Matrix stiffness induced gallbladder fibroblasts activation and paracrine SEMA7A promotes gallbladder cancer cell epithelial-mesenchymal transition and cancer stem cell-like properties by modulating AKT/p300 signalling

Authors

Liankang Sun

Huanhuan Wang

Hanqi Li

Ruida Yang

Xuyuan Dong

Danfeng Dong

Yangwei Fan

Enxiao Li

Hao Wang

Yinying Wu

Yu Shi

Doi

10.1186/s13062-025-00683-2

PMID: 24997986 · DOI: 10.1186/s13062-025-00683-2 · Journal: Biology Direct (2025)

TL;DR

Sun and colleagues at Xi’an Jiaotong University investigate how the stiff desmoplastic stroma of gallbladder cancer (GBC) drives tumor progression through cancer-associated fibroblast (CAF) biology. Using primary human gallbladder fibroblasts (GFs) isolated from 86 patient samples and cultured on silicon or polyacrylamide hydrogels of defined stiffness (0.5 kPa soft vs. 16 kPa stiff), they show that stiff matrix activates GFs via YAP1/WWTR1 (TAZ)/TEAD1 signaling, which transcriptionally upregulates semaphorin 7A (SEMA7A). Paracrine SEMA7A then binds ITGB1 (integrin β1) — not its canonical receptor PLXNC1 — on GBC cells, activating an AKT1/EP300 cascade (p-AKT S473 → p-p300 S1834) that drives EMT and cancer stem-like traits. Co-injection xenografts confirm the cascade in vivo, and Kaplan-Meier analysis on the cohort shows stromal SEMA7A is associated with shorter overall survival.

Cohort & data

  • Primary human samples: 86 GBC tissues and adjacent nontumor tissues collected at the First Affiliated Hospital of Xi’an Jiaotong University; patients had not received chemotherapy before surgery.
  • Primary cell models: Gallbladder fibroblasts (GFs) isolated from patients with gallbladder stones and cultured within 5 passages on silicon (0.5 / 16 kPa) or polyacrylamide (0.4–25.6 kPa) hydrogels.
  • GBC cell lines: GBC-SD and NOZ.
  • Generated transcriptomics: Bulk RNA-seq of primary human GFs on 0.5 vs 16 kPa silicon gels (NCBI SRA PRJNA1182410).
  • Public bulk transcriptomics (re-analyzed): GEO GSE76633 (9 GBC tumor / normal pairs); GEO GSE139682 (10 tumor / 10 adjacent — SEMA7A vs P300 correlation); GEO GSE276931 (7 tumor / 5 adjacent — GSVA AKT pathway score, GSEA).
  • Public scRNA-seq (re-analyzed): scRNA-seq from National Omics Data Encyclopedia OEP00001237 (13 GBC patients; 16 cell types identified including ACTA2+ and MFAP5+ CAF subpopulations).
  • In vivo: Subcutaneous xenografts in 4-week-old male BALB/c nude mice (n=6 per group) co-injecting 5×10⁵ GBC-SD with GFs (sh-NT or sh-SEMA7A) at 1:1.
  • Assays: Masson trichrome and IHC staining, transwell invasion, tumorsphere formation, ELISA, Western blot, Co-IP, ChIP-qPCR, RT-qPCR.

