CBX7 Protein Research Articles

RNF2 promotes chondrosarcoma progression by regulating ubiquitination and degradation of CBX7

ObjectiveChondrosarcoma (CHS) is resistant to conventional chemotherapy and radiotherapy and currently lacks effective treatment options when in advanced stages. Accordingly, this research investigated the mechanism of RNF2/CBX7 in CHS to drive the development of molecularly targeted drugs for CHS.MethodsRNF2 and CBX7 levels were detected in CHS cells and tissues. RNF2 and CBX7 expression was modulated through cell transfection to examine their effects on cell proliferation, apoptosis, migration, and angiogenesis. The correlation between RNF2 and CBX7 levels was determined, and the ubiquitination level of CBX7 was tested. Protein synthesis was blocked in RNF2-knockdown/overexpressing cells with CHX to assess the effect of RNF2 on CBX7 stability. JJ012 cells transfected with LV-sh-RNF2 were subcutaneously injected into nu/nu nude mice to ascertain the action of RNF2 in the growth and metastasis of CHS.ResultsRNF2 was highly expressed in CHS cells and tissues. RNF2 knockdown curbed CHS cell proliferation, migration, and angiogenesis while promoting apoptosis. RNF2 knockdown in JJ012 cells upregulated CBX7 protein levels and reduced CBX7 ubiquitination, whilst RNF2 had no effect on CBX7 mRNA expression. CBX7 knockdown partially nullified the repressing effects of RNF2 knockdown on CHS cell proliferation, migration, and angiogenesis, and CBX7 overexpression partially abolished the promotional effects of RNF2 overexpression. LV-sh-RNF2 prominently restricted tumor growth and weight and declined lung metastatic nodules and Ki-67-positive cells in mice.ConclusionRNF2 fosters CHS progression by elevating CBX7 degradation via the ubiquitination pathway.

P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7.

Background: It is well known that P16 INK4A , P14 ARF , P15 INK4B mRNAs, and ANRIL lncRNA are transcribed from the CDKN2A/2B locus. LncRNA P14AS is a lncRNA transcribed from antisense strand of P14 ARF promoter to intron-1. Our previous study showed that P14AS could upregulate the expression level of ANRIL and P16 INK4A and promote the proliferation of cancer cells. Because polycomb group protein CBX7 could repress P16 INK4A expression and bind ANRIL, we wonder whether the P14AS-upregulated ANRIL and P16 INK4A expression is mediated with CBX7. Results: In this study, we found that the upregulation of P16 INK4A , P14 ARF , P15 INK4B and ANRIL expression was induced by P14AS overexpression only in HEK293T and HCT116 cells with active endogenous CBX7 expression, but not in MGC803 and HepG2 cells with weak CBX7 expression. Further studies showed that the stable shRNA-knockdown of CBX7 expression abolished the P14AS-induced upregulation of these P14AS target genes in HEK293T and HCT116 cells whereas enforced CBX7 overexpression enabled P14AS to upregulate expression of these target genes in MGC803 and HepG2 cells. Moreover, a significant association between the expression levels of P14AS and its target genes were observed only in human colon cancer tissue samples with high level of CBX7 expression (n = 38, p < 0.05), but not in samples (n = 37) with low level of CBX7 expression, nor in paired surgical margin tissues. In addition, the results of RNA immunoprecipitation (RIP)- and chromatin immunoprecipitation (ChIP)-PCR analyses revealed that lncRNA P14AS could competitively bind to CBX7 protein which prevented the bindings of CBX7 to both lncRNA ANRIL and the promoters of P16 INK4A , P14 ARF and P15 INK4B genes. The amounts of repressive histone modification H3K9m3 was also significantly decreased at the promoters of these genes by P14AS in CBX7 actively expressing cells. Conclusions: CBX7 expression is essential for P14AS to upregulate the expression of P16 INK4A , P14 ARF , P15 INK4B and ANRIL genes in the CDKN2A/2Blocus. P14AS may upregulate these genes’ expression through competitively blocking CBX7-binding to ANRIL lncRNA and target gene promoters.

CBX7, a Potential Prognostic Biomarker in Lung Adenocarcinoma.

