Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: 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

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: High Content Screening, Knockdown, Migration, Immunohistochemical staining, Immunohistochemistry, Expressing, Derivative Assay, Gene Expression

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: HNF4G upregulation is caused by SMAD4 deficiency in PDAC. (A) HNF4G mRNA levels in PDAC as function of SMAD4 copy-number variation. Data were derived from the TCGA database. (B) The relationship between HNF4G and SMAD4 protein levels in PDAC determined by IHC staining. Left panel shows representative IHC images of HNF4G and SMAD4 in serial sections of PDAC tissue array ( n = 185). Scale bar in left images = 600 μm. Scale bar in right images = 200 μm. Right panel shows HNF4G levels as function of SMAD4 levels both expressed as IHC scores: low, 0; medium, 1–4; and high, 6–12. (C) The expression levels of HNF4G RNA (left) and protein (right) in 4 PDAC cell lines with SMAD4 deficiency and 3 cell lines without SMAD4 deficiency. Data represent mean ± SEM from 3 independent determinations and each had triplicates. (D and E) The effects of SMAD4 knockdown (D) or overexpression (E) on HNF4G RNA (upper panel) and protein levels (lower panel) in PDAC cells. Data are mean ± SEM from 3 independent determinations and each had triplicates. (F and G) Relative expression levels of reporter gene bearing the HNF4G promoter region in T3M4 cells with or without SMAD4 overexpression (F) and in MIAPaCa-2 cells with or without SMAD4 knockdown (G). (H) Relative expression levels of reporter gene bearing the mutated HNF4G promoter region in PDAC cells. Each promoter harbors a mutated SBE. Mutation in SBE 1 had the most significant impact on reporter gene expression compared with vector control and the mutation in other SBEs. Results are mean ± SEM from 3 experiments and each had 6 replicates. (I and J) Chromatin immunoprecipitation assays showing binding of SMAD4 to HNF4G promoter region SBE 1 in PDAC cells (I) and knockdown of SMAD4 expression in these cells substantially decreased the binding (J). Fold enrichment represents DNA levels associated with HNF4G or IgG (as control) relative to an input control from 3 independent experiments. Data are mean ± SEM of 3 experiments. Statistical significance: *, P < 0.05, **, P < 0.01, ***, P < 0.001 and ****, P < 0.0001 of Student’s t -test, χ 2 test or Wilcoxon rank-sum test. ns, not significant

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Derivative Assay, Immunohistochemistry, Expressing, Knockdown, Over Expression, Mutagenesis, Gene Expression, Plasmid Preparation, Control, Chromatin Immunoprecipitation, Binding Assay

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: HNF4G overexpression promotes PDAC cell invasiveness and activates the cell-cell junction pathway. (A) HNF4G overexpression promoted migration and invasion of PDAC cells in vitro . Left panel shows representative images of transwell assays and right panel shows quantification statistic. Data are mean ± SEM from 3 independent experiments and each had duplicate. (B) HNF4G overexpression promoted migration and invasion of PDAC cells transplanted in the pancreas of mice ( n = 3). Left panel shows representative bioluminescence images taken at 7 and 40 days of implantation; Right panel shows quantitative fluorescent intensity of the transplanted PDAC. (C) Representative H&E staining pictures of the pancreas, liver and lung from mice implanted orthotopically with PDAC cells with or without HNF4G overexpression. Scale bars: 100 μm. (D) The work flow schematic for analyzing the candidate genes targeted by HNF4G. (E) Functional enrichment of the 293 HNF4G-targeted genes by Metascape. (F) The expression levels of some downstream genes of HNF4G in PDAC cells with or without HNF4G overexpression. 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 or Wilcoxon rank-sum test. ns, not significant

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Over Expression, Migration, In Vitro, Staining, Functional Assay, Expressing

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: Metformin activates AMPK that induces HNF4G phosphorylation-ubiquitination coupled degradation. (A) Effect of Metformin (10 μmol/L) on HNF4G and AMPKα phosphorylation in T3M4 cells. (B) Metformin (10 μmol/L) promoted AMPKα 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 AMPKα knockdown treated with Metformin (10 μmol/L). (F and G) Immunoblot analysis of phosphorylated HNF4G and AMPKα 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

