Journal: Cancer Science

Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

doi: 10.1111/cas.13467

Figure Lengend Snippet: Low 15‐hydroxyprostaglandin dehydrogenase (15‐ PGDH ) expression is implicated in poor pancreatic ductal adenocarcinoma ( PDAC ) prognosis. A, Workflow diagram of patients who underwent pancreatic resection and contributed samples for immunohistochemical ( IHC ) analysis. B, Representative IHC staining of 15‐ PGDH expression in 107 PDAC tissues. Scale bar = 100 μm. C,D, Relationship between 15‐ PGDH expression and relapse‐free survival (C) or overall survival (D) using the Kaplan‐Meier method

Article Snippet: The human PDAC cell lines PK‐8 and S2‐013 were obtained from the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan) and RIKEN Bioresource Center Cell Bank (Tsukuba, Japan).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Cancer Science

Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

doi: 10.1111/cas.13467

Figure Lengend Snippet: 15‐Hydroxyprostaglandin dehydrogenase (15‐ PGDH ) downregulation by interleukin‐1β ( IL ‐1β) enhances pancreatic ductal adenocarcinoma cell growth. A,B, Expression of HPGD (the gene coding 15‐ PGDH protein, upper panel) or 15‐ PGDH (lower panel) in PK ‐8 cells (A) or S2‐013 cells (B) after treatment with si RNA targeting 15‐ PGDH or with control si RNA , evaluated by quantitative RT ‐ PCR (upper panel) or Western blot analysis (lower panel). Data are presented as the treated/control cell ratio. C,D, PK ‐8 cells (C) or S2‐013 cells (D) transfected with si RNA s targeting 15‐ PGDH or with control si RNA were incubated for up to 96 hours and assayed for cell number; data are presented as the treated/control (time = 0) cell ratio. E,F, Expression of 15‐ PGDH in PK ‐8 cells or S2‐013 cells after IL ‐1β (E) or tumor necrosis factor‐α ( TNF ‐α) (F) treatment for 24 and 48 hours and distilled water treatment for 48 hours as a control was evaluated by Western blotting. G, Column graph showing relative 15‐ PGDH levels in PK ‐8 cells or S2‐013 cells after IL ‐1β and TNF ‐α treatment for 24 and 48 hours, and distilled water treatment for 48 hours as a control, were evaluated using ImageJ software. H, Expression of HPGD and IL 1B in six PDAC patients determined by quantitative RT ‐ PCR . Data were normalized to the ACTB mRNA level and are shown as the mean ± SD of three independent experiments. **P < .01

Article Snippet: The human PDAC cell lines PK‐8 and S2‐013 were obtained from the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan) and RIKEN Bioresource Center Cell Bank (Tsukuba, Japan).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Transfection, Incubation, Software

Journal: Cancer Science

Article Title: Downregulation of 15‐hydroxyprostaglandin dehydrogenase by interleukin‐1β from activated macrophages leads to poor prognosis in pancreatic cancer

doi: 10.1111/cas.13467

Figure Lengend Snippet: Tumor‐associated macrophages are inversely correlated with pancreatic ductal adenocarcinoma ( PDAC ) cells harboring high 15‐hydroxyprostaglandin dehydrogenase (15‐ PGDH ) expression. A,B, Representative immunohistochemical ( IHC ) staining of 15‐ PGDH (upper panel) and CD 163 (lower panel) expression in high 15‐ PGDH (A) and low 15‐ PGDH (B) serial PDAC specimens. Scale bar = 200 μm. C, Graph showing Pearson's correlation between the expression of 15‐ PGDH and the number of CD 163‐positive cells in 107 PDAC patients. D, Schematic representation of the findings of this study. IL ‐1βR, interleukin‐1β receptor

Article Snippet: The human PDAC cell lines PK‐8 and S2‐013 were obtained from the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan) and RIKEN Bioresource Center Cell Bank (Tsukuba, Japan).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Cancer research

Article Title: Cancer associated fibroblasts in pancreatic cancer are reprogrammed by tumor-induced alterations in genomic DNA methylation

doi: 10.1158/0008-5472.CAN-15-3264

Figure Lengend Snippet: A, The schema of candidate gene selection process. B, Promoter methylation status of 8 genes as indicated was checked in 3 paired primary PDAC cells and CAFs by Methylation Specific PCR (MSP)(Supplemental Table 2). C, IHC of SOCS1 was performed on paraffin-embedded slides of the seven human PDAC specimens as described (41). Representative IHC images of paratumoral areas(upper panel) and intratumoral areas(lower panel) from the same slide were shown. In upper panel, arrowhead indicates the normal acinar cells; in lower panel, arrowhead indicates the PDAC tumor cells. In both panels, arrows indicate stromal fibroblasts. Scale bar, 20 µm. D, protein expression was quantified by the Image Analysis Software(Aperio) as the total pixel number of positive staining signals in stroma divided by the total area size of the stroma, as previously described (42). The comparison between intratumoral stroma(Tumoral Stroma) and paratumoral normal stroma (Normal Stroma) was conducted by a paired t-Test(p=0.0487). E, SOCS1 methylation was quantified by MethySYBR real-time PCR. SOCS1 mRNA expression was quantified by real-time RT-PCR(qPCR); and GAPDH was used for normalization. SOCS1 mRNA expression were significantly correlated with SOCS1 promoter methylation in the simple linear regression analysis(p=0.021).

