The Association Of The SNP Genotypes And Haplotypes Of Novel Tumor Suppressor HTPAP With Metastatic Potential Of Human Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is one of the most common and aggressive malignancies worldwide.It has been ranked the third in terms of cancer mortality worldwide and the 2nd cancer killer in China since 1990s.Despite clinical advances in surgical resection,the prognosis of HCC still remains dismal.Metastatic recurrence after HCC resection is one of the major obstacles to prolonging survival.Therefore, there is a tremendous interest and urgency to search for molecules related to HCC metastasis that would provide new predictors for HCC metastasis as well as new targets for intervention.HTPAP is a newly-discovered tumor-related gene,its official symbol is PPAPDC1B(Phosphatidic acid phosphatase type 2 domain containing 1B).It is mapped on chorosome 8p12 with seven exons and eight introns.Its full length is 4459bp with a cDNA of 826bp encoding 175 amino acid protein.The abnormal expression of HTPAP has been found in several human tumors,and has been showed a potential functional role of inhibiting the proliferation,invasion and metastasis of cancer cells.In our previous study,it has been demonstrated both in vitro and in vivo that HTPAP could suppress the invasion and metastasis of HCC,which indicate that HTPAP might be a new metastatic suppressor.In our previous study on the polymorphisms of HTPAP in HCC,we found allele C to G change on the rs1149.The risk associated with GG+GC genotype(61%) and G allele(39%) at rs1149 in HCCs with intrahepatic metastasis and vascular invasion is high than the without(39%,24%respectively)(P=0.046,P=0.026 respectively).In HCC,the genotype GG+GC was correlated with tumor number(P=0.026),TNM stage(P=0.026),5-year disease-free survival(DFS)(P=0.005),and 5-year overall survival(OS)(P=0.014).The allele C to G change at rs1149 might have an effect on HTPAP expression level and protein function and structure,consequently conferring up-regulating metastasis potential.Based on the above findings,we conducted the following study on the polymorphisms of HTPAP in HCC tissues using laser captured tissue microdissection and single nucleotide polymorphisms(SNPs) analysis.We sequence across the HTPAP region and detect all the SNPs,and evaluate the associations of SNP genotypes and haplotypes with metastasis potential,postoperative disease-free survival(HCC recurrence),and investigate the possible mechanisms.PARTⅠReconstruction of the haplotypes for HTPAP gene and their association with the metastatic potential of hepatocellular carcinoma.Objective:In our previous studies,HTPAP was found to be a candidate metastasis suppressor gene for HCC.In this study,we want to analyze the haplotypes of HTPAP through identifying SNPs on it,and their relations to invasion ability of HCC.Methods:We detected SNPs by direct sequencing of 7.5kb including HTPAP(4.5kb) and its 3',5'-flanking region(3kb),and then analyzed genotypes of the SNPs in DNA samples from 864 cases of microdiessected fresh HCC tissues.The 864 cases were enrolled to haplotype reconstruction by PHASE software.We calculate the genotypes, haplotypes and their relations to metastatic potential of hepatocellular carcinoma.Results:Six SNPs were detected(-1053 A/G,+64G/C,+357C/G(also as rs1149), +1648-/TAAG(we renamed the deletion genotype as T,and insertion genotype as G, as follows),+1838A/G,+3528C/T).A total of 12 haplotypes were found,four of them (A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C,G-C-G-G-A-T) occurred most frequently(94.62%),and accounted for 793 cases(91.78%).Five SNPs were found to be in a strong linkage disequilibrium(LD)(r~2=0.85-1.0),and their association between them can then be explained by tag SNP+357C/G..The other one at +1838A/G had a historical recombination,and were correlated with them with r~2 range of 0.32-0.36. Association analysis revealed that carriers of alleles G at tag SNP +357C/G.increased higher metastatic potential risk in a dose-dependent manner with a OR of 1.77 (95%C.I.=1.32-2.36,P=0.0001)from CC to GC,and a OR of 2.11(95%C.I.= 1.28-3.48,P=0.003) from CC to GG.No significance was found between +1838A/G genotype and higher metastatic potential risk(GA:OR=1.09(0.81-1.46),P=0.564,: GG:OR=1.39(0.91-2.12),P=0.126).The haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk(OR=1.50(1.18-1.91),P=0.001),but none with haplotypes of G-C-G-G-A-T,A-G-C-T-A-C and A-G-C-T-G-C. Conclusions:The tag SNP+357 GG+GC genotype and.haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk in these Chinese HCC patients,and the functional analysis of these different genotypes and haplotypes of HTPAP might provide a new way in the prediction of patients' prognosis and tumor recurrence.PARTⅡThe mechanism of different genotypes and haplotypes of HTPAP involved in the regulation of HCC metastasis potential1.HTPAP expression and its association with haplotypcs of HTPAPObjective:Evaluating the effect of haplotypes on mRNA expression.Methods:After construction of pEGFP-HTPAP the recombinated plasmids,we evaluate the HTPAP expression level(its three known isoforms) in HCCs composed of four common HTPAP haplotype