Animal model of non-alcoholic liver disease and composition of diagnosis, prevention or treatment for non-alcoholic liver disease
11692222 · 2023-07-04
Assignee
Inventors
Cpc classification
C12Q1/6883
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides a method of manufacturing an animal model of non-alcoholic liver disease by using correlation among metabolic dysregulations through AKT regulation by Hippo signaling, and an animal model prepared by the method above, and a screening method of a therapeutic agent by using the animal model.
Claims
1. An animal model of non-alcoholic liver disease in which the Pten and Sav1 genes are deleted specifically in a liver, wherein the non-alcoholic liver disease is selected from the group consisting of non-alcoholic fatty liver, non-alcoholic hepatosteatitis, cirrhosis, and liver cancer.
2. The animal model of non-alcoholic liver disease of claim 1, wherein the animal model is obtained from embryos with accession number of KCTC 13522BP.
3. A method of screening for a therapeutic agent for non-alcoholic liver disease comprising: (i) treating an animal model according to claim 1 with a candidate substance for a therapeutic agent for non-alcoholic liver disease; (ii) measuring the expression level or the activity level of at least one protein selected from the group consisting of YAP, TAZ, IRS2 and AKT proteins in the liver tissue of the animal model treated with the candidate substance; and (iii) determining the candidate substance as the therapeutic agent for liver disease when the expression level or the activity level measured in (ii) is lower than the expression level or the activity level of the same one or more proteins when measured in a control liver tissue of the animal model untreated with the candidate substance.
4. The method of claim 3, wherein the step of measuring the expression level or the activity level of protein is performed by measuring the transcription level of mRNA.
5. The method of claim 4, wherein the measuring transcription level of mRNA is performed by at least one method selected from the group consisting of PCR, reverse transcription PCR (RT-PCR), real-time PCR, RNase protection assay (RPA), microarray, and northern blotting.
6. The method of claim 3, wherein the measuring the expression level or the activity level of protein is performed by at least one method selected from the group consisting of western blotting, radioimmunoassay (RIA), radioimmunodiffusion, ELISA, immunoprecipitation (IP), flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and protein chip.
7. The method of claim 3, wherein the candidate substance comprises a primer, probe, aptamer, antisense oligonucleotide, polymeric compound, protein, peptide, nucleic acid molecule, virus, or antibody.
8. The method of claim 3, wherein the candidate substance inhibits the activity of AKT protein.
9. A method of manufacturing an animal model for non-alcoholic liver disease in which the Pten and Sav1 genes are deleted specifically in a liver, comprising: obtaining a first-generation animal by mating an animal of Pten.sup.f/f genotype and an animal of Albumin-Cre genotype expressing CRE; obtaining a second-generation animal by mating the first-generation animal and an animal of Sav1.sup.f/f genotype; and obtaining a third-generation animal comprising Pten.sup.f/f;Sav1.sup.f/f;Albumin-Cre genotype by mating between the second-generation animals.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
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MODE FOR INVENTION
(60) Hereinafter, the present invention will be described by Preparation Examples and Examples in detail. However, the following Preparation Examples and Examples are intended to illustrate the present invention, and should not be construed as limiting the present invention.
<Preparation Example 1> Generation of Liver-Specific Knockout and Transgenic Mice
(61) (1) Generation of Liver-Specific Knock Mouse and DKO Mouse
(62) Sav1.sup.f/f(PNAS, May 4, 2010. 107 (18) 8248-8253), Pten.sup.f/f(The journal of Clinical Investigation, 2004; 113(12):1774-1783), Yap.sup.f/f(Science Signaling, 2011 Oct. 25; 4(196):ra70), Taz.sup.f/f(PNAS, Aug. 20, 2013. 110(34) 13839-13844), MSTwt.sup.Tg, and MSTkd.sup.Tg(PLoS One. 2009 Nov. 24; 4(11):e8011) mice were generated as previously described in each paper. Mice were crossed as indicated to obtain the desired genotypes.
(63) For example, DKO(PTEN.sup.−/−, Sav1.sup.−/−) mice was obtained from the 2.sup.nd generation mice that obtained by mating of the 1.sup.st generation mice (Pten.sup.f/+;Sav1.sup.f/+;Albumin-Cre and Pten.sup.f/+;Sav1.sup.f/+) that obtained by mating of Pten.sup.f/f;Albumin-Cre mice and Sav1.sup.f/f mice. Only male mice were used for the present invention, and mice with the DKO genotype had 100% liver disease phenotype.
