid formation of ovarian cancer cells, respectively. Collectively, our findings define a CD83-MAPK pathway in the regulation of proliferation and stemness in ovarian cancer cells, with potential therapeutic applications in blocking their progression.

MicroRNA-196a promotes cell proliferation and inhibits apoptosis in human ovarian cancer by directly targeting DDX3 and regulating the PTEN/PI3K/AKT signaling pathway.

MicroRNAs (miRNAs/miRs) are small noncoding RNAs 19–24 nucleotides in length, which can play an important role in tumor development. However, the influence of miRNAs on the tumorigenicity of ovarian cancer cells has not been fully elucidated. Previously, DEAD box protein (DDX) 1 has been shown to play a role in tumor suppression of ovarian cancer progression. However, the functions of other DDX members in ovarian cancer development remain largely unknown. In the present study, it was demonstrated that overexpression of miR-196a promoted ovarian cancer cell proliferation. In addition, DDX3 was significantly downregulated in ovarian cancer cell lines relative to the normal ovarian cell line. Moreover, DDX3 was identified as a direct target of miR-196a in ovarian cancer cells. In addition, a preliminary mechanistic investigation indicated that downregulation of DDX3 promoted ovarian cancer cell proliferation through the PTEN/PI3K/AKT pathway. Taken together, this confirmed an association between DDX3 and miR-196a in ovarian cancer, and showed that miR-196a promoted the proliferation of ovarian cancer cells and attenuated apoptosis by downregulating the expression of DDX3 through the PTEN/PI3K/AKT pathway. Overall, the results of the present study suggested that DDX3 could be a novel target for ovarian cancer treatment.

ACTL6A regulates follicle-stimulating hormone-driven glycolysis in ovarian cancer cells via PGK1

Enhanced glycolysis has been identified as a hallmark of cancer. As a novel oncogene, ACTL6A is aberrantly amplified in several types of human cancers and has been shown to regulate tumor growth and progression. However, the roles of ACTL6A in the development of ovarian cancer and the regulation of cancer glucose metabolism are mostly unknown. Here we show that ACTL6A is overexpressed in ovarian cancers compared with adjacent non-tumor tissues, and that ACTL6A overexpression correlates with poor prognosis. Silencing of ACTL6A in vitro inhibits proliferation, clonal growth, and migration, and decreases glucose utilization, lactate production, and pyruvate levels of ovarian cancer cells. We found a positive correlation between ACTL6A and PGK1 expression in ovarian cancer tissues. Enforced ACTL6A expression increased PGK1 expression, whereas knockdown of ACTL6A had the opposite effect. Altered ACTL6A expression inhibits the tumorigenicity of ovarian cancer cells in vivo by downregulating PGK1. In addition, the expression of ACTL6A is regulated by follicle-stimulating hormone (FSH) stimulation via PI3K/AKT pathway. Importantly, ACTL6A regulates FSH-enhanced glycolysis in ovarian cancer. Taken together, our findings highlight the critical role of ACTL6A in ovarian cancer development and identify its contribution to glucose metabolism of cancer cells.

CD44v6 may influence ovarian cancer cell invasion and migration by regulating the NF-κB pathway.

Ovarian cancer (OC) has the worst prognosis among all malignancy types in females worldwide according to epidemiological studies in 2017. Although radiotherapy, chemotherapy and surgical treatment are the most common treatment methods, their curative effects are not satisfactory. The present study aimed to examine the role of cluster of differentiation 44 variant 6 (CD44v6) in the molecular mechanism of the proliferation and tumorigenicity of OC cells, and provide a novel target for the clinical treatment of OC. A total of 46 clinical samples were collected, including 24 malignant ovarian tumor tissue samples and 22 benign ovarian tissue samples. Expression of CD44v6 and nuclear factor-κB (NF-κB) in these samples was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. The A2780 OC cell line was used to establish a normal control group, a negative control group and a CD44v6-small interfering (si)RNA transfection group. The expression of CD44v6 and NF-κB mRNA was detected in each group by RT-qPCR. The proliferation, invasion and migration abilities of the cells were then assessed by Transwell and colony formation assays. Additionally, immunofluorescence was used to detect nuclear NF-κB expression. CD44v6 and NF-κB mRNA expression levels were significantly increased in malignant ovarian tumor tissues, compared with normal ovarian tissues (P<0.01), and immunohistochemistry demonstrated similar results. In the CD44v6-siRNA group, NF-κB mRNA expression was significantly reduced, compared with the control and negative control (both P<0.01) groups. Transwell and colony formation assays demonstrated that the migration, invasion and colony formation abilities of OC cells in the CD44v6-siRNA group were significantly reduced, compared with the control and negative control (both P<0.01) groups. Immunofluorescence results demonstrated that the expression of NF-κB in the cytoplasm and nucleus of the CD44v6-siRNA group was also markedly reduced, compared with the other two groups. In conclusion, CD44v6 may participate in the proliferation of OC cells through activation of the NF-κB pathway and these observations may provide a novel therapeutic target for the clinical treatment of OC.