BackgroundLung adenocarcinoma (LUAD) is a major type of NSCLC and has high morbidity and mortality. The identification of useful prognostic biomarkers for LUAD is important. CBX7 has been reported in various cancers yet its expression level and potential roles have not been fully understood.MethodsGEPIA, Oncomine, TCGA, KM plotter and OSluca databases were used to explore the expression profile and prognostic effects of CBX7 mRNA expression in patients with LUAD. TIMER was used to explore the relationship between CBX7 and immune infiltrating cells. GSEA was used to further explore the potential biological process and pathways regulated by CBX7 in LUAD. Lastly, IHC detection of CBX7 in 95 samples was used to validate the result.ResultsWe found CBX7 was downregulated in LUAD in GEPIA, Oncomine and TCGA databases. TCGA, KM plotter and OSluca databases suggested that CBX7 was associated with poor clinical outcomes and low survival rate. Using TIMER, we found that CBX7 might be associated with immune infiltration. Via gene set enrichment analysis, we found that tumor-associated biological processes and signaling pathways were enriched in the CBX7 downregulated group. Using clinical samples, we found that CBX7 protein has low expression in LUAD and was associated with poor survival.ConclusionCBX7 might serve as a promising biomarker and potential molecular target in LUAD.

Regulation of circGOLPH3 and its binding protein CBX7 on the proliferation and apoptosis of prostate cancer cells.

To clarify the mechanism of circGOLPH3 regulation on prostate cancer cells, we performed an overexpression and interference circGOLPH3 assay in prostate cancer cells PC-3 and then evaluated cellular viability, proliferation, cell cycle, and apoptosis of prostate cancer cells by MTT, CCK8, Edu stain, TUNEL stain, and flow cytometry. Binding proteins of CircGOLPH3 were identified by RNA pull-down, mass spectrometry, and RNA-binding protein immunoprecipitation (RIP) assays. The expressions of CircGOLPH3 and CBX7 were measured by qRT-PCR. The results showed that after overexpression of circGOLPH3, the proliferative capacity and the viability of PC-3cells were significantly improved, whereas apoptosis was inhibited. CircGOLPH3 could bind to the CBX7 protein that was highly expressed in the PC-3 cell. Additionally, a functional test on CBX7 showed that the CBX7 overexpression notably improved the proliferative capacity and the viability of PC-3 cells and decreased cellular apoptosis, which was consistent with the effects of circGOLPH3. The validated the present study that circGOLPH3 and its binding protein CBX7 can promote prostate cancer cell proliferation and inhibit apoptosis.

Роль белка Cbx7 в процессах регенерации поврежденного эпидермиса

The understanding of alterations of epigenetic mechanisms in the development of many disorders, such as impaired wound healing, cancer and aging is increasingly seen as a priority in biomedical research. This project aimed to investigate the expression of Polycomb group protein Cbx7 in the wound epithelium and its role in the migration of keratinocytes after wounding. Cbx7 expression was determined in mouse wound samples collected at early stages of wound healing and analyzed using PCR and immunofluorescent detection methods. For cell migration analysis, HaCaT cell line was treated with a specific small molecule inhibitor MS37452 followed by the scratch assay. Analyses of Cbx7 expression revealed a significant downregulation of Cbx7 mRNA and protein levels in the wound epithelium in comparison to the unwounded or adjacent epidermis. Inhibition of Cbx7 using MS37452 showed a significant acceleration of cell migration in vitro. These data suggest that a decrease in Cbx7 is necessary for the increased epidermal keratinocytes migration immediately after skin injury. The results of this work could be useful for the development of new therapeutic approaches for improvement of skin wound healing.

MiRNA-19 promotes non-small-cell lung cancer cell proliferation via inhibiting CBX7 expression.

BackgroundmiR-19 is a critical carcinogenic miRNA that participates in important biological processes of human malignancies. CBX7 plays a key role in lung cancer development and progression. In the present study, for the first time, we investigated the correlation between miR-19 and CBX7 in non-small-cell lung cancer (NSCLC).MethodsmiR-19 expression in NSCLC tissues and lung cancer cell lines was detected using quantitative reverse transcriptase PCR (qRT-PCR). Luciferase reporter assay, qRT-PCR, Western blot, and immunohistochemical assay were conducted to identify the target reaction of miR-19 and CBX7. Moreover, the influence of miR-19 on lung cancer cell proliferation, migration, and invasion was studied including cell counting kit-8 assay, scratch assay, transwell assay, flow cytometry assay, and staining assays.ResultsmiR-19 was overexpressed in NSCLC tissues and lung cancer cell lines. Luciferase reporter assay demonstrated that miR-19 could inhibit CBX7 expression via binding to the 3′-UTR of CBX7. Furthermore, miR-19 remarkably decreased CBX7 protein and mRNA expression. Additionally, overexpression of miR-19 could significantly enhance lung cancer cell proliferation and migration.ConclusionmiR-19 functions as a tumor accelerator promoting lung cancer cell proliferation through targeting CBX7 and inhibiting its expression.

