PTEN-WDR66 Axis Regulates Cell Biological Characteristics Of Salivary Adenoid Cystic Carcinoma

Objective:Salivary adenoid cystic carcinoma(SACC)is a highly malignant glandular epithelial neoplasm originating in the head and neck.It has the biological characteristics of slow growth,neurotropic invasion,local infiltration,hematogenous dissemination and distant metastasis.At present,there is no effective treatment for this tumor.The characteristics of recurrence and metastasis of SACC make it insensitive to radiotherapy and chemotherapy,which is the main cause of death.Due to the lack of specific tumor markers,it is difficult to achieve good therapeutic effect by molecular biological means.Therefore,the study on the mechanism of recurrence and metastasis of SACC is the key to overcome the difficulties in the treatment of SACC.Previous studies of our research group have confirmed that PTEN expression deficiency is common in SACCs,particularly in the solid pattern of SACCs with high malignancy,poor prognosis and high hematogenous metastasis.PTEN inhibits the proliferation of SACC cells mainly through PI3K/Akt/m TOR signaling pathway,and then exerts its anti-cancer effect.Although PI3K/m TOR inhibitors can significantly inhibit the growth of the subcutaneous transplanted tumor of human SACC in nude mice,the inhibition of lung metastasis is not very significant.Therefore,we speculate that PTEN deficiency regulates the recurrence and metastasis of SACC not through PI3K/Akt/m TOR signaling pathway,but through other molecular mechanism.Thepurpose of this study was to screen out the key genes with significant changes in SACC-83 cell model with PTEN knockdown by gene expression profiling analysis.The expression and significance of the downstream target genes of PTEN in SACC tissues and their effects on the biological characteristics of SACC cells were detected,and the regulation of SACC by PTEN and its downstream target genes was further explored.The potential mechanism of recurrence and metastasis of SACC provides a new and reliable theoretical basis for the clinical treatment and prognosis evaluation of SACC.This study was divided into three parts.In the first part,the target genes SMC4 and WDR66 of PTEN downstream were screened out by analyzing the differential expression genes in SACC-83 cell model with PTEN knockdown.In the second part,we investigated the expression and significance of SMC4 and WDR66 in normal salivary gland and SACC tissue,and discussed their roles in the recurrence and metastasis of SACC.In the third part,we analyzed the effects of WDR66,a target gene of PTEN,on the proliferation,invasion and migration of SACC-83 cells,EMT process and the expression of cancer stemness markers,and revealed that PTEN negatively regulates WDR66 expression and participates in the regulation mechanism of distant metastasis of SACC,which provides a new theoretical basis for clinical treatment and prognosis evaluation of SACC.Methods:1.Establishment of the SACC83 cell model with PTEN knockdown,screening and validation of differentially expressed genes The SACC83 cell model with PTEN knockdown was established,and the gene expression profile was analyzed by using Agilent human whole gene 4*44K microarray.The differentially expressed genes were screened out,and GO function analysis,KEGG signal pathway enrichment analysis and Cluster heat map analysis were performed.The deletion or mutation of differentially expressed genes in human Head and Neck tumors and glandular epithelial tumors were examined by c Bio Portal for Cancer Genomics,and some target genes of PTEN were screened out and validated.Some downstream target genes of PTEN were identified by real-time fluorescence quantitative PCR.2.Expression of target genes of PTEN,SMC4 and WDR66 and correlation with PTEN expression in SACC The expression and localization of target genes of PTEN,SMC4 and WDR66 in 20 normal salivary glands(SGs)and 51 salivary adenoid cystic carcinoma(SACC)tissues were examined by immunohistochemistry(IHC)staining respectively.The correlation between new targets and PTEN expression was analyzed.The effect of new targets on the survival rate of patients with SACC was also analyzed.3.Negative regulation of WDR66 by PTEN on cell biological characteristics of SACC Firstly,the molecular mechanism of PTEN in regulating WDR66 was evaluated by specifically blocking of PI3 K signaling pathway and Co-immunoprecipitation assay.Subsequently,si RNA interference technique was used to knock down the expression of WDR66 and PTEN in human SACC-83 cells respectively or simultaneously.CCK-8,Transwell chamber and Wound healing assay were used to detect the effects of WDR66 on proliferation,invasion and migration of SACC-83 cells.Real-time fluorescence quantitative PCR and Western Blot were used to detect the effects of WDR66 on EMT process and expression of cancer stemness markers in SACC-83 cells.Results:1.Screening and bioinformatics analysis of differentially expressed genes in SACC-83 cell model with PTEN knockdown and validation of some target genes We successfully established SACC-83 cell model with PTEN knockdown and screened out differentially expressed genes in the cell model by gene expression microarray.Based on the criteria of Fold change > 3 times and Fluorescence value >300,the microarray data revealed a total of 248 m RNA which are differentially expression,including 64 m RNA with up-regulated and 184 m RNA with down-regulated.We investigated the function of these differentially expressed genes through a series of bioinformatics analysis.GO functional analysis showed that differentially expressed genes were mainly located in cell parts and organelles,participated in various biological processes,including cell processes,biological regulation and accounted for a large proportion in binding and catalytic activity.Enrichment analysis of KEGG signaling pathway suggested that differentially expressed genes associated with PTEN accounted for a large proportion of signaling pathways,immune system and cancer.This study focused on differentially expressed genes associated with recurrence(DNA damage repair)and metastasis(cell invasion and migration).Cluster heat map analysis showed that some differentially expressed genes related to DNA damage repair were enriched in SACC-83 cell lines with PTEN knockdown,such as SMC4,POLQ,CDC25 A,CDC6,etc.The loss of PTEN expression could cause the