The Effect And Mechanism Of Hepatocyte-specific β-catenin Deletion On Hepatocarcinogenesis

BackgroundPrimary liver cancer is one of the most common malignant diseases and has been escalated to be the second leading cause for cancer-related mortality in the world.Hepatocellular carcinoma（HCC）accounts for approximately 80%of primary liver cancers,and the multi-step development of HCC is likely driven by both hepatocyte-intrinsic and hepatic microenvironmental factors,which remain to be elucidated.Currently,tumor resection and liver transplantation are curative treatments for early-stage patients suffering from HCC.However,many patients are diagnosed in advanced stages and as the only approved drug available for the treatment of unresectable HCC,Sorafenib only extended survival from 7.9 months to 10.7 months.Therefore,the most urgent need is elucidate the molecular mechanisms underlying the development of HCC if we want to develop more efficient therapies.β-catenin is a central player in the Wnt signaling pathway,and mutations in CTNNB1were detected in 20%–40%of HCC patients.Indeed,CTNNB1,c-Met and telomerase are on top of the most frequently mutated gene list identified in large scale genome-wide screening of human HCC specimens.In mouse models,mutations in CTNNB1 and altered expression were detected in approximately 25%of HCCs.Consistently,transgenic mice expressing a dominant active mutant ofβ-catenin exhibited increased susceptibility to HCC development induced by chemical carcinogen,diethylnitrosamine（DEN）.However,it was also reported that removingβ-catenin specifically in hepatocytes resulted in elevated susceptibility to DEN-induced HCC development in mice.A more recent study also demonstrated activating Wnt/β-catenin signaling could suppress HCC formation by inhibiting the positive feedback loop between YAP/TAZ and Notch signaling.Together,these studies revealed previously unrecognized complexity in the pathogenesis of liver cancer development,which remains to be elucidated.Recently,researchers developed a new technology named as hydrodynamic transfection to build mouse model for HCC investigation.Co-activation of c-Met andβ-catenin often occurs in human HCC samples.Co-delivery of c-Met（MET）and constitutively activeβ-catenin（ΔN90-β-catenin,CAT）into mouse livers using the Sleeping Beauty transposon system by hydrodynamic transfection also efficiently and rapidly induces primary hepatic tumors.Hence,this method gives us a chance to study the genetic interactions in hepatocarcinogenesis via combination with traditional transgenic mouse models.ObjectiveTo specifically determine a putative effect on HCC development of the hepatic microenvironement induced byβ-catenin deficiency in hepatocytes,we used hepatocyte-specificβ-catenin knockout mice(β-cat^Δhep,β-cat KO)and induced tumor development via MET/CAT plasmids hydrodynamic injection.We investigated the effect and mechanism of hepatocyte-specificβ-catenin deletion on hepatocarcinogenesis by molecular biological and bioinformatic methods,which may provide a promising therapeutic target of HCC.Method1.Mice and treatment.（1）We breededβ-cat^Δhep mice.Firstly,β-cat^flox/flox mice from C57BL/6 strain were bred with Albumin-Cre⁺transgenic mice to generateβ-cat^flox/+,Albumin-Cre⁺mice.Thenβ-cat^flox/flox mice were bred withβ-cat^flox/+,Alb-Cre⁺mice to generate hepatocyte-specificβ-cat KO mice(β-cat^flox/flox,Alb-Cre⁺mice).Wild type mice were used as control mice（WT）.（2）We established MET/CAT-induced HCC model.We selected male WT andβ-cat^Δhep mice and separated them into two groups.The first group contained 8-12 mice in each genotype.They were sacrificed at 8 weeks old（0 day）.The second group contained16-18 mice in each genotype.20.8μg of total plasmids,encoding the Sleeping Beauty transposase（HSB2）and transposons with oncogenes MET/CAT（10μg pT3-EF1α-c-Met（human）+10μg pT3-EF1α-ΔN90-β-catenin（human）+0.8μg HSB2）,were injected hydrodynamically into these mice at 8 weeks old.The injection procedure was completed in 7 seconds.Mice were maintained on the standard diet and sacrificed at 3 days or 7weeks after MET/CAT injection.Liver,spleen,blood and tail samples were collected.The genotypes of mice were checked by genotyping and Western blot using tail and liver samples.2.The role ofβ-cat KO on liver and hepatocarcinogenesis.（1）We observed the influence ofβ-cat KO on liver development.The liver weight,body weight and liver weight/body weight ratio（LW/BW）were measured.The histology of livers was detected by H&E staining.The proliferation was checked by Ki67immunofluorescence（IF）staining and the apoptosis was checked by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end labeling（TUNEL）staining.