Introduction

Gastric cancer (GC) remains a major global health burden and is especially prevalent in East Asia. While tumor-intrinsic genetic alterations have been well studied, the tumor microenvironment (TME) has emerged as a key driver of cancer progression. Among the components of the TME, cancer-associated fibroblasts (CAFs) play diverse roles in tumor growth, invasion, and therapy resistance. Despite this, the molecular mechanisms that regulate CAF function remain poorly defined. In particular, the role of YAP1 signaling and its upstream and downstream mediators in CAFs is not fully understood.

Aims & Methods

We aimed to investigate the regulatory mechanism and function of the NF-κB/YAP1/COL12A1 axis in CAFs and its role in gastric cancer progression. To achieve this, we combined single-cell RNA sequencing, multiplex immunohistochemistry, western blotting, CHIP-qPCR, and luciferase reporter assays. CAFs were isolated from gastric cancer tissues and subjected to in vitro manipulation, including YAP1 and COL12A1 knockdown or overexpression. Patient-derived organoids and mouse xenograft models were used to assess tumor-promoting capabilities. Additionally, conditional knockout (CKO) mice were utilized to examine the impact of YAP1 deletion on collagen expression in vivo.

Results

YAP1 was found to be highly active in matrix-producing CAFs (matCAFs), a subtype associated with poor prognosis in gastric cancer. Co-culture of fibroblasts with H. pylori revealed activation of NF-κB, which in turn directly upregulated YAP1 expression. CHIP-qPCR and luciferase assays confirmed NF-κB binding to the YAP1 promoter. YAP1 knockdown in CAFs impaired proliferation and invasion and promoted cellular senescence. COL12A1 was identified as a direct downstream target of YAP1 via CHIP-seq, and its expression was positively correlated with YAP1 in human samples. Functional studies revealed that COL12A1 deletion significantly reduced CAF viability and invasiveness, and impaired the growth and migration of co-cultured gastric cancer cells. In patient-derived organoid and in vivo models, COL12A1 promoted tumor growth and metastasis. Integrin α1/β1 was identified as a potential COL12A1 receptor mediating stromal-epithelial crosstalk.

Conclusion

Our study defines a novel molecular axis in the TME: NF-κB/YAP1/COL12A1. We demonstrate that NF-κB activation—triggered by H. pylori infection—drives YAP1 expression in CAFs, which in turn regulates COL12A1 expression. COL12A1 not only maintains CAF aggressiveness but also promotes gastric cancer progression through interaction with integrin receptors on tumor cells. These findings provide new insights into tumor-stroma interactions and suggest that targeting COL12A1 or the upstream NF-κB/YAP1 pathway may represent a promising therapeutic strategy for gastric cancer.

References

1. Chen, B., Chan, W.N., Xie, F., Mui, C.W., Liu, X., Cheung, A.H.K., Lung, R.W.M., Chow, C., Zhang, Z., Fang, C., et al. (2023). The molecular classification of cancer-associated fibroblasts on a pan-cancer single-cell transcriptional atlas. Clin Transl Med 13, e1516. https://doi.org/10.1002/ctm2.1516
2. Chen, B., Liu, X., Yu, P., Xie, F., Kwan, J.S.H., Chan, W.N., Fang, C., Zhang, J., Cheung, A.H.K., Chow, C., et al. (2023). H. pylori-induced NF-κB-PIEZO1-YAP1-CTGF axis drives gastric cancer progression and cancer-associated fibroblast-mediated tumour microenvironment remodelling. Clin Transl Med 13, e1481. https://doi.org/10.1002/ctm2.1481.
3. Zhang, J., Chen, B., Li, H., Wang, Y., Liu, X., Wong, K.Y., Chan, W.N., Chan, A.K., Cheung, A.H., Leung, K.T., et al. (2023). Cancer-associated fibroblasts potentiate colorectal cancer progression by crosstalk of the IGF2-IGF1R and Hippo-YAP1 signaling pathways. J Pathol 259, 205–219. https://doi.org/10.1002/path.6033.
4. Wong, K.Y., Cheung, A.H.-K., Chen, B., Chan, W.N., Yu, J., Lo, K.W., Kang, W., and To, K.F. (2022). Cancer-associated fibroblasts in nonsmall cell lung cancer: From molecular mechanisms to clinical implications. Int J Cancer 151, 1195–1215. https://doi.org/10.1002/ijc.34127.

