Deubiquitinase USP5 promotes non-small cell lung cancer cell proliferation by stabilizing cyclin D1.

BACKGROUND: Cyclin D1 (CCND1) is overexpressed in non-small cell lung cancer (NSCLC) and contributes to its tumorigenesis and progression. Accumulating evidence shows that ubiquitin-specific protease 5 (USP5), an important member of the USP family, acts as a tumor promoter by deubiquitinating and stabilizing oncoproteins. However, neither the mechanism for dysregulated turnover of CCND1 protein nor the association of CCND1 with USP5 in NSCLC is well understood. METHODS: The association of USP5 with CCND1 in human NSCLC cells and clinical tissues was determined by immunoprecipitation/immunoblotting, immunohistochemistry (IHC), and The Cancer Genome Atlas database analyses. The effect of USP5 knockdown or overexpression on NSCLC cell proliferation in vitro was assessed by Cell Counting Kit-8, flow cytometry-based cell cycle, and colony formation assays. The effect of the USP5 inhibitor EOAI3402143 (G9) on NSCLC proliferation in vitro was analyzed by CCK-8 assay. The effect of G9 on NSCLC xenograft tumor growth was also examined in vivo, using athymic BALB/c nude mice. RESULTS: USP5 physically bound to CCND1 and decreased its polyubiquitination level, thereby stabilizing CCND1 protein. This USP5-CCND1 axis promoted NSCLC cell proliferation and colony formation. Further, knockdown of USP5 led to CCND1 degradation and cell cycle arrest in NSCLC cells. Importantly, this tumor-suppressive effect elicited by USP5 knockdown in NSCLC cells was validated in vitro and in vivo through chemical inhibition of USP5 activity using G9. Consistently, G9 downregulated the protein levels of CCND1 in NSCLC cells and xenograft tumor tissues. Also, the expression level of USP5 was positively associated with the protein level of CCND1 in human clinical NSCLC tissues. CONCLUSIONS: This study has provided the first evidence that CCND1 is a novel substrate of USP5. The USP5-CCND1 axis could be a potential target for the treatment of NSCLC.

Fibroblast growth factor (FGF), FGF receptor (FGFR), and cyclin D1 (CCND1) DNA methylation in head and neck squamous cell carcinomas is associated with transcriptional activity, gene amplification, human papillomavirus (HPV) status, and sensitivity to tyrosine kinase inhibitors.

BACKGROUND: Dysregulation of fibroblast growth factor receptor (FGFR) signaling pathway has been observed in head and neck squamous cell carcinoma (HNSCC) and is a promising therapeutic target for selective tyrosine kinase inhibitors (TKIs). Potential predictive biomarkers for response to FGFR-targeted therapies are urgently needed. Understanding the epigenetic regulation of FGF pathway related genes, i.e. FGFRs, FGFs, and CCND1, could enlighten the way towards biomarker-selected FGFR-targeted therapies. METHODS: We performed DNA methylation analysis of the encoding genes FGFR1, FGFR2, FGFR3, FGFR4, FGF1-14, FGF16-23, and CCND1 at single CpG site resolution (840 CpG sites) employing The Cancer Genome Research Atlas (TCGA) HNSCC cohort comprising N = 530 tumor tissue and N = 50 normal adjacent tissue samples. We correlated DNA methylation to mRNA expression with regard to human papilloma virus (HPV) and gene amplification status. Moreover, we investigated the correlation of methylation with sensitivity to the selective FGFR inhibitors PD 173074 and AZD4547 in N = 40 HPV(-) HNSCC cell lines. RESULTS: We found sequence-contextually nuanced CpG methylation patterns in concordance with epigenetically regulated genes. High methylation levels were predominantly found in the promoter flank and gene body region, while low methylation levels were present in the central promoter region for most of the analyzed CpG sites. FGFRs, FGFs, and CCND1 methylation differed significantly between tumor and normal adjacent tissue and was associated with HPV and gene amplification status. CCND1 promoter methylation correlated with CCND1 amplification. For most of the analyzed CpG sites, methylation levels correlated to mRNA expression in tumor tissue. Furthermore, we found significant correlations of DNA methylation of specific CpG sites with response to the FGFR1/3-selective inhibitors PD 173074 and AZD4547, predominantly within the transcription start site of CCND1. CONCLUSIONS: Our results suggest an epigenetic regulation of CCND1, FGFRs, and FGFs via DNA methylation in HNSCC and warrants further investigation of DNA methylation as a potential predictive biomarker for response to selective FGFR inhibitors in clinical trials.

