H. pylori-induced NF-κB-PIEZO1-YAP1-CTGF axis drives gastric cancer progression and cancer-associated fibroblast-mediated tumour microenvironment remodelling

Abstract

Background: Gastric cancer (GC) is one of the most common tumours in East Asia countries and is associated with Helicobacter pylori infection. H. pylori utilizes virulence factors, CagA and VacA, to up-regulate pro-inflammatory cytokines and activate NF-κB signaling. Meanwhile, the PIEZO1 upregulation and cancer-associated fibroblast (CAF) enrichment were found in GC progression. However, the mechanisms of PIEZO1 upregulation and its involvement in GC progression have not been fully elucidated.

Methods: The CAF enrichment and clinical significance were investigated in animal models and primary samples. The expression of NF-κB and PIEZO1 in GC was confirmed by immunohistochemistry staining, and expression correlation was analysed in multiple GC datasets. GSEA and Western blot analysis revealed the YAP1-CTGF axis regulation by PIEZO1. The stimulatory effects of CTGF on CAFs were validated by the co-culture system and animal studies. Patient-derived organoid and peritoneal dissemination models were employed to confirm the role of the PIEZO1-YAP1-CTGF cascade in GC.

Results: Both CAF signature and PIEZO1 were positively correlated with H. pylori infection. PIEZO1, a mechanosensor, was confirmed as a direct downstream of NF-κB to promote the transformation from intestinal metaplasia to GC. Mechanistic studies revealed that PIEZO1 transduced the oncogenic signal from NF-κB into YAP1 signaling, a well-documented oncogenic pathway in GC progression. PIEZO1 expression was positively correlated with the YAP1 signature (CTGF, CYR61, and c-Myc, etc.) in primary samples. The secreted CTGF by cancer cells stimulated the CAF infiltration to form a stiffened collagen-enrichment microenvironment, thus activating PIEZO1 to form a positive feedback loop. Both PIEZO1 depletion by shRNA and CTGF inhibition by Procyanidin C1 enhanced the efficacy of 5-FU in suppressing the GC cell peritoneal metastasis.

Conclusion: This study elucidates a novel driving PIEZO1-YAP1-CTGF force, which opens a novel therapeutic avenue to block the transformation from precancerous lesions to GC. H. pylori-NF-κB activates the PIEZO1-YAP1-CTGF axis to remodel the GC microenvironment by promoting CAF infiltration. Targeting PIEZO1-YAP1-CTGF plus chemotherapy might serve as a potential therapeutic option to block GC progression and peritoneal metastasis.

Keywords: CTGF; H. pylori; NF-κB; PIEZO1; YAP1; cancer-associated fibroblast; gastric cancer.

FIGURE 1

H. pylori infection increases the abundance of α‐SMA⁺ cancer‐associated fibroblasts (CAFs) in gastric intestinal metaplasia. (A) The representative immunohistochemistry images on the left depict distinct variations in α‐SMA expression between H. pylori ⁺ and H. pylori ⁻ gastric cancer (GC) patients. Specifically, α‐SMA⁺ CAF density was assessed in 103 H. pylori ⁻ and 121 H. pylori ⁺ GC patients (scale bar, 200 μm). (B) Expression levels of α‐SMA mRNA were examined in stage I GC patients who did not receive concurrent adjuvant chemotherapy, as per the ACRG dataset, which included 7 H. pylori ⁻ and 8 H. pylori ⁺ GC patients. (C) A UMAP plot illustrates the distribution of α‐SMA mRNA expression in GC patients based on a scRNA‐seq dataset. (D) Violin and bubble plots reveal elevated expression levels of α‐SMA in scRNA‐seq samples of GC intestinal metaplasia, particularly in the H. pylori ⁺ sample (#6342). (E) A schematic diagram outlines a GC mouse model, which demonstrates the formation of intestinal metaplasia in situ in C57BL/6 mice after oral administration of MNU. A second group of mice was treated with H. pylori (n = 10 for each group). (F) Representative immunohistochemical images display α‐SMA staining in both H. pylori ⁻ and H. pylori ⁺ GC intestinal metaplasia in mice (scale bar, 50 μm). Accompanying scatter plots quantify the α‐SMA⁺ CAF density in both sets of mice. (G) IHC images of low‐density and high‐density α‐SMA⁺ CAF cases in the Hong Kong and Beijing GC cohorts (scale bar, 200 μm). (H‐I) α‐SMA⁺ CAF density predicted poor survival in GC patients (Hong Kong cohort, n = 278, p = .012; Beijing cohort, n = 162, p = .025). (J) Upregulation of CAF biomarkers, including Vimentin, FAP, α‐SMA, and NG2 was associated with unfavorable clinical outcomes (TCGA cohort, n = 375).