HER2 decreases drug sensitivity of ovarian cancer cells via inducing stem cell-like property in an NFκB-dependent way.

Increasing evidence shows that cancer stem cells are responsible for drug resistance and relapse of tumors. In breast cancer, human epidermal growth factor receptor 2 (HER2) induces Herceptin resistance by inducing cancer stem cells. In the present study, we explored the effect of HER2 on cancer stem cells induction and drug sensitivity of ovarian cancer cell lines. First, we found that HER2 overexpression (HER2 OE) induced, while HER2 knockdown (HER2 KD) decreased CD44+/CD24− population. Consistently, HER2 expression was closely correlated with the sphere formation efficiency (SFE) of ovarian cancer cells. Second, we found that NFκB inhibition by specific inhibitor JSH23 or siRNA targetting subunit p65 dramatically impaired the induction of ovarian cancer stem cells by HER2, indicating that NFκB mediated HER2-induced ovarian cancer stem cells. Third, we found that HER2 KD significantly attenuated the tumorigenicity of ovarian cancer cells. Further, we found that HER2 inhibition increased drastically the sensitivity of ovarian cancer cells to doxorubicin (DOX) or paclitaxel (PTX). Finally, we examined the correlation between HER2 status and stem cell-related genes expression in human ovarian tumor tissues, and found that expressions of OCT4, COX2, and Nanog were higher in HER2 positive tumors than in HER2 negative tumors. Consistently, the 5-year tumor-free survival rate of HER2 positive patients was dramatically lower than HER2 negative patients. Taken together, our data indicate that HER2 decreases drug sensitivity of ovarian cancer cells via inducing stem cell-like property.

Upregulation of Circular RNA VPS13C-has-circ-001567 Promotes Ovarian Cancer Cell Proliferation and Invasion.

Circular RNAs (circRNAs) comprise a class of noncoding RNA molecules that play an important role in several normal cellular functions, as well as tumorigenesis in humans. However, the expression patterns and biological functions of circRNAs in ovarian cancer (OC) remain unclear. Therefore, we investigated the expression profiles and biological functions of certain circRNAs in OC tumor tissues. The expression of three circRNAs (VPS13C-has-circ-001567, RAD50-has-circ-00718, and SPECC1-has-circ-000013) was detected by real-time polymerase chain reaction in OC cell lines, and also in tumor and pericarcinous tissues obtained from 20 patients with OC. The function of VPS13C-has-circ-001567 in SKOV3 and OV-1063 cells was investigated by knockdown of VPS13C-has-circ-001567 and then analyzing any resultant effects on the cell cycle, cell proliferation, apoptosis, and cell invasion ability. E-cadherin and N-cadherin expressions were analyzed by immunofluorescence and western blotting. Finally, the tumorigenicity of OC cells was assessed in nude mice. The results showed that VPS13C-has-circ-001567 was expressed at significantly higher levels in OC tumor tissues compared with pericarcinous tissues, and this overexpression was associated with tumor node metastasis stage and lymph node metastasis. We found that knockdown of VPS13C-has-circ-001567 significantly promoted apoptosis and inhibited the proliferation of SKOV3 and OV-1063 cells in vitro. Knockdown of VPS13C-has-circ-001567 led to cell cycle arrest at G1 phase and decreased the percentage of S1 phase cells. Additionally, knockdown of VPS13C-has-circ-001567 decreased the invasion ability of SKOV3 and OV-1063 cells, and also changed the levels of E-cadherin and N-cadherin expressions. Knockdown of VPS13C-has-circ-001567 significantly reduced the tumorigenicity of OC cells. Taken together, our results suggest that VPS13C-has-circ-001567 plays a role in the development of OC and might be a prognostic marker and therapeutic target for OC.