Article Snippet: Materials and Methods Tissue specimens and cultures Primary cultures of PDAC tumor cells, including 3.27T, 1.30T, 3.30T, 9.05T, 3.29T and 7.07T, and CAFs, including 2.15F, 10.29F, 5.101F, 2.01F, 7.02F, 10.09F, 9.07F, 9.28F, 5.10F, 3.16F, 7.09F, 3.05F, 3.27F, 1.30F, 3.30F, 9.05F, 2.29F, 7.12F, 1.23F and 7.21F, were established from banked, surgically resected PDAC specimens between 2008 and 2014 and the Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board(JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described( 17 , 18 ).

Techniques: Selection, Methylation, Expressing, Software, Staining, Comparison, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cancer research

Article Title: Cancer associated fibroblasts in pancreatic cancer are reprogrammed by tumor-induced alterations in genomic DNA methylation

doi: 10.1158/0008-5472.CAN-15-3264

Figure Lengend Snippet: Summary of the results of SOCS1 methylation in multiple primary PDAC tumor cell cultures and primary CAFs analyzed by MSP and those in stroma dissected from PDAC tissues analyzed by pryosequencing

Article Snippet: Materials and Methods Tissue specimens and cultures Primary cultures of PDAC tumor cells, including 3.27T, 1.30T, 3.30T, 9.05T, 3.29T and 7.07T, and CAFs, including 2.15F, 10.29F, 5.101F, 2.01F, 7.02F, 10.09F, 9.07F, 9.28F, 5.10F, 3.16F, 7.09F, 3.05F, 3.27F, 1.30F, 3.30F, 9.05F, 2.29F, 7.12F, 1.23F and 7.21F, were established from banked, surgically resected PDAC specimens between 2008 and 2014 and the Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board(JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described( 17 , 18 ).

Techniques: Methylation

Journal: Cancer research

Article Title: Cancer associated fibroblasts in pancreatic cancer are reprogrammed by tumor-induced alterations in genomic DNA methylation

doi: 10.1158/0008-5472.CAN-15-3264

Figure Lengend Snippet: The CROC analysis of genomic clustering of the genes both methylated and downregulated in CAFs upon interacting with PDAC tumor cells

Article Snippet: Materials and Methods Tissue specimens and cultures Primary cultures of PDAC tumor cells, including 3.27T, 1.30T, 3.30T, 9.05T, 3.29T and 7.07T, and CAFs, including 2.15F, 10.29F, 5.101F, 2.01F, 7.02F, 10.09F, 9.07F, 9.28F, 5.10F, 3.16F, 7.09F, 3.05F, 3.27F, 1.30F, 3.30F, 9.05F, 2.29F, 7.12F, 1.23F and 7.21F, were established from banked, surgically resected PDAC specimens between 2008 and 2014 and the Panc10.05 cell line was established in 1998 in accordance with the Johns Hopkins Medical Institution Institutional Review Board(JHMI IRB)-approved protocols and authenticated by DNA and gene expression profiling as previously described( 17 , 18 ).

Techniques: Methylation, Expressing

Journal: OncoTargets and therapy

Article Title: Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model

doi: 10.2147/OTT.S322276

Figure Lengend Snippet: Endoglin is highly expressed on CAFs in human pancreatic tumors. ( A ) Representative images of human pancreatic cancer (representative from n = 25 PDAC patients) and normal pancreas stained for α-SMA, endoglin, cytokeratin, and vimentin. Endothelial cells (black arrow) and endoglin expressing CAFs (white arrow). ( B ) Immunofluorescent double staining for α-SMA and endoglin in human PDAC tumors. ( C ) Endoglin mRNA expression by human cells; ECRF endothelial cells, MIA PaCa-2, PANC-1 and BxPC-3 PDAC cells and 8 patient derived primary pancreatic CAFs. ( D ) Endoglin protein expression on human pancreatic fibroblasts. Basal and BMP9-induced downstream signaling (pSMAD1) was inhibited with TRC105 (full-length blot shown in Supplementary figure 4A – C ).

Article Snippet: The mouse PDAC cell line KPC-3 (Kras G12D/+ LSL-Trp53 R172H/+ Pdx-1-Cre), (kindly supplied by the department of Immunology, LUMC) with a targeted insertion of codon-optimized Luc-2 (pGL4.10) [luc2] (Promega Leiden, the Netherlands), mouse MC38 cells (kindly supplied by the department of Immunology, LUMC) and primary fibroblasts were all cultured in DMEM/F12 glutamax medium (Invitrogen, Landsmeer, the Netherlands), with 10% fetal bovine serum (FBS) (Gibco, Bleiswijk, the Netherlands), 0.01 M HEPES, 0.1 μg/mL Gentamycin, 40U/mL Penicillin and 40 μg/mL Streptomycin (all Invitrogen Landsmeer, the Netherlands) at 37°C and 5% CO2.