Long non-coding RNA XIST promotes the progression of esophageal squamous cell carcinoma through sponging miR-129-5p and upregulating CCND1 expression.

Long non-coding RNA (lncRNA) X inactive specific transcript (XIST) has been identified as an oncogenic lncRNA in a series of human cancers, including esophageal squamous cell carcinoma (ESCC). In this study, we aimed to further explore the underlying mechanism of XIST on ESCC progression. qRT-PCR assay was used to determine the levels of XIST and miR-129-5p. Western blot analysis was performed to assess cyclin D1 (CCND1) expression. Bioinformatic analysis was performed using starBase v2.0 software. Dual-luciferase reporter and RNA immunoprecipitation assays were employed to confirm the interaction between XIST and miR-129-5p or miR-129-5p and CCND1. Cell cycle progression and apoptosis were measured by flow cytometric analysis, and cell migration and invasion were detected by transwell assay. Mouse studies were used to observe the effect of XIST silencing on tumor growth in vivo. Our results indicated that XIST was upregulated and miR-129-5p was downregulated in ESCC. XIST silencing or miR-129-5p overexpression repressed cell cycle progression, proliferation, migration, invasion, and promoted the apoptosis in ESCC cells. Moreover, XIST directly interacted with miR-129-5p and repressed miR-129-5p expression. MiR-129-5p mediated the regulatory effect of XIST on ESCC cell progression in vitro, and XIST promoted CCND1 expression by sponging miR-129-5p. Additionally, XIST silencing inhibited tumor growth in vivo. Our findings suggested that XIST silencing repressed the progression of ESCC at least partly through regulating the miR-129-5p/CCND1 axis. Targeting XIST might be a potential therapeutic strategy for ESCC treatment.

miR-361-5p reverses oxaliplatin resistance of gastric cancer SGC-7901 cells by targeting CCND1 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects of miR-361-5p on the oxaliplatin (OXA) resistance of gastric cancer SGC-7901 cells and its mechanism. Methods: The expression of miR-361-5p in gastric cancer cells (MKN-45, MGC80-3 and SGC-7901) and drug-resistant SGC-7901/OXA cells was detected by qPCR. The SGC-7901/OXA cells were transfected with miR-361-5p mimics/inhibitor or sh-CCND1 by using Liposome transfection technology. Then, cell proliferation, apoptosis and cell cycle of SGC-7901/OXA cells were measured by CCK-8 assay and Flow cytometry, respectively. The targeting relationship between miR-361-5p and CCND1 was examined by Dual luciferase report gene assay. The expression level of CCND1 in SGC-7901/OXA cells was detected by WB. Results: miR-361-5p was down-regulated in multiple gastric cancer cells and SGC-7901/OXA cells (P<0.05 or P<0.01). Over-expression of miR-361-5p significantly promoted the apoptosis, induced G0/G1 cell cycle arrest and inhibited cell proliferation of SGC-7901/OXA cells (P<0.05 or P<0.01). Dual luciferase reporter gene results verified that miR-361-5p targeted CCND1 and negatively regulated its expression (P<0.01). Further experiments showed that targeted down-regulation of CCND1 induced apoptosis and G0/G1 cell cycle arrest and inhibited CCND1 expression and proliferation of SGC-7901/OXA cells (P<0.05 or P<0.01). Over-expression of miR-361-5p targetedly down-regulated CCND1 and further promoted cell apoptosis, induced G0/G1 cell cycle arrest and inhibited cell proliferation of SGC-7901/OXA cells (P<0.05 or P<0.01). Conclusion: miR-361-5p over-expression can reverse the resistance of SGC-7901/OXA cells to OXA, and the mechanism may be related to its targeted down-regulation of CCND1 expression.

