miR-590-3p Targets Cyclin G2 and FOXO3 to Promote Ovarian Cancer Cell Proliferation, Invasion, and Spheroid Formation

Ovarian cancer is the leading cause of death from gynecological cancers. MicroRNAs (miRNAs) are small, non-coding RNAs that interact with the 3′ untranslated region (3′ UTR) of target genes to repress their expression. We have previously reported that miR-590-3p promoted ovarian cancer growth and metastasis, in part by targeting Forkhead box A (FOXA2). In this study, we further investigated the mechanisms by which miR-590-3p promotes ovarian cancer development. Using luciferase reporter assays, real-time PCR, and Western blot analyses, we demonstrated that miR-590-3p targets cyclin G2 (CCNG2) and Forkhead box class O3 (FOXO3) at their 3′ UTRs. Silencing of CCNG2 or FOXO3 mimicked, while the overexpression of CCNG2 or FOXO3 reversed, the stimulatory effect of miR-590-3p on cell proliferation and invasion. In hanging drop cultures, the overexpression of mir-590 or the transient transfection of miR-590-3p mimics induced the formation of compact spheroids. Transfection of the CCNG2 or FOXO3 plasmid into the mir-590 cells resulted in the partial disruption of the compact spheroid formation. Since we have shown that CCNG2 suppressed β-catenin signaling, we investigated if miR-590-3p regulated β-catenin activity. In the TOPFlash luciferase reporter assays, mir-590 increased β-catenin/TCF transcriptional activity and the nuclear accumulation of β-catenin. Silencing of β-catenin attenuated the effect of mir-590 on the compact spheroid formation. Taken together, these results suggest that miR-590-3p promotes ovarian cancer development, in part by directly targeting CCNG2 and FOXO3

Wnt/β-catenin signalling in ovarian cancer: Insights into its hyperactivation and function in tumorigenesis

Abstract Epithelial ovarian cancer (EOC) is the deadliest female malignancy. The Wnt/β-catenin pathway plays critical roles in regulating embryonic development and physiological processes. This pathway is tightly regulated to ensure its proper activity. In the absence of Wnt ligands, β-catenin is degraded by a destruction complex. When the pathway is stimulated by a Wnt ligand, β-catenin dissociates from the destruction complex and translocates into the nucleus where it interacts with TCF/LEF transcription factors to regulate target gene expression. Aberrant activation of this pathway, which leads to the hyperactivity of β-catenin, has been reported in ovarian cancer. Specifically, mutations of CTNNB1, AXIN, or APC, have been observed in the endometrioid and mucinous subtypes of EOC. In addition, upregulation of the ligands, abnormal activation of the receptors or intracellular mediators, disruption of the β-catenin destruction complex, inhibition of the association of β-catenin/E-cadherin on the cell membrane, and aberrant promotion of the β-catenin/TCF transcriptional activity, have all been reported in EOC, especially in the high grade serous subtype. Furthermore, several non-coding RNAs have been shown to regulate EOC development, in part, through the modulation of Wnt/β-catenin signalling. The Wnt/β-catenin pathway has been reported to promote cancer stem cell self-renewal, metastasis, and chemoresistance in all subtypes of EOC. Emerging evidence also suggests that the pathway induces ovarian tumor angiogenesis and immune evasion. Taken together, these studies demonstrate that the Wnt/β-catenin pathway plays critical roles in EOC development and is a strong candidate for the development of targeted therapies

Epidermal growth factor promotes cyclin G2 degradation via calpain-mediated proteolysis in gynaecological cancer cells

<div><p>Cyclin G2 (CCNG2) is an atypical cyclin that functions to inhibit cell cycle progression and is often dysregulated in human cancers. We have previously shown that cyclin G2 is highly unstable and can be degraded through the ubiquitin/proteasome pathway. Furthermore, cyclin G2 contains a PEST domain, which has been suggested to act as a signal for degradation by multiple proteases. In this study, we determined if calpains, a family of calcium-dependent proteases, are also involved in cyclin G2 degradation. The addition of calpain inhibitors or silencing of calpain expression by siRNAs strongly enhanced cyclin G2 levels. On the other hand, incubation of cell lysates with purified calpains or increasing the intracellular calcium concentration resulted in a decrease in cyclin G2 levels. Interestingly, the effect of calpain was found to be dependent on the phosphorylation of cyclin G2. Using a kinase inhibitor library, we found that Epidermal Growth Factor (EGF) Receptor is involved in cyclin G2 degradation and treatment with its ligand, EGF, induced cyclin G2 degradation. In addition, the presence of the PEST domain is necessary for calpain and EGF action. When the PEST domain was completely removed, calpain or EGF treatment failed to trigger degradation of cyclin G2. Taken together, these novel findings demonstrate that EGF-induced, calpain-mediated proteolysis contributes to the rapid destruction of cyclin G2 and that the PEST domain is critical for EGF/calpain actions.</p></div

Cyclin G2 is a target of calpain-mediated proteolysis.

