Central Role And Mechanism Study Of MTORC1 Downstream Of YAP/TAZ In Hepatoblastoma Development

Objective:Hepatoblastoma(HB)is a rare liver malignant tumor,although it seldom occurs in adults,it has a relatively high incidence in children.It is one of the most common hepatic malignancies in children.HB usually occurs in the patients younger than 3 years old,although it can also be presented in infancy or adolescence.There is a tight link between age and prognosis in HB,usually younger patients,such as younger than 3 years old,may achieve a relatively good prognosis than that of the older patients.Treatment of HB is a great success in the past forty years,the performance of adjuvant and neoadjuvant chemotherapy had brought a survival rate from 30%to 70-80%.This process was not only achieved by advanced drug therapy,but also relied on the new surgical procedures(such as liver transplantation,ultrasonic dissection),a more accurate disease risk stratification and multicenter cooperation.To date,chemotherapy and thoroughly tumor resection are still the mainstream ways for the treatment of HB.Though they have greatly improved the survival rate overall,there are lots of limitations to the late stage patients,such as patients associated with extrahepatic tumor metastasis,PRETEXT IV stage or patients with extremely AFP concentration.Most of these patients can only achive an average survival rate of 20-30%.It eagerly calls for an individualized and novel targeted therapy.To achieve this goal,a better characterization of the molecular genetics and signaling pathways underlying HB pathogenesis is imperative.Gene alteration and chromatin instability commonly related to tumorigenesis.Wnt/p-catenin signaling is a driver pathway in the process of HB formation.CTNNB1(which coding ?-catenin),APC and Axin gene mutations can be detected in over 80%of HB patients,which indicated the important role of Wnt/?-catenin cascade in the process of HB formation.However,overexpression either full length,N terminal deletion(AN90)or point mutation(S45Y)of ?-catenin alone is not sufficient to induce liver tumorigenesis in mice.It showed that besides Wnt/p-catenin signaling,there might be some other pathways involved in the process of HB formation,these pathways synergized with activated ?-catenin to generate tumorigenesis.Hippo signaling is a well-known signaling which played a wide role in regulating organ size and maintaining homeostasis.Yes-associated protein(YAP)and transcriptional coactivator with PDZ-binding motif(TAZ)are two major downstream effectors of the Hippo pathway,they function via regulating gene transcription of downstream factors(such as CTGF,CYR61).Previously we found that co-activation of YAP and ?-catenin triggering HB formation in mice,however,the mechanism is not clear yet.A study revealed that Hippo/YAP pathway induced tumorigenesis by activating mammalian target of rapamycin(mTOR)signaling.mTORC1 pathway is a central regulator of tissue growth and homeostasis.Deregulation of the mTORC1 pathway is frequently found in human cancers(including hepatocellular carcinoma and intrahepatic cholangiocarcinoma),obesity and diabetes.Targeting the mTOR signaling has been considered a promising strategy for the treatment of HCC.However,studies on the functional contribution of the mTOR pathway to HB development are lacking.Therefore,we plan to perform a series of in-vitro and in-vivo experiments to study the function and detailed mechanism of mTORC1 in the process of YAP and ?-catenin induced HB formation,hoping to find some novel therapeutic targets for the treatment of HB.Methods:1.Co-transfecting YAPS127A and ?-cateninAN90 by sleeping beauty transposon mediated hydrodynamic tail injection to generate HB formation in mice.2.Western blotting was used to detect the levels of YAP/TAZ,P-catenin and mTORCI signaling in HB cell lines(HepG2,Hep293TT)and tumor tissues of HB mice.3.In vitro,MLN0128(the second generation of mTOR inhibitor)was performed in HepG2 and Hep293TT.48 hours later,we measured the IC50 of MLN0128 by crystal violet staining.Furthermore,we detected relative cell proliferation and cell apoptosis after various concentrations(0,25,50,100,200 and 400nM)or various time points(0,2,8,24 hours)of MLN0128 treatment.Molecularly,levels of mTORC1 signaling and apoptosis related proteins were further validated by western blotting.4.In vivo,because Raptor is the unique and functional part of mTORC1,ablation of Raptor is sufficient to block mTORC1.Using Cre/loxP mediated Raptor gene knockout technology in Raptorf~flfl mice,we studied the function of mTORCl in YAP/?