The Effects And Mechanisms Of CSN6 On The Proliferation And Metastasis In Melanoma

Malignant melanoma(MM)is becoming the most lethal type of cutaneous carcinoma because of its rapid progression,tendency to metastasize and poor clinical prognosis.Although early-stage melanomas are usually curable via surgical resection,advanced metastatic melanomas respond poorly to radiation and chemotherapy.In the past 10 years,the development of targeted therapy and immunotherapy has greatly improved the prognosis of patients with metastatic melanoma;however,secondary drug resistance affects their long-term efficacy.Therefore,further exploration of the pathogenesis of melanoma,and identification of new potential biomarkers and targets,providing a basis for improving the prognosis of melanoma patients,are urgently needed.The constitutive photomorphogenic 9(COP9)signalosome(CSN)complex is highly evolutionarily conserved and ubiquitous in all eukaryotes.It consists of nine subunits,including CSN1-CSN8 and the newly discovered subunit CSN acidic protein(CSNAP)5.The CSN complex is an important regulator of cullin RING-ubiquitin ligases(CRLs)and modifies CRLmediated protein degradation.In recent years,CSN6 has been reported to exhibit upregulated expression and play vital roles in tumorigenesis and progression in lung cancer,glioblastoma,colorectal cancer,breast cancer,thyroid papillary cancer,cervical cancer,and pancreatic cancer,suggesting that CSN6 may be a possible prognostic marker and therapeutic target in a variety of cancers.It was reported that CSN6 plays critical roles in controlling protein ubiquitination and degradation by regulating the auto-ubiquitination and degradation of several E3 ligases.However,the expression level and biological function of CSN6 in melanoma are still unknown.The research objective of this paper is to explore the role of CSN6 in melanoma and explain the molecular mechanism of CSN6 regulation of melanoma proliferation and metastasis.The main research results are as follows:(1)CSN6 is overexpressed in melanoma cell lines and tissue and is a poor prognostic indicator in melanoma patientsTo identify the expression pattern of CSN6 in melanoma,an immunohistochemistry assay was conducted and confirmed that CSN6 was overexpressed in melanoma compared with benign nevi.Next,we examined the expression level of CSN6 in the A375,MV3,and Skmel28 melanoma cell lines and the immortalized melanocyte cell line PIG1.We found that CSN6 was overexpressed in the melanoma cell lines compared with the PIG1 cell line.To further determine whether the expression of CSN6 is associated with the prognosis of melanoma patients,survival database analysis of the R2:Genomics Analysis and Visualization Platform was performed and found that high expression of CSN6 was associated with poor overall survival,whereas a low CSN6 level was implicated in prolonged overall survival.Furthermore,the CSN6 level remarkably increased in an incremental manner with the progression of tumor pathological stage.Taken together,these findings indicate that CSN6 expression is upregulated in melanoma cell lines and tissue and might be a meaningful prognostic marker.(2)CSN6 is essential for the proliferation,colony formation and tumorigenesis of melanoma cellsTo explore the role of CSN6 in the proliferation of melanoma cells,CSN6 expression was knocked down in the A375 and MV3 melanoma cell lines by using lentivirus-mediated sh RNA technology.To investigate the cell viability of CSN6-knockdown melanoma cells,MTT assay was conducted and revealed that CSN6 knockdown remarkably inhibited melanoma cell proliferation in vitro.Brd U assay showed that the DNA synthesis ability in the CSN6-knockdown group was significantly decreased.Next,flow cytometry analysis demonstrated that CSN6 knockdown resulted in G1 arrest in melanoma cells.Furthermore,the expression of some cell cycle-related proteins was measured.We found that the expression of CDK1,CDK4,and cyclin D1 was reduced,while the expression of p21 and p27 was increased in CSN6-knockdown cells.To further confirm the involvement of CSN6 in melanoma proliferation,CSN6 expression was recovered through transfection of a full-length CSN6 sequence resistant to sh RNA#1 targeting into CSN6-knockdown melanoma cells.MTT and Brd U assays revealed that recovery of CSN6 expression rescued cell growth and proliferation.Furthermore,Western Blot revealed that CSN6 recovery led to significant increases in CDK1,CDK4 and cyclin D1 expression and decreases in p21 and p27 expression.In order to evaluate the role of CSN6 in colony formation in melanoma cells,soft agar colony formation assays were performed,and the results showed that the colonies formed by CSN6-knockdown melanoma cells were smaller and fewer in number than those of control cells.Furthermore,to evaluate the role of CSN6 in tumorigenesis in melanoma cells,subcutaneous xenograft experiments were carried out with nude mice.The results revealed that CSN6 knockdown remarkably inhibited melanoma cell tumorigenesis in vivo.These results suggested that the CSN6 is required for the tumorigenesis of melanoma cells.Taken together,these findings demonstrate that CSN6 is critical for the proliferation,colony formation and tumorigenesis of melanoma cells.