The Role And Mechanism Of Mitochondrial Dysfunction And Antioxidants In Sarcomagenesis Driven By Ptpn11 Activating Mutation

Sarcomas,including soft tissue sarcomas（STSs）and osteosarcomas,are one of the most lethal solid tumors,and they have higher heterogeneity.Traditional treatment is rarely effective in sarcoma therapy on.Besides,ineffective therapy might be associated with sarcoma recurrence and metastasis.Emerging therapy strategies,such as targeted therapy and immunotherapy,are revolutionizing the clinical management of advanced cancers and show a promising future in cancer treatment.However,substantial findings revealed that neither targeted mutation inhibition nor immune checkpoint（IC）lines of defense improved prognosis;instead they left a series of side effects.Thus,better understanding the mechanism of sarcoma development is imperative.Genetic mutation is a crucial for tumorigenesis.Different tumors have different genetic mutations,or even different sites of the same gene.Sarcomas also have a number of genetic mutations,including PTPN11.PTPN11,as an oncogene,is involved in the occurrence and development of a variety of cancers.PTPN11 encodes the SHP2,which is a non-receptor protein tyrosine phosphatase.SHP2 regulates cell proliferation and differentiation by activating downstream RAS/ERK signaling pathways.Activating mutant SHP2 continuously activates the RAS/ERK signaling pathway and regulates cell malignant proliferation.In addition,SHP2 is involved in regulating mitochondrial functions,including metabolism and production of reactive oxygen species,but the mechanism is unclear.Recent studies have shown that activating mutations of PTPN11 are also present in rhabdomyosarcoma.However,the role of PTPN11 in sarcomagenesis and its mechanism remains unclear.In addition,PTPN11 is closely associate with reactive oxygen species（ROS）which have carcinogenic effects,but PTPN11-ROS interplay in sarcomatogenesis is still unknown.Cell or animal models are important platform to explore the mechanism of tumorigenesis and development.Developing a proper model of sarcoma is critical for oncogenic pathway identification and drug development.Immortalized human sarcoma cells cannot fully exhibit the dynamic alteration of oncogenic signaling pathways from initiation to propagation and have even provided confusing results due to the xenogenous microenvironment.Animal models of sarcoma,which are characterized by the occasional occurrence of other tumors and undetermined signaling pathway regulation during progressive transformation,showed little value in assessing drug efficacy in preclinical therapy.Mesenchymal stem cells（MSCs）,which typically possess tri-lineage differentiation capacity,are reported to be valuable for dissecting mechanisms driving sarcoma development and accelerating the production of novel therapeutic strategies.In this study,the mouse MSCs expressing Ptpn11^E76K/+ was used to examine the ability whether this mutant MSCs underwent malignant transformation.In addition,we would further investigate the mechanism,especially for mitochondrial function,during malignant transformation of MSCs.It will provide a new perspective for the treatment of PTPN11-related sarcoma.Objective:To investigate whether mouse MSCs with Ptpn11^E76K/+ mutation undergo malignant transformation and explore the potential molecular mechanisms.Methods:The purity and identity of MSCs in the compact bone and bone marrow from Mx1-Cre;Ptpn11^E76K/+ and Mx1-Cre;Ptpn11+/+ transgenic mice was determined by flow cytometry and osteogenic/adipogenic differentiation assay.The proliferation ability of MSCs was detected by Ki-67 staining.The unanchored-growth ability and malignant transformation of MSCs were detected by soft agar colony assay and xenograft tumor formation assay in nude mice.Protein mass spectrometry,seahorse metabolism assay,ATP assay,ROS assay,immunofluorescence staining,mitochondrial membrane potential assay,glucose uptake,glutamate/branched-chain amino acid content assay,and mitochondrial complex activity assay were used to detect mitochondrial functional changes associated with malignant transformation of MSCs.The expression of metabolism-related molecules,mitochondrial complexes and immune checkpoint molecules were detected by immunofluorescence and western blotting.The causal relationship between the target molecule and sarcomagenesis was verified by intraperitoneal injection of small molecule inhibitors or intravenous injection of neutralizing antibodies of immunocheckpoint molecule.Immunohistochemistry and flow cytometry were used to detect the number and proportion of immune cells in sarcoma tissues and peripheral blood of tumor-bearing mice.Results:1.WT and Ptpn11^E76K/+-mutant MSCs expressed CD140 a,CD150 and CD90.Additionally,both MSCs occupied the capacity of osteogenesis and adipogenesis.2.Compared to WT MSCs,Ptpn11^E76K/+-mutant MSCs occupied enhanced capacity of proliferation and adipogenesis.3.MSCs expressing Ptpn11^E76K/+ underwent malignant transformation and initiated sarcomagenesis.4.MSCs expressing Ptpn11^E76K/+,compared to WT MSCs,exhibited mitochondrial hypermetabolism and excessive ROS.5.MSCs expressing Ptpn11^E76K/+,compared to WT MSCs,exhibited inactive immunoregulatory signaling pathway involved in microenvironmental response.6.ERK and m TOR signaling pathways were activated in MSCs expressing Ptpn11^E76K/+.7.SHP2E76 K impaired interaction with mitochondrial complex I and III,which was closely associated with mitochondrial dysfunction and excessive ROS.8.Inhibition of CI and CIII could alleviate Ptpn11^E76K/+-induced sarcomagenesis.9.Antioxidants promoted Ptpn11^E76K/+-induced sarcomagenesis.10.Antioxidants potentiated immunosuppressive microenvironment of Ptpn11^E76K/+-induced sarcomagenesis.11.Antioxidants promoted hedgehog production in MSCs harboring Ptpn11^E76K/+ mutation.12.Hedgehog promoted the expression of inhibitory immune checkpoint molecules in Ptpn11^E76K/+-induced sarcomagenesis.Conclusion:1.MSCs expressing Ptpn11^E76K/+ undergo malignant transformation and initiates sarcomagenesis.2.Activating mutant Ptpn11 controls mitochondrial dysfunction and promotes malignant transformation of MSCs.3.Antioxidants amplifies Hedgehog expression from MSCs expressing Ptpn11^E76K/+,which potentiates immunosuppressive microenvironment by enhancing the expression of inhibitory immune checkpoint molecules,and ultimately accelerates sarcomagenesis.