FIGURE 2

H. pylori‐induced activation of NFKB1/RELA directly regulates the transcriptional expression of PIEZO1 in gastric cancer (GC). (A) A Volcano plot illustrates elevated expression levels of inflammation‐associated genes in H. pylori‐activated GC. (B) KEGG enrichment analysis of DEGs highlights inflammation‐related pathways, notably the NF‐κB signaling pathway. (C) JASPAR‐predicted NFKB1/RELA binding motif is identified in the PIEZO1 promoter, suggesting a regulatory relationship. (D) ChIP‐qPCR using RELA antibody pulldown. (E) Duo‐luciferase assays provide evidence that NF‐κB directly interacts with the PIEZO1 promoter to activate its expression. (F) RELA ChIP‐sequencing in Detroit 562 cells reveals a peak at the PIEZO1 promoter, further corroborating the binding of RELA to this region. (G) Pseudotime analysis indicates that NFKB1/RELA and PIEZO1 are co‐upregulated along with proliferation‐associated genes in the early stages of GC, followed by the upregulation of metastasis‐related genes. (H) A schematic diagram encapsulates the finding that NFKB1/RELA serves as the transcription factor for PIEZO1. (I) Exposure to H. pylori augments the phosphorylation level of p65, along with the expression of PIEZO1. (J) Immunofluorescence staining showed that H. pylori treatment increased the mean fluorescence of PIEZO1 (green) intensity (DAPI, blue; scale bar, 50 μm). (K and L) Knocking down NFKB1 and RELA decreased PIEZO1 expression in MKN28 and NCI‐N87. (M and O) The protein levels of NFKB1/RELA and PIEZO1 demonstrated a positive correlation in primary samples (scale bar, 200 μm for low magnification and 10 μm for high magnification). (P‐Q) PIEZO1 expression positively correlated with NFKB1 (P) and RELA (Q) from TCGA cohort, respectively.

FIGURE 3

PIEZO1 exerts oncogenic functions in gastric cancer (GC) cancer cells and regulates the downstream Hippo pathway. (A) In TCGA cohorts, PIEZO1 mRNA expression was found to be elevated in tumour tissues compared to normal tissues. (B) PIEZO1 was notably up‐regulated in tumour samples when juxtaposed with their paired adjacent non‐tumour samples, highlighting its potential role in tumourigenesis. (C) Positive correlation was observed between the expression of PIEZO1 and metastasis‐associated genes in data extracted from TCGA. (D) GSEA demonstrated that high expression of PIEZO1 was positively correlated with tumour metastatic pathways. (E) Yoda1, an activator of PIEZO1, was found to escalate the expression of genes linked to both tumour metastasis and proliferation. Conversely, silencing PIEZO1 led to a suppression of these gene expressions. (F) GO enrichment bubble diagram demonstrating the pathway of DEG enrichment after Yoda1 intervention. (G) Overexpression of PIEZO1 in the NCI‐N87 cell line was found to spur GC peritoneal metastasis, implying its functional role in the metastatic process. (H) In the TCGA cohort, a high PIEZO1 expression group exhibited an activated YAP1 signature, indicating a possible regulatory relationship in GC samples. (I) GSEA results showed that elevated PIEZO1 expression positively correlates with the downstream pathways regulated by YAP1. (J) Knockdown of IEZO1 downregulated YAP1 expression in GC cells. (K) Yoda1 significantly induced Hippo pathway activation in a time‐dependent manner. (L) Protein expression of PIEZO1 and YAP1 was co‐localized in IHC‐stained samples from GC patients (scale bar, 200 μm for low magnification and 10 μm for high magnification). (M) Downstream genes of PIEZO1 and YAP1 were co‐expressed at the single‐cell level. (N) siYAP1 significantly suppressed the migration of GC cells, and its knockdown can entirely abolish the stimulatory effects of PIEZO1 overexpression.

FIGURE 4

CTGF, a downstream gene of YAP1, promotes the viability of α‐SMA⁺ cancer‐associated fibroblasts (CAFs), thereby contributing to the gastric cancer (GC) progression. (A) GSEA indicated that elevated CTGF expression is positively associated with fibroblast migration and proliferation, suggesting its role in stromal remodeling within the tumour microenvironment. (B) CTGF was shown to significantly enhance the proliferative, migratory, and invasive capacities of CAFs in a dose‐dependent manner, reinforcing its role in tumour stroma dynamics. (C) CTGF upregulated the expression of proliferation‐related genes does‐dependently. (D–F) The protein levels of α‐SMA were positively correlated with PIEZO1 and CTGF in the original samples, respectively (scale bar, 200 μm for low magnification and 20 μm for high magnification). (G–J) Overexpression of YAP1 and CTGF in cancer cells significantly promoted GC peritoneal metastasis. (K) IHC of peritoneal nodes showed overexpression of YAP1 and CTGF, leading to an increase in α‐SMA. (L) Protein‐protein interaction plot indicated that CTGF is associated with the Wnt and Hippo pathways, of which TGFBR2 and LRP1 are receptors of CTGF. (M) scRNA‐seq analysis screening CTGF receptors revealed that fibroblast highly expresses LRP1 in GC. (N) Further single‐cell sequencing revealed that among the LRP family members, LRP1 and LRP10 were prominently expressed in fibroblasts. Moreover, a positive correlation between the expression levels of CTGF and LRP1 was found in TCGA GC samples.