(64) The mice were genotyped by PCR analysis using the primers listed in Table 1 (Mice genotyping).
(65) (2) Generation of Transgenic Mice
(66) Adenovirus for overexpression of CRE, YAP5SA, TAZ4SA, or GFP (control) and AAV-saCas9-sgIrs2 or sgCtl in the mouse liver were injected into the tail vain of 6- and 5-e-old mice, respectively. The mice were deprived of food for 16 hours at day 4 (YAP5SA, TAZ4SA, and GFP) or week 4 (CRE and GFP) after adenovirus injection. MK-2206 (Selleckchem; S1078) was prepared in 30% Captisol (CyDex Pharmaceuticals; RC-0C7) and administered to DKO mice by intraperitoneal injection at a dose of 66 mg/kg every other day for 2 weeks, beginning at 3 or 12 weeks of age.
(67) TABLE-US-00001 TABLE 1 SEQ SEQ ID ID Gene Sense NO Antisense NO RT-qPCR Srebp1a 5′-GGCCGAGATG 1 5′-TTGTTGATGAGCTGG 2 TGCGAACT-3′ AGCATGT-3′ Srebp1c 5′- 3 5′-CAGGAAGGCTTCCAG 4 GGAGCCATGGATT AGAGG-3′ GCACATT-3′ Srebp2 5′-GCGTTCTGGA 5 5′-ACAAAGTTGCTCTGA 6 GACCATGGA-3′ AAACAAATCA-3′ Fasn 5′- 7 5′-GCCATGGTACTTGGC 8 GCCTACACCCAGA CTTG-3′ GCTACCG-3′ Acc1 5′- 9 5′-TTCTGCATTGGCTTT 10 CAACGAGATTTCA AAGGTCT-3′ CTGTGGCT-3′ Scd1 5- 11 5′-TAGCCTGTAAAGATT 12 CCGGAGACCCCTT TCTGCAAACC-3′ AGATCGA-3′ Tnfa 5′-CATCTTCTCA 13 5′-TGGGAGTGACAAGGT 14 AAATTCGAGT-3′ ACAA-3′ I16 5′-TCCACGATTT 15 5′-AGTTGCCTTCTTGGG 16 CCCAGAGAAC-3′ ACTGA-3′ Irs2 5′-CAAGAGCCCT 17 5′-CCGCGGATGCCAGTA 18 GGCGAGTACA-3′ GTG-3′ Acta2 5′-ATGCTCCCAG 19 5′-GTGGTGCCAGATCTT 20 GGCTGTTTTCCCA TTCCATGTCG-3′ T-3′ Desmin 5′-CTAAAGGATG 21 5′-GAAGGTCTGGATAGG 22 AGATGGCCCG-3′ AAGGTTG-3′ Tgfb 5′-CTCCCGTGGC 23 5′-GCCTTAGTTTGGACA 24 TTCTAGTGC-3′ GGATCTG-3′ Hmox1 5′-GCTCGAATGA 25 5′-GTTCCTCTGTCAGCA 26 ACACTCTGG-3′ TCAC-3′ Gadd 153 5′-CTGGAAGCCT 27 5′-CAGGGTCAAGAGTAG 28 GGTATGAGGAT- TGAAGGT-3′ 3′ Luciferase reportor structure TBS1 5′-CTTATCTGGC 29 5′-CACACCCTCGCACAC 30 AGCAGGAAGGAGA ATATCCCTC-3′ G-3′ TBS2 5′-CACCCTTGCA 31 5′-ACCGTGTTCACCCAG 32 CACGTAGAGACGC CACCCGGG-3′ T-3′ TBS3 5′-CCTGGCAGTG 33 5′-CAGCTGCTGCTTCTT 34 TCCCATAGTTGA- TAGGGG-3′ 3′ TBS4 5′-GCAGCAGCTG 35 5′-GCTGCTTTCCTCTCA 36 AAGTGCTAAAGA- TTGCTC-3′ 3′ TBS5 5′-GCCTCTGAGC 37 5′-TATGACCTCCCACCC 38 CAACATCTCTCT- ACTTCA-3′ 3′ TBS6 5′-CATGGCTCGT 39 5′-GAGTACTCAGGCCCA 40 TTCTCCTTCTGG- GGATGC-3′ 3′ ChIP-qPCR TBS1 5′-ATCTATGGTC 41 5′-CCAGAGTTTATCTTA 42 TTCAGAATCACA CAATTTAACCT-3′ C-3′ TBS2 5′-CACAGTTTAC 43 5′-GCTCTGGATGCGTAA 44 ACAAAGGGTAAAG ACAAAACA-3′ CA-3′ TBS4 5′-GCAGCAGTTG 45 5′-ACAATGGGGCAGGGA 46 ATTCCCATCCT- AGGTA-3′ 3′ TBS5 5′-GTAAAACCCA 47 5′-TGCTCTGCTTCTCTC 48 GAAACCCCACTTT ACTAGGA-3′ C-3′ Mice genotyping Sav1 5′-TGGTTTGCTT 49 5′-TGCTGGTTTTGTCTC 50 TTTAGTGGCC-3′ ACTAA-3′ Pten 5′-CTCCTCTACT 51 5′-ACTCCCACCAATGAA 52 CCATTCTTCCC- CAAAC-3′ 3′ Yap1 5′-ACATGTAGGT 53 5′-AGGCTGAGACAGGAG 54 CTGCATGCCAGAG GATCTCTGTGAG-3′ GAGG-3′ Taz 5′-GGCTTGTGAC 55 5′-CCCACAGTTAAATGC 56 AAAGAACCTGGGG TTCTCCCAAGACTGGG- CTATCTGAG-3′ 3′ Cre 5′-GTGTTGCCGC 57 5′-CACCATTGCCCCTGT 58 GCCATCTGC-3′ TTCACTATC-3′ MSTTg 5′-GCTCTAGAGC 59 5′-CCAGGGACCAGATGT 60 CTCTGCTAACCA- CTGC-3′ 3′ Adenovirus-sgIrs2 Ex1-1 5′-CACCGATCGC 61 5′-AAACCCTTGGTGTAG 62 CCTCTACACCAAG AGGGCGATC-3′ G-3′ Ex1-2 5′-CACCGCCGCC 63 5′-AAACGCCGCGGAGGC 64 GCAGCCTCCGCGG TGCGGCGGC-3′ C-3′
<Preparation Example 2> Histology, Immunostaining, and TUNEL Staining
(68) For immunohistochemical staining, 4-μm liver sections on slides were serially rehydrated with xylene and ethanol before heat-induced antigen retrieval (10 mM sodium citrate, pH 6.0; Duchefa). The antigen retrieval step was skipped for slides stained with the anti-IRS2 antibody. Blocking was performed with 0.3% BSA in PBS for the p-AKT-specific antibody and 5% goat serum in 3% BSA including 0.3% Triton-X for all other antibodies. After quenching endogenous peroxidases with hydrogen peroxide (Merck), the samples were incubated with a primary antibody in blocking solution. After washing and incubation with an HRP-conjugated anti-rabbit secondary antibody (Jackson Immunoresearch; 1:500), DAB (Vector Laboratories) was added for antigen detection. Finally, the slides were counterstained with hematoxylin. The antibodies used for immunohistochemical and immunohistofluorescence staining included those specific to Ser473 phosphorylated AKT (p-AKT) (Cell Signaling Technology; 4060); TAZ (Millipore Sigma; HPA007415); YAP (Cell Signaling Technology; 4912); Ki-67 (Abcam; 16667); pan-CK (Dako; Z0622); CK19 (Abcam; 15464); F4/80 (Abcam; 105155); PIPS (Echelon; Z-P345); and IRS2 (Abcam; 84906). For Oil red O staining, cryosections (10-μm thickness) of liver tissue were fixed with cold 10% formalin, dehydrated with 100% propylene glycol (MilliporeSigma; 398039), washed with 85% propylene glycol, and then stained with 0.5% Oil red O (MilliporeSigma; O0625). The sections were counterstained with Mayer's hematoxylin (MilliporeSigma; MHS1). Staining with Picrosirius red was performed with a solution of 0.1% Direct Red (MilliporeSigma; 365548) and 0.1% Fast Green (MilliporeSigma; F7252) in picric acid and subsequently incubated in 0.5% acetic acid. For PAS staining, the sections were incubated with 0.5% periodic acid followed by the Schiff reagent (MilliporSigma; 3952016). TUNEL staining was performed with the In Situ Cell Death Detection Kit (Roche; 11684795910).
