Mutation Frequency And Prognosis Of RNF43 In Chinese Colorectal Cancer Cohort And The Mechanism Of BET Inhibitor Surpressing Cancer-associated Fibroblasts

Backgroud and purpose Colorectal cancer?CRC?derived from the epithelial cells of the gastrointestinal tract is a malignant carcinoma that has become a major public health issue in China.According to the published epidemiological data,the current incidence and mortality of colorectal cancer in men and women are top five of the most commonly diagnosed carcinomas.Tumor microenvironment?TME?was stimulated by malignant tumor cells that transformed from mutated epithelial cells.Studies have shown that the invasion and metastasis of CRC depends not only on the properties of cancer cells,but also closely related to the function of TME.The mutated epithelial cells and support of the stromal have always have been involved in the whole process of CRC initial and progress.As a negative regulator of the classical Wnt pathway,RNF43 can degrade Frizzled receptor by ubiquitinating its lysine residues and play a role in inhibiting tumorigenesis and progression.Cancer-associated fibroblasts?CAFs?,as the main component of TME,can synthesize a large number of collagen fibers,secrete a variety of factors to participate in the progress of cancer,while protect CRC cells from the attack of immunochemotherapy drugs.Based on the theory that metastasis and invasion of CRC are inseparable from tumor matrix,targeted regulation or influence of CAFs function may provide a new direction for CRC treatment and research.Several studies have shown that the mutation frequency of tumor suppressor gene ring finger protein 43?RNF43?was high in endometrial cancer?EC?,gastric cancer?GC?and CRC patients.However,it is not confirm whether there is a high frequency mutation in Chinese CRC corhort,because several researches have shown that the frequency of gene mutation was also affected by ethnic.In previous studies,we found that there were Arg and His in the 117th amino acid of RNF43,single nucleotide polymorphism?SNP?in X117 of RNF43.It was proved that the occurrence and prognosis of cancer may affected by different amino acid.JQ1,an inhibitor of BET protein?Bromodomain and extra-terminal?,can specifically bind to the bromine-containing domain.In recent years,it has been found that JQ1 could treat breast cancer,leukemia and other tumors by repressing cell cycle and promoting apoptosis.The therapeutic effect of JQ1 in previous had been focus on cancer cells for a long time,but the effect of JQ1 on CAFs has not been studied.Therefore,the first aim of this research was to verify the mutation rate of RNF43 and investigate the influence of single nucleotide polymorphisms?SNPs?in RNF43 in a Chinese CRC cohort.Secondly,we need master the techniques of isolation,primary culture and identification of CAFs from cancer tissues of CRC patients.The third propurse was to study a new treatment mechanism of JQ1,a small molecule inhibitor of BET,by inhibiting the HGF/MET pathway in CRC cells and CAFs.Methods We randomly selected 177 paired CRC tissue and blood samples from CRC patients in our hospital sample library from February 2010 to December 2012.DNA from 177diagnosed CRC patients were extracted,amplified,and sequenced.The clinicopathological and prognostic data including name,sex,age,tumor stage and survival time were collected.Sequencing results were independently reviewed by three researchers,and the mutation rate of RNF43 and the relationship between SNP at X117 locus of RNF43 and prognosis were analyzed.Finally,the mechanism of SNP affecting the prognosis of CRC patients was studied by overexpression of RNF43^R117R117R and RNF43^R117H in HCT116 with Arg117 mutation.In order to study the effect of JQ1 on HGF/MET pathway,we first verified the effect of JQ1 on MET?MET proto-oncogene,also known as HGFR?in four CRC cell lines?sw480,sw620,RKO,HCT116?using qPCR and Western blot.After NFs and CAFs were obtained from surgical specimens of CRC patients by enzymatic digestion.We studed whether the CAFs'function was affected by JQ1 and analyzed the expression of hepatocyte growth factor?HGF?in two kinds of cells by using qPCR and ELISA.Finally,HCT116 was stimulated by fibroblasts'conditioned medium to study whether CAFs could promote CRC migration.Results Four novel RNF43 mutations?only including G659 and R117?were validated in 177 CRC patients?351 sequencing reaults?,accounting for 2.26%of the corhot;all events were somatic frameshift mutations.Furthermore,we also found that an SNP of the RNF43 X117site was associated with overall survival?OS,HR 1.894,95 CI 1.033-3.829,P<0.05?instead of disease free survival?DFS?.G homozygote subjects were significantly correlated with poor overall survival compared to the other groups.Univariate and multivariate Cox regression analysis showed that tumor location was an independent predictor of OS and DFS.Subsequently we rescued RNF43^R117R and RNF43^R117H expression in the HCT116cells and analyzed the targets of the Wnt/?-catenin pathway.The expression of CCND1and c-Myc were decreased.So the prognosis of CRC patients could be affected by the different functions of SNPsIn order to study the effect of JQ1 on HGF/MET pathway,we first treated the four CRC cell lines with JQ1 and found that both the protein and mRNA level of MET were decreased.We successfully isolated NFs and CAFs from CRC tissue samples by enzyme digestion,alpha SMA and Vimentin immunofluorescence showed the purity of fibroblasts can meet the experimental requirements.After qPCR and ELISA detectio,our team indicated that the expression of HGF in CAFs was significantly up-regulated compared with that in NFs.Meanwhile we also proved that JQ1 could down-regulate HGF synthesis and secretion in CAFs.We stimulated HCT116 cells with supernatants of NFs,CAFs and CAFs+JQ1 conditioned medium.The transwell results showed that fibroblasts,especially CAFs,could promote the migration of CRC cells.According to the reduction of gel contraction area after JQ1 treatment we suggest that JQ1 could inhibit the biological function of CAFs.Intrigingly,flow cytometry analysis showed that JQ1could inhibit the proliferation of CAFs by downregulating the classical star molecule c-Myc without increasing apoptosis.Conclusions In conclusion,our results implied that the mutation of RNF43 in different ethnic groups was positive,but the frequency was not coincidental.The rate of RNF43 mutations was lower?2.26%?,at least for G659 and R117 loci in Chinese patients with colorectal adenocarcinoma.More broadly,we suggest that the multiformity of wild type RNF43X117,in terms of survival rate,was indeed distinct in CRC patients.Subtype G homozygote of the R117 locus was significantly related to slightly poorer overall survival.The prognosis of cancer should be affected by the different functions of SNP subtypes.BET protein inhibitor JQ1 can down-regulate MET expression in CRC cancer cells,while BRD4 was the key molecule in this process.We successfully obtained primary fibroblasts from CRC patients'tissues by enzymatic digestion,and found that the expression of HGF in CAFs was significantly higher than that in NFs.Our results demonstrated that JQ1 can not only inhibit CRC cancer cells,but also indirectly affect the migration ability of cancer cells by down-regulating c-Myc expression,inhibiting the CAFs proliferation,affecting the biological function of CAFs and reducing the secretion of HGF.In conclusion,JQ1 can inhibit both cancer cells and CAFs and interfere with HGF/MET signaling pathway,which is an ideal choice for CRC treatment.