Role Of ATG 10 Expression Quantitative Trait Loci In Non-small Cell Lung Cancer Survival

Background:Lung cancer is one of the most common malignant cancer worldwide.An estimated 1.8 million new lung cancer cases occurred in 2012,accounting for about 13%of total cancer diagnoses.In China,the incidence and motality of lung cancer have been rapidly increasing and the ratios were higher than the average of the world.So lung cancer is now a great threat to the life and health of our residents and is also a public health problem to be solved.Non-small cell lung cancer(NSCLC)accounts for about 85%of all lung cacers.Surgical operation is still the main treatment for the early NSCLC cases.In spite of recent advances in clinical therapy,5-year survival rate of NSCLC patients is lower than 20%in the developed and developing world.Clinical stage,histological type and smoking status have been widely used to predict prognosis and response to therapy.Discovery and application of biomarkers that incorporate with traditional cancer staging could help to improve patient care.However,even if the patients have the same clinical stage,their prognosis differs greatly.It's necessary to continue evaluating molecular events or intermediate biomarkers in treatment response and prognosis of patients with lung cancer and thus to guide therapeutic decisions.Autophagy is a cellular recycling mechanism by which cells capture,degrade,and recycle intracellular proteins and organelles in lysosomes.Autophagy is a dynamic evolution process regulated by a series of autophagy related gene.Central to this process is the formation of autophagosomes,double-membrane vesicles responsible for delivering long-lived proteins and excess or damaged organelle into the lysosome for degradation and reuse of the resulting macromolecules.Autophagosome formation requires two crucial ubiquitin-proteasome systems,the ATG12 and the microtubule-associated protein 1 light chain 3 B(LC3B)systems,which are correlated with expansion of autophagosomal membrane.More than 34 autophagy-related genes has been found,including ATG3,ATG5,ATG7,ATG10,ATG12.Emerging evidence has highlighted the importance of above autophagy-related genes(ATGs)in cancer pathogenesis,clinical therapy and prognosis.However,the regulatory mechanisms and clinical values of these key genes have not been extensively explored in NSCLC.The expression quantitative trait loci(eQTL)are genetic variants that influence gene expression levels and subsequently modulate the biological function of genes,which are important for the understanding of genetic contribution to cancer development and prognosis.Moreover,the increasing number of studies has evaluated the influence of genetic variation on DNA methylation level at specific sites,defined as methylation quant:itative trait loci(meQTL),which construct the functional connection between genetic and epigenetic information.Therefore,eQTL and meQTL analyses integrate polymorphisms,DNA methylation and gene expression and provide powerful method to improve the understanding of disease-associated genetic variants.Purpose:We hypothesized that polymorphisms of autophagy-related genes were associated with the prognosis of NSCLC patients in China.To validate our hypothesis,we investigated the associations of SNPs in autophagy pathway with NSCLC survival.Methods:1;341 patients with histologically or cytologically confirmed primary NSCLC were recruited from the Cancer Hospital of Jiangsu Province,and the First Affiliated Hospital of Nanjing Medical University in China since July 2003 as previously described.The patients were followed-up by personal or family contacts from the time of enrollment until death or the last follow-up.The survival time was obtained by calculating from the time of diagnosis until death or the latest follow-up(August 2013).The last date of follow-up was August 2013,and 1001 cases(74.6%)with tihe median survival time(MST)of 26.0 months had sufficient information to permit survival analysis.Five core autophagy genes(ATG3,ATG5,ATG7,ATG10,ATG12)were selected in our study.Using the public HapMap SNP database(phase ?+? Feb 09,on NCBI B36 assembly,dbSNP bl26)and the HaploView 4.2 software,common SNPs(MAF>0.05 in Chinese Han population)of these genes were screened in gene regions(including 10-kb up-stream region of each gene).After the prediction by using SNPinfo Web Server(http://snpinfo.niehs.nmh.gov/),14 potentially functional loci were selected for genotyping by using Illurmina Infinium(?)Human Exome BeadChip.By using GTEx lung tissues and TCGA Lung Adenocarcinoma(LUAD)dataset,we performed eQTL and meQTL analyses to examine the effects of SNPs on mRNA expression.Spearman's rank correlation was used to evaluate genotype-expression,genotype-methylation and methylation-expression associations.Kaplan-Merier test were used to compare the survival time in different subgroups.Cox regression analysis were performed to estimate the crude or adjusted hazard ratio(HR)and their 95%confidence intervals(CIs).Different groups with various treatments were compared by Student's t-test.Real-time quantitative reverse transcription PCR(RT-PCR)was performed to detect the relative expression levels of ATG10.Cell proliferation assay was measured by Cell Counting Kit-8(CCK8)and xCELLigence Real-Time Cell Analyzer(RTCA)DP system.Cell migration assay was measured by Costar Transwell plates and RTCA migration assay.Results:The results showed that only rs1864183 in ATG10 was found to be significantly associated with the survival time of lung cancer(Log-rank P<0.05 in additive model and dominant model).After adjusting for age,gender,smoking status,sxurgical operation,clinical stage,chemotherapy or radiotherapy status and histology,Cox regression analysis further showed that three SNPs in ATG10(rs10514231,rs1864182,rs1864183)had association with the poor prognosis of lung cancer under the additive genetic model,with adjusted HRs of 1.33(95%CI,1.07-1.65),1.43(95%CI,1.13-1.81),and 1.38(95%CI,1.14-1.68),respectively.Moreover,eQTL analysis revealed that all variant alleles(rs10514231-G,rs1864182-C and rsl864183-G)showed increased ATG10 mRNA expression(P=8 8x10-12,7x10-11 and 0.02,respectively)from GTEx and TCGA datasets.The elevated expression of ATG10 also predicted shorter survival time in lung cancer patients(HR = 2.10,95%CI = 1.33-3.29).meQTL analysis found that after adjusting for age,gender and clinical stage,the hyper-methylation of cg17942617 was significantly associated with the unfavorable outcome of patients(adjusted HR = 1.77,95%CI = 1.06-2.97,P = 0.029).Moreover,the variants of rsl0514231 and rs1864182 were associated with the increased methylation levels of cg17942617,which in turn contributed to the elevated ATG10 expression and decreased survival time.Further functional assays revealed that overexpression of ATG10 facilitated lung cancer cell growth and migration;conversely,siATG1 inhibited lung cancer cell growth and migration.Conclusion:Our findings supported the view that genetic variants of ATG10 affected NSCLC survival via the genetic or epigenetic mechanism.It is the first attempt to analyze the effect of genetic variants in autophagy core genes on NSCLC survival and clarify the underlying mechanism by the bioinformatics analyses and preliminary functional experiments.Further population-based prospective and functional studies are warranted to confirm our findings and elucidate the underlying mechanism.