<Preparation Example 3> Immunoblot Assay
(69) Liver or AML12 cell lysates were prepared with Proprep Lysis Buffer (Intron Biotechnology) and NETN buffer (20 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 mM EDTA, 0.5% nonidet P-40), respectively. For nuclear/cytoplasmic fractionation analysis, frozen liver tissue was added to lysis buffer (10 mM HEPES [pH 7.8], 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, and protease inhibitors) for cytoplasmic extraction. After grinding the tissue with a hand pestle, the tissues were mixed with 0.3% NP-40 by vortexing for 5 seconds, and the cytoplasmic fraction was obtained from the supernatant after centrifugation. After 2 washes in PBS, the pellet was boiled in sample buffer and used as the nuclear fraction. The primary antibodies for the immunoblot analyses included those specific to p-AKT (catalog 4051 or 4056), AKT2 (catalog 3063), p-GSK3β (catalog 9336), p-S6K (catalog 9205), FAS (catalog 3189), p-ACC (catalog 3661), PTEN (catalog 9559), MST1 (catalog 3682), p-YAP (catalog 4911), YAP (catalog 4912), TAZ (catalog 4883), LATS2 (catalog 5888), p-LATS (catalog 8654), and p-ERK (catalog 4376) (all from Cell Signaling Technology); to IR (catalog 07-724), the p85 subunit of PI3K (catalog 06-195), IRS1 (catalog 06-248), and IRS2 (catalog 06-506) (all from MilliporeSigma); to SREBP1 (catalog 28481), FAS (catalog 196854), ACC (catalog 45174), and GAPDH (catalog 125247) (all from Abcam); to lamin B (catalogs 6217), CTGF (catalog 14939), and CYR61 (catalog 13100) (all from Santa Cruz Biotechnology); to β-actin (catalog A5316; MilliporeSigma); and to α-tubulin (catalog LF-PA0146; Abfrontier). The SAV1-specific antibody was developed in our laboratory. See complete unedited blots in the supplemental material.
<Preparation Example 4> Adenovirus and AAV Preparation
(70) The CRE, human TAZ4SA, and YAP5SA cDNAs were cloned separately into the pAdtrack-CMV-GFP vector. The resulting vectors were then recombined with the pADEasy-1 vector in BJ5183-AD-1 electroporation-competent cells (Agilent Technologies; 240005 and 200157). The recombinant DNA was linearized with Pad and introduced into 293AD cells by transfection with polyethyleneimine (Polyscience; 23966). After checking the cells for GFP expression, we pelleted them with centrifugation, resuspended them with 10% glycerol in PBS, and lysed them with 4 freeze-thaw cycles (LN.sub.2 and a 47° C. water bath) to release their viruses. To amplify the adenoviruses, the present inventors repeated this step with increasing numbers of cells. The adenoviruses were finally purified by ultracentrfifugation at 46,000×g for 2 hours at 4° C. on a discontinuous gradient from 2.2 to 3.0 M CsCl (Amresco) in 10 mM HEPES (MilliporeSigma). The adenovirus-containing layer was removed with a syringe needle, and the viruses were washed twice in a solution containing 10 mM Tris-HCl (pH 8.0) and 2 mM MgCl2 using an Amicon Ultra Centrifugal Filter (MilliporeSigma; UFC810024). Virus titration was performed by counting exposed 293AD or target cells positive for GFP with a fluorescence microscope. A total of 1×10.sup.9 to 1×10.sup.10 PFU were used for tail-vein injections. For the generation of an AAV encoding saCas9-sgRNA against Irs2, we used the pX602-AAV-TBG::NLS-SaCas9-NLS-HA-OLLAS-bGHpA;U6::BsaI-sgRNA vector, which was purchased from Addgene (plasmid 61593). The steps for AAV generation, concentration, and purification were performed as previously described (Nature, v.520, p.186-191). Genomic copies of AAV (2×10.sup.10 to 2×10.sup.11) were used for tail-vein injections into 5-week-old mice that were analyzed 7 weeks later. The oligonucleotide sequences for sgIrs2 are listed in Table 1.
