Activation of STAT3 signaling is mediated by TFF1 silencing in gastric neoplasia

Abstract

TFF1, a secreted protein, plays an essential role in keeping the integrity of gastric mucosa and its barrier function. Loss of TFF1 expression in the TFF1-knockout (KO) mouse leads to a pro-inflammatory phenotype with a cascade of gastric lesions that include low-grade dysplasia, high-grade dysplasia, and adenocarcinomas. In this study, we demonstrate nuclear localization of p-STAT^Y705, with significant overexpression of several STAT3 target genes in gastric glands from the TFF1-KO mice. We also show frequent loss of TFF1 with nuclear localization of STAT3 in human gastric cancers. The reconstitution of TFF1 protein in human gastric cancer cells and 3D gastric glands organoids from TFF1-KO mice abrogates IL6-induced nuclear p-STAT3^Y705 expression. Reconstitution of TFF1 inhibits IL6-induced STAT3 transcription activity, suppressing expression of its target genes. TFF1 blocks IL6Rα-GP130 complex formation through interfering with binding of IL6 to its receptor IL6Rα. These findings demonstrate a functional role of TFF1 in suppressing gastric tumorigenesis by impeding the IL6-STAT3 pro-inflammatory signaling axis.

Fig. 1

Loss of TFF1 promotes STAT3 nuclear localization in the TFF1-KO mice gastric tissues. a Representative immunohistochemistry images in the upper panels show nuclear p-STAT3 (Y705) in hyperplasia, dysplasia, and cancer from the antropyloric region of glandular stomach in TFF1-knockout (TFF1-KO), but not in TFF1-wild-type (TFF1-WT) normal gastric mucosa. The lower panels demonstrate the expression of TFF1, original magnification (×200) and insets (×400). b Immunohistochemistry staining of p-STAT3 of gastric mouse tissues from TFF1-WT and TFF1-KO mice at the age of 2, 6, and 10 months detected p-STAT3 in the nucleus at all ages in TFF1-KO mice. p-STAT3 staining was absent in tissues from matched ages in TFF1-WT mice with normal glands. The TFF1-KO mice showed dysplastic lesions at 6 and 10 months of age. Original magnification (×400) and insets (×600)

Fig. 2

Loss of TFF1 promotes mRNA expression of STAT3 target genes in gastric tumourigenesis. a Quantitative real-time PCR analysis demonstrated upregulation of mRNA expression of STAT3 target genes (Vegf, c-Myc, Birc5, and Il17A) in gastric tissues from the TFF1-KO mice (n = 36) as compared with normal gastric tissues from TFF1-WT mice (n = 15). b Expression levels of STAT3 target genes in nondysplastic and dysplastic gastric mucosa from TFF1-KO, as compared to normal gastric mucosa from TFF1-WT. c Expression levels of STAT3 target genes in TFF1-KO gastric mucosa at 2–4 and >6 months of age, as compared to age-matched TFF1-WT gastric mucosa. Horizontal bars indicate the mean values. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 by two-tailed Student’s t test (for two groups) and ANOVA Newman–Keuls multiple comparison test (for multiple groups)

Fig. 3

TFF1 suppresses STAT3 nuclear localization in ex vivo and 3D organoid cultures. a Ex vivo immunofluorescence staining of p-STAT3 (Y 705) in gastric epithelial cells isolated from the antropyloric region in TFF1-wild type (TFF1-WT) or TFF1-knockout (TFF1-KO) mice. As shown, there was absence of nuclear p-STAT3 staining in TFF1-WT cells (arrowheads in upper panels), whereas strong nuclear p-STAT3 was detected in TFF1-KO (arrows in lower panels). Quantification of nuclear p-STAT3 positive staining in at least 200 counted cells from three different fields is presented as percentage ± SEM (right panel); ^***P < 0.001 by two-tailed Student’s t test. b 3D organoid cultures derived from antropyloric glands of TFF1-WT (upper two panels) and TFF1-KO (lower two panels) showing nuclear localization of p-STAT3 in TFF1-KO organoids (arrows), but not in TFF1-WT (arrowheads). c Immunofluorescence assay performed on organoids derived from the antrum of TFF1-KO mouse stomach. Cells showing nuclear staining of p-STAT3 in organoids treated with conditioned media from AGS-pcDNA cell line (Ctrl CM) for 24 h (upper panel). Organoids treated with TFF1 conditioned media from AGS-TFF1 cell line (TFF1 CM) displayed loss of nuclear staining (arrowheads) of p-STAT3 (middle panel). Organoids treated with recombinant TFF1 protein (400 ng mL⁻¹) showed absence of p-STAT3 (arrowheads) nuclear staining (lower panels). The merge of the representative full organoid gland and its H&E staining is presented in the right panels. Scale bar 2 µm. Original magnification is ×600. Zo1 (red) was used as an epithelial cell marker, and DAPI (blue), as a nuclear counterstain. Graph showing the quantification of nuclear p-STAT3-positive cells in at least 4 counted organoid glands (at least 25–50 cells in each organoid) presented as percentage ± SEM (right upper panel); ^***P < 0.001 by two-tailed Student’s t test. H&E staining and bright field images (BF) of representative organoids are shown on the right of panels (b) and (c)

