The Role Of Gain-of-function E76K Mutant SHP2 In Colorectal Tumorigenesis And Progression

Background : SHP2 is a classical,non-receptor PTP encoded by the Ptpn11 gene,consisting of two SH2 domains,a PTP domain and a C-terminal region.In the wild-type SHP2,the backside of the N-SH2 domain binds to the PTP domain,resulting in autoinhibition of the PTP catalytic activity,while activated SHP2 gain of function mutations disrupting the autoinhibitory mechanism have been found in human hematopoietic malignancies and solid tumors.Colorectal cancer is a common malignant tumor of the digestive system,with increasing incidence in these years.The rate of SHP2 mutations in colorectal cancer is the highest rate among the solid tumor,second to hematopoietic malignancies.The rate of SHP2 mutations is different in variety of malignant tumor.Activated mutations were found in the blood malignancies firstly,and the mutation rate is 7.52%.Then SHP2 mutations were also found in solid tumor.Researches associated with common solid tumors have been reported in lung cancer,liver cancer,and breast cancer.But we found that the rate of SHP2 mutation in these solid tumors is very low according to COSMIC(catalogue of somatic mutations in cancer)database.The mutation rate of lung cancer is 1.79%,liver cancer is 0.47%,and breast cancer is 0.15%.The mutation rate of colorectal cancer is 5.75% is second only to Hematological malignancies 7.52%.SHP2E76 K is a common mutation site among the E76 location.E76 K mutation is also found in a clinical case according to COSMIC database.Colorectal cancer is a common malignancy of the digestive system.The incidence is greatly different in different regions of the world.North America,Oceania have a higher incidence,Europe have a lower incidence,followed by Asia and Africa.But with the change of our national diet,intaking of high-fat and high-protein food,the incidence of colorectal cancer in China has showed an increasing trend.South China,especially the southeast coast,is significantly higher than the North.We have a in-depth understanding about the evolution process of colorectal cancer.The evolution of colorectal cancer is normal intestinal epithelial cells-proliferative changes/mall adenoma-early adenoma-interim adenoma-late adenoma-cancer-metastasis.There are different molecular mechanisms in different stages of the evolution.Progression and metastasis of colorectal cancer are closely related to EMT,which play a pivotal role in the process of tumor invasion and metastasis.During EMT,cells lose its polarity,become weaker connections,gain the ability of invasion and migration,lead to local invasion and distant metastasis of tumor.EMT is also associated with a variety of signaling pathway,like Notch signaling,TGF signaling,and Wnt signaling.Wnt signaling pathway is one of the classic pathway which is necessary for embryonic development.People come to realize its importance.It determines many important aspects of organism.Such as cell migration,polarization,neurogenesis and formation of organ during embryonic development.However,with further research,the researchers found that it has a close relationship between abnormal activation of the Wnt signaling pathway and the development of a variety of tumors.And it has also been reported,Wnt/?-catenin signaling pathway is an inducible factor of the occurrence of EMT.Objective: to learn more about the role of SHP2E76 K in tumorigenesis and progression of colorectal cancer,and further ananlysis the mechinasm of EMT induecd by SHP2E76 K and SHP2 WT.Research contents:Part I.SHP2E76 K and SHP2 WT promote the malignant biological behavior of colon cancer cells1.Construction of SHP2 relevant plasmid and packaged into a lentivirus.2.HT29 and HCT116 were infected with the virus suspension and screened with puromycin to establish stably transfected cell lines of SHP2E76 K and SHP2 WT.3.Changes of colon cancer cells' malignant behavior were observed by series of experiments in vitro.1)MTT assay detected proliferation of HT29 and HCT116 and draw the growth curve.2)Transwell for observing the migration ability of HT29 and HCT116.3)Colony for observing the clone forming ability of HT29 and HCT116.4)Soft agar colony forming assay for observing the capacity of anchor-independent growth of HT29 and HCT116.4.Stably transfected cell lines of HT29 and HCT116 were inoculated to mice subcutaneously.Tumorigenic capacity was observed in nude mice.Part II.Mechanisms of SHP2E76 K and SHP2 WT promote the malignant biological behavior of colon cancer cells1.EMT occurence can be observed by cell morphology.2.Experiments verified the occurrence of EMT1)Western blot for detecting the changes of EMT-related markers.2)Immunofluorescence for detecting the changes of EMT typical markers.3.Western blot for detecting wether Wnt/?-catenin signaling pathway is involved in the occurrence of EMT.4.Nude mice experiments for observing the liver metastases of stably transfected cell lines in vivo.Part III.The role of SHP2E76 K in colitis-associated colorectal cancer1.The establishment of Ptpn11E76 K knock in mouse model2.Breeding and genotyping the double transgenic mice of Ptpn11E76 K and p Villin-Cre3.induced Colitis-associated colorectal cancer model induced with AOM/DSSResults:Part I.SHP2E76 K and SHP2 WT promote the malignant biological behavior of colon cancer cells1.The plasmid was constructed successfully,and the stable cell lines of HT29 and HCT116 transfected with SHP2E76 K and SHP2 WT were established successfully.2.SHP2E76 K activating mutations and SHP2 WT promote the proliferation,clone formation,migration ability of CRCs and promote the the proliferation capacity of the stable cell lines in nude mice.Part II.Mechanisms of SHP2E76 K and SHP2 WT promote the malignant biological behavior of colon cancer cells1.EMT was involoved with the progression of CRCs.2.Wnt/?-catenin signaling pathway participated in EMT induced by SHP2E76 K and SHP2 WT.3.SHP2E76 K and SHP2 WT promote the CRCs proliferation in vivo and metatasis of liver.Part III.SHP2E76 K paly an important role in colitis-associated colorectal cancer1.Successfully established the Ptpn11E76 K gene knock in mouse model;2.Successfully breeding and genotyping the double transgenic mice of Ptpn11E76 K and p Villin-Cre;3 SHP2E76 K could aggravate inflammation in mice and promote the tumorigenesis of colitis-associated colorectal cancerConclusions: SHP2E76 K and SHP2 WT could increase the malignant biological behavior of CRCs in vitro and in vivo,and induce Wnt/?-catenin mediated-EMT;furthermore,SHP2E76 K promote the tumorigenesis of colitis-associated colorectal cancer.