<Preparation Example 5> Generation of Stable Knockdown or Overexpression of Cell Lines
(71) To generate knockdown constructs, the plko.1 vector was digested with EcoRI and AgeI and ligated with annealed oligonucleotides encoding SAV1 or PTEN shRNAs (5′-CCGGCGGCTACATCTCTAGGGAATTCTCGAGAATTCCCTAGAGATGTAGCCGTTT TT-3′ (SEQ ID NO: 65) and 5′-CCGGCAACCGATACTTCTCTCCAAACTCGAGTTTGGAGAGAAGTATCGGTTGTTT TT-3′ (SEQ ID NO: 66), respectively). The shRNA constructs were transfected into 293T cells, together with psPAX2 and pMD2G. After 2 days, viral particles were harvested from the culture media by filtration. The viruses were then used to infect AML12 cells in the presence of polybrene (6 μg/ml) (MilliporeSigma; H9268), and stable cell lines were obtained via antibiotic selection with 10 μg/ml puromycin (Gibco, Thermo Fisher Scientific; A11138-03) or 50 μg/ml hygromycin B (Thermo Fisher Scientific; 1068 7010). The present inventors cloned TAZ4SA, TAZ4SA/S51A, and YAP5SA into pMSCV-puro vector (catalog 631461; Clontech), or purchased IRS2 construct (catalog DU4859; MRC PPU Reagents) for generating AML12 stable cell line expressing those genes, respectively. Next, the resulting constructs were used to prepare recombinant retroviruses for infection and subsequent puromycin selection of infected cells.
<Preparation Example 6> Knockdown of IRS2 with siRNA in AML12 Cells
(72) An AML12 cell line stably expressing shPten and shSav1 was transfected with 20 nM siRNA (ST Pharm Oligo Center) using RNAiMAX (Invitrogen, Thermo Fisher Scientific; 13778-150) according to the manufacturer's instructions. Two days later, the cells were deprived of serum for sixteen hours and then treated with insulin (100 nM). The oligonucleotide sequence information was provided by Calvin J. Kuo (Stanford University School of Medicine, Calif., USA) (Nature Medicine, 2013; 19(10):1331-1337). An AML12 cell line was purchased from ATCC and maintained in DMEM (Gibco, Thermo Fisher Scientific; Ser. No. 12/100,046) containing 10% FBS (Gibco, Thermo Fisher Scientific; Ser. No. 12/483,020), 1% (w/v) penicillin-streptomycin (Gibco, Thermo Fisher Scientific; Ser. No. 15/140,122), 0.005 mg/ml insulin (Gibco, Thermo Fisher Scientific; Ser. No. 12/585,014), 0.005 mg/ml transferrin (MilliporeSigma; T8158), 5 ng/ml selenium (MilliporeSigma; S5261), and 40 ng/ml dexamethasone (MilliporeSigma; D4902) in a humidity-controlled environment (37° C., 5% CO.sub.2). The cell line was confirmed to be mycoplasma free with a Mycoplasma PCR Detection Kit (Intron; 25233).
<Preparation Example 7> Luciferase Assay
(73) The indicated portions of the Irs2 genomic locus, including 6 potential TBSs, were cloned into the pGL3-Basic vector (Promega). Each mutant construct was generated by deletion of specific TBSs. 293T cells were cotransfected with a Renilla plasmid, a TEAD-encoding plasmid, and the constructs of interest. Twenty-four hours later, the cells were harvested, lysed, and assayed with the Dual Luciferase Reporter Assay System (Promega; E1960).
<Preparation Example 8> ChIP-qPCR Analysis
(74) Two days after retrovirus infection, AML12 cells were fixed with 1% (v/v) formaldehyde for 10 minutes and then neutralized with 12 5 mM glycine for 5 minutes at room temperature. The cells were washed with PBS and then lysed with ChIP dilution buffer (50 mM HEPES [pH 7.5], 155 mM NaCl, 1% (v/v) Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA) containing 1% (w/v) SDS. The DNA in the cell lysates was fragmented by sonication using a Bioruptor sonicator. The cell lysates were centrifuged at 20,000×g for 15 minutes at 4° C., and the resulting supernatants were further diluted with ChIP dilution buffer. The supernatants were then incubated overnight at 4° C. with either the TAZ antibody (MilliporeSigma; HPA007415) or IgG (Santa Cruz Biotechnology). The next day, protein A/G beads (Gendepot) were added, and the samples were incubated for an additional 3 hours at 4° C. The beads were then isolated with centrifugation, washed with ChIP wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA), and suspended in SDS lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS) for overnight incubation at 65° C. The beads were then removed, and the remaining material was incubated for 2 hours at 55° C. with proteinase K (20 mg/ml) and glycogen (20 mg/ml). After a final 1-hour incubation with RNaseA at 37° C., the DNA was purified using standard procedures and analyzed by qPCR using the primers listed in Table 1.
