Fig 1: PA serves as a ligand that activates HNF4A(A) Exogenous expressions of HNF4A2 and HNF4A8 in HLF cells, detected via immunoblotting using a FLAG tag. (B–D) H4 luciferase (B), cell growth assay (C), and mRNA expression level of HNF4A P1, SLCO1B3, ALB, and TTR (D) in HLF cells with persistent expression of HNF4A P1 and P2. (E) Immunoblot analysis of HNF4A in HLF cells with persistent high expression of HNF4A2 (HNF4A2 overexpression) compared with HLF cells generated via an empty vector as a negative control. Cell proliferation of control HLF cells or those overexpressing HNF4A treated with the indicated concentration of PA for 72 h (n = 4). (F) Docking of PA in the X-ray structure of LBD of HNF4A, predicting PA to bind the hydrophobic groove of the LBD in both open (docking score: −11.938 kcal/mol) and closed conformations (docking score: −12.053 kcal/mol). Receptor residues within 4 Å of the PA molecule are shown in line representation. The hydrogen bond/salt bridge to Thr181 and Arg226 is depicted. (G) Quantitation of LA or PA eluted from recombinant HNF4A or RXRA (n = 4 in each group). (H) Quantitation of PA eluted from recombinant HNF4A with wild-type (WT), V255M, E285Q, or I314F mutations (n = 7 in each group). (I) Protein levels of HNF4A measured 72 h after transfection of HLF cells with empty (control), HNF4A2 WT, or HNF4A2 V255M. (J) H4 luciferase activity values 48 h after the addition of 40 and 80 μM of PA to HLF cells overexpressing control, HNF4A2 WT, and HNF4A2 V255M (n = 3 in each group). (K) DR1 WT luciferase activity and DR1 mut-luciferase activity after 48 h of overexpression of control, HNF4A2 WT, and HNF4A2 V255M in HLF cells, followed by the addition of 40 and 80 μM of PA (n = 3 in each group). Data are presented as the mean (SD) (in B–E and G–J); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, as determined using the one-way analysis of variance. LBD, ligand-binding domain; PA, polyprenoic acid; HNF4A, hepatocyte nuclear factor 4 alpha; DN, dysplastic nodule; WT, wild-type; RXR, retinoid X receptor.
Fig 2: HNF4α mediates sensitivity to PA in an in vivo DN model(A) Experimental schedule of LNP-small interfering RNA and PA administration in 32-week-old male PDGF-C Tg mice. (B) Representative magnetic resonance images of PDGF-C Tg livers treated with siRNA control or siRNA Hnf4a before and after treatment with PA. (C) Tumor volumes in the liver before and after PA treatment for siRNA control (n = 5) and siRNA Hnf4a (n = 4). (∗p < 0.05, as determined using paired t test.) (D) Quantitative reverse transcription polymerase chain reaction and immunoblot analyses of PDGF-C Tg mouse liver treated with siRNA Control or siRNA Hnf4a All (n = 3). (E) Immunoblot analysis of livers from PDGF-C Tg mice following siRNA and PA administration according to the schedule shown in (A). (F) Number of mutated genes in liver tumors of mice treated with siRNA control (n = 2) or siRNA Hnf4a All (n = 2) groups. (G and H) Representative photomicrographs of hematoxylin and eosin staining and immunohistochemistry for Ki-67, OATP1, AFP, CD31, and HNF4A in tumors of PDGF-C Tg mice treated with siRNA control and PA (G) siRNA Hnf4a All and PA for 8 weeks (H). Scale bars, 100 μm. (I) mRNA expression levels of Ttr, Slco1a1, Alb, and Afp in liver tumors from PDGF-C Tg mice receiving PA with continuous tail injection of siRNA control (n = 5) or siRNA Hnf4a All (n = 4). Data are presented as the mean (SD). ∗p < 0.05, as determined using the Mann-Whitney U test. PDGF-C, platelet-derived growth factor C; PA, polyprenoic acid; HNF4A, hepatocyte nuclear factor 4 alpha; Tg, transgenic.
Fig 3: Reduced expression of HNF4A in liver tumors abrogates the antitumor effects of PA(A) Experimental timeline of the treatment course of 32-week-old male hPDGF-C Tg/Alb-creERT2/Hnf4a flox/+ (heterozygous) mice, with intraperitoneal administration of 1 mg TAM/mice and oral 80 mg/kg/day. (B) EOB-magnetic resonance images before and after PA oral administration (vehicle n = 9, PA n = 8). Red arrows indicate a liver tumor. (C) Tumor mass measurements before and after PA oral administration. Data are presented as the mean (SD) (∗p < 0.05, as determined using paired t test). (D) Protein levels of HNF4A P1, P2, and ACTB in liver tissues of mice administered vehicle or PA. (E) mRNA levels of Afp, Alb, Slco1a1, and Ttr in liver tumor tissues from vehicle and PA-treated groups. Data are presented as the mean (SD) (n = 8) (∗p < 0.05, as determined using Mann-Whitney U test.). (F) Immunohistochemistry analysis of hematoxylin and eosin, HNF4A, OATP1, and Ki67 in liver tissues from vehicle- and PA-treated groups. Scale bars, 100 μm. PA, polyprenoic acid; HNF4A, hepatocyte nuclear factor 4 alpha; Tg, transgenic.
