Cyclin G2 regulates canonical Wnt signalling via interaction with Dapper1 to attenuate tubulointerstitial fibrosis in diabetic nephropathy.

Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression and is often dysregulated in human cancers. Cyclin G2 in the occurrence and development of diabetic nephropathy (DN), one of the most severe diabetic complications, has not been fully identified. In this study, we investigated the function and regulatory mechanism of cyclin G2 in DN. In vivo studies revealed that a deficiency of cyclin G2 significantly increased albuminuria and promoted tubulointerstitial fibrosis in established DN. Cyclin G2 regulated the expression of fibrosis-related proteins via the canonical Wnt signalling pathway in renal tubular epithelial cells. Moreover, the binding of cyclin G2 to Dapper1 (Dpr1/DACT1), a protein involved in Wnt signalling, decreased the phosphorylation of Dpr1 at Ser762 by casein kinase 1 (CK1) and suppressed the Wnt signalling pathway. These findings reveal that cyclin G2 can protect against renal injury and fibrosis associated with DN and, thus, is a new target for the prevention and treatment of diabetic complications.

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 ß-catenin signaling, we investigated if miR-590-3p regulated ß-catenin activity. In the TOPFlash luciferase reporter assays, mir-590 increased ß-catenin/TCF transcriptional activity and the nuclear accumulation of ß-catenin. Silencing of ß-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.

Morin inhibited lung cancer cells viability, growth, and migration by suppressing miR-135b and inducing its target CCNG2.

Lung cancer is one of the most severe threats with the highest mortality rate to humans in the world. Recently, morin has been reported to have anti-tumor properties observed in several types of cancers. However, its mechanism is still unclear. We assessed the influences of morin on cell viability, colony formation, and migration ability of A549 and employed microRNA array to identify the microRNAs affected by morin. We found that morin-treated A549 cells showed statistically decreased cell viability, colony formation, and migration rate when comparing with the dimethyl sulfoxide-treated cells. Microarray results showed that with the treatment of morin, the expression level of miR-135b significantly reduced compared the control group, suggesting that morin may exert its anti-cancer property by suppressing the expression of miR-135b. In addition, we found a potential binding site of miR-135b within 3' untranslated region of CCNG2-encoding cyclin homolog cyclin-G2. We evidenced that miR-135b directly targets CCNG2, which could be a potential biomarker of lung cancer prognosis. Morin exerts its anti-tumor function via downregulating the expression of miR-135b that directly targets and represses CCNG2.

MicroRNA-378a-5p targets cyclin G2 to inhibit fusion and differentiation in BeWo cells.

MicroRNAs are expressed abundantly in the placenta throughout pregnancy. We have previously reported that microRNA (miR)-378a-5p promoted trophoblast migration and invasion. To further understand the role of miR-378a-5p during placental development, we investigated whether it may regulate the differentiation of syncytiotrophoblast (STB). Using a choriocarcinoma cell line, BeWo, we found that miR-378a-5p was down-regulated during forskolin-induced STB differentiation. Transfection of a miR-378a-5p mimic into BeWo cells decreased the formation of multinucleated STB, increased E-cadherin, and decreased the expression level of STB marker genes. On the other hand, transfection of anti-miR-378a-5p resulted in an increase in formation of multinucleated STB and expression of STB marker genes, as well as the loss of E-cadherin. Bioinformatic analysis revealed that miR-378a-5p has four potential binding sites at the 3' untranslated region (UTR) of cyclin G2 (CCNG2). Using luciferase reporter assays, we showed that miR-378a-5p decreased the luciferase activity of reporter constructs that contain CCNG2 3' UTR. In addition, miR-378a-5p decreased, whereas anti-miR-378a-5p increased, CCNG2 mRNA levels. Overexpression of CCNG2 increased the expression of syncytin-1 and fusion index and reversed the inhibitory effects of miR-378a-5p. In contrast, silencing of CCNG2 using siRNA increased E-cadherin and decreased syncytin-1 levels. These findings provide initial evidence that CCNG2 promotes STB differentiation and suggest that miR-378a-5p exerts an inhibitory role in STB differentiation, in part, by down-regulating CCNG2 expression, in the BeWo cell model.