The molecular selectivity of UNC3866 inhibitor for Polycomb CBX7 protein from molecular dynamics simulation

Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains, which plays a key role in the development of numerous cancers. UNC3866, is a recently reported peptide-based inhibitor of the methyllysine (Kme) reading function of CBX chromodomains (CBX2, 4 and 6–8). The previous experiments showed that UNC3866 bound the chromodomains of CBX7 strongly, with ∼20-fold selectivity over other CBX chromodomains. However, the potential mechanism of UNC3866 preferentially binding to CBX7 is still unknown. In this study, we performed two pairs of microsecond molecular dynamic simulations (CBX2 (-UNC3866)) and (CBX7 (-UNC3866)) to study the inhibition and isoform-selective mechanism of UNC3866 to CBX7. The conformational analysis of apo- and holo- CBX2 and CBX7 indicated that the aromatic cage of CBX7 protein was more prone to be induced by UNC3866 relative to CBX2 protein. The results of predicted binding free energy suggested the binding affinity of UNC3866 with CBX7 was stronger than that with CBX2, because of the lower binding free energy of the former. Furthermore, the energetic origin of UNC3866 selective for CBX7 protein mainly came from the higher van der Waals contributions. The binding mode analysis showed that Asn47 of CBX2 formed a hydrogen bond with the OH group of C-terminal cap of UNC3866, inducing the conformational changes of diethyllysine of UNC3866 that is obviously different from that in CBX7. Additionally, His39 in CBX2 chromodomain interrupted the structured aromatic cage, partly explaining the reason for UNC3866 preferring for binding to CBX7. The proposal of this selective mechanism could be helpful for the rational design of novel selective inhibitors of the Polycomb CBX protein.

Critical evaluation of Cbx7 downregulation in primary colon carcinomas and its clinical significance in Chinese patients.

BackgroundCBX7 is a Polycomb group protein that shows variable expression changes in various cancers that are often contradictive. A mouse knockout experiment has validated the tumor suppressor role in carcinogenesis. The purpose of this study is to verify the tumor suppressor role of Cbx7 in human colon carcinomas (CC).MethodsFrozen CC and the surgical margin (SM) tissue samples from patients (n = 97) were obtained from the Peking University Cancer Hospital. All patients had follow-up data for at least three years. The level of Cbx7 mRNA and protein was determined by quantitative RT-PCR, immunohistochemistry and Western blot, respectively. The association between Cbx7 mRNA level and clinicopathological characteristics of CC patients was then statistically analyzed.ResultsCBX7 expression changes detected through immunohistochemistry and Western blot in 10 pairs of representative CC samples significantly correlated with their corresponding mRNA levels when Alu, but not GAPDH, was used as the endogenous reference control in quantitative RT-PCR. The Alu-normalized Cbx7 mRNA levels were significantly increased in SM tissues when compared with CC tissues or colon biopsies taken from non-cancer patients (Student’s t-test, P < 0.036 or 0.007). Furthermore, decreased levels of Cbx7 mRNA positively correlated with lymph metastasis (P = 0.029). Overall survival (OS) of CC patients classified as Cbx7 expression-low was considerably shorter than those classified as Cbx7 expression-high (Hazard ratio = 2.97, 95% CI [1.68 ~ 5.25]; P <0.001). Multiple variant analyses showed that the Cbx7 expression-low was an independent predictor of short OS (Hazard ratio = 3.16, 95% CI [1.58-6.30]; P < 0.001).ConclusionCbx7 is downregulated in CCs, and Cbx7 expression-low tumors correlated with lymph metastasis and poor overall survival of CC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1172-6) contains supplementary material, which is available to authorized users.

Up-regulation of miR-9 target CBX7 to regulate invasion ability of bladder transitional cell carcinoma.