<Preparation Example 9> Gene Expression Profiling and GSEA
(75) Total RNA was extracted from livers with Ribo-EX (GeneAll) and subjected to microarray analysis with MouseRef-8, version 2.0 BeadChip (Illumina). To identify differentially expressed genes, the raw values from all groups of mice were normalized to the mean values for each gene set and log 2 transformed. For the heatmap, the values of 25,697 genes were rank-ordered by their average value for the Pten.sup.−/− Sav1.sup.−/− samples, and 500 high-rank and 500 low-rank values were selected. The Multi-Experiment Viewer program was used to generate the heatmap, and representative genes from the GO_Insulin receptor signaling pathway, KEGG_Insulin signaling pathway, GO_Glucose homeostasis, Reactom_Metabolism of carbohydrates, or Cordenonsi_YAP conserved signature gene clusters were selected from the Broad Institute's Molecular Signatures Database. GSEA was performed using the GenePattern tool from the Broad Institute, with version 5.2 of the Molecular Signature Database libraryies. Pten.sup.−/− Sav1.sup.−/− mice were compared with Pten.sup.−/-mice, and representative upregulated genes with a nominal P value of less than 0.05 and a FDR q of less than 0.25 are presented.
<Preparation Example 10> Statistics
(76) Because of individual variation between mice, we used at least 3 to 5 mice per group for all experiments (e.g., immunoblot analyses, qPCR, H&E staining, Oil red O staining, Picrosirius red staining, IHC, etc.). We used only 2 mice per group for the microarray analyses. Group sizes were determined in accordance with previous studies, and no mice were excluded. For the comparison of liver specific mutant mice with WT mice, we used WT littermates for all experiments. We did not randomize the mouse experiments. The H&E staining experiments and microarray analyses were conducted by researchers who were blinded to the mouse genotypes. All data met the assumptions of each statistical test, and the variations between groups were similar. All in vitro experiments were replicated at least 3 times, except for the luciferase assay, which was replicated 5 times. Data are presented as the mean±SEM and analyzed using 1-way ANOVA followed by Tukey's multiple comparisons test, a 2-tailed Student's t test, or a χ2 test, as appropriate. Statistical analyses were performed with GraphPad Prism 7 (GraphPad Software). P values of less than 0.05 were considered statistically significant. All graphs were generated with GraphPad Prism 7.
Example 1: Deletion of Both Pten and Say 1 in the Liver Promotes NAFLD, NASH, and Tumorigenesis
(77) To investigate any potential crosstalk between the Hippo and AKT pathways in vivo, the present inventors first generated liver-specific PTEN and SAV1 double-knockout mice (Pten.sup.−/− Sav1.sup.−/−, referred to herein as DKO mice) (
(78) The livers from 5-month-old mice of each genotype are shown in
(79) Given that Pten−/− liver cancer proceeds through NAFLD and NASH, the present inventors examined young DKO mice for phenotypic changes occurring prior to tumor development. The results of tissue staining for each age of indicated genotypes are shown in
(80) Acute deletion of Pten and Sav1 in the adult stage using a CRE-encoding adenovirus also consistently led to the development of NAFLD (
(81) The liver-to-body weight ratio for mice atg 2 and 4 months of age (left), the liver enzymes (AST and ALT) in the serum of the indicated genotypes (right top), and the graph of quantification of apoptotic cells and macrophages following the TUNEL and F4/80 staining (right bottom) are shown in
Example 2: Increased AKT Signaling Accelerates the Development of Fatty Liver in Pten.SUP.−/−.;Sav1.SUP.−/− Mice
(82) The present inventors performed gene expression profiling on the DKO mice to explore the mechanism underlying their early development of NAFLD. A heatmap of differentially expressed genes in the livers of 3-month-old mice as revealed by microarray analysis is shown in