A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C and G-C-G-G-A-T by quantitive real-time PCR.Result:We constructed the recombinated plasmids successfully.HTPAP expression in 170 HCC cases were calculated by quantitive real-time PCR.Among them,21cases belonged to GCGGGT homozygotes carriers,65 AGCTAC homozygotes,52 AGCTAC/GCGGGT heterozygotes,13 AGCTAC/GCGGAT heterozygotes and 19 AGCTAC/AGCTGC heterozygotes.The expression levels in groups of AGCTAC homozygotes and AGCTAC/AGCTGC heterozygotes were higher than those of GCGGGT homozygotes,AGCTAC/GCGGGT and 13 AGCTAC/GCGGAT heterozygotes.Overally,no significance was found between them(P＞0.05).Conclusions:Different HTPAP haplotypes can influence the expression level,but none of them reach significance.2.HTPAP promoter haplotypes and their transcriptional activitiesObjective:Evaluating the effect of three HTPAP promoter haplotypes on their transcriptional activities.Methods:We generated the three HTPAP promoter haplotype luciferase reporter vectors(pGL3-basic) spanning from-1764 to +315 bp of the HTPAP promoter region, and transiently transfect these promoters into MHCC-97H,HepG2 and Hela cells,and detect the luciferase index.Result:Compared with void pGL3-basic vectors,three recombinated promoter plasmids observed obvious transcriptional activity.-1053AA/+64GG haplotype promoter manifested significantly higher luciferase index than that of -1053 AG/+64 GC and -1053 GG/+64 CC promoter(P=0.005,0.009 respectively).However,no significance was observed between -1053AG/+64 GC and -1053 GG/+64 CC promoter(P=0.26)..Conclusions:The different haplotype promoters could affect the transcriptional activity signficantly.3 The investigation of mechanism regulating the metastasis potential with different HTPAP genotype and haplotype by Affymetrix gene-expression biochipObjective:To analyse the different expression pattern between HCC of different HTPAP haplotype,and investigate possible mechanism regulating the metastasis potential.Methods:We randomly selected 16 HCCs in GCGGGT homozygote and AGCTAC homozygote each for genechip analysis by Affymetrix Human Genome U133.By SAM(Significant Analysis of Microarray),we analysed the different expression between them,and reevaluate.the different expression by Real-time PCR.To broaden our research,we again randomly selected 4 cases in each group(AGCTAC/GCGGGT heterozygotes,AGCTAC/GCGGAT heterozygotes,AGCTAC/AGCTGC heterozygotes,GCGGGT homozygote and AGCTAC homozygote) for further SAM and Cluster&TreeView analysis.Result:By SAM analysis(Significant Analysis of Microarray),we obtained 41significantly different genes(including 2 transcripts)(P＜0.05,fold change＞2) between GCGGGT and AGCTAC homozygote carriers(the whole 41 genes or transcripts were overexpressed in GCGGGT homozygote carriers).By uncentered correlation and average linkage analysis,we accurately classified 16 GCGGGT homozygotes tumors(88%,with 2 sample missed) from 16 AGCTAC homozygotes with the above 41 significant genes(transcripts) in the classifier.Further evaluation of the classifier in randomly selected 4 cases in each group(AGCTAC/GCGGGT heterozygotes carrier,AGCTAC/GCGGAT heterozygotes carrier, AGCTAC/AGCTGC heterozygotes carrier,GCGGGT homozygotes carrier and AGCTAC homozygotes carrier) showed the five groups of HCC could be distinguish correctly into two large groups(groupⅠ:GCGGGT homozygotes, AGCTAC/GCGGGT heterozygotes,AGCTAC/GCGGAT heterozygotes,and groupⅡ:AGCTAC homozygotes and AGCTAC/AGCTGC heterozygotes)(83%,with 2 sample missed).When we applied the classifier in the all 52 HCCs,we found the 52 HCC samples could also be distinguish correctly into two large groups as above (92%,with only two missed).IL-8(with fold change over 8) and TLR-2(over 3) were the most overexpressed in GCGGGT homozygote HCC group in the classifier.By RT-PCR and real-time quantitative PCR,we found IL-8 and TLR-2 were overexpressed in tumors of GCGGGT homozygotes,AGCTAC/GCGGGT heterozygotes,and AGCTAC/GCGGAT heterozygotes more than in those of AGCTAC/AGCTGC heterozygotes and AGCTAC homozygotes.When we study the phenotype in the 52 HCC patients,19 of 28 patients in groupⅠand 8 out of 24 in groupⅡwere found high metastasis potential(P=0.025),indicating different haplotype carriers might contribute different risk of metastatic potential.Conclusions:The different expression patterns might occurred between different HTPAP haplotype HCC.These significantly different genes could functionally associated with different HTPAP haplotype,contributedly rendered changes in HCC phenotype such as invasion ability and metastasis potential,indicating that the different HTPAP haplotypes might be the prognostic predicator of HCC metastasis potential.4 The association of HTPAP genotype and haplotype with protein expression and postoperative disease-free survival in HCCObjective:We evaluate the relationship between the protein expression and HTPAP genotype and haplotype by immuno-histochemical analysis on expression of HTPAP in paraffin enbedded HCC tissues.Also,we investigate the association between postoperative disease-free survival(DFS)(HCC recurrence) and HTPAP genotype and haplotype by detecting 665 cases of HCC with 5-year