Exosomal derived miR-1246 from hydroquinone-transformed cells drives S phase accumulation arrest by targeting cyclin G2 in TK6 cells.

BACKGROUND: Hydroquinone (HQ), a major metabolite of benzene and known hematotoxic carcinogen. MicroRNA 1246 (miR-1246), an oncogene, regulates target genes in carcinogenesis including leukemia. This study investigates the impact of exosomal derived miR-1246 from HQ-transformed (HQ19) cells on cell-to-cell communication in recipient TK6 cells. METHODS: RNA sequencing was used to identify differentially expressed exosomal miRNAs in HQ19 cells and its phosphate buffered solution control cells (PBS19), which were then confirmed using qRT-PCR. The impact of exosomal miR-1246 derived from HQ-transformed cells on cell cycle distribution was investigated in recipient TK6 cells. RESULTS: RNA sequencing analysis revealed that 34 exosomal miRNAs were upregulated and 158 miRNAs were downregulated in HQ19 cells compared with PBS19 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses predicted that their targets are enriched in cancer development-related pathways, such as MAPK signaling, microRNAs in cancer, apoptosis, PI3K-Akt signaling, cell cycle, Ras signaling, and Chronic myeloid leukemia. Eleven miRNAs were confirmed to have differential expression through qRT-PCR, with 6 upregulated (miR-140-3p, miR-551b-3p, miR-7-5p, miR-1290, miR-92a-3p, and miR-1246) and 5 downregulated (miR-183-5p, miR-26a-5p, miR-30c-5p, miR-205-5p, and miR-99b-3p). Among these, miR-1246 exhibited the highest expression level. HQ exposure resulted in a concentration-dependent increase in miR-1246 levels and decrease Cyclin G2 (CCNG2) levels in TK6 cells. Similarly, exosomes from HQ19 exhibited similar effects as HQ exposure. Dual luciferase reporter gene assays indicated that miR-1246 could band to CCNG2. After HQ exposure, exosomal miR-1246 induced cell cycle arrest at the S phase, elevating the expression of genes like pRb, E2F1, and Cyclin D1 associated with S phase checkpoint. However, silencing miR-1246 caused G2/M-phase arrest. CONCLUSION: HQ-transformed cells' exosomal miR-1246 targets CCNG2, regulating TK6 cell cycle arrest, highlighting its potential as a biomarker for HQ-induced malignant transformation.

Cyclin G2 in macrophages triggers CTL-mediated antitumor immunity and antiangiogenesis via interferon-gamma.

BACKGROUND: IFN-γ is a key mediator of tumor immunity that can induce macrophage polarization to suppress tumor growth. Cyclin G2 functions as a tumor suppressor in various cancer cells; however, its role in macrophages remains unclear. This study aimed to investigate the role and underlying mechanisms of cyclin G2 in macrophages in vitro and in vivo. METHODS: Mouse tumor models were used to determine the effect of cyclin G2 in macrophages on tumor growth in vivo following IFN-γ treatment. Immunohistochemistry staining, immunofluorescence staining and flow cytometry were used to evaluate the number of cytotoxic T lymphocytes (CTLs) and blood vessels in the mouse tumors. Moreover, the biological roles of cyclin G2 in macrophages with regard to CTL chemotaxis, cytotoxic function, and vascular endothelial cell tube formation were assessed using in vitro functional experiments. Immunoprecipitation (IP), real-time PCR, and enzyme-linked immunosorbent assays (ELISAs) were conducted to investigate the underlying mechanisms by which cyclin G2 regulates CTLs and vascular endothelial cells. RESULTS: We found that cyclin G2 expression was upregulated in macrophages after IFN-γ treatment. Upregulated cyclin G2 inhibited lung and colon cancer growth by increasing the secretion of its downstream effector CXCL9, which promoted CTL chemotaxis and suppressed vascular endothelial cell tube formation. Moreover, cyclin G2 increased CXCL9 mRNA levels by promoting STAT1 nuclear translocation. In addition, cyclin G2 promoted the activation of the STAT1 signaling pathway, which was dependent on PP2Ac. CONCLUSIONS: Cyclin G2 is upregulated by IFN-γ in macrophages, promotes the secretion of CXCL9 to increase CTL chemotaxis and inhibit angiogenesis to suppress tumor growth. Our findings suggest that targeting cyclin G2 could benefit future immunotherapy.

