Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A is methylated and suppressed in pancreatic cancer. (A) Global DNA methylation analysis (Illumina Human Methylation 450K array) in 20 human pancreatic cancer and 11 normal tissues (Stanford University Medical Center, USA). Heatmap of methylation beta values for the top 7242 differentially regulated loci in cancer vs control ( P < .05, FC ≥ 1.5). (B) Venn diagram showing 2717 unique methylation sites identified in our study. (C) Heatmap of methylation beta values across the HNF4A locus. The HNF4A promoter area with increased DNA methylation (high beta values) corresponds to probes covering the proximal promoter area (from −200 nt to −3 nt, TSS200). (D) HNF4A expression as assessed by RT-qPCR, in pancreatic cancer and control tissues. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to control tissues (set as 1). (E) Immunohistochemical analysis for HNF4A in normal (N) and pancreatic cancer tissues (red, HNF4A; blue, nuclei staining). Scale bar: 1 mm and 100 μm (left and right panel, respectively).

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Methylation, DNA Methylation Assay, Control, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Verification of HNF4A CpG methylation sites through bisulfite sequencing. (A) HNF4A expression as assessed by RT-qPCR, in 8 pancreatic cancer cell lines. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to the low HNF4A expressing cell line, MIA PaCa-2 (set as 1). (B) Diagram illustrating the generation of HNF4A isoforms through transcriptional regelation by P1 and P2 promoters and alternative splicing. Different primers were designed to recognize the 4 subgroups of the 12 HNF4A isoforms. (C) HNF4A P1a isoforms expression in pancreatic and liver (SNU-475 and Hep-3B) cancer cell lines as assessed by RT-qPCR. Expression was normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to the high HNF4A expressing cell line, Hep-3B (set as 1). (D) Diagram illustrating the experimental design for the evaluation of HNF4A methylation through bisulfite sequencing. (E) Heatmaps of the methylation ratio across the HNF4A locus, at the single CpG site level, for untreated (Cont) or 5-AZA-CdR–treated MIA PaCa-2 cells (5-Aza). Cells were treated with 5-Aza (1μM), for 48 h. Analysis was performed in 40 CpG sites spanning from a distal locus upstream of +1 position to exon 3.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: CpG Methylation Assay, Methylation Sequencing, Expressing, Quantitative RT-PCR, Alternative Splicing, Methylation

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: DNA methylation at the proximal promoter area regulates HNF4A transcription in pancreatic cancer. (A) HNF4A expression restoration following pancreatic cancer cell treatment with 5-AZA-CdR (5-Aza). Low HNF4A-expressing cells (MIA PaCa-2 and PANC-1) were treated with different concentrations (1 and 2 μM) for 48 or 96 h. HNF4A expression was assessed through RT-qPCR, normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to MIA PaCa-2 untreated cells (set as 1). (B and C) HNF4A isoforms expression in MIA PaCa-2 and BxPC-3, respectively. Cells were treated with different concentrations (1 and 2 μM) of 5-Aza, for 48 h. RT-qPCR data were normalized to GAPDH and β-actin levels. (D) HNF4A expression in high (Capan-1 and HPAF-II) HNF4A-expressing pancreatic cancer cell lines. Cells were treated with different concentrations (1 and 2 μM) of 5-Aza, for 48 h. HNF4A expression was assessed through RT-qPCR, normalized to GAPDH and β-actin levels and results were expressed as mean ± SEM compared to untreated cells (set as 1). (E–G) Pearson’s correlation analyses between HNF4A site-specific DNA methylation and gene expression, in human pancreatic cancer tissues. Correlations in TSS200 (proximal promoter area, from −200 nt to −3 nt), distal promoter, and the gene body. (H) Pearson’s correlation analyses between HNF4A site-specific DNA methylation (TSS200 area) and gene expression, in pancreatic cancer cell lines. DNA methylation is expressed in normalized beta values and gene expression assessed by RT-qPCR is expressed in comparison to MIA PaCa-2 cells (set as 1). (I) Methylation of the HNF4A promoter area using the pCpGfree-basic- Lucia reporter plasmid. Upper panel : Diagram illustrating the HNF4A cloned promoter fragment containing 8 CpG sites, highlighted in red on the sequence (represented as lollipops). M.SssI and M.HpaII enzymes were used to methylate the HNF4A promoter region. Lower panel : HEK293T cells were transiently transfected with unmethylated, M.SssI or M.HpaII methylated reporter constructs and luciferase activity was measured in cell supernatants at 48 and 72 h after transfection. The cells were co-transfected with pCMV-Cypridina Luc Vector which secretes a variant of Cypridina luciferase and was used for normalization. Results were expressed as mean ± SEM of Lucia / Cypridina activity compared to the unmethylated (Un) respective control (set as 1). Asterisks denote statistically significant differences, ∗∗∗ P < .001, Student’s t -test.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Comparison, Methylation, Plasmid Preparation, Clone Assay, Sequencing, Transfection, Construct, Luciferase, Activity Assay, Variant Assay, Control