Fig. 4

Reconstitution of TFF1 inhibits IL6-mediated STAT3 activation. a In vitro immunofluorescence assay of AGS pcDNA control cells (Ctrl) and AGS cells stably expressing pcDNA-TFF1. Cells were cultured and treated with or without IL6 (100 ng mL⁻¹). Nuclear localization of STAT3 is shown in green (arrows). DAPI (blue) was used as a nuclear counterstain. Original magnification is at ×400. b Quantification of nuclear STAT3-positive staining in at least 200 cells from three images is presented as a percentage in the right panel. Data are graphed with mean ± SEM. c, d Protein-expression levels from the nuclear and cytosolic fractions of AGS (c) and STKM2 (d) cells infected with control or TFF1 adenoviruses (5 MOI). After 48 h, cells were treated or not with IL6 (100 ng mL⁻¹) for 30 min. Similar amounts of protein were applied to SDS-PAGE for immunoblotting with total and p-STAT3 (Y705) antibodies. Anti-NaKATPase antibody was used as a loading control for cytosol fraction, and anti-LAMIN B1 for nuclear fraction. The relative intensity ratio of nuclear p-STAT3/Lamin B1 is calculated and graphed using Image-lab software from BioRad (lower panels). e, f The luciferase reporter assay using a STAT3–Luc reporter plasmid. The results are expressed as mean ± SEM of at least three independent experiments. AGS cells (e) and STKM2 cells (f) infected with control or TFF1 adenoviruses were transfected with STAT3–luciferase reporter and stimulated with IL6 (100 ng mL⁻¹) for 3 h. The results are expressed as mean ± SEM of at least three independent experiments; ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 by ANOVA Newman–Keuls multiple comparison test

Fig. 5

TFF1 suppresses expression of STAT3 target genes in vitro. a–c Quantitative real-time PCR analysis demonstrated expression levels of several STAT3 target genes (VEGF, C-MYC, CXCL10, and IL17A) in vitro. a AGS cell lines were infected with either TFF1 or control adenoviruses (5 MOI) for 48 h and then treated with or without IL6 (100 ng mL⁻¹) for 2 h. b AGS cells were treated with or without TFF1 recombinant protein (400 ng mL⁻¹) for 24 h and either stimulated or not with IL6 for 2 h. c AGS cells were treated with conditioned media from AGS-pcDNA or AGS-TFF1 stable cell lines for 24 h. The results are expressed as mean ± SEM of at least 3 independent experiments; ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 by two-tailed Student’s t test (for two groups) and ANOVA Newman–Keuls multiple comparison test (for multiple groups)

Fig. 6

ChIP assay confirms the decreased binding of STAT3 to its target genes after TFF1 reconstitution. a–c ChIP assay in AGS cells infected with control or TFF1 adenoviruses and stimulated with or without IL6 (100 ng mL⁻¹), followed by quantitative real-time PCR with primers designed for STAT3 binding site on VEGF (a) and IL6 (b) promoter regions and control primers (c)