Cyclin G2 reverses immunosuppressive tumor microenvironment and potentiates PD-1 blockade in glioma.

BACKGROUND: Expression of aberrant cyclin G2 is a key factor contributing to cancer biological processes, including glioma. However, the potential underlying mechanisms of cyclin G2 in the glioma tumor immune microenvironment remain unclear. METHODS: Co-immunoprecipitation (co-IP), in situ proximity ligation assay (PLA), and in vitro kinase assay were conducted to reveal the underlying mechanism by which cyclin G2 regulates Y10 phosphorylation of LDHA. Further, the biological roles of cyclin G2 in cell proliferation, migration, invasion capacity, apoptosis, glycolysis, and immunomodulation were assessed through in vitro and in vivo functional experiments. Expressions of cyclin G2 and Foxp3 in glioma specimens was determined by immunohistochemistry. RESULTS: In this study, we found that cyclin G2 impeded the interaction between LDHA and FGFR1, thereby decreasing Y10 phosphorylation of LDHA through FGFR1 catalysis. Cyclin G2 inhibited proliferation, migration, invasion capacity, and glycolysis and promoted apoptosis glioma cells via suppressing Y10 phosphorylation of LDHA. Moreover, we further verified that cyclin G2 reversed the immunosuppressive to antitumor immune microenvironment through inhibiting lactate production by glioma cells. Besides, cyclin G2 potentiated PD-1 blockade and exerted strong antitumor immunity in the glioma-bearing mice model. CONCLUSIONS: Cyclin G2 acts as a potent tumor suppressor in glioma and enhances responses to immunotherapy. Our findings may be helpful in selecting glioma patients for immunotherapy trials in the future.

Cyclin G2 promotes the formation of smooth muscle cells derived foam cells in atherosclerosis via PP2A/NF-κB/LOX-1 pathway.

BACKGROUND: To investigate the role and underlying mechanism of cyclin G2 (G2-type cyclin) in the formation of vascular smooth muscle cells (VSMCs) derived foam cells. METHODS: The levels of α-SMA (alpha-SM-actin), p-NF-κB (phosphorylation nuclear transcription factors kappa B), and LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) were measured by immunohistochemistry and western blotting. The mouse aortic root smooth muscle cell line MOVAS was transfected to over-express cyclin G2, which were then stimulated with 80 µg/mL ox-LDL (oxidized low-density lipoprotein) to induce foam cell formation. DT-061 an activator of PP2A (protein phosphatase 2A) agonist was used to verify the role of PP2A in the process. RESULTS: Knocking out the Ccng2 gene in Apoe-/- mice alleviated aortic lipid plaque, foam cell formulation, ameliorative body weight, and LDL-cholesterol. We observed that the number of α-SMA positive cells was significantly decreased in Apoe-/-Ccng2-/- mice compared to Apoe-/- mice. Also, the protein levels of p-NF-κB and LOX-1 were markedly reduced in the aortic root of Apoe-/-Ccng2-/- mice. Upon stimulation with ox-LDL, upregulated cyclin G2 increased the intracellular lipid accumulation in MOVAS cells. Also, it suppressed the activity of PP2A but up-regulated LOX-1. Additionally, the cell nuclear translocation of p-NF-κB was increased. Interestingly, DT-061 intervention, re-activating the activity of PP2A, reduced the levels of nuclear p-NF-κB and LOX-1. This led to decreased lipid endocytosis reducing the formation of VSMCs- derived foam cells. CONCLUSIONS: Cyclin G2 increases the nuclear translocation of p-NF-κB by reducing the enzymatic activity of PP2A and upregulating LOX-1, thereby promotes the formation of VSMCs -derived foam cells in atherosclerosis.