FIGURE 5

PIEZO1 acts as an essential bridge between cancer cells and α‐SMA⁺ cancer‐associated fibroblasts (CAFs) to regulate gastric cancer (GC) peritoneal metastasis. (A) Representative multiplex fluorescent immunohistochemical staining depicts the tumour microenvironment of H. pylori ⁺ and H. pylori ⁻ GC. PIEZO1 represents green, YAP1 represents red, α‐SMA represents pink, and DAPI represents blue (scale bar, 50 μm for low magnification and 20 μm for high magnification). (B) Correlation bubble plots of cytoplasmic PIEZO1 expression levels with cytosolic YAP1 expression levels in H. pylori ⁺ and H. pylori ⁻ GC, respectively. (C) The expression of PIEZO1 is positively correlated with the Hippo pathway and CAF biomarkers from TCGA cohort. (D) Detailed scatter plot of the correlation between CAF biomarkers and YAP1, CTGF, PIEZO1 (n = 375). (E) Regulation of fibroblast proliferation was positively correlated with Yoda1 treatment (p < .001). (F and G) PIEZO1 knockdown combined with 5‐FU achieved a synergistic effect in controlling tumour dissemination in a peritoneal metastasis model. (H) The representative IHC images of PIEZO1, YAP1, proliferation marker Ki‐67 and CAF marker α‐SMA in xenografts (scale bar, 50 μm). (I–L) Quantitative IHC indices of PIEZO1, YAP1, Ki‐67, and a‐SMA. **p < .01; ***p < .001.

FIGURE 6

CTGF is a poor prognostic gene for gastric cancer (GC), and Procyanidin C1 is expected to be an effective CTGF inhibitor. (A) Representative images of IHC staining of CTGF in GC tissue microarrays from the Hong Kong cohort (scale bar, 200 μm for low magnification and 20 μm for high magnification). CTGF was mainly distributed in the cytoplasm and nearby extracellular matrix of cancer cells, while showing negative expression in the adjacent epithelial tissue. The high expression of CTGF was associated with unfavorable clinical outcomes (Hong Kong cohort, n = 278, P = .017; Beijing cohort, n = 162, p = .037; TCGA cohort, n = 375, p = .002; ACRG cohort, n = 300, p = .001). (B) Schematic diagram of the treatment strategy for GC patients. Blocking the interaction between tumour cells and cancer‐associated fibroblasts (CAFs) by inhibiting CTGF in the GC tumour microenvironment. (C) Scatter plot of computer‐aided drug screening using CTGF as a target (n = 4114, binding energy < 0). The top‐ranked Binding energy is Procyanidin C1. (D) The 3D structure of CTGF and Procyanidin C1 binding domain. (E) CCK‐8 examination of the viability of Procyanidin C1‐treated cancer cells and CAFs. (F) Schematic diagram of Procyanidin C1 treatment of NCI‐N87 and CAF co‐culture. (G) Procyanidin C1 inhibited CTGF and cell‐cycle‐related genes dose‐dependently. (H) Procyanidin C1 intervention with co‐cultured NCI‐N87 and CAF resulted in a dose‐dependent inhibition of CAF growth.

FIGURE 7

Targeting CTGF synergizes with 5‐FU to arrest peritoneal metastasis of gastric cancer (GC). (A) Schematic diagram of the in vivo experiment. (B) Photographs of peritoneal metastases in the vehicle group, 5‐FU group, Procyanidin C1 group, Pamrevlumab group, and co‐drug groups. (C) Photon quantification of GC peritoneal metastasis in NSG mice inoculated with NCI‐N87 cells and cancer‐associated fibroblasts (CAFs). (D) Survival curves of NSG mice in each intervention group. (E) The overall schematic presentation of the project. Based on the molecular features of cancer cells and enrichment of CAFs in GC, blocking the CTGF effectively disrupts the mutual remodeling of cancer cells and CAFs, inhibits the GC peritoneal metastasis, and serves as a potential novel therapeutic strategy to prolong GC patient survival.