follow-up.Methods:By immuno-histochemical analysis,we observed the expression of HTPAP in 377 cases,and evaluate the relationship between the protein expression and HTPAP genotype and haplotype.665 cases of HCC from Jan.2002 to June 2003 were enrolled for examining the SNPs by prosequencing 96,the association between postoperative DFS and HTPAP genotype and haplotype were investigated.Results:By immuno-histochemical analysis in 377 cases of HCC,we observed the positive staining in 177 case(46.9%).Overally,two patterns of positive immunohistochemical staining have been occurred in cytoplasm.We observed intensified granular staining in cancer cells of the whole nodules,while the scattered positive staining of HCC in cancer nodules was also frequently present.Meanwhile, we found strong HTPAP staining in hyperplastic bile duct epithelial cell,but no positive staining in normal hepatic cells,liver cirrhotic cells,and infiltrating lymphocyte.Positive protein expression occurred more frequently in HCC cases with high metastasis potential compared with that with low potential(P=0.028).When we analysed the association of HTPAP genotypes and haplotypes with protein expression, we found the tag SNP +357(GG+GC) genotype and haplotypes(GCGGGT+GCGGAT) were negatively associated with HTPAP expression(P=0.002,P=0.001,respectively).No significance of HTPAP staining was found between tag SNP+1838(AG+GG) and AA genotype HCCs(P=0.130).Kaplan-Meier analysis showed that the tag SNP+357 GG and GC was significantly associated with a decreased probability of postoperative DFS compared with +357CC genotype(P＜0.001),but we found no significance of postoperative DFS between the tagSNP+1838 GG,AG and AA genotype HCCs(P=0.282).We found the poorer postoperative DFS in HCCs of GCGGGT homozygotes and heterozygotes than that of others(P＜0.001),showing that GCGGGT homozygotes or heterozygotes was associated with a decreased probability of postoperative DFS compared with other haplotypes.We recalculated the HRs for each tag SNP+357G/C genotyoes,and for haplotype of GCGGGT homozygotes,heterozygotes and others/others following adjustments for clinical characteristics.This analysis showed that only the +357 GG+GC genotype and GCGGGT homozygotes and heterozygotes were negatively associated with postoperative DFS(for+357GG genotype,P=0.021,HR(Hazard Ratios)=1.51, 95%C.I.=1.07-2.15,for+357GC genotype,P=0.030,HR=1.29,95%CI=1.03-1.62; for GCGGGT homozygotes,P=0.009,HR=1.59,95%C.I.=1.12-2.27,and for GCGGGT heterozygotes,P=0.022,HR=1.32,95%C.I.=1.04-1.67).Conclusions:Our results show that variants at tumor suppressor have different effects on HCC metastasis potential.The tag SNP +357(GG+GC) genotype and haplotypes(GCGGGT+GCGGAT) were negatively associated with HTPAP expression. +357 GG+GC genotype and GCGGGT homozygote and heterozygotes of HTPAP are independently associated with a poor postoperative DFS in HCC patients after surgical resection,which indicates that +357 GG+GC genotype and GCGGGT homozygotes and heterozygotes might have great value in prediction of HCC metastasis potential(postoperative recurrence) and prognosis..Conclusions1.The tag SNP +357 GG+GC genotype and.haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk in these Chinese HCC patients.2.Different HTPAP haplotypes can influence the expression level,but none of them reach significance.The SNP genotype and haplotype in promoter could affect the transcriptional activity signficantly.3.The different expression patterns might occurred between different HTPAP haplotype HCC associated with different HTPAP haplotype,and contributedly render changes in HCC phenotype such as invasion ability and metastasis potential indicating that the different HTPAP haplotypes could be the prognostic predicator of HCC metastasis potential.4.Our results show that variants at tumor suppressor have different effects on HCC metastasis potential.+357GG+GC genotype and GCGGGT homozygote and heterozygotes are independently associated with a poor prognosis in HCC patients after surgical resection,which indicates they might have great value in prediction of HCC metastasis potential and prognosis Potential application of this study1.The tag SNP +357 GG+GC genotype and.haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk,and might be a new biomarker of tumor metastasis.2.Genotype +357 GG+GC and GCGGGT homozygote and heterozygotes are independently associated with a poor prognosis in HCC patients after surgical resection,which indicates they might have the potential of being a new genetic marker in prediction of metastasis and prognosisNovelties1.To the best of our knowledge,this is the first report investigating the association between the variants at HTPAP gene and HCC metastasis potential.2.To the best of our knowledge,this is the first time to analyse different expression pattern between different HTPAP haplotype carriers in HCC by global gene-expression array to investigate the mechanism involved in the regulation of metastasis.3.In addition,our data for the first time show that Genotype +357 GG+GC and GCGGGT homozygote and heterozygotes of HTPAP are independently associated with a decreased probability of postoperative DFS in HCC patients.