Methylated Regulation,Function,Mechanism And Clinical Significance Of SOX30 In Lung Cancer

Background:Lung cancer is the most common cancer and the leading cause of cancer death globally.However,the pathogenesis of lung cancer is still unclear with limited clinical treatment and lack of effective screening/early-diagnosis markers.Carcinogenesis is a complex multistep process with a variety of genetic and epigenetic abnormalities.Aberrant epigenetic changes were the most frequent events and were regarded as important mechanisms in carcinogenesis.Moreover,methylation profiles have been used as potential biomarkers for early diagnosis,prognosis and screening in some cancers.Recently,accumulating evidence demonstrated that DNA hypermethylation of tumor suppressors associated with gene silencing has an essential role in carcinogenesis.Increasing numbers of tumor suppressors associated with epigenetic alterations have been identified in various human cancers.So,identification of new useful biomarkers and new genes functionally involved in tumor development may provide novel approaches for prognostic evaluation and effective therapeutic target.Through whole genome methylation-sensitive representational difference analyses,we have identified a novel preferentially methylated gene,SRY-box containing gene 30(SOX30),in human lung cancer.SOX proteins play key roles in a wide variety of embryonic and postnatal developmental processes,such as sex determination,neural development,skeletal development,and especially the definitive endoderm development that gives rise to various organs including liver,pancreas,intestine and lung.Moreover,mounting evidence indicates that SOX genes are altered in a variety of human diseases,highlighting their involvement in these diseases,particularly in malignancies.SOX30,as a relatively new SOX gene,has been considered to be involved in spermatogonial differentiation and spermatogenesis.However,it remains unclear whether SOX30 has any role in human cancer.In our previous study,we first found that SOX30 may be involved in lung cancer.Here,we further determine the epigenetic regulation,major biological functions,molecular mechanisms and possible clinical and prognostic significance of SOX30 in lung cancer.Contents:1.Identification SOX30 as a novel preferentially methylated gene and analysis the epigenetic regulation of SOX30 expression in lung cancerUsing genome-wide methylation-sensitive representational difference analyses,we identified a novel preferentially methylated gene SOX30 in human lung cancer.SOX30 methylation was verified in lung cancer cell lines and tissues using methylation-specific polymerase chain reaction(MSP)and bisulfite genomic sequencing PCR(BSP).SOX30 expression in different methylated lung cancer cell lines and tissues was tested to determine the relationship between its expression and methylation by RT-PCR,qRT-PCR,WB and IHC analyses.This relationship was further confirmed by de-methylation assay with 5-aza-2'-deoxycytidine.Moreover,the possibility of genetic mutation and deletion in SOX30 was excluded using direct sequencing.Finally,the clinical characteristics of SOX30 methylation were analyzed in lung cancer patients.2.The studies on main biological functions of SOX30 in lung cancerThe effects of SOX30 on the growth,proliferation,apoptosis,migration and invasion of tumor cells were analyzed by MTS,Clone formation,EdU,Flow cytometry,DNA ladder and transwell assays in vitro.The effects of SOX30 on tumor formation,growth and distant metastasis were analyzed by constructing a model of differential expression transplantation nude mice.The effects of SOX30 on the ability of tumor formation and lung metastasis were analyzed by constructing gene knockout animal model of chemical-induced and transplantable tumor.The effects of SOX30 on lung metastasis were further analyzed by constructing induced recovery gene knockout animal model of transplantation tumor in vivo.3.The studies on molecular mechanisms of SOX30 function in lung cancerThe possible downstream target genes and signaling pathways of SOX30 was screened by expression profile array,and were confirmed in the different expression cell and animal models via qRT-PCR,WB,IHC,etc.The expression associations between SOX30 and downstream target genes or signaling pathways were analyzed in cell,animal and human samples.The possible binding of SOX30 to downstream target genes or signaling pathway related genes was predicted by bioinformatic analyses.At the transcriptional level,we determined whether SOX30 can(directly)bind to the promoters of the downstream target gene by Luciferase reporter,ChIP,EMSA and Site-directed mutagenesis experiments.At the protein level,we determined whether SOX30 can(directly)interact with the downstream target protein via Co-IP,FRET and Structure prediction.4.The analyses on clinical and prognostic significance of SOX30 in lung cancerThe clinical characteristics and possible prognostic significance of SOX30 expression in lung cancer and different subtypes of lung cancer were analyzed by Chi-square test,Kaplan-Meier and Co-regression methods,The differences of function and molecular mechanism for the different clinical characteristics and prognostic significance in different subtypes of lung cancer were analyzed by bioinformatics,network rich concentration,transwell,Luciferase reporter,ChIP,etc.Results:1.The methylation of SOX30 leads to its inactivation in lung cancer(1)SOX30 gene is methylated in most lung cancer tissue(70.83 %,85/120),whereas is not methylated in adjacent normal tissues(8.0%,2/25)and normal tissues(0.0%,0/20),suggesting SOX30 methylation as a characteristic event for cancer tissues.(2)SOX30 methylation is associated with the clinical stage of lung cancer patients.(3)The methylation of SOX30 is closely related to its transcription inactivation.