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

Hepatocellular carcinoma (HCC) constitutes a major global health burden, with its high metastatic potential and heterogeneous metastatic sites serving as critical determinants of postoperative survival outcomes. While recent research has elucidated the involvement of ncRNAs in the metastatic progression of HCC, the precise mechanisms underlying their regulation within the tumor immune microenvironment remain incompletely understood. This study employs cutting-edge spatial transcriptomics to investigate the spatial dynamics of these interactions in HCC.

Aims & Methods

To define the role of ncRNAs in facilitating metastasis within the HCC, we selected key ncRNAs for further investigation through BLAST homology analysis and differential expression profiling between primary and metastatic liver cancer specimens. Subsequently, utilizing spatial single-cell transcriptomic data, we conducted cell-cell communication and neighborhood analysis to explore the complex interactions among tumor cells, immune cells, and stromal cells.

Results

Our findings reveal that cancer-associated fibroblasts (CAFs) promote tumor metastasis through exosome-mediated communication with tumor cells and tumor-associated macrophages (TAMs), driving epithelial-mesenchymal transition (EMT) and enhancing the migratory and invasive capacities of cancer cells. Specifically, through the spatial interaction map, we identified hsa-miR-548d-5p and POT1-AS1 as critical molecular nodes involved in the metastatic process. Tumor cells exposed to both CAFs and TAMs exhibited significantly altered gene expression profiles that favored metastasis, with CAF-derived signals priming TAMs to release pro-metastatic factors that ultimately reprogrammed neighboring cancer cells. Furthermore, we validated our findings through transwell experiments and analysis of ncRNA sequencing.

Conclusion

This study utilized spatial transcriptomics to elucidate how CAFs influence tumor cells and TAMs through exosome-mediated ncRNA signaling, promoting EMT and enhancing the migratory and invasive potential of tumor cells. These findings suggest novel therapeutic strategies targeting CAFs and their interactions with TAMs, providing new avenues for optimizing HCC immunotherapy.