LncRNA PTAR promotes EMT and invasion-metastasis in serous ovarian cancer by competitively binding miR-101-3p to regulate ZEB1 expression

BackgroundOvarian cancer (OvCa) is one of the most common malignant diseases of the female reproductive system in the world. The majority of OvCa is diagnosed with metastasis in the abdominal cavity. Epithelial-to-mesenchymal transition (EMT) plays a key role in tumor cell metastasis. However, it is still unclear whether long non-coding RNA (lncRNA) is implicated in EMT and influences cell invasion and metastasis in OvCa.ResultsIn this study, using bioinformatcis analysis, we constructed a lncRNA-mediated competing endogenous RNA (ceRNA) network for mesenchymal OvCa and identified lncRNA AP000695.4, which we named pro-transition associated RNA (PTAR). PTAR was significantly up-regulated in the mesenchymal subtype samples compared with the epithelial subtype samples from the TCGA OvCa data sets. In addition, our study showed that PTAR expression was positively correlated with the expression level of ZEB1 in the mesenchymal OvCa samples. Meanwhile, we found that silencing miR-101 promoted cell migration, whereas the overexpression of miR-101 suppressed EMT and cell migration in OvCa cell lines through the regulation of ZEB1. Further analysis showed that enhanced expression of PTAR promoted EMT and metastasis through the regulation of miR-101, whereas silencing PTAR led to the attenuation of TGF-β1-induced tumorigenicity in ovarian cancer cells. Mechanistically, we found that PTAR acted as a ceRNA of miR-101, as forced expression of PTAR reduced the expression and activity of miR-101. More importantly, the knockdown of PTAR reduced tumorigenicity and metastasis in vivo.ConclusionsTaken together, the results from our study highlight a role for the PTAR-miR-101-ZEB1 axis in OvCa, which offers novel strategies for the prevention of metastasis in OvCa.

Abstract 897: PEA-15 regulates epithelial-mesenchymal transition and invasive behavior through its phosphorylation in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype with no proven active targeted therapies available. Patients with TNBC have a very poor prognosis because the disease often metastasizes. Newer approaches to preventing metastasis and inhibiting tumor growth, are crucial to improving prognosis for these patients. Previously it has been shown that PEA-15 binds to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. Our previous studies showed that overexpression of wild type PEA-15 inhibited ERK activity and reduced tumor volume in a TNBC xenograft model. We also showed that PEA-15 being unphosphorylated at both Ser104 and Ser116 inhibited ovarian cancer cell tumorigenicity. However, the function and impact of PEA-15 phosphorylation on TNBC is not well understood. From these observations, we hypothesized that unphosphorylated PEA-15 will prevent metastasis in TNBC through the inhibition of EMT. To study the effect of the phosphorylation status of PEA-15 on metastasis in TNBC, we established stable cell lines overexpressing nonphosphorylatable (PEA-15-AA) and phosphomimetic (PEA-15-DD) mutants in MDA-MB-468 cells. The clonogenic growth of PEA-15-AA was significantly reduced by 80% compared with vector control, PEA-15-V. Anchorage-independent growth of PEA-15-AA was inhibited by 60% compared with PEA-15-V (P&amp;lt;0.05). PEA15-AA upregulated the expression of E-cadherin, a key epithelial molecular marker and decreased expression of vimentin suggesting that PEA-15-AA reverses EMT. Moreover, compared with PEA-15-V, migration and invasion of PEA-15-AA were significantly reduced. To determine the mechanism of how PEA-15 regulates EMT and metastasis, we performed RT-PCR immune and metastasis arrays. We found that expression levels of IL-6, IL-8 and PDGF-BB were greatly decreased in PEA-15-AA. Upon stimulation of the PEA-15-AA with these cytokines, mesenchymal characteristics were partially rescued indicating that PEA-15-AA may inhibit the expression of these cytokines, thereby reversing EMT. Further, to determine the in vivo effect of PEA-15-AA, we injected stable transfectants of MDA-MB-468 cells into the mammary fat pad of NOD/SCID mice. The PEA-15-DD-injected group showed an increase (P&amp;lt;0.05) in tumor volume compared with PEA-15-V and PEA-15-AA groups. Additionally, immunohistochemical data revealed that vimentin and p-β-catenin (Ser675) expression was greatly decreased in PEA-15-AA tissues, suggesting that PEA-15-AA has antitumor effects. Further studies are warranted to evaluate the impact of PEA-15 phosphorylation on metastasis in vivo using mice bearing highly metastatic human breast tumor and determine the mechanism underlying decreased cytokine expression in PEA-15-AA. Together, this study highlights the potential for overexpression of unphosphorylated PEA-15 as an approach for TNBC-targeted therapy. Citation Format: Jihyun Park, Evan N. Cohen, Gaurav Chauhan, Jangsoon Lee, Naoto T. Ueno, Debu Tripathy, James M. Reuben, Chandra Bartholomeusz. PEA-15 regulates epithelial-mesenchymal transition and invasive behavior through its phosphorylation in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2017-897

OCT4 controls mitotic stability and inactivates the RB tumor suppressor pathway to enhance ovarian cancer aggressiveness.