Fig 4: PA activates HNF4A P1 signaling and restores hepatocyte homeostasis in DNs of PDGF-C transgenic mice(A) Representative pathological images of two DN cases (left) newly added to the two HCC cases (right) among the 70 HNF4A-stained samples reported by Yamashita et al. (Hepatology, Vol. 60, No. 5, 2014). (B) HNF4A P1 and P2 protein levels in 32-week-old DN and 52-week-old HCC tissues. (C) Protein expression of HNF4A P1 and P2 in fetal and adult liver tissue. (D) Total protein levels of HNF4A P1 and P2 in DN tumor tissues treated with vehicle or PA for 8 weeks. (E) Nuclear HNF4A P1 levels in DN tumor tissues following vehicle or PA treatment, with LAMIN A/C used as controls for nuclear fractions. Data are presented for DN + vehicle (n = 7) and DN + PA (n = 6). (F) mRNA expression levels of hepatocyte-maturation-related genes Alb, Ttr, Slco1a1, Atf7, Stat6, Otc1, and Pck1 in DN + vehicle (n = 7), DN + PA (n = 6), HCC + vehicle (n = 8), and HCC + PA (n = 8) tumor tissues. (G) mRNA expression levels of the hepatocyte immaturity markers Afp, Vim, and Sall1 in DN + vehicle (n = 7), DN + PA (n = 6), HCC + vehicle (n = 8), and HCC + PA (n = 8) tumor tissues. (H) Protein expression levels of total ERK1/2, p-ERK1/2 (Thr202/Tyr204), and AKT, p-AKT (Ser473). ACTB was used as a loading control. Data are presented for DN + vehicle (n = 7) and DN + PA (n = 6). Data in Figures 4E–4H are presented as mean ± SD. Statistical significance was assessed using the Mann-Whitney U test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). PDGF-C, platelet-derived growth factor C; Tg, transgenic; HCC, hepatocellular carcinoma; PA, polyprenoic acid; DN, dysplastic nodule; MRI, magnetic resonance imaging; HNF4A, hepatocyte nuclear factor 4 alpha.
Fig 5: Antitumor effect of PA is enhanced by fatty acids(A) Quantitation of HNF4A bound to HBEs in DN-like primary HCC cells with no treatment or treated with LA (20 μM), PA (20 μM), ATRA (20 μM), or 9-cis RA (20 μM) for 72 h using ELISA (n = 3). (B) Quantitation of HNF4A bound to HBEs in DN-like primary HCC cells treated with the indicated concentration of PA for 72 h using ELISA (n = 3). (C) Quantitation of HNF4A bound to HBEs in PA-treated HLF cells transfected with wild-type (WT) or V255M mutant HNF4A (80 μM, 48 h) using ELISA (n = 3). (D) Immunoblotting protein levels of HNF4A, RXRα, RXRα ΔN197, RARα, and ACTB in HCC cell lines Huh7, HLE, and HLF. (D) Quantitative reverse transcription polymerase chain reaction analysis of selected genes in DN-like primary HCC cells treated with siRNA control, siRNA HNF4A, or siRNA RXRα for 48 h (n = 3). (E) Cell proliferation of KH cells transfected with small interfering RNAs treated with PA (40 μM) or ATRA (40 μM) for 96 h (n = 4). (F) mRNA levels of HNF4A P1, P2, ALB, and TTR in DN-like primary HCC cells treated with PA (20 μM) for 1, 3, 6, and 12 h. (G) Protein levels of HNF4A P1 and P2 following treatment with PA (20 μM). (H) H4-Luciferase following 12 h of treatment with PA. (I) Chromatin immunoprecipitation followed by sequencing analysis using an anti-HNF4A antibody in DN-like primary HCC cells treated with 40 μM PA for 12 h, integrated with the RNA sequencing results. (J) Hallmark pathway enrichment analysis of genes activated by HNF4A binding and transcription following treatment with PA. Data are presented as the mean (SD) (in A–B, D–F, and H). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, as determined using one-way analysis of variance. HCC, hepatocellular carcinoma; HBEs, HNF4A-binding elements; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; HNF4A, hepatocyte nuclear factor 4 alpha; TTR, transthyretin.
Supplier Page from OriGene Technologies for HNF 4 alpha (HNF4A) Human shRNA Plasmid Kit (Locus ID 3172)