BackgroundBladder urothelial carcinoma is the most common genitourinary system cancer in China. The objective of this study was to investigate whether the miR-9 can regulate the invasion ability of human bladder transitional cell carcinoma cells by down-regulation of CBX7.Material/MethodsThe expression of miR-9 was detected by quantitative real-time PCR in bladder transitional cell carcinomas (TCC) and normal bladder transitional cell (NBTC) samples. Bioinformatics software was used to predict some potential target genes of miR-9. T24 cells were transfected with pre-miR-9, and the CBX7 protein expression was detected by Western blot. Luciferase activities assay was selected to verify that CBX7 was a direct and specific gene of miR-9. T24 cells were transfected with pcDNA-CBX7, and the expression of CBX7 gene was detected. Then, the transwell assay was used to detect the invasion ability of T24 cells with CBX7 over-expression.ResultsThe expression of miR-9 increased significantly in human TCC specimens compared to that in NBTC specimens. TargetScan and PicTar software programs predicted CBX7 gene was a target gene of miR-9. The pre-miR-9 could up-regulate the miR-9 expression and down-regulate CBX7 protein expression. The luciferase activities assay verified that CBX7 gene was a direct and specific target gene of miR-9. The pcDNA-CBX7 transfection could up-regulate the CBX7 protein expression, and the invasion ability of T24 cells with CBX7 over-expression decreased significantly.ConclusionsAberrantly expressed miR-9 contributes to T24 cells invasion, partly through directly down-regulating CBX7 protein expression in TCC. This miRNA signature offers a new potential therapeutic target for TCC.

CBX7 Expression in Oncocytic Thyroid Neoplastic Lesions (Hürthle Cell Adenomas and Carcinomas)

Background: Previous analysis of CBX7 expression in a large number of thyroid adenoma and carcinoma samples revealed a progressive reduction of CBX7 levels that was well related with the malignant grade of thyroid neoplasias. Hürthle cell tumors are unusual thyroid neoplasms characterized by the presence of particular cells called oncocytes. Objectives: In order to develop new tools for a more accurate diagnosis of Hürthle cell tumors of the thyroid, we evaluated CBX7 protein levels to verify the possible presence of an expression signature. Methods: CBX7 expression was evaluated by immunohistochemistry in a panel of thyroid tissue sections including normal thyroids, goiters, follicular adenomas and oncocytic lesions. Results: CBX7 expression was low or null in 68% of Hürthle adenomas, whereas it was comparable to normal thyroid tissue in Hürthle hyperplasias and follicular adenomas. Conclusions: Reduced expression of CBX7 suggests a more aggressive identity of Hürthle adenomas with respect to non-Hürthle ones.

CBX7 is a tumor suppressor in mice and humans

The CBX7 gene encodes a polycomb group protein that is known to be downregulated in many types of human cancers, although the role of this protein in carcinogenesis remains unclear. To shed light on this issue, we generated mice null for Cbx7. Mouse embryonic fibroblasts derived from these mice had a higher growth rate and reduced susceptibility to senescence compared with their WT counterparts. This was associated with upregulated expression of multiple cell cycle components, including cyclin E, which is known to play a key role in lung carcinogenesis in humans. Adult Cbx7-KO mice developed liver and lung adenomas and carcinomas. In in vivo and in vitro experiments, we demonstrated that CBX7 bound to the CCNE1 promoter in a complex that included HDAC2 and negatively regulated CCNE1 expression. Finally, we found that the lack of CBX7 protein expression in human lung carcinomas correlated with CCNE1 overexpression. These data suggest that CBX7 is a tumor suppressor and that its loss plays a key role in the pathogenesis of cancer.

Abstract 2027: CBX7 protein, whose expression is decreased in human carcinomas, positively regulates E-cadherin expression by interacting with the HDAC2 protein

Abstract CBX7 is a chromobox family protein encoding a novel Polycomb protein whose expression shows a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, CBX7 protein levels decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins among which we selected Histone deacetylase 2 that is well known to play a key role in neoplastic cell transformation and to downregulate E-cadherin expression, whose loss is a critical event in the Epithelial-Mesenchymal Transition (EMT). We confirmed by co-immunoprecipitation that CBX7 physically interacts with the Histone deacetylase 2 protein and is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter, and that CBX7 is able to upregulate E-cadherin expression. Consistent with these data we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we demonstrated that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression by interacting with Histone deacetylase 2 and inhibiting its activity on the E-cadherin promoter would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype in thyroid cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2027. doi:10.1158/1538-7445.AM2011-2027

Identification of a New Pathway for Tumor Progression: MicroRNA-181b Up-Regulation and CBX7 Down-Regulation by HMGA1 Protein.