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Immunoprecipitation, Control, Western Blot, Knockdown, Knock-Out, Expressing

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: Metformin suppress HNF4G-induced PDAC metastasis depending on SMAD4 status. (A) Metformin treatment significantly repressed in vitro migration and invasion of SMAD4-deficient T3M4 cells but not SMAD4-efficient PANC-1. Left panels show representative images of transwell assays and right panels represent quantitative statistic. Data are mean ± SEM from 3 independent experiments and each had triplicate. Shown are the results in cells treated with or without Metformin (10 μmol/L); see also Fig. S5 for the entire and detailed dose-dependent results. (B) Knockdown of SMAD4 expression significantly promoted Metformin to suppress the migration and invasion of PDAC cells. Upper panel are representative transwell images and lower panel are quantitative data (mean ± SEM from 3 independent experiments and each had triplicate). (C) Metformin treatment significantly repressed the spread of HNF4G-overexpressing T3M4 cells implanted in mouse pancreas ( n = 4). Left panel shows bioluminescence images of mice and the right panel shows quantitative fluorescent intensities. (D) Metformin treatment significantly prolonged survival time of mice implanted with PDAC in the pancreas as compared with vehicle control. (E) Metformin treatment significantly reduced PDAC metastases in the liver compared with vehicle control. Left panel shows representative tumor nodes staining by H&E and right panel shows quantitative statistic. Scale bars: 100 μm (40×) and 10 μm (400×). (F) Representative IHC pictures showing that Metformin treatment substantially reduced HNF4G but increased p-AMPKα expression levels in serial sections of both pancreas and liver from mice with PDAC implantation as compared with vehicle control. Scale bars: 100 μm (100×) and 25 μm (400×). Statistical significance: **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 of Student’s t -test or Wilcoxon rank-sum test. ns, not significant

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: In Vitro, Migration, Knockdown, Expressing, Control, Staining

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: 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 χ 2 test. ns, not significant

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Immunohistochemistry

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: 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

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Expressing, Phospho-proteomics, Ubiquitin Proteomics

Journal: Molecular Therapy Oncology

Article Title: Enhancing cancer killing and natural killer cell persistence by targeting NOXA, a predictor of poor patient survival

doi: 10.1016/j.omton.2026.201152

Figure Lengend Snippet: Cancer-derived conditioned media induces apoptosis-related gene expression and dysfunction in NK cells Human primary NK cells were incubated with control media (RPMI), AsPC-1-CM, or Capan-2-CM, followed by functional and molecular analyses. (A) NK cells were treated with CM for 72 h and stained with Annexin V/7-aminoactinomycin D (7-AAD) to assess apoptosis. (B) Metabolic activity in NK cells after 24 h of CM exposure was measured by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). (C) CM-pretreated NK cells were co-cultured with AsPC-1-Luc or Capan-2-Luc cancer cells for 24 h, and cancer-cell-killing activity was measured. (D) Expression of apoptosis-related proteins BIM and NOXA in CM-treated NK cells was analyzed using western blotting. Representative blots show NOXA and BIM levels following 24 h exposure to CM derived from normal pancreatic epithelial cells (HPDE) or pancreatic cancer cell lines (AsPC-1 and Capan-2). BIM and NOXA protein levels were quantified from three independent experiments and normalized to β-actin. (E) Western blots showing the expression of NOXA and BIM in NK cells following 20 h exposure to IL-6, IL-10, or TGF-β1. All data are presented as mean ± standard error of the mean (SEM). p values were calculated using one-way ANOVA with multiple comparison tests (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001).