Techniques: Staining, Expressing, Double Staining, Derivative Assay

Journal: Oncotarget

Article Title: Perspectives of TGF-β inhibition in pancreatic and hepatocellular carcinomas

doi:

Figure Lengend Snippet: At the cellular level, TGF-β induces proliferation and survival of PDAC cells in the late phase of PDAC carcinogenesis (after SMAD4 inactivation), and promotes epithelial-to-mesenchymal transition (EMT), invasion, and metastasis. At the microenvironment level, TGF-β is a key mediator of the dialogue between cancer and stellate cells (fibrotic cells), involved in the production of a dense fibrotic stroma and the resulting low vascularization of PDAC. TGF-β also deregulates the immune microenvironment toward immunosuppression and inappropriate inflammation.

Article Snippet: Consistent with this, Bachem et al.[ ] showed that PDAC cell lines stimulated PSC proliferation.

Techniques:

Journal: Clinical & experimental metastasis

Article Title: Genome-wide in vivo RNAi screen identifies ITIH5 as a metastasis suppressor in pancreatic cancer

doi: 10.1007/s10585-017-9840-3

Figure Lengend Snippet: A, Schematic diagram of the in vivo shRNA forward screen to identify genes regulating liver metastasis in PDAC. Briefly, following transduction, S2-028 PDAC cells were injection intrasplenically. After 4 wk, liver metastases were isolated, cultured and re-injected into the spleens of athymic mice. Hepatic metastases were isolated and established in cell culture before isolating DNA and identification of shRNA integrated. After the second round of intrasplenic injections, two target genes were identified from 2 cell clones, HMP19 and ITIH5. B, Representative images of livers from mice injected with control S2-028 or shRNA library-infected S2-028. Scale bar, 10 mm.

Article Snippet: Cell lines and cell culture Five human PDAC cell lines (S2-007, S2-028, MIAPaCa-2, BxPC-3 and Panc-1) were obtained from Dr. M. Anthony Hollingsworth (Eppley Cancer Center).

Techniques: In Vivo, shRNA, Transduction, Injection, Isolation, Cell Culture, Clone Assay, Control, Infection

Journal: Clinical & experimental metastasis

Article Title: Genome-wide in vivo RNAi screen identifies ITIH5 as a metastasis suppressor in pancreatic cancer

doi: 10.1007/s10585-017-9840-3

Figure Lengend Snippet: A, The relative expression ITIH5 levels of in a panel of PDAC cell lines and HPNE, as determined by immunoblotting. Relative metastatic potential is provided for comparison. B, C Mixed pools of S2-007 and MIAPaCa-2 cells transduced with ITIH5 cDNA over-express ITIH5 and the number of liver metastasis are compared following intrasplenic injections. Representative livers are shown. Arrows highlight liver foci. Results are means + SE of liver nodules from positive mice. * P < 0.05 and ** P < 0.001; Mann-Whitney U test.

Article Snippet: Cell lines and cell culture Five human PDAC cell lines (S2-007, S2-028, MIAPaCa-2, BxPC-3 and Panc-1) were obtained from Dr. M. Anthony Hollingsworth (Eppley Cancer Center).

Techniques: Expressing, Western Blot, Comparison, Transduction, MANN-WHITNEY

Journal: Clinical & experimental metastasis

Article Title: Genome-wide in vivo RNAi screen identifies ITIH5 as a metastasis suppressor in pancreatic cancer

doi: 10.1007/s10585-017-9840-3

Figure Lengend Snippet: A-C, Images of pancreatic tumors and average tumor volume following orthotopic injections of PDAC cells in which ITIH5 expression levels have been altered by transduction of shRNA (in S2-028 cells) or cDNA (in S2-007 or MIAPaCa-2 cells). Graphical representation of orthotopic tumor size at time of euthanasia. Results are means + SE of the orthotopic tumor volume. * P < 0.05 and ** P < 0.01; Mann-Whitney U test.

Article Snippet: Cell lines and cell culture Five human PDAC cell lines (S2-007, S2-028, MIAPaCa-2, BxPC-3 and Panc-1) were obtained from Dr. M. Anthony Hollingsworth (Eppley Cancer Center).

Techniques: Expressing, Transduction, shRNA, MANN-WHITNEY

Journal: Clinical & experimental metastasis

Article Title: Genome-wide in vivo RNAi screen identifies ITIH5 as a metastasis suppressor in pancreatic cancer

doi: 10.1007/s10585-017-9840-3

Figure Lengend Snippet: Representative photographs showing migration (A-C) and invasion (D-F) are inhibited when ITIH5 levels are high. Results are means + SE of the % wound closure and number of invaded cells, respectively. G-I, Growth curves of PDAC cell lines altered ITIH5 expression levels. * P < 0.01 and ** P < 0.001; Mann-Whitney U test. Scale bar, 200 μm.

Article Snippet: Cell lines and cell culture Five human PDAC cell lines (S2-007, S2-028, MIAPaCa-2, BxPC-3 and Panc-1) were obtained from Dr. M. Anthony Hollingsworth (Eppley Cancer Center).

Techniques: Migration, Expressing, MANN-WHITNEY

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