The Role And Mechanism Of Activating Mutant Ptpn11 In The Transformation Of Mesenchymal Stem Cells （MSCs） Into Cancer Stem Cells （CSCs）

[Backgrounds]Cancer stem cells（CSCs）are a subgroup of tumor cells,also known as tumor initiation cells,which are found in most cancers,including blood cancer and solid tumor.So far,CSCs can be isolated from almost all solid tumors and have the ability of self-renewal,maintenance of stem cell characteristics and differentiation into many types of cancer cells,which play an important role in tumorigenesis,drug resistance and recurrence after treatment.At present,it is generally believed that the origin of CSCs is mutated adult stem cells or dedifferentiated cancer cells.In addition,epithelial mesenchymal transformation of（EMT）and its reverse process（MET）are important sources of CSCs have been confirmed in breast cancer and other cancers.PTPN11 is a proto-oncogene,the SHP2 protein encoded by PTPN11 is widely distributed in human tissues.SHP2 contains two SH2 domains（N-SH2 and C-SH2）and a protein tyrosine phosphatase（PTP）domain.SHP2 maintains a self-inhibitory conformation when the PTP domain interacts with the N-SH2 domain,and SHP2 gains catalytic activity when the PTP domain is exposed.SHP2 plays an important role in human development and the cancer.It regulate cells proliferation,differentiation,apoptosis and survival by participating in cell signal transduction.Abnormal expression or mutation of SHP2 is closely related to human developmental disorders,leukemia and solid tumors.There is a view that SHP2 promotes the progression of solid cancer by promoting the expansion of CSCs,but the specific mechanism is not clear.It is worth noting that current studies have shown that SHP2 can induce or promote EMT in prostate cancer,breast cancer,colorectal cancer,glioma and other tumors.Mesenchymal stem cells（MSCs）are typical adult stem cells.In our laboratory’s previous studies,we found that MSCs expressing activated mutant Ptpn11 can form tumors in vivo,while wild-type MSCs do not have the ability to form tumors.Based on the above background,we are full of curiosity about MSCs that activate mutant Ptpn11.We guess that MSCs with activating mutation of Ptpn11 acquire tumorigenesis ability by transforming into CSCs.In order to verify our conjecture and explore the mechanism,we isolated primary MSCs from MX1-cre/Ptpn11^+/+and MX1-cre/Ptpn11^E76K/+mice and passed in vitro function experiments,in vivo tumor formation experiments and a series of markers to explore whether the MSCs expressing the activating mutation Ptpn11 are transformed into CSCs and its mechanism.[Research methods]1.MX1-cre/Ptpn11^+/+and MX1-cre/Ptpn11^E76K/+mice are generated by crossing MX1-cre tool mice with Ptpn11^E76K-neo/+mice,Intraperitoneal injection of PI-PC induces the specific expression of Cre enzyme in bone marrow MSCs to induce the Ptpn11-E76K activation mutation of bone marrow MSCs in mice,Then isolate the primary MSCs in vitro to obtain the MX1-cre/Ptpn11^+/+（wild type）and MX1-cre/Ptpn11^E76K/+（Ptpn11activating mutations）MSCs.2.The primary wild-type and Ptpn11-activated mutant MSCs were continuously subcultured in vitro,and the cell growth status was observed and the cell behavior differences were compared by detecting cell proliferation-related Ki67 and senescence-relatedβ-galactosidase.Furthermore,the malignant degree of the two kinds of cells and the tumor-forming ability of the same cells in different generations were compared by tumor formation experiment in vitro.3.The expression differences of stemness markers of wild-type and Ptpn11-activated MSCs were detected by immunofluorescence,Western blot and flow cytometry in vitro,and the existence of CSCs population was verified by a series of transplantation experiments in vivo.4.Through proteomic difference analysis,we are looking for biomarkers that can specifically describe CSCs populations,and specific cell subpopulations are sorted by FACS,and tumor microsphere culture is used to test whether the subpopulations are CSCs.5.The mechanism was explored by detecting the specific markers of mesenchymal and epithelial cells and the changes of CSCs subsets before and after inhibiting the open conformation of SHP2.[Research results]1.Wild-type MSCs gradually aged and died with passage,and the Ptpn11^E76K/+activating mutation makes MSCs escape aging,their proliferation activity and tumor-forming ability gradually increases with continuous subculture.2.The biomarkers of CSCs in Ptpn11^E76K/+-activated mutated MSCs are gradually up-regulated and the series transplantation experiments have confirmed the existence of CSCs subsets.3.The proteomics difference analysis determined that the possible CSCs biomarker was PDPN.The PDPN positive subgroups sorted by FACS can form tumor spheres under serum-free conditions,confirming that this subgroup is a real CSCs.4.As for the mechanism,by detecting the markers of epithelial and mesenchymal cells and comparing the proportion of PDPN positive subsets and expression of epithelial cell markers before and after inhibition of SHP2 open conformation,it can be concluded that Ptpn11^E76K/+activated mutation promotes MSCs to undergo"partial mesenchymal epithelial transformation"and then transform into CSCs.[Conclusion]The Ptpn11^E76K/+activating mutation promotes"partial mesenchymal epithelial transformation"of MSCs,transforming them into an intermediate phenotype with both epithelial and mesenchymal characteristics,namely PDPN-positive CSCs.