Fig 2: A proposed working model for aberrant SMAD4-HNF4G in PDAC invasiveness and the effect of Metformin. In PDAC cells where SMAD4 is sufficient, the expression of the downstream oncogene HNF4G that promotes PDAC invasiveness is physiologically inhibited by the SMAD complex. In PDAC cells where SMAD4 is deficient, the expression of HNF4G is over-activated, which evokes cancer cell invasion and metastasis. Metformin may act as a target drug repressing PDAC invasion and metastasis by activating AMPK that induces phosphorylation-ubiquitination coupled HNF4G degradation

Fig 3: HNF4G is an important player in PDAC progression and invasiveness. (A) Scheme of reanalyzing PDAC susceptibility genes using combined GWAS data. (B) High content screening strategy for 36 candidate genes in PDAC cells. Scale bar: 100 µm. (C) Heatmap showing the results of high content screening with a siRNAs library in PDAC cells. (D) The effect of siRNA knockdown of indicated genes on migration ability of PANC-1 cells. Data represent mean ± SEM from 3 experiments. (E) Immunohistochemical (IHC) staining of HNF4G in tissue array consisting of 65 PDAC samples. Left panel, representative IHC images, Scale bar: 700 µm (left images) and 60 µm (right images); right panel, quantification statistic. (F) Scatter dot plots showing HNF4G expression levels in PDAC tumor and normal samples. Data are derived from the Gene Expression Profiling Interactive Analysis (GEPIA). (G) Scatter dot plots showing HNF4G expression levels in early and latter stage of PDAC. Data were derived from the TCGA PDAC dataset. (H) Kaplan-Meier plots of overall survival of patients derived from the TCGA PDAC dataset stratified by HNF4G expression. The best performing threshold is used as a cutoff. HR, hazard ratio; CI, confidence interval. Statistical significance: *, P < 0.05, **, P < 0.01 and ****, P < 0.0001 of Student’s t-test or Wilcoxon rank-sum test. ns, not significant

Fig 4: Metformin treatment improves clinical outcomes of patients with SMAD4-deficient PDAC. (A) Representative images of IHC staining of SMAD4 and HNF4G proteins in PDAC. Scale bar = 100 µm. (B) The correlation between HNF4G and SMAD4 protein levels in PDAC determined by IHC score. SMAD4-, IHC score = 0; SMAD4+, IHC score > 0. HNF4G Low level, scores 0–4; HNF4G high level, scores 6–12. (C) The distribution of patients by PDAC tumor stage and SMAD4 status as function of Metformin treatment. (D) Kaplan-Meier estimate of survival time in 118 patients with PDAC by SMAD4 status and Metformin treatment. Hazard ratio (HR) and 95% confidence interval (CI) were calculated with age, sex, tumor stage as covariates. SMAD4-, IHC score = 0; SMAD4+, IHC score > 0. Statistical significance: *, P < 0.05 and**, P < 0.01 of ?2 test. ns, not significant

Fig 5: Metformin activates AMPK that induces HNF4G phosphorylation-ubiquitination coupled degradation. (A) Effect of Metformin (10 µmol/L) on HNF4G and AMPKa phosphorylation in T3M4 cells. (B) Metformin (10 µmol/L) promoted AMPKa phosphorylation and HNF4G degradation in T3M4 cells. (C) Metformin promoted HNF4G degradation but not inhibited its synthesis in T3M4 cells. Left panel, Metformin treatment substantially decreased the HNF4G levels with time in cells exposed to protein synthesis inhibitor cycloheximide (CHX; 20 µg/mL) compared with cells exposed to vehicle; right panel, Metformin treatment no longer substantially decreased HNF4G level in cells exposed to proteasome inhibitor MG132 (5 µmol/L). (D) Metformin promotes HNF4G ubiquitination. T3M4 cells were treated with or without Metformin (10 µmol/L). Cell lysates were immunoprecipitated (IP) with either control IgG or antibody against HNF4G and analyzed by immunoblotting with a ubiquitin (Ub)-specific antibody. Bottom panels, input from cell lysates. (E) Immunoblot analysis of HNF4G phosphorylation status in T3M4 cells with or without AMPKa knockdown treated with Metformin (10 µmol/L). (F and G) Immunoblot analysis of phosphorylated HNF4G and AMPKa in T3M4 cells, cells with HNF4G knockout or cells with ectopic expression of S382A-mutated HNF4G exposed to Metformin (10 µmol/L) for different times. (H) Metformin treatment significantly decreased the expression levels of some oncogenes in PDAC cells compared with vehicle controls. Results are mean ± SEM from 3 independent determinations and each had triplicate. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 of Student’s t-test