OCT4 (Octamer-binding transcription factor 4) is essential for embryonic stem cell self-renewal. Here we show that OCT4 increases the aggressiveness of high-grade serous ovarian cancer (HG-SOC) by inactivating the Retinoblastoma tumor suppressor pathway and enhancing mitotic stability in cancer cells. OCT4 drives the expression of Nuclear Inhibitor of Protein Phosphatase type 1 (NIPP1) and Cyclin F (CCNF) that together inhibit Protein Phosphatase 1 (PP1). This results in pRB hyper-phosphorylation, accelerated cell proliferation and increased in vitro tumorigenicity of ovarian cancer cells. In parallel, OCT4 and NIPP1/CCNF drive the expression of the central Chromosomal Passenger Complex (CPC) components, Borealin, Survivin and the mitotic kinase Aurora B, promoting the clustering of supernumerary centrosomes to increase mitotic stability. Loss of OCT4 or NIPP1/CCNF results in severe mitotic defects, multipolar spindles and supernumerary centrosomes, finally leading to the induction of apoptosis. These phenotypes were recapitulated in different cancer models indicating general relevance for human cancer. Importantly, activation of these parallel pathways leads to dramatically reduced overall survival of HG-SOC patients. Altogether, our data highlights an unprecedented role for OCT4 as central regulator of mitotic fidelity and RB tumor suppressor pathway activity. Disrupting this pathway represents a promising strategy to target an aggressive subpopulation of HG-SOC cells.

Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech.

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68+ and HmAIF-1+ macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

ARHGAP10, downregulated in ovarian cancer, suppresses tumorigenicity of ovarian cancer cells.

Rho GTPase-activating proteins (RhoGAPs) are implicated in the development and progression of ovarian cancer. ARHGAP10 is a member of RhoGAP proteins and inactivates Cdc42 by converting GTP-bound form to GDP-bound form. Here, we aimed to evaluate ARHGAP10 expression profile and functions in ovarian cancer. The decreased expression of ARHGAP10 was found in 77.3% (58/75) of ovarian cancer tissues, compared with their non-tumorous counterparts. Furthermore, overall survival in ovarian cancer patients with higher expression of ARHGAP10 was longer than those with lower expression. Ectopic expression of ARHGAP10 in two ovarian cancer cell lines with lower expression of ARHGAP10 (A2780 and HO-8910) dramatically suppressed cell proliferation in vitro. In nude mice, its stable overexpression significantly inhibited the tumorigenicity of A2780 cells. We further demonstrated that overexpression of ARHGAP10 significantly inhibited cell adhesion, migration and invasion, resulted in cell arrest in G1 phase of cell cycle and a significant increase of apoptosis. Moreover, ARHGAP10 interacted with Cdc42 and overexpression of ARHGAP10 inhibited the activity of Cdc42 in A2780 cells. Gene set enrichment analysis on The Cancer Genome Atlas dataset showed that KEGG cell cycle, replication and base excision repair (BER) pathways were correlatively with the ARHGAP10 expression, which was further confirmed in ovarian cancer cells by western blotting. Hence, ARHGAP10 may serve as a tumor suppressor through inactivating Cdc42, as well as inhibiting cell cycle, replication and BER pathways. Our data suggest an important role of ARHGAP10 in the molecular etiology of cancer and implicate the potential application of ARHGAP10 in cancer therapy.

MiR-1207 overexpression promotes cancer stem cell-like traits in ovarian cancer by activating the Wnt/β-catenin signaling pathway.