<p>A) OV2008 cells were transiently transfected with full-length cyclin G2 (CCNG2-V5), PEST-24, or ΔPEST and lysed in a buffer containing either calcium, purified calpain-1 (CAPN-1), or the combination of calcium and CAPN-1, with or without the calpain inhibitor, calpeptin. Calpain-1 induced the degradation of full length cyclin G2 and PEST-24 and this effect was attenuated by calpeptin. However, the level of ΔPEST was not affected by either calpain or calpeptin. B) Calpain promoted the degradation of cyclin G2 in ovarian cancer cells. ES2 and SKOV3.ip1 cells stably transfected with FLAG-CCNG2 were lysed in a buffer containing calcium, CAPN-1, and/or calpeptin, as indicated. The combination of calcium and calpain dramatically decreased cyclin G2 levels, whereas calpeptin reversed this effect. C) Inhibition of calpain activity enhanced cyclin G2 levels. OV2008 cells were transiently transfected with the FLAG-CCNG2 plasmid and treated with or without 50μM calpeptin for 2h or 6h. Western blot analysis demonstrated a protective effect of calpeptin on cyclin G2 stability. D) Silencing of calpains increased cyclin G2 levels. Top panel, OV2008 cells were transfected with siRNAs for calpain-1 (si-CAPN-1) or calpain-2 (si-CAPN-2) and casein zymography was performed to confirm the down-regulation and specificity of each siRNA for its respective calpain. Calpain-2 is identified by higher mobility on the gel. Bottom panel, OV2008 cells were transfected with si-CAPN-1 or si-CAPN-2 for 6 hours prior to overnight (16 hour) transfection of FLAG-CCNG2. Cells were recovered for 6 hours in the presence of CHX. Both calpain siRNAs increased cyclin G2 stability. NC, non-targeting control.</p

Proposed role of EGF in cyclin G2 degradation.

<p>EGF binds to its receptor, EGFR, to induce cyclin G2 phosphorylation, either directly or indirectly via downstream kinases. EGF may also activate calpain-2 to enhance its activity. Phosphorylated cyclin G2 is recognized and degraded by calpains.</p

Cyclin G2 is an unstable protein and can be degraded through a calcium-mediated mechanism.

<p>A) OV2008, SKOV3.ip1, or ES2 cells stably transfected with a FLAG-tagged cyclin G2 (FLAG-CCNG2) were seeded at equal densities and treated with 10μg/ml cychoheximide (CHX) to block <i>de novo</i> protein synthesis. Cells were lysed before CHX treatment (time 0) or at 1 to 5 hour after CHX treatment. Cyclin G2 levels were determined by Western blotting using an anti-FLAG antibody. B) Prediction of calpain cleavage site using CALPCLEAV. Many potential sites were found; however, the one located in the PEST domain between position 319 and 320 had the highest score. C) Lystaes of OV2008 or ES2 cells stably transfected with FLAG-CCNG2 were incubated in a buffer containing different concentrations of CaCl_2, for 1–2 hours (OV2008) or 1 hour (ES2). Cyclin G2 levels were analyzed by Western blotting. Increased calcium concentration resulted in decreased amounts of cyclin G2. D) Upper panel: Schematic representation of three cyclin G2 constructs, full-length-CCNG2 (CCNG2), PEST-24 (containing the first 24 amino acid of the PEST domain), and ΔPEST (complete removal of the PEST domain). Lower panel: Cyclin G2 wild type and deletion constructs were used to transfect OV2008 cells. Following transfection, cells were treated with either DMSO as a control or 1μM of the calcium ionophore, A23187, with or without pre-incubation with 20μM ALLN for 30 minutes. Cells were lysed and Western blot analyses were performed. Treatment with A23187 decreased the levels of full length and PEST-24 cyclin G2 while the protease inhibitor, ALLN, protected cyclin G2 from degradation. A23187 and ALLN had no effect on the level of ΔPEST.</p

MiR-590-3p promotes ovarian cancer growth and metastasis via a novel FOXA2-versican pathway

Abstract miRNAs play important roles in gene regulation, and their dysregulation is associated with many diseases, including epithelial ovarian cancer (EOC). In this study, we determined the expression and function of miR-590-3p in EOC. miR-590-3p levels were higher in high-grade carcinoma when compared with low-grade or tumors with low malignant potential. Interestingly, plasma levels of miR-590-3p were significantly higher in patients with EOC than in subjects with benign gynecologic disorders. Transient transfection of miR-590-3p mimics or stable transfection of mir-590 increased cell proliferation, migration, and invasion. In vivo studies revealed that mir-590 accelerated tumor growth and metastasis. Using a cDNA microarray, we identified forkhead box A2 (FOXA2) and versican (VCAN) as top downregulated and upregulated genes by mir-590, respectively. miR-590-3p targeted FOXA2 3′ UTR to suppress its expression. In addition, knockdown or knockout of FOXA2 enhanced cell proliferation, migration, and invasion. Overexpression of FOXA2 decreased, whereas knockout of FOXA2 increased VCAN mRNA and protein levels, which was due to direct binding and regulation of the VCAN gene by FOXA2. Interrogation of the TCGA ovarian cancer database revealed a negative relationship between FOXA2 and VCAN mRNA levels in EOC tumors, and high FOXA2/low VCAN mRNA levels in tumors positively correlated with patient survival. Finally, overexpression of FOXA2 or silencing of VCAN reversed the effects of mir-590. These findings demonstrate that miR-590-3p promotes EOC development via a novel FOXA2–VCAN pathway. Significance: Low FOXA2/high VCAN levels mediate the tumor-promoting effects of miR-590-3p and negatively correlate with ovarian cancer survival. Cancer Res; 78(15); 4175–90. ©2018 AACR.</jats:p