changes in the expression of genes related to cell invasion and migration,such as WDR66,VIM,Zeb1,Zeb2,etc.The expression of differentially genes regulated by PTEN in human Head and Neck tumors were examined by c Bio Portal for Cancer Genomics.We found that SMC4 and WDR66 were amplified to some extent in SACC.Trans-cancer genome analysis showed that gene amplification,mutation or deletion of WDR66 and SMC4 were detected in most of glandular epithelial tumors,such as gastric adenocarcinoma,lung adenocarcinoma,colorectal cancer,breast cancer,adenoid cystic carcinoma and so on.The results of real-time quantitative fluorescence PCR showed that the m RNA level of SMC4 and WDR66 were significantly up-regulated,which was consistent with the results of gene microarray,and proved that the microarray data was real and reliable.2.The expression,localization and correlation with PTEN of target genes,SMC4 and WDR66 in SACC.In normal salivary gland tissues,SMC4 and WDR66 were specifically expressed in ductal epithelial cells,showing cytoplasmic expression.In cribriform and tubular pattern of SACC with high PTEN expression,SMC4 was expressed in different degrees of cytoplasm,the positive rate were 31.3% and 33.3% respectively,and WDR66 was almost not expressed.In solid pattern of SACC with PTEN deficiency,SMC4 was expressed in different degrees of cytoplasm,the positive rate was 40%,WDR66 was strongly expressed in cytoplasm and nucleus,the positive rate was as high as 70.6%.Spearman correlation analysis showed that there was no correlation between the expression of SMC4 and PTEN in SACC,while WDR66 and PTEN were negatively correlated in SACC.Kaplan-Meier survival analysis showed that patients of SACC with high expression of WDR66 were significantly correlated with the shorter survival rate.3.Negative regulation of WDR66 by PTEN on cell biological characteristics of SACC The results showed that PTEN did not regulate the expression of WDR66 by specifically blocking PI3 K signaling pathway.There was a direct interaction between WDR66 and PTEN.The results of CCK-8 cell proliferation experiment showed that compared with the control group,the proliferation of SACC-83 cells in si WDR66 transfection group was inhibited to some extent,while the proliferation of SACC-83 cells in si PTEN transfection group was significantly enhanced.The results of cell clone formation experiment showed that compared with the control group,the number of cell clone formation in si WDR66 transfection group decreased slightly,while the number of cell clone formation in si PTEN transfection group increased significantly.The results of Transwell invasion assay in vitro showed that compared with the control group,the number of cells passing through the ventricular membrane in si WDR66 transfection group decreased significantly,while the number of cells in si PTEN transfection group increased significantly.The results of Transwell migration assay in vitro showed that compared with the control group,the migration ability of cells in si WDR66 transfection group was significantly reduced,while the migration ability of cells in si PTEN transfection group was significantly enhanced.The results of wound healing assay showed that compared with the control group,the migration rate of cells in si WDR66 transfection group decreased significantly,while the migration rate of cells in si PTEN transfection group increased significantly.When PTEN was knocked down in SACC-83 cells with WDR66 knocked down simultaneously,the biological characteristics of SACC-83 cells could be restored to a certain extent to its normal level.The results of Real-time fluorescence quantitative PCR showed that compared with the control group,the m RNA level of E-cadherin in si WDR66 transfection group significantly increased,the m RNA level of Vimentin slightly decreased,while the m RNA level of E-cadherin in si PTEN transfection group significantly decreased,and the m RNA level of Vimentin significantly increased.Compared with the si PTEN transfection group,the m RNA level of E-cadherin in the si PTEN + si WDR66co-transfection group increased significantly,the m RNA level of Vimentin slightly increased.The results of Western Blot test was the same as above.The results of Real-time fluorescence quantitative PCR showed that compared with the control group,the m RNA level of NANOG,OCT4,ALDH1 and SOX2 of SACC-83 cells in si WDR66 transfection group significantly decreased,while the m RNA level of NANOG,OCT4,ALDH1 and SOX2 of SACC-83 cells in si PTEN transfection group significantly increased.Compared with the si PTEN transfection group,the m RNA level of NANOG,OCT4,ALDH1 and SOX2 of SACC-83 cells in the si PTEN + si WDR66co-transfection group significantly decreased.The results of Western Blot test was the same as above.Conclusion:1.We screened out the differentially expressed genes in SACC-83 cell model with PTEN knocking down by gene expression microarray.The microarray data revealed a total of 248 m RNA which are differentially expression,including 64 m RNA with up-regulated and 184 m RNA with down-regulated.The target genes of PTEN downstream,SMC4 related to DNA damage repair and WDR66 related to invasion and metastasis,were screened out by bioinformatics and c Bio Portal for Cancer Genomics analysis.2.The expression of SMC4 in three patterns of SACCs showed a certain degree of cytoplasmic expression,which was not correlated with PTEN expression and prognosis of patients.SMC4 did not play an important role in the recurrence of SACC.The high expression of WDR66 in SACCs is negatively correlated with the prognosis of patients.WDR66 may be closely related to the metastasis of SACC.The expression of WDR66 in solid pattern of SACCs was significantly higher than that in cribriform/tubular pattern,which was negatively correlated with PTEN expression,suggesting that as a downstream target gene of PTEN,WDR66 may play a more important role in solid pattern of SACCs and may be related to PTEN.3.WDR66 could significantly promote the invasion and migration of SACC-83 cells,induce EMT,and make SACC-83 cells acquire the characteristics of cancer stemness.WDR66 could maintain the expression of EMT phenotypes and cancer stemness markers in SACC-83 cells with PTEN deficiency.WDR66 was a downstream target gene by PTEN deficiency,which mediated carcinogenesis and could be used as a new target for the treatment and prognosis of SACC.