（2）We explored the impact ofβ-cat KO on hepatocarcinogenesis.The liver weight,body weight and liver tumor loads of each mouse at 3 days or 7 weeks after injection were measured.The pathological characteristics of tumors were analyzed by H&E staining.Ki67 IF staining and TUNEL staining were also detected.We also detected the expression levels ofγ-catenin,E-cadherin and N-cadherin by Western blot to investigate the involvement ofβ-cat KO in adherens junctions（AJs）.3.We sought the impact ofβ-cat KO on gene expression profile.We detected liver samples by RNA-seq analysis.Then we used GSEA and IPA software to find related pathways and upstream or downstream regulators.4.We investigated the impact ofβ-cat KO on some HCC-related signaling pathways.IF staining was performed to check the delivery of MET and CAT plasmids after three days of injection.Western blot was performed to explore the classical molecules of Wnt/β-catenin,MAPK/ERK and PI3K/Akt pathways.Western blot,Real-time PCR and IHC were performed to detect the transcriptional factor Sox9.5.We detected the influence ofβ-cat KO on topical inflammation.The distribution status of F4/80,Ly6G,B220 and CD3 was measured by immunohistochemical（IHC）or IF staining.The expression levels of IL-12,TGF-β1,TNF-α,IL-1βand IL-6 were measured by Real-time PCR.Results1.Ablatingβ-catenin slightly tempered liver development.The liver weight and LW/BW was lower inβ-cat^Δhep mice compared to WT mice at 8 weeks old.But there was no difference in H&E,Ki67 or TUNEL staining,suggesting thatβ-cat KO did not affect histology,proliferation or apoptosis in mouse liver.2.β-catenin loss in hepatocytes promoted MET/CAT-induced hepatocarcinogenesis.After 7 weeks of hydrodynamic injection,the tumor numbers,maxim tumor diameter and LW/BW ofβ-cat^Δhep mice were much higher than those of control mice.H&E staining further demonstrated lower differentiation cells and more disordered structures in the liver ofβ-cat^Δhep mice.TUNEL staining showed much higher apoptosis in tumors inβ-cat^Δhep mice.In 8 week-oldβ-cat KO mice,protein level ofγ-catenin was increased compared to WT mice,while E-cadherin protein expression was lower by western blot assays.After 3days of MET/CAT hydrodynamic injection,γ-catenin protein expression was decreased while E-cadherin protein level was enhanced.It seems thatβ-catenin deletion did not influence adherens junctions.3.RNA sequencing analysis revealed several potential mechanisms.Extracellular matrix and immune response related pathway inβ-cat^Δhep mice at 8 week-old increased.At3 days after injection,E2F1 downstream and cell cycle targets were found higher in WT mice.At 7 weeks after injection,inflammation related pathway,hypoxia related pathway,HIF1αtranscription network,c-Jun downstream targets,DNA damage positive regulation dataset and lymphocyte apoptosis negative regulation dataset genes are identified in KO mice.Furthermore,expression levels of Sox9,IL-6 and genes regulated by IL-6 signaling via Stat3 were higher inβ-cat^Δhep at three different time points.4.MET/CAT expression promoted multiple proliferative signaling pathways inβ-catenin-deficient liver such as Wnt/β-catenin,MAPK/ERK and PI3K/Akt pathways and transcriptional factors Cyclin D1 and Sox9.5.β-catenin deficiency induced a tumor-promoting hepatic inflammatory microenvironment.Immunostaining assays implied more F4/80 and CD3 positive cells inβ-cat KO mice at 3 days after injection and more Ly6G and B220 positive cells at 7 weeks after injection.The gene expression levels of TGF-β1 and IL-6 inβ-cat KO mice were higher at 8 weeks old.At 3 days after injection,there were significantly higher expressions of IL-12,TGF-β1,TNF-α,IL-1βand IL-6 in theβ-cat KO mice.IL-12,IL-1βand IL-6gene expression levels were still higher in theβ-cat KO mice at 7 weeks after injection.ConclusionWe observed hepatocyte-specificβ-catenin knockout promoted MET/CAT-induced hepatocarcinogenesis.The tumor-promoting effect was associated with more severe inflammation,increased activation of Wnt/β-catenin,MAPK/ERK and PI3K/Akt pathways,overexpression of transcriptional factors Cyclin D1 and Sox9,and increased tumor related positive regulation genes and pathways.These observations thus suggest thatβ-catenin removal creates a complexly hepatic microenvironment that is conducive to cancer initiation and development.