Cyclin G2 Inhibits Oral Squamous Cell Carcinoma Growth and Metastasis by Binding to IGFBP3 and Regulating the FAK-SRC-STAT Signaling Pathway.

The cell cycle protein cyclin G2 is considered a tumor suppressor. However, its regulatory effects and potential mechanisms in oral cancers are not well understood. This study aimed to investigate the effect of cyclin G2 on oral squamous cell carcinoma (OSCC). The data from 80 patients with OSCC were utilized to predict the abnormal expression of cyclin G2. The proliferation and metastasis were determined by a cell counting Kit-8 assay, flow cytometry, a wound-healing assay, and a cell invasion assay. The expression of key proteins and genes associated with the cyclin G2 signaling pathways was determined by western blotting and real-time PCR, respectively. The orthotopic nude mice model was established by a mouth injection of SCC9 cells overexpressing cyclin G2. We showed that the low level of cyclin G2 in OSCC, which is negatively correlated with clinical staging, was a negative prognostic factor for the disease. We also found that cyclin G2 inhibited the proliferation, metastasis, and blocked the cell cycle at G1/S of OSCC cells, suggesting that cyclin G2 has an inhibitory effect in OSCC. Mechanistically, cyclin G2 inhibited the growth and metastasis of OSCC by binding to insulin-like growth factor binding protein 3 (IGFBP3) and regulating the focal adhesion kinase (FAK) -SRC-STAT signal transduction pathway. Cyclin G2 competed with integrin to bind to IGFBP3; the binding between integrin and IGFBP3 was reduced after cyclin G2 overexpression, thereby inhibiting the phosphorylation of FAK and SRC. These results showed that cyclin G2 inhibited the progression of OSCC by interacting with IGFBP3 and that it may be a new target for OSCC treatment.

Cyclin G2 Inhibits the Warburg Effect and Tumour Progression by Suppressing LDHA Phosphorylation in Glioma.

Cyclin G2 has been identified as a tumour suppressor in several cancers. However, its regulatory roles and underlying mechanisms in tumours are still unknown. In this study, we demonstrated that cyclin G2 was expressed at low levels in glioma, which was as a poor prognostic factor for this disease. We also found that, cyclin G2 could suppress cell proliferation, initiate cell apoptosis and reduce aerobic glycolysis, suggesting that cyclin G2 plays a tumour suppressive role in glioma. Mechanistically, cyclin G2 could negatively regulate tyrosine-10 phosphorylation of a critical glycolytic enzyme, lactate dehydrogenase A, through direct interaction. Taken together, these results indicate that cyclin G2 acts as a tumour suppressor in glioma by repressing glycolysis and tumour progression through its interaction with LDHA.

Cyclin G2 suppresses Wnt/ß-catenin signaling and inhibits gastric cancer cell growth and migration through Dapper1.