CCND1 Amplification Contributes to Immunosuppression and Is Associated With a Poor Prognosis to Immune Checkpoint Inhibitors in Solid Tumors.

Cyclin D1 (CCND1) amplification relevant to malignant biological behavior exists in solid tumors. The prevalence and utility of CCND1 amplification as a biomarker for the clinical response to treatment with immune checkpoint inhibitors (ICIs) are unknown. Our study is a preliminary investigation mainly focused on the predictive function of CCND1 amplification in the tumor microenvironment (TME) in the aspect of genome and transcriptome. We examined the prevalence of CCND1 amplification and its potential as a biomarker for the efficacy of ICI therapy for solid tumors using a local database (n = 6,536), The Cancer Genome Atlas (TCGA) database (n = 10,606), and the Memorial Sloan Kettering Cancer Center (MSKCC) database (n = 10,109). Comprehensive profiling was performed to determine the prevalence of CCND1 amplification and the correlation with the prognosis and the response to ICIs. A CCND1 amplification occurs in many cancer types and correlates with shorter overall survival and inferior outcomes with ICI therapy. Transcriptomic analysis showed various degrees of immune cell exclusion, including cytotoxic cells, T cells, CD8+ T cells, dendritic cells (DCs), and B cells in the TME in a TCGA CCND1 amplification population. The gene set enrichment analysis suggested that CCND1 amplification correlates with multiple aggressive, immunosuppressive hallmarks including epithelial-mesenchymal transition, transforming growth factor (TGF)-ß signaling, KRAS signaling, phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling, p53 pathway, and hypoxia signaling in solid tumors. These findings indicate that CCND1 amplification may be a key point related to immunosuppression in TME and multiple malignancy hallmarks, and it hinders not only the natural host immune responses but also the efficacy of ICIs.

lncRNA HOTAIR promotes malignant biological behaviors of breast cancer SKBR3 cells through miR-519d-3p/CCND1 axis / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the effect of lncRNA HOTAIR/miR-519d-3p/cyclin D1 (CCND1) axis on the proliferation and metastasis of breast cancer cells and its underlying mechanism. Methods: A total of 50 pairs of breast cancer tissues and corresponding para-cancer tissues resected from breast cancer patients in the Department of Breast Surgery, the Third Hospital of Nanchang from March 2017 to February 2019 were collected for this study. The expression level of HOTAIR in breast cancer tissues and paired paracancer tissues was detected by qPCR, in addition, the expressions of HOTAIR and miR-519d-3p in normal breast epithelial cells and breast cancer cell lines were also detected. Breast cancer SKBR3 cells were divided into NC group (without any treatment), si-HOTAIR group, mir-519d-3p mimics group, miR-519d-3p mimic+pcHOTAIR group, miR-519d-3p mimic+pcCCND1 group, and si-HOTAIR+ pcCCND1 group. The proliferation ability of SKBR3 cells was detected by CCK-8. Invasion and migration of SKBR3 cells were detected by Transwell. The expression levels of E-cadherin, N-cadherin, Vimentin and CCND1 in SKBR3 cells were detected by Western blotting. The targeting relationship between HOTAIR and miR-519d-3p, miR-519d-3p and CCND1 was detected by Dualluciferase reporter gene system. Results: HOTAIR was highly expressed in breast cancer tissues and cell lines, with the highest expression in SKBR3 cells. HOTAIR knockdown significantly inhibited the proliferation, invasion and migration of SKBR3 cells, as well as increased the expression level of E-cadherin and decreased the expression levels of N-cadherin and Vimentin. Dual-luciferase reporter gene assay showed that HOTAIR targetedly down-regulated the expression of miR-519d-3p, and miR-519d-3p targetedly downregulated the expression of CCND1. Further studies showed that knockout of HOTAIR inhibited the EMT, proliferation, invasion and migration of SKBR3 cells through enhancing the inhibitory effect of miR-519d-3p on CCND1 expression (all P<0.05). Conclusion: HOTAIR knockdown inhibits proliferation and metastasis of SKBR3 cells by regulating the axis of miR-519d-3p/CCND1.

