Gαi3-mediated RTKs Signaling Drives Tumorigenesis And Progression Of Osteosarcoma

Research backround and objectiveOsteosarcoma(OS)is a malignant bone tumor originating from mesenchymal tissue,which mainly occurs in children and adolescents,and is characterized by high degree of malignancy and proneness to local recurrence or distant metastasis.Although the current clinical diagnosis and treatment technology of OS has made great progress,the overall prognosis of OS patients is still poor.Therefore,it is necessary to find potential targets for effective OS treatment.More and more studies have indicated that the abnormal expression and activation of receptor tyrosine kinases(RTKs)and their dowstream signals promote the initiation and progression of tumors.In recent years,studies have confirmed that Gαi(G protein a inhibitory subunit)plays an important role in mediating multiple RTKs signaling.Thus,Gαi may be an important oncogene and therapeutic target of human cancers.However,whether Gαi is involved in the initiation and progression of OS and the possible mechanisms involved remain unclear.We first studied the expression of Gαi3 in OS and its biological effects.Based on the above results,we initially explored the molecular mechanism of Gαi3 involved in the initiation and progression of OS.At last,we researched the effect of Gαi3 on the tumorigenicity of OS cells in vivo,so as to provide new ideas for finding possible therapeutic targets for OS.MethodsPart One:TCGA(https://portal.gdc.cancer.gov/)(The Cancer Genome Atlas)database was searched to analyze Gαi3 expression in human osteosarcoma tissues,and Kaplan-Meier survival analysis was used to study the correlation between high Gαi3 expression and poor prognosis in patients with osteosarcoma.The OS tissues and surrounding normal tissues were separated,and Western blotting and qRT-PCR were used to detect the expressions of Gαi3 protein and mRNA in the separated tissues and OS cells,respectively.Immunohistochemistry(IHC)was performed to further confirm the expression of Gαi3 in the separated OS tissues.Part Two:shRNA method,CRISPR/Cas9 system and ectopic overexpression strategy were used to negatively/positively regulate the expression of Gαi3 in OS cells.The effect of Gαi3 expression on OS cell apoptosis was determined by Western blotting,TUNEL staining and Annexin V/7-AAD.ssDNA ELISA,JC-1 staining and Cell ROX staining were applied to observe the effects of Gαi3 expression on single strand DNA accumulation,mitochondrial depolarization,reactive oxygen species(ROS)accumulation of OS cells.The effects of Gαi3 expression on OS cell proliferation,cell cycle progression,migration and invasion were observed by CCK-8,EdU staining,PI-FACS,Transwell and Matrigel Transwell separately.Part Three:shRNA method,CRISPR/Cas9 system,dominant negative(DN)mutant strategy and ectopic overexpression strategy were used to negatively/positively regulate the expression of Gαi3 in OS cells.The correlation between Gαi3 and RTKs in human OS tissues and OS cells was investigated by Co-IP assay,and the expression of Akt-mTOR cascade was detected by Western blotting.Then,the effects of Gαi3 expression on the proliferation,migration and invasion of OS cells were explored by EdU staining,Transwell and Matrigel Transwell.Aktl adenovirus constructs(Ad-caAktl)was employed to restore the expression of Aktl in Gαi3-KO pOS-1 cells.After Akt1/2 shRNA method were used to knock down the expression of Aktl,Gαi3 was knocked down or overexpressed in pOS-1 cells.Then,the expressions of Gαi3 and AkT-mTOR proteins and mRNAs were detected by Western blotting and qRT-PCR,respectively.The effects of Akt expression on the proliferation,migration and invasion of OS cells were observed by EdU staining,Transwell and Matrigel Transwell.Part Four:pOS-1 cells were subcutaneously injected into nude mice,and the xenografted tumors were established.Then,shRNA-Gαi3 and sh-NC were injected into xenografted tumors respectively for comparative analysis.pOS-1 cells of Gαi3-KO were subcutaneously injected into nude mice to establish another xenografted model.Mice body weight and tumor weight and volume were observed.Tumors were isolated from each group,and the levels of Gαi3 expression and downstream Akt-mTOR activation were detected by qRT-PCR and Western blotting.The effect of Gαi3 expression on the apoptosis of OS tissues was detected by Western blotting.ResultsPart One:TCGA database showed that Gαi3 transcript was significantly elevated in OS tissues,and high Gαi3 expression correlated with low survival probability in osteosarcoma patients.Gαi3 expression was significantly increased in human OS tissues and cells,but low in peritumoral normal tissues and human osteoblasts.Part Two:After the expression of Gαi3 in OS cells was knocked down or knocked out,the apoptosis of OS cells was activated,with caspase activity and the expressions of apoptosis-related proteins,DNA accumulation,mitochondrial depolarization and ROS accumulation significantly increased.Meanwhile,the proliferation,migration,invasion and cell cycle progression were significantly inhibited.Conversely,the overexpression of Gαi3 significantly enhanced the proliferation,migration and invasion of OS cells.The expression level of Gαi3 had no significant effect on the biological behavior of osteoblasts.Part Three:Western blotting and Co-IP analysis revealed that Gαi3 associates with RTKs in human OS tissues and OS cells.After the expression of Gαi3 was knocked down,the expression of Akt-mTOR significantly decreased.In contrast,overexpression of Gαi3 significantly increased Akt-mTOR expression.Knockdown of Gαi3 by DN mutant strategy inhibited the interaction between Gαi3 and RTKs and the transduction of downstream AktmTOR cascade,thereby significantly inhibiting proliferation,migration and invasion of OS cells.Ad-caAktl relieved the inhibitory effects of Gαi3 knockdown on the proliferation,migration and invasion of OS cells,and reduced or overexpressed Gαi3 did not affect the proliferation and migration of pOS-1 cells with Akt1/2 knockdown.Part Four:In the xenografted tumors established by subcutaneous injection of pOS cells,intratumor injection of Gαi3-shRNA can significantly inhibit the growth of xenograft tumors,decrease the expression of Gαi3,suppress the transduction of downstream AktmTOR cascade,and increase the expression of apoptosis-related proteins.In addition,pOS1 cells with Gαi3-KO were subcutaneously injected into nude mice to establish another xenografted model.In the model,the growth of tumors was significantly inhibited.The expression of Gαi3 and Akt-mTOR decreased significantly,and the expression of apoptosisrelated proteins increased significantly.ConclusionsGαi3 expression is significantly elevated in human OS tissues and cells,and high Gαi3 expression correlates with low survival probability in OS patients;Gαi3 is involved in the regulation of OS cell proliferation,migration,invasion,apoptosis and other malignant biological behaviors;As a key adaptor protein,Gαi3 associates with RTKs to mediate the activation of downstream Akt-mTOR cascade,thereby promoting malignant biological behaviors of OS cells;Knockdown or knockout of Gαi3 significantly inhibits the tumorigenicity of OS cells in vivo,and promotes the apoptosis of OS.