BACKGROUND: Gastric cancer is one of the most common malignant tumors. Cyclin G2 has been shown to be associated with the development of multiple types of tumors, but its underlying mechanisms in gastric tumors is not well-understood. The aim of this study is to investigate the role and the underlying mechanisms of cyclin G2 on Wnt/ß-catenin signaling in gastric cancer. METHODS: Real-time PCR, immunohistochemistry and in silico assay were used to determine the expression of cyclin G2 in gastric cancer. TCGA datasets were used to evaluate the association between cyclin G2 expression and the prognostic landscape of gastric cancers. The effects of ectopic and endogenous cyclin G2 on the proliferation and migration of gastric cancer cells were assessed using the MTS assay, colony formation assay, cell cycle assay, wound healing assay and transwell assay. Moreover, a xenograft model and a metastasis model of nude mice was used to determine the influence of cyclin G2 on gastric tumor growth and migration in vivo. The effects of cyclin G2 expression on Wnt/ß-catenin signaling were explored using a TOPFlash luciferase reporter assay, and the molecular mechanisms involved were investigated using immunoblots assay, yeast two-hybrid screening, immunoprecipitation and Duolink in situ PLA. Ccng2-/- mice were generated to further confirm the inhibitory effect of cyclin G2 on Wnt/ß-catenin signaling in vivo. Furthermore, GSK-3ß inhibitors were utilized to explore the role of Wnt/ß-catenin signaling in the suppression effect of cyclin G2 on gastric cancer cell proliferation and migration. RESULTS: We found that cyclin G2 levels were decreased in gastric cancer tissues and were associated with tumor size, migration and poor differentiation status. Moreover, overexpression of cyclin G2 attenuated tumor growth and metastasis both in vitro and in vivo. Dpr1 was identified as a cyclin G2-interacting protein which was required for the cyclin G2-mediated inhibition of ß-catenin expression. Mechanically, cyclin G2 impacted the activity of CKI to phosphorylate Dpr1, which has been proved to be a protein that acts as a suppressor of Wnt/ß-catenin signaling when unphosphorylated. Furthermore, GSK-3ß inhibitors abolished the cyclin G2-induced suppression of cell proliferation and migration. CONCLUSIONS: This study demonstrates that cyclin G2 suppresses Wnt/ß-catenin signaling and inhibits gastric cancer cell growth and migration through Dapper1.

Cyclin G2: A novel independent prognostic marker in pancreatic cancer.

Unlike other cyclins that positively regulate the cell cycle, cyclin G2 (CCNG2) regulates cell proliferation as a tumor suppressor gene. A decreased CCNG2 expression serves as a marker for poor prognosis in several types of cancer. The aim of the present study was to clarify the correlation of CCNG2 expression with overall survival and histopathological factors in pancreatic cancer patients. This retrospective analysis included data from 36 consecutive patients who underwent complete surgical resection for pancreatic cancer and did not undergo any preoperative therapies. The association between prognoses and the expression of CCNG2 was assessed using immunohistochemical staining. Multivariate analysis identified that the expression of CCNG2 is an independent prognostic factor. In addition, the Kaplan-Meier curve for overall survival revealed that decreased expression of CCNG2 was a consistent indicator of poor prognosis in pancreatic cancer patients (P=0.0198). A decreased CCNG2 expression significantly correlated with venous invasion in tumor specimens and the tumor invasion depth. In conclusion, CCNG2 expression inversely reflected cancer progression and may be a novel, independent prognostic marker in pancreatic cancer.

Modulation of insulin degrading enzyme activity and liver cell proliferation.

Diabetes mellitus type 2 (T2DM), insulin therapy, and hyperinsulinemia are independent risk factors of liver cancer. Recently, the use of a novel inhibitor of insulin degrading enzyme (IDE) was proposed as a new therapeutic strategy in T2DM. However, IDE inhibition might stimulate liver cell proliferation via increased intracellular insulin concentration. The aim of this study was to characterize effects of inhibition of IDE activity in HepG2 hepatoma cells and to analyze liver specific expression of IDE in subjects with T2DM. HepG2 cells were treated with 10 nM insulin for 24 h with or without inhibition of IDE activity using IDE RNAi, and cell transcriptome and proliferation rate were analyzed. Human liver samples (n = 22) were used for the gene expression profiling by microarrays. In HepG2 cells, IDE knockdown changed expression of genes involved in cell cycle and apoptosis pathways. Proliferation rate was lower in IDE knockdown cells than in controls. Microarray analysis revealed the decrease of hepatic IDE expression in subjects with T2DM accompanied by the downregulation of the p53-dependent genes FAS and CCNG2, but not by the upregulation of proliferation markers MKI67, MCM2 and PCNA. Similar results were found in the liver microarray dataset from GEO Profiles database. In conclusion, IDE expression is decreased in liver of subjects with T2DM which is accompanied by the dysregulation of p53 pathway. Prolonged use of IDE inhibitors for T2DM treatment should be carefully tested in animal studies regarding its potential effect on hepatic tumorigenesis.