Wnt/β-catenin signaling pathway is strictly controlled by multiple negative regulators. However, how tumor cells override the negative regulatory effects to maintain constitutive activation of Wnt/β-catenin signaling, which is commonly observed in various cancers, remains puzzling. In current study, we reported that overexpression of miR-1207 in ovarian cancer activated Wnt/β-catenin signaling by directly targeting and suppressing secreted Frizzled-related protein 1 (SFRP1), AXIN2 and inhibitor of β-catenin and TCF-4 (ICAT), which are vital negative regulators of the Wnt/β-catenin pathway. We found that the expression of miR-1207 was ubiquitously upregulated in both ovarian cancer tissues and cells, which inversely correlated with patient overall survival. Furthermore, overexpression of miR-1207 enhanced, while silencing miR-1207 reduced, stem cell-like traits of ovarian cancer cells in vitro and in vivo, including tumor sphere formation capability and proportion of SP+ and CD133+ cells. Importantly, upregulating miR-1207 promoted, while silencing miR-1207 inhibited, the tumorigenicity of ovarian cancer cells. Hence, our results suggest that miR-1207 plays a vital role in promoting the cancer stem cell-like phenotype in ovarian cancer and might represent a potential target for anti-ovarian cancer therapy.

Knockdown of RhoA expression alters ovarian cancer biological behavior in vitro and in nude mice.

RhoA regulates cell proliferation, migration, angiogenesis and gene expression. Altered RhoA activity contributes to cancer progression. The present study investigated the effects of RhoA knockdown on the regulation of ovarian cancer biological behavior in vitro and in nude mice. The expression of RhoA was knocked down using a lentivirus carrying RhoA short hairpin RNA (shRNA) in ovarian cancer cells and was confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The altered ovarian cancer biological behaviors were assayed by cell viability, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL), migration, invasion, and nude mice tumorigenicity assays, while the altered gene expression was detected by RT-qPCR and western blot analysis. The results showed that lentivirus-carrying RhoA shRNA significantly suppressed RhoA expression in ovarian cancer cells, which suppressed tumor cell viability, migration, invasion and adhesion in vitro. RhoA silencing also inhibited the tumorigenicity of ovarian cancer cells in nude mice, which was characterized by the suppression of tumor xenograft formation and growth and induction of tumor cell apoptosis. The results of the present study demonstrated that knockdown of RhoA expression had a significant antitumor effect on ovarian cancer cells in vitro and in nude mice, suggesting that RhoA may be a target for the development of a novel therapeutic strategy in the control of ovarian cancer.

Upregulation of miR-572 transcriptionally suppresses SOCS1 and p21 and contributes to human ovarian cancer progression.

Ovarian cancer is a gynecological malignancy with high mortality rates worldwide and novel diagnostic and prognostic markers and therapeutic targets are urgently required. The suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibitor 1A (p21(KIP)) are known to regulate tumor cell proliferation. However, the mechanisms that regulate these genes have not yet been completely elucidated. In the present study, analysis of a published microarray-based high-throughput assessment (NCBI/E-MTAB-1067) and real-time PCR demonstrated that miR-572 was upregulated in human ovarian cancer tissues and cell lines. Kaplan-Meir analysis indicated that high level expression of miR-572 was associated with poorer overall survival. Ectopic miR-572 promoted ovarian cancer cell proliferation and cell cycle progression in vitro and tumorigenicity in vivo. SOCS1 and p21 were identified as direct targets of miR-572 and suppression of SOCS1 or p21 reversed the inhibiting-function of miR-572-silenced cell on proliferation and tumorigenicity in ovarian cancer cells. Additionally, the expression of miR-572 correlated inversely with the protein expression levels of SOCS1, p21 and positively with Cyclin D1 in ovarian carcinoma specimens. This study demonstrates that miR-572 post-transcriptionally regulates SOCS1 and p21 and may play an important role in ovarian cancer progression; miR-572 may represent a potential therapeutic target for ovarian cancer therapy.

Abstract 1433: Omental derived adipose stromal cells regulate nitric oxide homeostasis to maintain tumorigenicity in ovarian cancer cells