(4)SOX30 expression is restored after demethylation in lung cancer cell lines.(5)No genetic mutations and deletion are detected in lung cancer cell lines and lung cancer tissues.2.SOX30 significantly inhibits tumor formation,growth and distant metastasis(1)Overexpression of SOX30 can significantly promote apoptosis and inhibit proliferation,migration and invasion of tumor cells;(2)Knockdown of SOX30 expression by RNAi inhibits apoptosis and promotes proliferation,migration and invasion of tumor cells.(3)SOX30 significantly inhibits tumor formation and distant metastasis in nude mice transplanted with tumor.(4)Chemical induced carcinogenesis is significantly accelerated in Sox30 knockout mice.(5)Tumor formation and growth is promoted in Sox30 knockout mice transplanted with tumor.(6)The lung metastasis of Sox30 knockout mice transplanted with tumor is strengthened.(7)There is a significant decrease in the number of metastatic tumors when restoring Sox30 expression.in the lung of Sox30 knockout mice3.SOX30 transcriptionally activates P53 and its signaling pathway by direct binding to P53 promoter to induce apoptosis and inhibit proliferation of tumor cells(1)P53 and its signaling pathway may be an important downstream target gene or signaling pathway of SOX30 function by transcriptional screening.(2)SOX30 can significantly activate the activity of P53 and its signaling pathway downstream molecules P21,BAX and PMAIP1.(3)SOX30 activates P53 transcriptional expression by directly binding to its promoter to enhance its signaling pathway activity.(4)The HMG-box of SOX30 is required for direct binding to P53 promoter to activate P53 transcriptional expression,and the C-C-T-G sequences in P53 promoter are the key binding sites for SOX30 binding to P53 promoter.(5)P53 is a functional and important downstream target gene for SOX30 promoting apoptosis and inhibiting proliferation of tumor cells.4.SOX30 inhibits Wnt/?-catenin signaling to prevent metastasis by directly transcriptional repression of and directly competitive interaction with CTNNB1(1)CTNNB1 and the Wnt signaling pathway may be another important downstream target gene or signaling pathway of SOX30 function by transcriptional screening.(2)SOX30 significantly inhibits the activity of the CTNNB1 and the Wnt signaling pathway.(3)SOX30 represses Wnt/?-catenin signaling pathway and its downstream molecules MYC,CCND1,MMP7 and MMP2 by direct binding to CTNNB1 promoter.(4)SOX30 also inhibits Wnt/?-catenin signaling pathway activity by direct interaction with CTNNB1 protein.(5)SOX30 competitively binds to CTNNB1 with TCF through direct protein interaction.(6)The C-terminal rather than the HMG-box of SOX30 is the key region for its binding to CTNNB1 promoter,and the N-terminal of SOX30 is the key region for its interaction with CTNNB1 protein.(7)CTNNB1 is a functional and important target gene for SOX30 inhibiting tumor metastasis.5.SOX30 specifically inhibits Wnt signaling in ADC to prevent tumor metastasis and improve patient prognosis(1)In 516 clinical samples of lung cancer,SOX30 expression is associated with the patient's lung cancer subtype,clinical stage and tumor metastasis.(2)In 275 ADC cases,SOX30 expression is closely related to patient's clinical stage,tumor metastasis and tumor size,whereas in 228 SCC cases,SOX30 expression is not associated with clinical stage,tumor metastasis and tumor size.(3)SOX30 expression is not associated with the survival of total lung cancer patients.It is an independent favorable prognostic factor in ADC,but is an independent unfavorable prognostic factor in SCC.(4)SOX30 expression is an independent favorable prognostic marker only in the ADC patients at early stage especially at early stage I.(5)SOX30 expression can significantly improve the survival of the nude mice and knockout mice transplanted with tumor.(6)SOX30 is an important and specific tumor suppressor in ADC,whereas SOX30 has no effect on tumor in SCC.(7)SOX30-related genes are enriched for metastasis and Wnt signaling pathway in ADC,and SOX30 is negatively associated with CTNNB1 expression only in ADC.(8)SOX30 can directly repress CTNNB1 to inhibit Wnt/?-catenin signaling activity only in ADC.Conclusions:(1)SOX30 is a new gene directly silenced by DNA methylation in lung cancer,and SOX30 methylation,which is associated with clinical stage of patients,is a unique phenomenon of cancer tissues suggesting a potential epigenetic biomarker in lung cancer.(2)SOX30 is a novel key tumor suppressor that controls tumor growth and distant metastasis by promoting tumor cell apoptosis,inhibiting tumor cell proliferation,migration,and invasion in lung cancer.(3)For the first time,SOX30 is found to play tumor suppressed role by promoting apoptosis and inhibiting proliferation/growth of tumor cells through directly transcriptional activation of P53 and its downstream signaling pathway.(4)For the first time,SOX30 is found to prevent metastasis of tumor cells by inhibiting Wnt/?-catenin signaling pathway activity through directly transcriptional inhibition of CTNNB1 and directly competitive bind to CTNNB1 with TCF.(5)SOX30 expression is a specific and independent favorable prognostic factor for ADC patients,and is an independent favorable prognostic marker only for the ADC patients at early stage,especially at the early stage I.(6)SOX30 can specifically induce apoptosis,inhibit proliferation,migration and invasion of tumor cells suppressing tumor formation/growth and preventing tumor metastasis,and eventually prolong the patient's survival time in ADC.In this study,we found a new pathogenic mechanism of lung cancer occurrence,development and metastasis,and highlighted the specific role,mechanism and prognostic value of SOX30 in ADC.This study will provide new targets for the clinical treatment of specific subtype patients of lung cancer,and effective indicators for the accurate assessment of specific patient's prognosis.