Key findings

  • GBC stroma exhibits substantial collagen deposition by Masson staining and α-SMA+ (ACTA2) CAF infiltration; primary human GFs cultured on 16 kPa hydrogel adopted a spread, α-SMA-high myofibroblast morphology while 0.5 kPa GFs remained quiescent PMID:24997986.
  • High stiffness (16 kPa vs 0.5 kPa) increased SEMA7A mRNA in primary human GFs 3.21-fold by RNA-seq (PRJNA1182410); SEMA7A was the most strongly upregulated SEMA in an RT-qPCR screen of the family.
  • SEMA7A was higher in GBC than normal gallbladder in GSE76633, and in scRNA-seq (OEP00001237, 13 patients) co-expressed with YAP1 and the CAF markers ACTA2, MFAP5, and COL1A1 in CAF subclusters.
  • Stromal SEMA7A correlated positively with fibrillar collagens (COL1A1, COL1A2, COL4A1, COL4A2, COL8A1), ACTA2, PDGFRA, TGFBR1, and TGFBR2.
  • High stromal SEMA7A by IHC was associated with larger tumor size (P = 0.006), lymph node metastasis (P = 0.001), and shorter overall survival (Kaplan-Meier, log-rank).
  • Stiffness drives nuclear translocation of YAP1; YAP1 shRNA, WWTR1 (TAZ) siRNA, or verteporfin abolished stiffness-induced GF activation and SEMA7A secretion. ChIP confirmed TEAD1 binding at the SEMA7A promoter, enriched under 16 kPa.
  • Conditioned medium from stiff-cultured GFs increased GBC-SD/NOZ invasion and tumorsphere formation; this was abrogated by SEMA7A knockdown in GFs and rescued by recombinant human SEMA7A.
  • SEMA7A signals through ITGB1, not PLXNC1: an integrin β1 neutralizing antibody and an RGD peptide (blocking the SEMA7A integrin-binding motif) blocked rh-SEMA7A-induced invasion, EMT and stemness, whereas PLXNC1 shRNA or anti-PLXNC1 antibody did not. Co-IP showed HA-SEMA7A binding to ITGB1 was lost when the RGD motif was mutated to RGE.
  • Downstream of SEMA7A/ITGB1, rh-SEMA7A induced concurrent phosphorylation of AKT1 at S473 and EP300 at S1834; PI3K inhibition by LY294002 blocked p-EP300 S1834 induction. A p300 S1834A phosphodead mutant failed to rescue invasion, EMT, and stemness in p300-depleted GBC cells, while wild-type p300 did.
  • p300 inhibition by C646 or shRNA abrogated rh-SEMA7A-induced invasion, EMT, and tumorsphere formation. ChIP showed rh-SEMA7A increased p300 occupancy and H3K27ac at SNAI1 (Snail) and ZEB1 promoters; the p300 S1834A mutation abolished H3K27ac.
  • In vivo: GBC-SD + GF co-injection xenografts had larger tumors and elevated Ki-67, α-SMA, vimentin (VIM), CD44, EPCAM and reduced E-cadherin (CDH1) compared with GBC-SD alone; SEMA7A knockdown in GFs partly reversed all of these (n=6 per group).

Genes & alterations

This is a functional/mechanistic study; no somatic mutations were profiled. The reported gene-level findings are expression, phosphorylation, and protein–protein interaction events:

  • SEMA7A — Upregulated in stiffness-activated GFs and in GBC stroma; secreted as a soluble paracrine factor; high stromal expression associates with poor overall survival, large tumor size, and nodal metastasis in the 86-patient cohort.
  • ITGB1 (integrin β1) — Functional SEMA7A receptor on GBC cells; binds via the SEMA7A RGD motif (lost in RGD→RGE mutant). Neutralizing antibody blocks downstream signaling and phenotypes.
  • PLXNC1 — Canonical SEMA7A receptor, not required for SEMA7A-induced GBC EMT/stemness; PLXNC1 knockdown blocked PAK phosphorylation but not invasion/EMT/stemness.
  • EP300 (p300) — Phosphorylated at S1834 downstream of SEMA7A/ITGB1/AKT. Phosphorylation is required for H3K27ac at SNAI1 and ZEB1 promoters; a p300 S1834A mutant is null.
  • AKT1 — Phosphorylated at S473 in response to rh-SEMA7A in GBC cells; PI3K inhibition by LY294002 blocks p-p300 S1834.
  • YAP1 / WWTR1 (TAZ) / TEAD1 — Mechanotransducers in GFs; required for stiffness-induced SEMA7A transcription. TEAD1 binds the SEMA7A promoter.
  • CAF activation markers up under high stiffness: ACTA2, CCN2 (CTGF), FN1, COL1A1, COL4A1.
  • EMT / stemness markers in GBC cells (downstream readouts): up — VIM, EPCAM, CD44, ALDH1A1, SNAI1, ZEB1; down — CDH1 (E-cadherin).
  • ERBB pathway (EGFR, ERBB2, ERBB3, ERBB4) — Discussed only in context: prior GBC whole-exome work (cited as ref. 48 [Li et al. Nat Genet 2014]) identified recurrent ERBB-pathway mutations that activate AKT, the same kinase the authors place upstream of p300 in their cascade.