Article Snippet: The pancreatic cancer cell lines Capan-2 and AsPC-1 were procured from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Derivative Assay, Gene Expression, Incubation, Control, Functional Assay, Staining, Activity Assay, Cell Culture, Expressing, Western Blot, Comparison

Journal: Molecular Therapy Oncology

Article Title: Enhancing cancer killing and natural killer cell persistence by targeting NOXA, a predictor of poor patient survival

doi: 10.1016/j.omton.2026.201152

Figure Lengend Snippet: In vitro evaluation of NK-mediated anti-tumor activities under standard and suppressive conditions (A) Single KO or DKO NK cells were co-cultured with AsPC-1-Luc and Capan-2-Luc cells. After 24 h of incubation, luminescence-based assays were performed to assess cancer-killing activity. (B) Cryopreserved NK cells were thawed and analyzed for their cancer killing activity using luminescence-based assays. (C) Cytotoxicity of NK cells pretreated with conditioned media (CM) was evaluated after 24 h of co-culture with cancer cells. (D) Effect of cytokines on NK cell cytotoxicity. NK cells were co-cultured with luciferase-expressing tumor cells at 0.5:1 E:T ratio for 24 h in the presence of IL-6 or TGF-β1 (20 ng/mL). Cytokines were added directly to the NK-tumor co-cultures and maintained throughout the assay. (E) Schematic illustration of a serial cancer killing assay. NK cells were co-cultured with luciferase-expressing cancer cells, and surviving NK cells were sequentially transferred into freshly cultured cancer cells at 24-h intervals. (F and G) Single-KO and DKO NK cells were sequentially challenged three times with freshly cultured tumor cells. Cancer-killing activity was evaluated using a luminescence-based assay with AsPC-1-Luc cells (F) and a cell viability assay with SNU-3947-TO-2D cells (G), both in the presence of IL-2. (H) Serial cancer killing assays were also conducted in the absence of IL-2 to assess NK cell functionality under cytokine-limited conditions. Error bars represent the mean ± standard SEM of three independent experiments. Statistical significance was calculated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The pancreatic cancer cell lines Capan-2 and AsPC-1 were procured from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: In Vitro, Cell Culture, Incubation, Activity Assay, Co-Culture Assay, Luciferase, Expressing, Luminescence Assay, Viability Assay

Journal: Molecular Therapy Oncology

Article Title: Enhancing cancer killing and natural killer cell persistence by targeting NOXA, a predictor of poor patient survival

doi: 10.1016/j.omton.2026.201152

Figure Lengend Snippet: Prolonged proliferation and enhanced metabolic activity in NOXA-KO NK cells (A) The effect of NOXA KO on proliferation marker Ki67 expression in NK cells was assessed via flow cytometry. (B) Proliferation assay of CFSE-labeled NK cells was analyzed by flow cytometry, showing enhanced cell proliferation in NOXA -KO NK cells. (C) Expression of the NK cell activation marker CD69 and the degranulation marker CD107a was measured after co-culture with AsPC-1 or Capan-2 cells for 6 h. (D) Expression of the NK-cell-activating receptors was analyzed using flow cytometry. (E) Cytokine secretion by NK cells was quantified using ELISA. NK cells were co-cultured with AsPC-1-luc or Capan-2-luc cells at 1:1 E:T ratio for 24 h, and supernatants were analyzed. (F and G) Mitochondrial metabolic activity in NK cells was evaluated using the Seahorse XF Cell Mito Stress Test, showing enhanced oxidative metabolism in NOXA -KO NK cells. Metabolic activities in NOXA -KO NK cells was assessed under normal conditions (F) and after 24 h of treatment with tumor-derived conditioned media (CM) (G). Statistical significance was determined using two-way analysis of variance (ANOVA) followed by multiple comparison test. ∗ p < 0.05, ∗∗∗ p < 0.001. (H and I) Metabolite analysis was conducted to assess pathways related to glycolysis, the tricarboxylic acid (TCA) cycle (H), and the pentose phosphate pathway (I). Bar graphs summarizing levels of each metabolite represent three independent experiments and are displayed as mean ± standard SEM (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001).

Article Snippet: The pancreatic cancer cell lines Capan-2 and AsPC-1 were procured from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Activity Assay, Marker, Expressing, Flow Cytometry, Proliferation Assay, Labeling, Activation Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay, Comparison