Comparative Proteomic Analysis Of Gastrointestinal Stromal Tumors Based-on Differential Risk Classification

Befo re the appea rance of ta rgeted the rapy,the p rognosis of gastrOintestinalstromal tumor(GISTs)was poo r.In gene rally,a 5-yea r ove ralI su rvival rate(OS)after acomplete su rgical resection is now only 32—63%.Prognosis fo r high risk patients iswo rse,with a 5-yea r recu rrence—free su rvival(RFS)as low as 20%; even after ROresection.And metastasis leads to treatment failu re and death of patients with GISTs.Based on ea rlie r clinical and pathological study data,tumo r size and mitoticindex a re significant prognostic facto rs fo r GISTs patients,and risk stratification is aneffective p redicto r fo r prognosis of patients with GISTs.The long—te rm outcome forGIST patients with diffe rent risk stratification shows significant diffe rences . Unfo rtunately,the molecula r basis of such differences is stilI unclear.P revious studieshad shown that GISTs may originate from gastrointestinal pacemaker celIs/inte rstitialCajal cells(ICCs),and mutations of the p roto—oncogene KIT and PDGFRc^,withactivation of the tyrosine kinase signaling pathway,may be an impo rtant molecula rmechanism.Because KIT gene mutations a re found in 75—80%of GISTs,it is thoughtthat mutations to KIT a re the majo r gene mutation in GISTs.Therefo re,we combined KIT expression along with diffe rent risk stratifications of GISTs as prognostic indicatorsin this study.We fi rst used Real time—PCR combined with di rect sequencing to analyzethe mutations of KIT and PDGFRcⁱn high and low risk types of GIST.A compa rativeproteomics strategy with two—dimensional gel electrOOhOresis and massspectrometry system were then used to screen and identify diffe rentially exp ressedproteins between high—risk and low—risk GIST patients.Bioinformatics analysis wasdone to explore the overalI molecula r cha racte ristics of diffe rentially exp ressedproteins.Finally,based on follow—uP data.we made tissue microwa rray and usedimmunohistochemical staining to dete rmine thei r me rits of prognosis predicting andmetastasis wa ring fo r GISTs with the levels of the diffe rently expressed proteins ofInte rest.In the first pa rt ofou r study 10 KIT-positive GISTs,that included five cases of highrisk GISTs and five cases with low risk GISTs,were en rolled.RT-PCR and di rectsequencing we re used to determine the mutations of exons 9,11,13 and 17 of theKIT proto—oncogene and exons 12 and 18 of the PDGFRe gene.Exon 11 was the mostcommonly mutated in the KIT gene in GISTs,and codons 550—560 of 5 terminal inexon 11 was a"hot mutation zone".A6 bp(TGGAAG)deletion co rresponding to thedeletion of Trp一557 and Lys一558 in exon 11 of KIT may be a unique mutation ofhigh—risk GISTs.We found that genotyping for mutations can add to the molecula r info rmatiOn of clinical and pathological data of risk st ratification based on theaccu racy of p rognosis.In the second pa rt.we used two—dimensional electropresis technique afte rtyping the gene mutations to identify p roteins diffe rentially expressed between highand low risk GISTs.By compa rative analysis of protein expression p rofiles we found41 diffe rentially expressed p rotein spots.Using the matrix—assisted lase r deso rptionionization time of flight mass spectrometry(MALDI—TOF—MS)technique fo r massspectrometry analysis of diffe rentially expressed protein spots we successfully identified 27 diffe rent p rotein molecules.Of these,14 p roteins included ALDHlA2,UQCRCl,CCT一2,PDHB,PRDX3,P