| Background: According to GLOBOCAN,prostate cancer(PCa)is said to be one of the most common malignant tumors in male,leading to severe damages to the health.More than 1.1 million cases of prostate cancer and 307,000 deaths were recorded around the globe in 2012,accounting for approximately 15% of new cancer cases in men and 6.6% of the total male cancer mortality,respectively.Recent studies suggested that the morbidity and mortality rates increased in China.However,the exact pathogenesis is unclear.As a multifactorial disease,PCa is said to be associated with environmental and genetic factors.Among them,heredity is important.Nowdays,as a powerful method,genome wide association study(GWAS)is useful in discovering the genetic factors of complex diseases.Based on this method,many PCa-related loci had been identified.Moreover,our own GWAS also discovered two new single nucleotide polymorphisms(SNPs)in Asian firstly.However,only focused on single SNP and the strict threshold of GWAS(P<10-8)might result in loss of genetic information.Moreover,most of the gene and loci discovered in GWAS were located in the intron,which had been considered to be meaningless.So,a comprehensive study with gene,pathway and functional experiment is needed.In order to solve these issues,based on GEO database,our previous study identified that genes associatited with PCa were located on the RTK/ERK pathway.In order to understand the function of this pathway in PCa,this study was conducted.Purpose: On the basis of Chinese Consortium for Prostate Cancer Genetics(China PCa),we aim to understand the function of RTK/ERK pathway in the development,advance and metastasis of PCa by combining with SNP,gene,pathway,natural selection and genetic molecular experiment,which will help us identify the pathogenesis of PCa.Our results will also pave a way for the further pathway study and the prediction,diagnosis and treatment of PCa.Part I.The assocaition between RTK/ERK pathway and the development of prostate cancerMethods: Based on 1417 PCa and 1008 controls from China PCa,this part was conducted to invetagiate the association between RTK/ERK pathway and the development of PCa on the level of SNP,gene and pathway with PLINK and adaptive rank-truncated product(ARTP)used respectively.Meanwhile,combined with some other atabases such as the Hap Map Project and Genevar,the expression of quantitative trait loci(e QTLs)analysis and natural selection analysis were involved,to identify poteintial key loci and genes significantly associated with PCa.Results: On the level of SNP,317 loci are identified to be significantly associated with PCa.Meanwhile,the pathway analysis confirrmed the positive relationship between PCa risk and several key genes,including erb-b2 receptor tyrosine kinase 2(ERBB2)?protein tyrosine kinase 2(PTK2)?fms related tyrosine kinase 1(FLT1)?Raf-1 proto-oncogene,serine/threonine kinase/ Raf-1(RAF1)?baculoviral IAP repeat containing 2(BIRC2)?kinase insert domain receptor(KDR)and Cyclin D1(CCND1).In order to verify these genes and loci,further natural selection and e QTL analyses were conducted,which confirmed that 4 genes(EGFR,ERBB2,PTK2,and RAF1)with five SNPs(rs11238349,rs17172438,rs984654,rs11773818,and rs17172432)especially rs17172432,might be pivotal factors in the development of PCa.These results indicated that the RTK/ERK pathway under natural selection was a key link in PCa risk.The genes and loci with positive selection might be one reason for the development of PCa.These factors might give insight into prevention and aid in predicting the success of potential therapies for PCa.Part II.Correlation of RTK/ERK pathway and the advance and metastasis of prostate cancerMethods: Based on the significant association between RTK/ERK pathway and PCa risk,we further tried to understand the function of this pathway in the advance and metastasis of PCa.Based on the 1417 PCa from Chin PCa in our part I,956 aggressive and 347 non-aggressive Pca were selected.For the genotype,760 tag SNPs in the RTK/ERK pathway(r2 ? 0.8,minor allele frequency ? 5%,CHB+JPT population in Hap Map)were collected.In the analysis,we applied three common genotype models,including dominant,recessive and additive model.Meanwhile,Cochran–Armitage trend test and condition analysis were also used to confirm those associations.The experiments of genes overexpression and inhibition were conducted in prostate cancer cell lines LNCa P,PC3 and DU145 to assay the cell proliferation.In addition,combining with immunohistochemistry and clinical features(including overall survival,biochemical recurrence,GS,PSA levels,and clinical T state),we tried to understand the function of these significant genes and loci in predicting the prognosis of PCa,on the protein and clinical levels.Results: In the traditional SNP-associated analysis,32 loci in additive model,31 for dominant model and 21 for recessive model were identified to be significantly assocaited with aggressive Pca(P < 0.05).In the additive and dominant model,rs603781/PDGF-D was the most significant(additive model: P=1.132 × 10-3;dominant model: P=9.800 × 10-4).As for recessive model,the lowest P value was 2.269 × 10-3 for rs11247380/IGF1 R.After combining with Cochran–Armitage trend test and conditional analysis,we found that only one SNP(rs3217869 in CCND2)was shown to be signifcantly associated with aggressive prostate cancer afer the analysis in the three models(Additive model: P = 4.653 × 10-3,OR = 1.511,95% CI = 1.135-2.010;Dominant model: P = 5.341 × 10-3,OR = 6.907,95% CI = 1.773-26.900;Recessive model: P= 2.156 × 10-2,OR = 1.435,95% CI = 1.055-1.954),Cochran–Armitage trend test(P = 5.188 × 10-3)and conditional analysis(Additive model: P = 7.263 × 10-3;Dominant model: P = 8.938× 10-3;Recessive model: P = 2.629× 10-2).The association of rs3217869 with aggressive prostate cancer was also confirmed in a Finnish cohort including 1729 aggressive and 693 non-aggressive cases.Meanwhile,on the m RNA level,our analysis revealed a statistically significant relationship between rs3217869 risk genotype and decreased CCND2 expression levels.Meanwhile,CCND2 downregulation was consistently observed in the advanced prostate cancer in 18 available clinical data sets with a total amount of 1,095 prostate samples,which suggested that CCND2 was an anti-oncogene.In order to verify the function of CCND2,genes overexpression and inhibition were conducted in LNCa P,PC3 and DU145 cell lines respectively.Similar to previous results,reduced expression of CCND2 promoted cell proliferation and its overexpression inhibited cell growth of prostate cancer.Additionally,on the protein level,comparing to non-aggressive PCa,the expressions of CCND2 protein were partly lost.Furthermore,the lower expression levels of CCND2 markedly correlated with prostate tumor progression to high Gleason score and elevated PSA levels,and served as an independent predictor of biochemical relapse and overall survival.In this part,we have identified an association between the RTK/ERK pathway and aggressive PCa.Meanwhile,CCND2 gene may be important in predicting the susceptibility and metastasis of PCa.Conclusions: Based on previous GWAS,we find that RTK/ERK pathway and its genes and loci were significantly associated with the development,advance and metastasis of PCa,by combining with SNP,gene,pathway and evolution analyses.Moreover,our study highlights the importance of CCND2 in PCa susceptibility and tumor progression to metastasis. |
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