Mechanism Study Of Mutant P53 Or Mutant Kras Accompanying With MTOR Hyperactivation Promoting Hepatocellular Carcinoma Proliferation And Metastasis

backgroundHepatocellular carcinoma(HCC)accounts for more than 90% of primary HCC.Due to the complexity and heterogeneity of HCC molecular pathological mechanisms,molecular targeted therapy and immunotherapy are only effective for part of HCC patients.Therefore,there is an urgent need to further explore the biological characteristics of HCC and further reveal the molecular mechanism of HCC pathogenesis.Accurate individualized treatment for HCC patients with different genetic backgrounds will be an important strategy to further improve the prognosis of HCC.mTOR(The mechanistic/mammalian target of rapamycin)pathway regulates a variety of intracellular metabolism and is involved in the regulation of cell proliferation,survival and metastasis of malignancies.mTOR is divided into mTORC1 and mTORC2 complexes.mTORC1 is mainly involved in the regulation of metabolism,while mTORC2 is mainly involved in the regulation of cytoskeleton.Studies have shown abnormal hyperactivation of mTOR in more than 50% of HCC patients.Among the many molecules that regulate mTOR,Tsc1/2 has the highest mutation rate.Deletion or mutation of Tsc1 or Tsc2 can affect the formation of tuberous sclerosis complex,leading to hyperactivation of mTOR.p53 plays an important role in responding to a variety of stresses inside and outside the cell,including DNA damage,oncogene activation,metabolic disorders,ribosomal stress,telomere erosion,cell senescence,induced cell death,and cell cycle arrest.Under normal circumstances,various stress signals in vivo and in vitro tend to stably activate p53,and the activated p53 plays a key role in tumor inhibition,thus avoiding the occurrence of various tumors.Functional loss of p53 is a prerequisite for the occurrence and proliferation of most tumors.In Chinese HCC,the mutation rate of p53 is up to 58%.Although the mutation rate of Kras in HCC is only 5%,the activation rate of Ras/raf/Mek/Erk pathway in HCC is 50-100%.Kras gene encodes a small membrane-bound GTPase,which is involved in signal transduction of Receptor Tyrosine Kinase(RTKs)to promote cell proliferation,cell differentiation or cell survival.Kras mutation can lead to continuous activation of downstream signaling pathways,thus leading to the occurrence of tumors.Although previous studies have explored the role and mechanism of mTOR activation,p53 mutation and Kras mutation in promoting HCC,respectively,it is not clear whether there is a synergistic effect between them.Part Ⅰ: The mechanism of p53 mutation and mTOR hyperactivation promoting hepatocellular carcinoma proliferationand metastasis Purposes1.Determine whether p53 mutation and mTOR activation have synergistic effects in promoting HCC proliferation and metastasis.2.Determine whether p53 mutation can furtherly activate mTOR and its specific molecular mechanism.3.Identify potential therapeutic targets of p53 mutation associated with mTOR hyperactivation of HCC.Methods1.Construction of transgenic mice: construction of transgenic mice with mTOR hyperactivation(Tsc1 knockout).Transgenic mice with p53(haplo)insufficiency were constructed by mutating a single strand of the p53 gene,since mice cannot survive complete p53 knockout.Transgenic mice with both p53 mutation and Tsc1 knockout were obtained by interbreeding.2.Construction of HCC model:(1)Spontaneous tumor formation model: the transgenic mice were sacrificed after 320 days of continuous observation without any treatment.The size and number of liver tumors,liver weight ratio and lung metastases of the transgenic mice were analyzed.(2)tumor-inducing model: 0.5mg/kg N-nitrosodiethylamine(DEN)was injected in transgenic mice on the 15 th day(intraperitoneal injection,i.p.),and CCl4(0.5ml/kg)was injected once a week from the 21 st day for 17 weeks(i.p.),followed observing for another 4 weeks.The mice were sacrificed and the size and number of liver tumors and liver weight ratio were counted.3.Hematoxylin-eosin staining,immunohistochemical staining,Sirius red staining,Western blot,q PCR,primary cell extraction,drug inhibition test and CCK-8 were used to determine whether there was a synergistic effect between p53 mutation and mTOR hyperactivation in promoting HCC proliferation and metastasis,and how to play the synergistic effect.4.The downstream substrates activated by p53 mutation with mTOR hyperactivation were identified by RNA-seq.5.The clinical correlation between the above-mentioned substrates and HCC was analyzed in combination with clinical case samples and public database.6.The function of the above substrates in HCC was determined by CCK-8 and Transwell experiments.7.To determine whether mTOR is a therapeutic target for p53 mutation with mTOR hyperactivation of HCC through transgenic mice.Results1.p53(haplo)insufficiency can further activate mTOR activation caused by Tsc1 deletion,thus promoting the proliferation and metastasis of HCC.2.p53(haplo)insufficiency further activates mTOR through the Pten/PI3K/Akt pathway.The simultaneous use of PI3 K and mTOR inhibitors can significantly inhibit the activity and proliferation of mTOR pathway in transgenic mouse liver primary cells