CCNG2 and CDK4 is associated with insulin resistance in adipose tissue.

BACKGROUND: The involvement of cyclin G2 (CCNG2) and cyclin-dependent kinase-4 (CDK4), cell cycle regulatory proteins, in adipose tissue metabolism and insulin resistance is still unknown. The objective of this study was to analyze CCNG2 and CDK4 levels in visceral (VAT) and subcutaneous adipose tissue (SAT) from nonobese and morbidly obese patients and their relationship with insulin resistance. METHODS: We studied the mRNA and protein levels of CCNG2 and CDK4 in VAT and SAT from 12 nonobese and 23 morbidly obese patients (11 with low [MO-L-IR] and 12 with high insulin resistance [MO-H-IR]). RESULTS: The nonobese patients had a significantly greater CCNG2 expression in VAT (P = .004) and SAT (P<.001) than the MO-L-IR and MO-H-IR patients. The MO-H-IR patients had a significantly lower CDK4 expression in VAT than the MO-L-IR (P = .026), but similar to the nonobese patients. CDK4 and CCNG2 expression correlated significantly in VAT (r = 0.511, P<.001) and SAT (r = .535, P = .001). In different multiple regression analysis models, CCNG2 and CDK4 expression in VAT was mainly predicted by glucose (P = .047 and P = .008, respectively), and CCNG2 expression in SAT was mainly predicted by body mass index (P = .041). No significant associations were found with CDK4 expression in SAT. Moreover, VAT CCNG2 expression was the main determinant of the improvement in the homeostasis model assessment of insulin resistance index at 3 months after bariatric surgery (B = -271.7, P = .026). CONCLUSION: Our data show for the first time that the human CCNG2 and CDK4 expression of VAT are inversely associated with glucose and insulin resistance.

Recruitment of cyclin G2 to promyelocytic leukemia nuclear bodies promotes dephosphorylation of γH2AX following treatment with ionizing radiation.

Cyclin G2 (CycG2) and Cyclin G1 (CycG1), two members of the Cyclin G subfamily, share high amino acid homology in their Cyclin G boxes. Functionally, they play a common role as association partners of the B'γ subunit of protein phosphatase 2A (PP2A) and regulate PP2A function, and their expression is increased following DNA damage. However, whether or not CycG1 and CycG2 have distinct roles during the cellular DNA damage response has remained unclear. Here, we report that CycG2, but not CycG1, co-localized with promyelocytic leukemia (PML) and γH2AX, forming foci following ionizing radiation (IR), suggesting that CycG2 is recruited to sites of DNA repair and that CycG1 and CycG2 have distinct functions. PML failed to localize to nuclear foci when CycG2 was depleted, and vice versa. This suggests that PML and CycG2 mutually influence each other's functions following IR. Furthermore, we generated CycG2-knockout (Ccng2 (-/-) ) mice to investigate the functions of CycG2. These mice were born healthy and developed normally. However, CycG2-deficient mouse embryonic fibroblasts displayed an abnormal response to IR. Dephosphorylation of γH2AX and checkpoint kinase 2 following IR was delayed in Ccng2 (-/-) cells, suggesting that DNA damage repair may be perturbed in the absence of CycG2. Although knockdown of B'γ in wild-type cells also delayed dephosphorylation of γH2AX, knockdown of B'γ in Ccng2 (-/-) cells prolonged this delay, suggesting that CycG2 cooperates with B'γ to dephosphorylate γH2AX. Taken together, we conclude that CycG2 is localized at DNA repair foci following DNA damage, and that CycG2 regulates the dephosphorylation of several factors necessary for DNA repair.