(83) TABLE-US-00002 TABLE 2 Upregulated genes in Pten.sup.−/−; Sav1.sup.−/− ES NOM (P value) FDR (q value) Collection TNFA_SIGNALING_VIA_NFKB 2.72 0 0 Hallmark ACYL_COA_METABOLIC_PROCESS 2.49 0 0.029 C5.BP:GO STEROID_BIOSYNTHETIC_PROCESS 2.43 0 0.039 C5.BP:GO GUO_TARGETS_OF_IRS1_AND_IRS2 2.37 0 0.015 C2.CGP LIPID_BIOSYNTHETIC_PROCESS 2.34 0 0.058 C5.BP:GO REGULATION_OF_CELL_GROWTH 2.29 0.004 0.069 C5.BP:GO REGULATION_OF_TOR_SIGNALING 2.29 0 0.067 C5.BP:GO AKT_UP.V1_UP 2.26 0.006 0.042 C6:OS FATTY_ACID_METABOLIC_PROCESS 2.09 0.002 0.119 C5.BP:GO LIPID_HOMEOSTASIS 2.00 0.010 0.165 C5.BP:GO PHOSPHATIDYLINOSITOL_3_PHOSPHATE_BINDING 1.85 0.023 0.244 C5.MF:GO CHOLESTEROL_HOMEOSTASIS 1.82 0.013 0.035 Hallmark IL6_JAK_STAT3_SIGNALING 1.69 0.029 0.060 Hallmark CORDENONSI_YAP_CONSERVED_SIGNATURE 1.58 0.042 0.243 C6:OS
(84) Based on the above results, the present inventors studied whether AKT acts as important factor in dysregulation of liver metabolism found in DKO mice.
(85) The immunoblot analysis result of AKT signaling components and lipogenesis-related proteins in the livers of 3-month-old mice is shown in
(86) In
(87) The result of qPCR analysis of relative mRNA levels for lipogenesis- or inflammation-related genes in the livers of 3-month-old mice is shown in
Example 3: YAP/TAZ Enhance AKT Activation and the Development of Fatty Liver in the Absence of PTEN
(88) To identify the mechanism underlying the enhanced AKT activation in DKO livers, the status of the various hippo pathway components were observed. The result of immunoblot analysis of Hippo pathway components in livers from 3-month-old mice of each genotype is shown in
(89) Consistent with the role of SAV1 as an upstream regulator of LATS, SAV1-deficient livers (from both Sav1−/− and DKO mice) showed reduced LATS activation (pLATS) that was also associated with increased levels of YAP but low levels of p-YAP (
(90)
(91) The result of quantification of YAP/TAZ localization from
(92) The increased expression of YAP/TAZ targets CTGF and CYR61 provided further confirmation of the upregulation of YAP/TAZ activity in DKO livers (
(93) To determine whether YAP and TAZ directly promote fatty liver development via AKT activation, the present inventors used an adenovirus to induce overexpression in the liver of active TAZ (TAZ4SA) or a version of YAP (YAP5SA) that cannot be inhibited by LATS. In WT mice, it was found that liver size and morphology remained unaffected 4 days after injection of the viruses inducing the expression of TAZ4SA or YAP5SA (
(94) On the other hand, in Pten−/− mice under the same experimental conditions, the expression of TAZ4SA or YAP5SA induced hepatomegaly, promoted the development of NAFLD without fibrosis or inflammation (
Example 4: Transcriptional Regulation of Irs2 by YAP/TAZ
(95) The next addressed question was how YAP and TAZ potentiate AKT activity in Pten.sup.−/− livers. The result of immunoblot analysis of insulin signaling molecules in the livers of 3-month-old mice is shown in
(96) In addition, the increase in Irs2 mRNA observed in DKO mice was more significant than the increase observed in Sav1.sup.−/− or Pten.sup.−/− mice (
(97) Next, the present inventors used the normal mouse hepatocyte cell line AML12 to further clarify the molecular relationship between YAP/TAZ and IRS2 in vitro. The result of IRS2 immunoblot analysis of when Pten and/or Sav1 expression are suppressed by infecting the cells with lentiviruses encoding shPten and/or shSav1 is shown in
(98) In
(99)
(100) Since the transcription factor TEAD binds YAP/TAZ for target gene transcription, it was asked whether YAP/TAZ can directly regulate Irs2 expression through TEAD. The result of immunoblot assay of cells that TAZ4SA or TAZ4SA/S51A was overexpressed, and in control (CTL) is shown in
(101)
(102) The result of luciferase reporter analysis for expression pattern of Luc in