Fig. 7

TFF1 negatively regulates IL6-induced STAT3 activation through GP130-IL6Rα axis. a Western blot analysis using AGS cell lines infected with control or TFF1 adenovirus. After stimulation with IL6 (100 ng mL⁻¹) for 30 min, TFF1 expressing cells showed a significant decrease of p-STAT3 (Y705), p-GP130 (S782), and p-JAK2 (Y1007/Y1008) protein levels as compared to control cells. b The relative intensity ratio of p-JAK2/β-Actin, p-GP130/β-Actin, and p-STAT3/β-Actin were calculated by Image-lab software from BioRad. The results are expressed as mean ± SEM of at least three independent experiments. ^***P < 0.001 by two-tailed Student’s t test. c Immunoprecipitation and Western blot analysis following IL6Rα pulldown using AGS cells infected with TFF1 or control adenoviruses (5 MOI), with or without treatment with IL6 (100 ng mL⁻¹) for 30 min. The first lane exhibits AGS control following immunoprecipitation with mouse IgG control antibody. All immunoprecipitations and their corresponding input samples were subjected to immunoblotting with rabbit polyclonal antibody against GP130 and IL6Rα. The expression of TFF1 and equal amounts of protein loading were confirmed in the input samples. d Punctate proximity ligation assay (SOURCE) was performed in accordance with supplier’s instructions in AGS cells infected with control or TFF1 adenovirus. The presence of red signals indicates positive ligation, indicative of interaction. Using IL6Rα and GP130 antibodies, the results indicated the presence of IL6Rα–GP130 interaction (red signals), following stimulation with IL6 in control cells (Ctrl). This interaction was not detected in AGS cells expressing TFF1 (upper left two panels). The lower panel displays immunofluorescence following the use of TFF1 and IL6Rα antibodies and demonstrates an interaction between TFF1 and IL6Rα (red signals), (lower right two panels). As a negative control for PLA background reaction, control cells were stimulated with IL6 and probed with a single antibody for GP130 (right single panel). Maximum intensity projection is presented in the upper and right side of each image

Fig. 8

IL6–IL6Rα soluble complex antagonizes TFF1-mediated suppression of STAT3 activation. a AGS cells were infected with control or TFF1 adenoviruses (5 MOI), transfected or not with GP130 expression plasmid (500 ng mL⁻¹) and stimulated or not with IL6 (100 ng mL⁻¹). AGS expressing TFF1 showed a decrease of p-STAT3 (Y705) after GP130 transfection and IL6 stimulation. b AGS cells were transfected or not with GP130 expression plasmid (500 ng mL⁻¹) and treated with recombinant TFF1 (400 ng mL⁻¹), then stimulated or not with IL6 (100 ng mL⁻¹). TFF1 treated cells showed a decrease of p-STAT3 (Y705) after GP130 transfection and IL6 stimulation. c AGS cells were infected with control or TFF1 adenoviruses (5 MOI) and treated or not with IL6–IL6Rα soluble complex (10 ng mL⁻¹). AGS-expressing TFF1 showed no effect of p-STAT3 (Y705) after IL6–IL6Rα treatment as compared to control. d AGS cells were treated or not with TFF1 recombinant protein (400 ng mL⁻¹) and, or IL6–IL6Rα soluble complex protein (10 ng mL⁻¹). AGS cells treated with TFF1 and IL6–IL6Rα showed no effect of p-STAT3 (Y705) as compared to control cells treated with IL6–IL6Rα alone. The relative intensity ratio p-STAT3/β-Actin were calculated by the Image-lab software from BioRad, and presented under each blot. The results are expressed as mean ± SEM of at least 3 independent experiments; ***P < 0.001 by two-tailed Student’s t test, and P > 0.05 is considered nonsignificant (ns)

Fig. 9

Human gastric cancer tissues demonstrate nuclear localization of STAT3 and loss of TFF1. a–f Representative immunohistochemical (IHC) staining images of TFF1 (upper panels a and b) and STAT3 (lower panels c and d) using human gastric cancer tissue microarrays that contained 108 cancer samples with their matching adjacent histologically normal stomach, original magnification is at ×200 and insets are at ×400. e, f Summary graphs of the IHC staining of TFF1 (e) and nuclear STAT3 (f) results using box-and-whisker blots to depict the smallest value, lower quartile, median, upper quartile, and largest value, (+) indicate the mean. g Schematic cartoon representing the role of trefoil factor 1 (TFF1) in regulating inflammatory signaling in gastric epithelial cells. Gastric cancer is associated with inflammation mediated by the release of cytokines such IL6 and TNFα from stromal and epithelial cells inducing activation of STAT3 and NFκB transcriptional factors. Loss of TFF1 promotes IL-6-mediated IL6Rα-GP130 complex formation, recruitment and phosphorylation of JAK2, with subsequent phosphorylation and nuclear localization of STAT3 to activate STAT3 transcription targets. The role of TFF1 in regulating NFκB has been described before