The Interaction And Molecular Mechanism Of Notch And AKT Signaling Pathway In Intrahepatic Cholangiocarcinoma

Aims: Abnormal activation of PI3K/AKT/mTOR signaling pathway can lead to a series of complex diseases,including type 2 diabetes and cancer.Notch gene plays a key role in liver development and is necessary for biliary differentiation.There is evidence that the proportion of abnormally activated AKT and Notch signaling in human intrahepatic cholangiocarcinoma(ICC)is greater than 50%.In addition,animal experiments also have shown that AKT can synergize with other oncogenes to drive the development of ICC,and Notch overexpression can also induce ICC.However,the role and molecular mechanisms of them has not been elucidated in ICC.Importantly,in previous studies,we learned that the AKT-induced hepatocellular carcinoma(HCC)was extremely dependent on the endogenous fatty acids synthesis pathway.Conversely,the AKT/NICD-induced ICC didn’t require endogenous fatty acids.We speculate that the different metabolic pathway of ICC may be the result of Notch acting on AKT signaling which need to be verified.Hence,this study explored the interaction and molecular mechanism of the two signaling pathways and the metabolic pathway in ICC animal models.Methods: In this study,we successfully established an ICC mouse model induced by AKT1/NICD1 viathe laboratory’s unique modeling method,high-pressure tail vein injection of oncogene plasmids.In this model,we first verified whether AKT regulates mTORC1 complex in ICC by knocking out RAPTOR.After knocking,we observed mice conditions and collected liver tissue for immunohistochemistry,western blotting and qRT-PCR to determined related molecule expression.In addition,we also administered ICC mice with the mTORC1 specific inhibitor Rapamycin,and then studyits effects of blocking and therapyof ICCvia immunohistochemistry,western blotting and qRT-PCR.Secondly,in order to study the time node of the ICC fatty acids synthesis pathway,we collected the early tissues of ICC mice for immunohistochemistry and oil red O staining,and detected the uptake of exogenous fatty acids in ICC cells.To further explore the molecular mechanism of Notch inhibition on fatty acids synthesis and the role of AKT in ICC,we performed RNA sequencing in WT,AKT and AKT/NICD mice.Then,we analyzed potential downstream target molecules of Notch and AKT signaling pathways in ICC using statistical methods.Results: First,we found that mTORC1 loss had a significant inhibitory effect on the development of ICC by knocking out RAPTOR.In addition,we detected a low expression of proteins involved in the fatty acids synthesis pathway in tissues,indicating that AKT regulated mTORC1 in ICC but didn’t activate endogenous fatty acids synthesis.Secondly,when we treated ICC mice with Rapamycin at early stage,liver tumor nodules were significantly reduced and tumor development was largely inhibited.However,the therapeutic effect on malignant ICC was very limited when we treated ICC mice with Rapamycin at later stage.Therefore,we treated ICC cells with AKT/mTOR and Notch inhibitors in vitro and found that human ICC cell growth was significantly inhibited.Importantly,the expression levels of proteins involved in the fatty acids synthesis pathway in tissues were still low which consistent with previous results.Next,we demonstrated that the fatty acids synthesis pathway was low expressed at early stage in ICC without lipid droplet accumulation.In addition,we found that ICC cells have strong uptake of exogenous fatty acids compared to HCC cells,and this process depended on fatty acid transporters.Finally,we found that the high expression of cell cycle and proliferation signaling and the low expression of glycolysis synergistically promoted the development of ICC via RNA-sequence,which we will verify later.In summary,this study demonstrated that AKT/mTORC1 and Notch signaling pathways have a driving role in the development of ICC and are expected to become targets for the treatment of ICC,and initially explored the metabolic characteristics of ICC.Conclusion: First,AKT signaling promotes tumorigenesis and development by activating mTORC1 in ICC,but at the same time mTORC1 does not regulate endogenous fatty acid synthesis,and promotes cell proliferation and growth through a kinase pathway to accelerate the development of ICC.Secondly,the metabolic characteristics of ICC do not follow the Warburg effect.The down-regulation of glycolysis pathway expression in ICC makes it impossible for cancer cells to obtain enough raw materials to synthesize endogenous fatty acids to satisfy their own proliferation needs,so they can only rely on the uptake of exogenous fatty acids for life activities.Innovation: This study revealed that the metabolic characteristics of ICC are different from that of HCC,which does not follow the Warburg effect.This may represent an abnormal lipid metabolism pathway in non-classical tumors,and thus become a useful supplement to the existing theory of tumour metabolism.