Clinical implications

  • Therapeutic target: The authors propose SEMA7A — or its receptor ITGB1, or the downstream AKT1/EP300 axis — as a candidate target to disrupt CAF–tumor crosstalk in GBC. Tool compounds active in their system: verteporfin (YAP inhibitor, blocks SEMA7A induction in GFs), LY294002 (PI3K inhibitor, blocks p-p300 S1834), and C646 (p300 inhibitor, blocks EMT/stemness).
  • Prognostic biomarker: High stromal SEMA7A by IHC associates with larger tumors (P = 0.006), lymph node metastasis (P = 0.001), and shorter overall survival in the 86-patient single-institution cohort.
  • No direct treatment recommendation is made; all compounds tested are preclinical tool inhibitors, not approved GBC therapies.

Limitations & open questions

  • Single-institution cohort (n = 86, Xi’an Jiaotong University); survival and clinicopathological associations are not externally validated. No multivariable analysis is reported in the main text.
  • Functional work uses two GBC cell lines (GBC-SD, NOZ); no patient-derived organoids or genetically diverse models.
  • Co-injection xenografts use immunodeficient nude mice — the immune contribution to SEMA7A biology (a known immune regulator) is not assessed.
  • The 16 kPa “stiff” condition is biologically motivated but a single point; a stiffness dose–response for SEMA7A induction is only shown in supplementary figures and not quantified across primary GFs from independent donors.
  • Membrane-bound vs soluble SEMA7A activities are not formally separated; the work emphasizes soluble SEMA7A but does not exclude a role for membrane-bound forms.
  • The bridge to human somatic-mutation data is indirect: the authors cite (ref. 48) the Li et al. Nat Genet 2014 whole-exome GBC study showing ERBB-pathway mutations that converge on AKT, but they do not stratify their own cohort or models by ERBB-pathway genotype. Whether GBC tumors with intrinsic AKT activation (e.g., ERBB-mutant) are differentially dependent on stromal SEMA7A is an open question.

Citations from this paper used in the wiki

  • “SEMA7A expression on 16 kPa stiffness was increased 3.21-fold in comparison to 0.5 kPa soft gel” (Results, Fig. 2A).
  • “high SEMA7A expression in GBC stroma was significantly associated with large tumour sizes (P = 0.006) and lymph node metastasis (P = 0.001)” (Results, Table S3).
  • “soluble SEMA7A, secreted by activated gallbladder fibroblasts under matrix stiffness microenvironment, directly interacts with integrin β1 receptor on GBC cells via its RGD motif” (Results).
  • “targeting PlexinC1 cannot effectively abrogate the rh-SEMA7A induced invasion, EMT and cancer stemness of GBC cells” (Results).
  • “the simultaneous phosphorylation of p300 at S1834 and Akt at S473” downstream of rh-SEMA7A (Results, Fig. 7C).
  • “Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the erbb pathway” — Li et al. Nat Genet 2014;46(8):872–6 (reference 48; the upstream cohort behind cBioPortal study gbc_shanghai_2014).

This page was processed by crosslinker on 2026-05-11.