Abstract The rate of obesity is increasing dramatically in the United States, resulting in a rising incidence of obesity associated diseases including colorectal, gallbladder, esophageal, renal, breast, endometrial and ovarian cancer. The location of excess adipose tissue is reportedly an important factor which influences the effect of obesity on cancer incidence. The tumor promoting effects of regional adipose may be attributed, at least in part, to the presence of mesenchymal stem cell (MSC) population in adipose tissue. Adipose derived stromal cells (ASCs) are a multi-potent population of MSCs, which support cancer progression in many models. These cells are uniquely tumor tropic, migrating into tumor stroma from regional sources where they support formation of tumor stroma elements and promote tumor progression. Thus, we hypothesize that the omentum provides a particularly hospitable microenvironment for OVCA metastasis due to the presence of omental ASCs (O-ASCs). One critically important, yet often overlooked contributor to ovarian tumor growth, progression and metastasis in obese individuals is nitric oxide (NO). Cancer cells' high affinity for NO and NO's characteristic high solubility in lipids could explain the proximity of many carcinomas to fatty tissue, and thus the high positive correlation between obesity and cancer. Here we report a novel NO mediated reciprocal coupling between O-ASCs and ovarian cancer which promotes cancer cell growth and energy metabolism. Our results show that O-ASCs supports viability of cancer cells by secreting arginine and cancer cells in turn induce adipogenesis in O-ASCs by secreting citrulline. We found that OASCs promote tumor growth and increase resistance to chemotherapy by modulating NO homeostasis. The inhibition of NO synthesis or scavenging of NO enhanced chemo-sensitivity. Using metabolic profiling we found that OASCs rewire metabolic pathways related to nitrogenous amino acids and reprogram energy metabolism. In summary, our data indicate that modulation of intrinsic NO substantially affects tumor growth and drug toxicity in ovarian cancer cells. Our study will lead to significant advances in the understanding of the omentum in altering cancer metabolism and lead to novel therapeutics. Citation Format: Bahar Salimian Rizi, Ann H. Klopp, Deepak Nagrath. Omental derived adipose stromal cells regulate nitric oxide homeostasis to maintain tumorigenicity in ovarian cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1433. doi:10.1158/1538-7445.AM2014-1433

Abstract 3874: Loss of DDB2 enhances the tumorigenicity of ovarian cancer cells through expanding cancer stem-like cell population

Abstract Ovarian cancer is an extremely aggressive disease associated with a high percentage of recurrence and chemotherapy resistance. DNA damage binding protein 2 (DDB2) is a 48-kDa protein originally identified as a component of the damage-specific DNA-binding heterodimeric complex DDB. Besides the known ability of DDB2 protein to bind ultraviolet (UV)-damaged DNA and serving as the initial damage recognition factor during nucleotide excision repair (NER), DDB2 was also reported to be involved in the promotion of apoptosis to reduce cisplatin resistance of ovarian cancer cells, control of cell migration, invasiveness and breast tumor progression, as well as suppression of metastasis of colon cancer through a transcriptional regulatory mechanism. Here we show that DDB2 is downregulated in human ovarian serous adenocarcinomas. DDB2 overexpression in human ovarian cancer cell line CP70 suppressed its capability to recapitulate tumors in athymic nude mice, as well as the growth of these cells cultured in semisolid media. Flow cytometry analyses indicate that DDB2 overexpression in CP70 cells reduced the abundance of ALDH+ cells, which are believed to be cancer stem-like cells (CSCs) in ovarian cancer. In contrast, downregulation of DDB2 in ovarian cancer cell line A2780 increased the percentage of ALDH+ cells. In addition, we analyzed the expression level of DDB2 in the putative CSCs isolated from various ovarian cancer cell lines, and demonstrated that DDB2 expression is downregulated in all tested CSCs in comparison with their parental bulk tumor cells. Overexpression of DDB2 in the ovarian CSCs reduced the expression of CSC marker Nanog, as well as inhibited the self-renewal of these CSCs. In addition, overexpression of DDB2 in CSCs isolated from ovarian cancer cell line SKOV-3 also decreased the tumorigenicity of these cells in NOD/SCID mice. Further analysis of publicly available gene expression array datasets revealed that low DDB2 expression correlates with poor outcomes among ovarian cancer patients, suggesting that loss of DDB2 may promote ovarian tumor relapse through expanding CSC population. Given that the DDB2 protein level is downregulated in ovarian tumor cells, targeting DDB2 expression could be a promising strategy to eradicate CSCs, and helps to halt ovarian cancer relapse. (Supported by NIH R01 grant CA151248 to QEW) Citation Format: Chunhua Han, Ran Zhao, Xingluo Liu, Amit Kumar Srivastava, Li Gong, Hsiaoyin Mao, Meihua Qu, Weiqiang Zhao, Jianhua Yu, Qi-En Wang. Loss of DDB2 enhances the tumorigenicity of ovarian cancer cells through expanding cancer stem-like cell population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3874. doi:10.1158/1538-7445.AM2014-3874