with p53 mutation and Tsc1 knockout.3.p53(haplo)insufficiency promotes HCC proliferation and metastasis in conjunction with Tsc1/mTOR activation of downstream molecule ABCC4.4.ABCC4 is significantly correlated with the prognosis of HCC patients with p53 mutation,and ABCC4 is closely correlated with tumor size,differentiation,TNM stage and vascular thrombosis in HCC patients.5.Inhibition of ABCC4 expression can significantly inhibit the proliferation and metastasis of p53 mutant HCC cells.6.mTOR inhibitors can significantly inhibit the proliferation of HCC in transgenic mice with p53 mutation and Tsc1 knockout.Conclusions1.p53 mutation plays a synergistic role with Tsc1/mTOR through Pten/PI3K/Akt/mTOR axis,which jointly activate the downstream substrate ABCC4 and promote the proliferation and metastasis of HCC.2.mTOR may be a clinically effective therapeutic target for p53 mutation with hyperactivation of mTOR.Part Ⅱ: The mechanism of Kras mutation and mTOR hyperactivation promoting hepatocellular carcinoma proliferationand metastasis Purposes1.To determine whether Kras mutation and mTOR activation have synergistic effects in promoting HCC proliferation and metastasis.2.To determine whether Kras mutation can furtherly activate mTOR and its specific molecular mechanism.3.To identify potential therapeutic targets of Kras mutation associated with mTOR hyperactivation of HCC.Methods1.Construction of transgenic mice: construction of transgenic mice with mTOR hyperactivation(Tsc1 knockout).Only heterozygous transgenic mice with Kras G12 D mutations were constructed because the homozygous mice with Kras G12 D mutations could not survive.Transgenic mice with both Kras G12 D mutation and Tsc1 knockout were obtained by interbreeding.2.Construction of HCC model: the transgenic mice were continuously observed for 280 days without any treatment.After the mice were sacrificed for spontaneous tumor formation in the liver,the size and number of liver tumors,the weight ratio of liver and the size and number of lung metastases in the transgenic mice were calculated.3.Hematoxylin-eosin staining,immunohistochemical staining,Sirius scarlet staining and Western staining were used Blot,q PCR,primary cell extraction technology,drug inhibition test,CCK-8 and bioinformatics analysis were used to determine whether Kras mutation and mTOR hyperactivation had synergistic effects in promoting HCC proliferation and metastasis and how to play synergistic effects.4.Bioinformatics analysis,fluorescence color development,flow cytometry and other experiments were used to determine how the mutant Kras played a synergistic role with mTOR in the case of complete loss of Tsc1.5.The downstream substrate molecules activated by Kras mutation with mTOR hyperactivation were identified by RNA-seq.6.The clinical correlation between the above-mentioned substrates and HCC was analyzed by combining clinical case samples and public databases.7.The function of the above substrates in HCC was determined by CCK-8 and Transwell experiments.8.Identify other proteins directly bound to the downstream substrate through immunocoprecipitation and other experiments,and explore the molecular mechanism.9.The effect of the above substrates on HCC proliferation and metastasis in vivo was determined by subcutaneous tumor formation model in nude mice and lung metastasis model injected by caudal vein.10.To determine whether mTOR can be used as a therapeutic target for Kras mutation with mTOR hyperactivation of HCC through in situ implantation tumor model.Results1.Kras mutation can further activate mTOR activation caused by Tsc1 deletion,thus promoting HCC proliferation and metastasis.2.Kras mutation further activates mTOR mainly through the Mek/Erk axis.Simultaneous application of Mek and mTOR inhibitors can significantly inhibit the activity and proliferation of mTOR pathway in primary hepatocytes of transgenic mice with Kras mutation and Tsc1 knockout.3.Kras mutations can lead to ROS accumulation,and high levels of ROS can mediate further activation of mTOR by the Kras/Mek/Erk axis,which is caused by Tsc1 deletion.4.Kras mutation in collaboration with Tsc1/mTOR will activate downstream PEG3 molecules.5.PEG3 is significantly correlated with the prognosis of HCC patients with Mek/Erk/mTOR axis activation,and PEG3 is closely correlated with tumor size and UICC stage in HCC patients.6.Inhibition of PEG3 expression in vivo and in vitro can significantly inhibit the proliferation and metastasis of Kras mutated HCC cells with Tsc1 knockout.7.PEG3 directly binds to STAT3,promoting the phosphorylation of STAT3Tyr705,thus increasing the expression of BEX2,and ultimately promoting the proliferation and metastasis of HCC.8.mTOR inhibitors can significantly inhibit the proliferation and metastasis of Kras mutation with Tsc1 knockout of HCC.Conclusions1.Kras mutation acts synergistically with Tsc1/mTOR through the Mek/Erk/ROS/mTOR axis,and both of them jointly activate the downstream substrate PEG3.PEG3 binds to STAT3 and induces phosphorylated activation of p-STAT3Tyr705,which promotes BEX2 gene expression and ultimately leads to proliferation and metastasis of HCC.2.mTOR may be a clinically effective therapeutic target for Kras mutation with mTOR hyperactivation of HCC.