High mobility group A (HMGA) overexpression plays a critical role in neoplastic transformation. To investigate whether HMGA acts by regulating the expression of microRNAs, we analyzed the microRNA expression profile of human breast adenocarcinoma cells (MCF7) transfected with the HMGA1 gene, which results in a highly malignant phenotype. Among the microRNAs induced by HMGA1, we focused on miR-181b, which was overexpressed in several malignant neoplasias including breast carcinomas. We show that miR-181b regulates CBX7 protein levels, which are down-regulated in cancer, and promotes cell cycle progression. We also demonstrate that CBX7, being negatively regulated by HMGA, is able to negatively regulate miR-181b expression. Finally, there was a direct correlation between HMGA1 and miR-181b expression and an inverse correlation between HMGA1 and CBX7 expression in human breast carcinomas. These data indicate the presence of a novel pathway involving HMGA1, miR-181b, and CBX7, which leads to breast cancer progression.

Loss of the CBX7 protein expression correlates with a more aggressive phenotype in pancreatic cancer

Polycomb group (PcG) proteins function as multiprotein complexes and are part of a gene regulatory mechanism that determines cell fate during normal and pathogenic development. Several studies have implicated the deregulation of different PcG proteins in neoplastic progression.Pancreatic ductal adenocarcinoma is an aggressive neoplasm that follows a multistep model of progression through precursor lesions called pancreatic intraepithelial neoplasia (PanIN).Aim of this study was to investigate the role of PcG protein CBX7 in pancreatic carcinogenesis and to evaluate its possible diagnostic and prognostic significance.We analysed by immunohistochemistry the expression of CBX7 in 210 ductal pancreatic adenocarcinomas from resection specimens, combined on a tissue microarray (TMA) including additional 40 PanIN cases and 40 normal controls. The results were evaluated by using receiver operating characteristic (ROC) curve analysis for the selection of cut-off scores and correlated to the clinicopathological parameters of the tumours and the outcome of the patients. Expression of E-cadherin, a protein positively regulated by CBX7, was also assessed.A significantly differential, and progressively decreasing CBX7 protein expression was found between normal pancreatic tissue, PanINs and invasive ductal adenocarcinoma. Loss of CBX7 expression was associated with increasing malignancy grade in pancreatic adenocarcinoma, whereas the maintenance of CBX7 expression showed a trend toward a longer survival. Moreover, loss of E-cadherin expression was associated with loss of CBX7 and with a trend towards worse patient survival.These results suggest that CBX7 plays a role in pancreatic carcinogenesis and that its loss of expression correlates to a more aggressive phenotype.

Chromobox Protein Homologue 7 Protein, with Decreased Expression in Human Carcinomas, Positively Regulates E-Cadherin Expression by Interacting with the Histone Deacetylase 2 Protein

Chromobox protein homologue 7 (CBX7) is a chromobox family protein encoding a novel polycomb protein, the expression of which shows a progressive reduction, well related with the malignant grade of the thyroid neoplasias. Indeed, CBX7 protein levels decreased in an increasing percentage of cases going from benign adenomas to papillary, follicular, and anaplastic thyroid carcinomas. To elucidate the function of CBX7 in carcinogenesis, we searched for CBX7 interacting proteins by a proteomic analysis. By this approach, we identified several proteins. Among these proteins, we selected histone deacetylase 2 (HDAC2), which is well known to play a key role in neoplastic cell transformation and down-regulation of E-cadherin expression, the loss of which is a critical event in the epithelial-to-mesenchymal transition. We confirmed by coimmunoprecipitation that CBX7 physically interacts with the HDAC2 protein and is able to inhibit its activity. Then, we showed that both these proteins bind the E-cadherin promoter and that CBX7 up-regulates E-cadherin expression. Consistent with these data, we found a positive statistical correlation between CBX7 and E-cadherin expression in human thyroid carcinomas. Finally, we showed that the expression of CBX7 increases the acetylation status of the histones H3 and H4 on the E-cadherin promoter. Therefore, the ability of CBX7 to positively regulate E-cadherin expression by interacting with HDAC2 and inhibiting its activity on the E-cadherin promoter would account for the correlation between the loss of CBX7 expression and a highly malignant phenotype.