(103)
Example 5: Deletion of Yap/Taz or Activation of Hippo Signaling Attenuates Fatty Liver Development by Downregulation of IRS2
(104) To confirm the role of YAP/TAZ in the DKO mouse phenotype, the DKO mice that also carried conditional Yap and/or Taz alleles were generated. A schematic diagram of method to generate Pten.sup.−/−;Sav1.sup.−/−;Yap.sup.−/−;Taz.sup.−/− mice is shown in A panel of
(105) The result of H&E and Oil red O staining of livers from 1-month-old mice is shown in
(106) QKO mice also had a reduced abundance of IRS2, p-AKT, p-GSK3β, and FAS (FIG. 5B), and less upregulation of Irs2 mRNA than did DKO mice (
(107) Liver-to-body weight ratio (B), Sirius red and CK19 immunohistochemical staining of the liver (C), and isolated serum color (D) for 1-month-old mice of the indicated genotypes are shown in
(108) Next, to determine whether the components of the Hippo pathway upstream of YAP/TAZ inhibit AKT signaling, we generated Pten−/− mice expressing transgenes encoding either a WT (MST-WT.sup.Tg) or kinase-dead mutant (MSTkd.sup.Tg) form of human MST1, which is a binding partner of SAV1 and an activator of the Hippo pathway.
(109) The mating method of above is shown in the A panel of
(110) Pten−/− MSTWTTg livers, but not Pten−/− MSTkdTg livers, were smaller and had less lipid droplet accumulation than did Pten−/− livers (
(111) Together, these data indicate that enhanced Hippo pathway activity inhibits AKT signaling, probably by inhibiting YAP/TAZ-mediated regulation of IRS2 and, consequently, attenuates the development of NAFLD.
Example 6: YAP/TAZ and IRS2/p-AKT are Positively Correlated in HCC Patients' Specimens
(112) To extend the results in mice to humans, it was examined that the hepatic expression of YAP, TAZ and IRS2 in HCC patients' samples. Scatter plots of log.sub.e (mRNA abundance) values for IRS2 versus TAZ or YAP1 in tissue specimens from each patients are shown in
(113) In addition, scatter plots of log 2 (mRNA abundance) values for IRS2 versus CTGF or CYR61 in the samples of
(114) The IHC result of TAZ and IRS2 of HCC patient sample is shown in
(115) To extend the result of NAFLD-HCC mice to the patients' tissue, the samples of HCC from NAFLD or from non-NAFLD were obtained and expression level was detected. The result images and a quantificated graph are shown in
(116) The comparison of IHC intensities of TAZ, YAP, IRS2, and pAKT(S473) protein levels between NAFLD-associated HCC and non-NAFLD HCC is shown in
Example 7: Inhibition of AKT Signaling of Silencing of IRS2 Attenuates the Development of Liver Cancer
(117) Given the lack of treatments for NASH, the present inventors next asked whether pharmacological inhibition of AKT could ameliorate fatty liver and slow cancer progression in DKO mice. To this end, the present inventors administered the pan-AKT inhibitor MK-2206 (phase II clinical trials) intraperitoneally to 3-week-old DKO mice. The schematic image of specific experimental method is shown in
(118) Macroscopic appearance of the liver (middle) as well as H&E and Oil red O staining (bottom) are shown in
(119) Further, to investigate the suppression of liver cancer, MK-2206 was injected to 12-week-old DKO mice that 80% of them had liver tumor (
(120) These effects were also accompanied by reduced expression of genes related to fibrosis (i.e., Acta2, desmin, and Tgfb), cell death or injury (i.e., Hmox1 and Gadd153), and inflammation (i.e., Tnfa) (
(121) To clarify the role that IRS2 plays in the development of NAFLD and cancer in DKO mice in vivo, we generated an adeno-associated virus (AAV) encoding Staphylococcus aureus Cas9 (saCas9) and single-guide RNA (sgRNA) against Irs2 (sgIrs2) to abrogate IRS2 expression in the DKO mouse liver. The analysis results after injection of AAV virus encoding Cas9 and an sgRNA against Irs2 (sgIrs2) or an sgCtl into 5-week-old DKO mice are shown in
(122) Specifically,