CCDN1 rs603965 polymorphism may serve as a genetic biomarker of brain tumor: A meta-analysis of 5,769 subjects.

INTRODUCTION: Some studies already tried to assess the associations between cyclin D1 (CCND1) polymorphisms and brain tumor. However, the results of these studies were not consistent. Thus, we performed the present meta-analysis to explore the relationship between CCND1 polymorphisms and brain tumor in a larger pooled population. METHODS: PubMed, Web of Science, Embase, and CNKI were searched for related articles. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the potential associations. RESULTS: Totally nine studies with 5,769 subjects were analyzed. A significant association with brain tumor susceptibility was observed for the rs603965 polymorphism in GG versus GA + AA (dominant comparison, p = 0.003, OR = 0.72, 95% CI 0.57-0.89, I2  = 64%), AA versus GG + GA (recessive comparison, p = 0.004, OR = 1.46, 95% CI 1.13-1.88, I2  = 67%), and G versus A (allele comparison, p = 0.0004, OR = 0.77, 95% CI 0.66-0.89, I2  = 66%) in overall population. Further subgroup analyses by ethnicity yielded similar positive results in both Asians and Caucasians. Moreover, in stratified analyses by type of disease, we noticed that the rs603965 polymorphism was significantly associated with the susceptibility to glioma, but such positive results were not detected in pituitary adenoma or meningioma. Additionally, a significant association with tumor grade was also observed for the rs603965 polymorphism in G versus A (allele comparison, p = 0.02, OR = 0.74, 95% CI 0.59-0.95, I2  = 26%). CONCLUSIONS: Our findings suggested that CCND1 rs603965 polymorphism may serve as a potential genetic biomarker of brain tumor, especially for glioma.

Clear cell sarcomas of the kidney are characterised by BCOR gene abnormalities, including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion.

AIMS: Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE-NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically. METHODS AND RESULTS: By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITDs, with positive BCOR immunoreactivity being found in four (36%) or eight (72%) cases, depending on the antibody clone. By reverse transcription polymerase chain reaction, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative polymerase chain reaction showed markedly elevated BCOR expression in this case, whereas BCOR ITD cases had lower levels of elevated BCOR expression. CONCLUSIONS: The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE-NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR-CCNB3 fusion sarcoma. BCOR-CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.

Fibroblast growth factor 9 (FGF9) regulation of cyclin D1 and cyclin-dependent kinase-4 in ovarian granulosa and theca cells of cattle.

To determine the mechanism by which fibroblast growth factor 9 (FGF9) alters granulosa (GC) and theca (TC) cell proliferation, cell cycle proteins that regulate progression through G1 phase of the cell cycle, cyclin D1 (CCND1) and cyclin-dependent kinase-4 (CDK4; CCND1's catalytic partner), were evaluated. Ovaries were obtained from a local abattoir, GC were harvested from small (1-5 mm) and large (8-22 mm) follicles, and TC were harvested from large follicles. GC and TC were plated in medium containing 10% fetal calf serum followed by various treatments in serum-free medium. Treatment with 30 ng/mL of either FGF9 or IGF1 significantly increased GC numbers and when combined, synergized to further increase GC numbers by threefold. Abundance of CCND1 and CDK4 mRNA in TC and GC were quantified via real-time PCR. Alone and in combination with IGF1, FGF9 significantly increased CCND1 mRNA expression in both GC and TC. Western blotting revealed that CCND1 protein levels were increased by FGF9 in TC after 6 h and 12 h of treatment, but CDK4 protein was not affected. A mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway inhibitor, U0126, significantly reduced FGF9-induced CCND1 mRNA expression to basal levels. For the first time we show that CCND1 mRNA expression is increased by FGF9 in bovine TC and GC, and that FGF9 likely uses the MAPK pathway to induce CCND1 mRNA production in bovine TC.