(3)CSN6 is essential for melanoma cell migration and invasionTo determine whether CSN6 is involved in melanoma metastasis,migration and invasion assays were conducted,and the results showed that CSN6-knockdown melanoma cells migrated much more slowly than control cells.In order to further clarify the effect of CSN6 on cell migration and invasion,we also detected the expression of some cell metastasis related proteins.The results revealed that the expression of N-cadherin and vimentin(mesenchymal markers)was decreased,while that of E-cadherin(epithelial marker)was upregulated in CSN6-knockdown cells.These results suggest that down-regulation of CSN6 expression can inhibit the migration and invasion ability of melanoma cells.Next,we found that recovery of CSN6 expression in sh CSN6 cells almost completely rescued their migratory and invasive abilities.Furthermore,Western Blot revealed that CSN6 recovery led to significant increases in N-cadherin,and vimentin expression and decreases in E-cadherin expression.Taken together,these findings demonstrate that CSN6 is critical for melanoma cell migration and invasion.(4)CSN6 interacts with CDK9 and regulates CDK9 stabilityTo identify possible protein interaction partners of CSN6 in melanoma cells,immunoprecipitation(IP)–mass spectrometry analysis was performed,and CDK9 was detected as a potential CSN6-interacting protein.A co-IP assay further validated that CSN6 interacted with CDK9 in A375 and MV3 melanoma cells.To investigate whether CDK9 expression is influenced by CSN6,a Western Blot assay was performed and demonstrated that the protein level of CDK9 was remarkably reduced in CSN6-knockdown melanoma cells and recovered after overexpression of CSN6 in CSN6-knockdown cells,while the CDK9 m RNA level was not significantly affected by the CSN6 status,indicating that CSN6 may regulate the expression of CDK9 via posttranscriptional regulation.Then,we explored how CSN6 affects the stability of CDK9 and found that overexpression of CSN6 decreased the turnover rate of CDK9 in melanoma cells by using the de novo protein synthesis inhibitor cycloheximide(CHX).Furthermore,we evaluated whether CSN6 regulates CDK9 expression by ubiquitination and degradation of CDK9 and found that the decrease in CDK9 protein expression in CSN6-knockdown melanoma cells was obviously rescued by using the proteasome inhibitor MG-132.In addition,to investigate whether CSN6 could stabilize CDK9 expression by regulating CDK9 ubiquitination and degradation,in vivo ubiquitination assays were performed and found that CSN6-overexpression decreased CDK9 ubiquitination levels in melanoma cells and 293 FT cells.In summary,these results indicate that CSN6 interacts with CDK9 and regulates CDK9 stability by reducing CDK9 ubiquitination and degradation.(5)CSN6 stabilizes CDK9 by regulating the E3 ubiquitin ligase UBR5 ubiquitination and degradationCSN6 has been found to control the steady state of several E3 ligases,such as ?-Trcp,E6 AP,Fbxw7.The E3 ubiquitin ligase UBR5 has shown the ability to ubiquitinate CDK9,so we hypothesized that CSN6 stabilizes the expression of CDK9 by regulating the E3 ligase UBR5.A co-IP assay was performed and demonstrated that CSN6 interacted with UBR5 in A375 and MV3 melanoma cells.Next,Western Blot analysis demonstrated that CSN6 increased CDK9 expression and decreased UBR5 expression in a dose-dependent manner in 293 FT cells.In addition,UBR5 expression was negatively correlated with CSN6 expression in melanoma cells.Interestingly,q RT-PCR showed that UBR5 m RNA levels were not affected by the CSN6 expression status,indicating that CSN6 may control the UBR5 level through posttranscriptional regulation.To further verify this hypothesis,we examined the UBR5 turnover rate using CHX and found that the turnover rate of UBR5 was decreased in the CSN6-knockdown group.Then,Western Blot analysis showed that the proteasome inhibitor MG132 could rescue CSN6-mediated UBR5 downregulation.In addition,to investigate whether CSN6 controls the ubiquitination and degradation of the E3 ligase UBR5,in vivo ubiquitination assays were performed and found that CSN6 increased UBR5 ubiquitination levels in melanoma cells and 293 FT cells.In summary,these results suggested that CSN6 improved CDK9 stability by elevating ubiquitin-mediated UBR5 degradation.(6)UBR5 deficiency in CSN6-knockdown cells counteracts the effects induced by CSN6 silencingWe found that CSN6 is essential for melanoma proliferation and metastasis and that CSN6 stabilizes CDK9 expression by elevating the ubiquitin-mediated degradation of UBR5,so we speculated that UBR5 may be the downstream effector of CSN6 in melanoma cells.We knocked down UBR5 expression in CSN6-knockdown melanoma cells,and found that CDK9 expression was recovered in the UBR5 and CSN6-knockdown melanoma cells compared with CSN6-knockdown cells.We performed an MTT assay and Transwell assay to found that UBR5-knockdown rescued cell growth ability,migratory and invasive abilities in CSN6-knockdown melanoma cells.Next,soft agar colony formation assays were performed,and the results showed that UBR5-knockdown rescued the colony formation ability of CSN6-knockdown melanoma cells.Furthermore,subcutaneous xenograft experiments revealed that the tumor formation ability was remarkably rescued in the UBR5-knockdown CSN6-knockdown group compared with CSN6-knockdown group.In summary,these results indicate that the CSN6-UBR5-CDK9 axis promotes the growth,migration,invasion and tumorigenesis of melanoma cells through CDK9-mediated signaling pathways.In conclusion,we found that high expression of CSN6 is associated with poor prognosis in melanoma patients.UBR5,a downstream factor of CSN6,is a key E3 ubiquitin ligase that regulates CDK9 ubiquitination and degradation.CSN6 activates the UBR5/CDK9 pathway,and promotes the proliferation,colony formation,tumorigenesis,migration and invasion of melanoma cells through CDK9 mediated signaling pathways in this study.Thus,this study illustrates the mechanism by which the CSN6-UBR5-CDK9 axis promotes melanoma development,and demonstrate that CSN6 may be a potential biomarker and and anticancer target in melanoma.