The Role Of CHERP In Regulation Of Proliferation And Apoptosis In Neuroblastoma Cells

Neuroblastoma is a common and malignant tumor that develops from the sympathetic nervous system in children,usually before the age of 5.Neuroblastoma symptoms may include abdominal mass,pain,diarrhea,or a general feeling of discomfort.Neuroblastoma deaths account for 12% of cancer-associated deaths in children.Understanding the exact mechanism involved in neuroblastoma occurrence and development will help to improve preventive measures and treatment strategies for this poorly understood cancer.Here,we show that calcium homeostasis endoplasmic reticulum protein(CHERP)is involved in neuroblastoma cell proliferation,apoptosis and tumorigenicity.In this study,we found that depletion of CHERP could inhibit AKT/mTOR phosphorylation and induce ER stress,leading to the activation of DR5.Additionally,treatment with the ER stress-specific inhibitor GSK2606414 partially rescued the DR5-dependent apoptosis triggered by CHERP depletion.In brief,we reveal that the down-regulation of CHERP induces the extrinsic apoptotic pathway in neuroblastoma cells by activating ER stress signaling.These results indicate that targeting CHERP might be a potential and novel therapeutic strategy for the neuroblastoma patient population.The main results are as follows:1.High CHERP expression in neuroblastoma patients is associated with poor prognosisTo investigate whether aberrant CHERP expression is associated with neuroblastoma patient prognosis,we used the Tumor Neuroblastoma public database,which is available from the online R2: Genomics Analysis and Visualization Platform.We selected three commonly used datasets(Versteeg,Kocak and Asgharzadeh),which contain data from 88,476 and 247 neuroblastoma patients,respectively,to evaluate the effects of CHERP on overall patient survival.Kaplan–Meier analysis showed that high CHERP expression was associated with poor prognosis,whereas low CHERP expression was associated with good prognosis.Moreover,according to the Versteeg dataset,in the death,older age(>18 month)and tumor-caused death groups,CHERP was expressed significantly more highly than in the control group.Furthermore,we analyzed whether the CHERP expression level was associated with MYCN in neuroblastoma patients.The results from the three datasets showed that CHERP expression increased significantly in the MYCN-amplification group.These results suggest that CHERP may be a diagnostic marker of neuroblastoma.To confirm this conclusion,we used another online database,the Neuroblastoma Prognosis Database,and Kaplan–Meier analysis revealed that high CHERP expression was prognostic for poor outcomes in the Oberthuer Lab and Seeger dataset.Moreover,high CHERP expression correlated significantly with advancing tumor stage with the data obtained from the Kocak dataset.In conclusion,higher CHERP expression is significantly associated with poor overall survival in neuroblastoma patients,indicating that CHERP is a prognostic marker for neuroblastoma.2.CHERP is generally expressed in neuroblastoma and is located in nucleiTo confirm the CHERP expression levels in six neuroblastoma cell lines,we performed immunoblot analysis and qRT-PCR assays and found that CHERP was commonly expressed in neuroblastoma cell lines,with a higher level in BE(2)-C lines and a lower level in SHEP1 cell lines,and which we chose these two cell lines for further studies.It was previously reported that in rat soleus muscle tissue,CHERP co-localized with RyR1 in the sarcoplasmic reticulum.However,recently reported data show that CHERP acts as a nucleoprotein and impacts cellular proliferation in HEK293 and SKBR3 cells.To confirm the definite location of CHERP in neuroblastoma cells,we extracted nuclear and cytoplasmic proteins and used western blot assays to determine the precise location of CHERP.We found that in the three neuroblastoma cell lines BE(2)-C,SK-N-DZ and SHEP1,endogenous CHERP was only detected in the nucleus,not in the cytoplasm.Moreover,immunofluorescence assays showed that CHERP only localized in the nucleus in BE(2)-C and SHEP1 cells.These observations demonstrate that CHERP is commonly expressed in neuroblastoma cells and is located in the nucleus.3.CHERP depletion inhibits neuroblastoma cell proliferation in vitroTo verify the importance of CHERP in neuroblastoma,we used a lentivirus system carrying small hairpin RNA(shRNA)to construct plasmids against target genes and then used these reconstructed lentiviruses(CHERPsi-1#,CHERPsi-2#,and GFPsi as control)to infect BE(2)-C and SHEP1 cells.Immunoblot analysis and qRT-PCR assays showed that infecting the cells with target lentivirus-expressing shRNAs resulted in significant down-regulation of CHERP.Cell number was dramatically decreased in the CHERPsi group compared with that the control group.This result was further supported by a growth curve assay,which revealed significant growth inhibition by CHERP down-regulation.These data demonstrate that CHERP plays an indispensable role in neuroblastoma cell proliferation.4.CHERP depletion induces cell cycle arrest at the G0/G1 phase in BE(2)-C and SHEP1 cellsThe aforementioned data show that CHERP depletion inhibits neuroblastoma cell proliferation,and as reported,cell proliferation is often associated with cell cycle progression.Thus,we used flow cytometric analysis to further analyze the cell cycle status in BE(2)-C and SHEP1 cells with or without CHERP knock-down.Cell cycle analysis of CHERP-depleted cells revealed a significant increase in the proportion of cells in the G0/G1 phase together with a significant reduction in S phase compared with the GFPsi cells.These data showed that CHERP depletion induced cell cycle