Insulin-like growth factor 1 regulates growth of endometrial carcinoma through PI3k signaling pathway in insulin-resistant type 2 diabetes.

Previous studies have shown that insulin-like growth factor 1 (IGF-1) may be responsible for the higher risk for developing endometrial carcinoma (EMC) in insulin-resistant type 2 diabetes mellitus (T2DM) patients. However, the underlying mechanisms are not understood. Here, we compared T2DM patients with or without EMC. We did not find difference in the serum levels of IGF-1, insulin-like growth factor 2 (IGF-2), IGF-1 binding protein 3, as well as the activation of IGF-1 receptor (IGF1R) in endometrial cells between T2DM patients with or without EMC. However, the levels of IGF2R activation and activation of PI3k, an IGF1R downstream factor, were significantly higher in endometrial cells in T2DM patients with EMC. In vitro analyses of activation of IGF1R, IGF2R, PI3k and CCND1 in EMC cells or IGF2R-overexpressing EMC cells by IGF-1 or IGF-2 suggest that increases in IGF2R in endometrial cells in T2DM may increase PI3k/CCND1-dependent cell growth through loss of competitive binding of IGF-2 to IGF1R, as a possible explanation for the higher risk for developing EMC in T2DM.

Association between cyclin D1 (CCND1) G870A polymorphism and gastric cancer risk: a meta-analysis.

Published data on the association between cyclin D1 (CCND1) G870A polymorphism and gastric cancer (GC) risk are inconclusive. Thus, we conducted a meta-analysis to evaluate the relationship between CCND1 G870A polymorphism and GC risk. We searched PubMed, EMBASE, Web of science and the Cochrane Library up to June 12, 2015 for relevant studies. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the strength of associations. Nine studies published from 2003 to 2014, with a total of 1813 cases and 2173 controls, were included in this meta-analysis. The pooled results showed that there was no association between CCND1 G870A polymorphism and GC risk in any genetic model. The subgroup analysis stratified by ethnicity showed an increased breast cancer risk in Caucasian based on heterozygote comparison (GA vs. GG: OR=1.49, 95% CI=1.06-2.10, P=0.02). We found the same association in population based (PB) stratified analyses by Source of controls (AA vs. GG: OR=1.39, 95% CI=1.01-1.93, 0.05). When stratifying by the type, Sex and H. pylori infection in dominant model, Interestingly, we found the opposite result in Male (AA + GA vs. GG: OR=0.5, 95% CI=0.33-0.76, P=0.001), there were no association between CCND1 G870A polymorphism and GC risk in any other subgroup. This meta-analysis suggests that CCND1 G870A polymorphism is a risk factor for susceptibility to GC in Caucasians and in general populations. While, CCND1 G870A polymorphism plays a possible protective effect in GC in Male. Further large scale multicenter epidemiological studies are warranted to confirm this finding.

Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation.

Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis.

Down regulation of miR-203 in radiation-induced thymic lymphoma promoted cells proliferation and inhibited apoptosis / 中华放射医学与防护杂志

Objective To investigate the role of miR-203 in radiation-induced thymic lymphoma (RITL).Methods A 60Co irradiator was used for total-body irradiation.MicroRNAs(miRNAs) level was assayed by qRT-PCR.Cell proliferation was assayed by MTT assay.Cell apoptosis was examined by fluorescence activated cell sorter (FACS).Dual luciferase reporter assay system was used to detect the 3'UTR reporter.Results MiR-203 was down-regulated in RITL tissues.Overexpression of miR-203 strongly inhibited the proliferation of both NIH3T3 cells and EL4 cells and vice versa.MiR-203 inhibited cells proliferation and induced apoptosis via TANK-binding kinase (TBK1),SLUG (SNAI2) and Cyclin D1 (CCND1).Conclusions Radiation down-regulated the level of miR-203 in thymic,which promoted radiation-induced thymic lymphoma by targeting TBK1,SNAI2 and CCND1.