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A loss-of-function and gain-of-function studies in pancreatic cancer cell lines. (A–C) Stable HNF4A knockdown was achieved by means of 2 different shRNAs (shHNF4A_1 and shHNF4A_2) in Capan-1 and HPAF-II, through lentiviral transduction. Cells transduced with shGFP were used as the control. (A) HNF4A protein levels were determined through western blot analysis and loading was assessed using an antibody against CREB. (B and C) Cell growth was assessed by the MTT and CellTiter-Glo luminescent cell viability assay. Data were expressed as mean ± SEM (the respective control cells, at day 2, were set as 100%). (D–F) HNF4A was stably overexpressed, through lentiviral transduction (HNF4A) in PANC-1 and MIA PaCa-2 cells. Cells transduced with the empty retroviral vector tagged with GFP (EV-GFP) were used as the control. (D) HNF4A protein levels were determined through western blot analysis and loading was assessed using an antibody against CREB. (E and F) Cell growth was assessed by the MTT and CellTiter-Glo luminescent cell viability assay. Data were expressed as mean ± SEM (the respective control cells, at day 2, were set as 100%). (G and H) Anchorage-independent cell growth was assessed by soft agar assays for 5 days. Data were expressed as the mean number of colonies ± SEM (respective control cells set as 100). Representative images were acquired at a 10× and 4× magnification, respectively, using an Evos microscope. (I) Spheroid formation assays using the ultra-low attachment 96-well plate method for PANC-1 (1000 cells/well) and the hanging drop method for MIA PaCa-2 (30,000 cells/20 μL drop). Representative images were acquired on day 7, at a 10× magnification, using an Evos microscope. Asterisks denote statistically significant differences, ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, Student’s t -test.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Knockdown, Transduction, Control, Western Blot, Cell Viability Assay, Stable Transfection, Retroviral, Plasmid Preparation, Microscopy

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A loss is an early event and promotes PDAC growth in vivo. (A–C) Effect of HNF4A on in vivo xenograft tumour growth. HNF4A was stably overexpressed, through lentiviral transduction (HNF4A) in MIA PaCa-2 and PANC-1 cells. Cells transduced with the empty retroviral vector tagged with GFP (GFP) were used as the control. Stable HNF4A knockdown was achieved by means of shRNA in HPAF-II cells, through lentiviral transduction and cells transduced with shControl were used as the control. Cells were injected subcutaneously in NOD-SCID mice and tumor growth was monitored for a total period of 4 weeks. Tumor volumes were calculated by the equation V (mm 3 ) = a × b 2 /2, where a is the largest diameter and b is the perpendicular diameter. (D) Representative images of tumours extracted from mice at the end of the experiment. (E) HNF4A is suppressed at early stages of pancreatic cancer growth in the KPC ( LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre ) mouse model. Tissues extracted from different stages of pancreatic cancer development were subjected to HNF4A immunohistochemical analysis (brown, HNF4A; blue, haematoxylin). PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal adenocarcinoma. For PANC-1, N = 3 mice/group. For MIA PaCa-2 and HPAF-II, N = 8 mice/group. Asterisks denote statistically significant differences, ∗ P < .05, ∗∗ P < .01, Student’s t -test.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: In Vivo, Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Control, Knockdown, shRNA, Injection, Immunohistochemical staining

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Discovery cohort of 168 pancreatic cancer patients reveals that HNF4A loss is an early event and correlates with poor overall survival. HNF4A expression was assessed by immunohistochemical analysis, in 168 pancreatic cancer and 38 normal (uninvolved) tissues (QMC: Queen’s Medical Centre, Nottingham, UK). (A) Staining and scoring of tissues was performed in Histopathology Department of QMC and results were expressed as mean ± SEM compared to normal tissues (set as 1). (B) Assessment of HNF4A staining in 168 pancreatic cancer tissues according to their tumor stage. Results were expressed as mean ± SEM compared to normal tissues (set as 1). (C) Survival analysis in 153 patients divided into low, intermediate (interm), and high HNF4A expression subgroups. Survival estimates were generated using the Kaplan-Meier method and compared using log-rank tests.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Expressing, Immunohistochemical staining, Staining, Histopathology, Generated

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Effect of HNF4A Expression on Overall Survival was Assessed in 153 Patients (QMC Cohort), Using the Univariate and Multivariate Cox Proportional Hazard Analyses

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Expressing

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: HNF4A loss indicates an increased risk of death in a validation cohort of 145 pancreatic cancer patients. HNF4A expression was assessed by immunohistochemical analysis, in 145 pancreatic cancer tissues (UCLA, USA). (A) Staining and scoring of the HNF4A immunostained tissues was performed in the Department of Pathology at UCLA Medical Center. Assessment of HNF4A staining in pancreatic cancer tissues according to their tumor stage. (B) Survival analysis in patients divided into low, intermediate (interm), and high HNF4A expression subgroups. Survival estimates were generated using the Kaplan-Meier method and compared using log-rank tests.

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Expressing, Immunohistochemical staining, Staining, Generated

Journal: Gastro Hep Advances

Article Title: Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

doi: 10.1016/j.gastha.2024.04.005

Figure Lengend Snippet: Effect of HNF4A Expression on Overall Survival Was Assessed in 145 Patients (UCLA Cohort), Using the Univariate and Multivariate Cox Proportional Hazard Modelling

Article Snippet: Briefly, 4 μg of the HNF4A promoter-Lucia reporter construct were incubated with 16 U of M.SssI or M.HpaII CpG methyltransferases and 640 μM S-Adenosylmethionine (B9003S, NEB) at 37 °C for 4 hours.

Techniques: Expressing