rohibitin,Pa2813,COX5A,CLPP,TuFM,GST01,HCRTRl.NM23一H1 and an unknown protein were upregulated in high—risk GISTs.13proteins included APOAl.Haptoglobin,HEBPl,Clo rfl23.HSPB6.SH3BGRL.GSTM2.TPMl,PDIA3,DDAHl,TTCl,HNRNPC and DJ一1 we re down regulated in high—riskGlSTs. The fourth part of this study,we applied muItiple online bioinfo rmatics toolsavailable to analyze data from the second pa rt of the experiment,to evaluate thediffe rentially exp ressed p roteins and the ch romosomal localization of thei r genes,thei r molecula r biology and signal transduction function.The online analysis ofdiffe rentially expressed proteins fu rther revealed thei r overalI biologicalcha racte ristics.We found that 76%of diffe rentially exp ressed p roteins dist ributed in the cytoplasm,of which the la rgest p ropo rtion we re cytoplasmic p roteins(45%),followed by mitochond rial p roteins(23%).There we re also nuclea r proteins andcytoskeletal proteins.Ch romOSOmal locations of those Oroteins a re widelv distributedamong ch romosomes 1,3,5,10,11,12,14,15,16,17,19 and the x ch romosome.Ch romosomes lp and 15q saw the highest concent ration(35%)of genes affected;including five p roteins coded fo r on lp(Clo rfl23,GSTM2,DDAHl,DJ一1 and HCRTRl) and fou r p roteins coded fo r on 15q(ALDHlA2 , TPMl , PDIA3 and COXSA).Biologicalfunction analysis showed these p roteins have dive rse biological functions ; related totumo r celI gene t ranscription ,translation regulation(31 % ) , celI p rolife ration(11 . 5 % ) ,apoptosis(11 . 5 % )and to G p rotein—COUpied recepto r signaling pathways(11.5%).Finally,according to KEGG pathway analysis,we found that the p roteins we reinvolved in signal t ransduction pathways,antigen p rocessing and p resentation,proteasomes,RNA splicing,oxidative phOsphOrylatiOn,PPAR signaling pathway andothers.The fou rth pa rt of this study,fu rthe red by the mass spectrometry, bioinformatics,mining and cOmp rehensive analysis of the lite ratu re,and tissuemicroa rray we re combined with immunOhistOchemist rv to dete rmine the clinicalsignificance of Ove rexO ressiOn of PRDX3 and Prohbitin in high—risk GISTs compa red tothe low—risk GISTs.Co rrelations of OverexO ressiOn of PRDX3 and Prohbitin with theclinicopathological pa ramete rs and recu rrence—free su rvival(FRS)of GISTs we reanalyzed.A ability of OverexO ressiOn of PRDX3 and Prohbitin to p redict recu rrence and metastasis of GISTs we re also analyzed.The results showed that,there we resignificant diffe rences of PRDX3 Ove rexOressiOn at the original site of the tumo r(X2=11.362,P=O.023),mitotic index(X2=4.740,P=O.029)and risk classification(x。=6.370,P=O.035)of GISTs.The 1,3,and 5-yea r RFS of patients with PRDX3Overexp ressiOn we re wo rse than patients without PRDX3 expression,71.7%vs.92.5%,58.3%vs.79.8%and 52.9%vs.72.O%,respectively(HR 2.564,95%CI 1.325—4.960; P=O.00s).The Prohibitin overexo ression shows a significant co rrelation with thegender of patients(X2=3.883,P=O.049),size of tumors(X2=28.900,P=O . 000) , o riginsite of tumo r(X2=9 . 896 , P=O . 019) , mitotic index(X2=13.086,P=O.000)and riskclassification(x2=13.740,P=O.001)of GISTs.The 1,3,5-yea r RFS of patients withProhibitin Ove rexOressiOn we re also worse than patients without ProhibitinOverexp ressiOn;60.1%vs.92₃%,43.2%vs.82.2%and 33.6%vs.74.8%,respectively (HR 2.120,95%CI 1.146—3.919;P=0.000).0ve rexp ression of PRDX3 and Prohbitin canbe used to p redicate the prognosis and can effectivelV wa rn of the metastasis ofGISTs and may be potential therapeutic ta rgets.