arrest at the G0/G1 phase in BE(2)-C and SHEP1 cells.Furthermore,we performed staining for the proliferation marker Ki67 and found that the Ki67-positive cell population was significantly decreased in CHERP-depleted cells relative to the controls,.To validate these results,we evaluated G1/S checkpoint-associated proteins by western blot analysis.The expression levels of CDK2 and Cyclin E were decreased in the CHERP-depleted cells,but there were no obvious changes in the expression levels of CDK1,CDK4 or Cyclin B1.These results showed that CHERP represses neuroblastoma cell proliferation by reducing CDK2 and Cyclin E expression and inducing cell cycle arrest at the G0/G1 phase.5.CHERP depletion induces cell apoptosis in BE(2)-C and SHEP1 cellsBecause apoptosis may reduce cell proliferation and inhibit tumor growth,we detected nuclear condensation in CHERP-depleted cells using Hoechst 33258 staining.The nuclei of the GFPsi group were large and round with a smooth nuclear membrane,whereas in the CHERPsi group,the nuclei exhibited fragmentation,indicating that CHERP depletion induced apoptosis and affected nuclear morphology.Quantitative analysis of cells with fragmented nuclei demonstrated that the proportion of fragmented nuclei cells(%)in the CHERPsi group was dramatically increased compared with that in the GFPsi group.Furthermore,FITC Annexin-V and PI staining followed by flow cytometry confirmed that the cell apoptosis rate was notably increased in the CHERP-depletion group.Next,we analyzed the expression levels of several apoptosis-associated proteins by western blot analysis.Cleaved caspase-3 and cleaved caspase-8 were up-regulated in CHERP depletion cells compared with the GFPsi cells.These results demonstrated that down-regulation of CHERP could induce neuroblastoma cell apoptosis in vitro.6.CHERP depletion results in alterations in the drug sensitivity and cell viability of neuroblastoma cells in response to DoxOne of the important properties of cancer cells is their resistance to drugs.We next investigated whether CHERP depletion could affect the response of neuroblastoma to doxorubicin(Dox)treatment.We treated CHERP-depleted cells with 2 mM Dox for the indicated time.Compared with the GFPsi group,clonogenic assays revealed that CHERP down-regulation in BE(2)-C and SHEP1 cells impaired Dox resistance in a time-dependent manner.In addition,we treated CHERP-depleted cells with 2 mM Dox and extracted the total protein for western blot assays.Consistent with these conclusions,cleaved caspase-3 and cleaved caspase-8 protein levels showed dramatic increases compared with those in the control group.These results showed CHERP down-regulation could attenuate the drug sensitivity and cell viability of neuroblastoma cells.7.CHERP depletion inactivates AKT/mTOR and induces apoptosis in neuroblastoma cells by CHOP-dependent DR5 inductionThus far,we have shown that CHERP depletion can induce neuroblastoma cell apoptosis and increase cleaved caspase 3/8 expression.Although most human neuroblastoma cells exhibit drug resistance because of their low expression of caspase-8,previous reports have shown that TRAIL-receptor 2(TRAIL-R2/DR5/TNFRSF10b)is an important upstream effector of caspase-8.Thus,we then examined the DR5 level in CHERP-depleted cells.Consistent with our expectations,DR5 was up-regulated significantly at both the mRNA and protein levels.We found that mTOR and AKT phosphorylation were attenuated,indicating that CHERP depletion induced neuroblastoma cell apoptosis via the AKT/mTOR pathway.ER stress could induce DR5 transcription by CHOP in human carcinoma cells.To determine whether DR5 transcription is regulated by ER stress in neuroblastoma cells,we examined the ER stress marker proteins BIP,ATF4 and CHOP.Depletion of CHERP in BE(2)-C and SHEP1 cells dramatically increased BIP,ATF4 and CHOP expression,which suggested that ER stress was involved in neuroblastoma cell apoptosis.To confirm this conclusion,we applied the ER stress inhibitor GSK2606414.After GSK2606414 treatment,cleaved caspase-3 and cleaved caspase-8 were reduced relative to the negative group.CHOP and DR5 also decreased with GSK2606414 treatment in CHERP-depleted BE(2)-C and SHEP1 cells.These results indicate that ER stress is involved in neuroblastoma cell apoptosis induced by CHERP depletion and that DR5 plays a key role in this process.8.CHERP depletion reduces neuroblastoma cell colony formation and represses tumorigenicity in vitro and in vivoTo investigate whether depletion of CHERP could affect tumorigenicity in neuroblastoma cells,we performed a soft-agar colony-formation assay.The size and number of the colonies were obviously reduced in CHERP-depleted cells.MYCN is a classical oncogene in neuroblastoma,and a high level of MYCN is a tumorigenic event in the development of malignant neuroblastoma in patients with poor clinical outcome.In Figure 1,according to the online data,we found that the expression of CHERP in the MYCN-amplification groups was significantly higher than that in the non-amplification groups.To verify this conclusion,we checked whether the expression of MYCN is associated with CHERP in BE(2)-C cells.In the CHERPsi group,we found that MYCN expression was coincident with CHERP and was also dramatically decreased.Furthermore,we investigated the role of CHERP in the tumorigenicity of neuroblastoma cells in vivo by subcutaneously implanting CHERP-depleted BE(2)-C cells into immunodeficient mice.As shown,CHERP depletion impaired tumor growth compared with the GFPsi group,and the CHERPsi-2# group did not form xenograft tumors.Taken together,these experiments indicate that CHERP plays a key role in the colony-formation ability and tumorigenicity of neuroblastoma.