Introduction

Src-family tyrosine kinases (SFKs) are mutated or aberrantly regulated in up to 80% of colorectal cancers and are central drivers of oncogenic signaling. Therapeutically targeting SFKs has proven difficult due to compensatory signaling and mechanisms that stabilize these proteins. Their membrane localization and stability are normally governed by N-terminal myristoylation, a co-translational modification of Gly2 (glycine) following initiator methionine removal. Loss of this modification disrupts subcellular localization and exposes Gly2 to N-degron pathways, which mediate proteasomal degradation based on the identity and modification state of the N-terminus.

Aims & Methods

We aimed to define how omission of N-terminal myristoylation affects SFK stability and degradation, and to identify the responsible E3 ligases. To this end, we employed peptide pulldowns combined with mass spectrometry, in vitro binding assays, and AlphaFold3-based interaction predictions. Functional validation included knockdown of NMT1/2 and DCAF10, CRISPR/Cas9-mediated knockout of LYN, and re-expression of N-terminal Lyn mutants. In vitro ubiquitination assays with purified DCAF10-DDB1-CUL4A complex confirmed E3 ligase activity toward acetylated Gly substrates.

Results

Loss of myristoylation led to increased turnover of Src-family kinases. Free N-terminal Gly residues were recognized by the known CUL2-ZYG11B/ZER1 complex. Unexpectedly, we also observed aberrant N-terminal acetylation of Gly2, which triggered degradation via the DCAF10-DDB1-CUL4A E3 ligase complex. Knockdown of DCAF10 stabilized acetylated Gly–Lyn in cells, and in vitro assays confirmed direct recognition and ubiquitination of this substrate. Both pathways acted in parallel, ensuring efficient clearance of non-myristoylated Lyn variants.

Conclusion

Our findings identify a dual N-degron surveillance mechanism targeting non-myristoylated SFKs: CUL2-ZYG11B/ZER1 acts on free Gly residues, while DCAF10-DDB1-CUL4A targets aberrantly acetylated Gly. This quality control system prevents the accumulation of mislocalized or dysfunctional SFKs. Beyond degradation, N-terminal modifications also regulate localization and signaling: while myristoylation anchors SFKs at membranes, non-myristoylated or G2A-mutant forms—typically N-terminally acetylated—accumulate in the nucleus when export is blocked, suggesting that myristoylation actively restricts nuclear localization. Given emerging evidence for nuclear SFK functions, our data suggest that alternative N-terminal modifications and their recognition by distinct degradation pathways fine-tune SFK stability, localization, and oncogenic output—highlighting new opportunities for targeted cancer therapies.

References

Timms, R. T. et al. A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation. Science 365, (2019).

Disclosure

We declare no conflict of interest

Introduction

Over 50% of Crohn’s disease (CD) patients develop intestinal fibrosis, often resulting in strictures. Although anti-inflammatory therapies have improved disease management, fibrotic complications continue to represent a major unmet clinical challenge. Emerging evidence suggests that the gut microbiota may contribute to fibrosis independently of inflammation¹, yet the microbial signatures associated with different stages of fibrosis remain poorly defined.

Aims & Methods

We performed a meta-analysis of RNA-seq data using the IBD TaMMA framework² on fibrotic CD and healthy tissue. Mucosal fibroblasts, epithelial, and endothelial cells were isolated via FACS and profiled transcriptomically. Epithelial-derived organoids and co-culture systems were used to model host–microbe interactions. Fibroblasts stimulated with Roseomonas mucosa lysates (10 µg/mL) were assessed for fibrosis markers (α-SMA, vimentin, FAP) by immunofluorescence and qRT-PCR (ACTA2, PDPN, FAP).

Results

Gene ontology analysis revealed enrichment of antimicrobial immune responses during fibrosis progression. Microbial profiling indicated a consistent enrichment of Roseomonas mucosa in CD samples across fibroblasts, epithelial, endothelial, and immune cell compartments. Notably, fibroblasts stimulated with R. mucosa lysates showed increased expression of fibrosis markers compared to controls.

Conclusion

These findings suggest that Roseomonas mucosa or its components may influence the transcriptional programming of mucosal cells and contribute to fibrosis development in CD. Ongoing spatial transcriptomics and RNA-scope analyses aim to define the spatial and temporal dynamics of microbial–host interactions in fibrotic tissue, advancing our understanding of microbiota-driven fibrosis.

References

1.  Watanabe, D. & Kamada, N. Contribution of the gut microbiota to intestinal fibrosis in crohn’s disease. Front Med (Lausanne) 9, 826240 (2022).
2. Massimino, L. et al. The Inflammatory Bowel Disease Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) framework. Nat. Comput. Sci. 1, 511–515 (2021).