-catenin induced HB mice model.pT3-Cre(experimental group)or pT3(control group),co-transfected YAP and P-catenin,were injected into mice.We observed the differences of liver tumor formation between two groups,mice were monitored and sacrificed in certain time points,liver tissues were collected and analyzed respectively for H&E and Ki67 staining.5.Next,to explore the crosstalk of mTORC1 signaling and YAP in HB formation,first we compared the levels of SLC38A1,SLC7A6,SLC7A5 and SLC1A5 in wild type(WT)and YAP/?-catenin induced HB tissues by qRT-PCR.Then amino acid deprivation assay were performed in HepG2 and Hep293TT cells to validate the central role of amino acid to mTORC1 signaling.Cells were treated with amino acid deprivation cell culture medium and collected after various time points(0,1,2,4,6 hours),protein levels of mTORC1 signaling were analyzed by western blotting.6.For gene silencing studies,cells were plated in 6-well plates and transfected with 30 pmol small interfering(siRNA)targeting YAP,TAZ,either alone or in combination.A scramble siRNA was used as negative control RNA.48-72h post transfection,cells were collected for protein and RNA analysis.ChIP-PCR was then performed to detect if YAP functioned by directly binding to the promoter region of SLC38A1.Meanwhile,we silenced P-catenin in these two cell lines,and analyzed the levels of SLC38A1 by western blotting and qRT-PCR.7.We assessed the levels of p-4EBP1 a surrogate marker of mTORC1 activation,and SLC38A1 in a collection of human HB specimens(n=28)by immunohistochemistry(IHC).Results:1.Co-transfecting YAPS127A and ?-cateninAN90 was sufficient to induce HB formation in mice.Histologically,the tumor resembled as human HB fetal or crowed fetal subtype.2.Western blotting and IHC results revealed that levels of YAP and TAZ were upregulated in HB cell lines and HB tumor tissues of mice.Meanwhile mTORC1 signaling was activated.3.MLN0128,which effectively blocking the mTORCl activity,dramatically decreased HB cell proliferation and increased cell apoptosis in a dose and time dependent manner.Consistently,pro-survival proteins were decreased whereas apoptosis proteins were upregulated respectively.4.In vivo,we performed Cre/LoxP mediated Raptor knockout by hydrodynamic tranfection in Raptor~flfl mice,ablation of Raptor strongly inhibited YAP/?-catenin-induced HB tumorigenesis in mice,which was represented by prolonged tumor formation and reduced liver weight.5.qRT-PCR results showed that levels of SLC38A1 and SLC7A6 were upregulated in HB liver tissues when compared with that of WT liver tissues,while there were no significant differences in levels of SLC7A5 and SLC1A5.6.We found that deprivation of amino acids strongly inhibited Hep293TT and HepG2 cell growth,and it led to the reduction of mTORC1 activation,which was indicated by decreased levels of p-mTOR,p-4EBP1,p-S6K and p-RPS6.And the inhibition can be detected as early as 2 hours after amino acids deprivation.7.YAP or TAZ silenced alone in HepG2 and Hep293TT cells was not sufficient to suppress mTORC1 activity,when YAP and TAZ were concomitantly silenced,p-RPS6 levels were significantly decreased.Furthermore,levels of CTGF,CYR61,and SLC38A1 downregulated correspondingly.Finally,using ChIP-PCR,we found that YAP functioned by binding to the promoter region of SLC38A1.While there was no significant changes in SLC38A1 mRNA and protein levels upon?-catenin silencing in HepG2 and Hep293TT cells.8.Among 28 cases of human HB species,strong immunoreactivity for p-4EBP1 and SLC38A1 proteins were detected in most human tumor tissues when compared with corresponding non-tumorous surrounding liver tissues.Conclusion:YAP and its paralog TAZ regulate the expression of the amino acid transporter SLC38A1,thus leading to mTORC1 activation and hepatoblastoma formation.