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.

Introduction

The inflammatory bowel disease (IBD) subgroup classification of Crohn’s disease (CD) and ulcerative colitis (UC) is based on clinical, endoscopic, histological and radiological findings. A non-invasive serological tool to aid the diagnosis of IBD is currently lacking. Extracellular matrix (ECM) proteins have been proposed as promising biomarkers of inflammatory activity, mucosal damage, and fibrosis development in IBD and may have diagnostic utility.

Aims & Methods

We aimed to investigate the diagnostic accuracy of 13 serum ECM protein epitopes representing inflammation and collagen formation and degradation. Serum from 518 newly diagnosed and treatment-naïve adult IBD patients (180 CD, 338 UC) and 205 symptomatic controls (SC) from the IBSEN III cohort (discovery cohort) were collected at diagnosis. The serum was analysed for a panel of ECM protein epitopes (C4M, C6M, C7M, PROC3, PROC4, PROC5, PROC6, PROC11, PROC16, PROC22, VICM, CTXIII, and CPa9HNE) using enzyme-linked immunosorbent assay (ELISA). We used log2-transformed protein concentrations to fit logistic models for IBD vs SC, CD vs SC, UC vs SC, and CD vs UC. We fitted models for each individual protein and for all 13 proteins combined. Backward regression with the Bayesian information criterion, starting from the full model with 13 proteins, identified models with three to four proteins.
The models fitted to the discovery cohort were validated in the independent NORDTREAT cohort of 130 newly diagnosed IBD patients (56 CD, 74 UC) and 103 SC using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve.

Results

The coefficients of models fitted to the discovery cohort identified C6M, C7M, C4M, PRO-C3, and PRO-C6 as the most important proteins for separating disease from controls. In the validation cohort, the combination of C6M, C7M and PRO-C3 reached the highest AUC of 0.65 for distinguishing IBD from SC. When fitting the model to differentiate CD from SC, the collagen VI epitope C6M was superior, reaching an AUC of 0.65 and increasing to 0.70 when combined with C7M, PRO-C3, and PRO-C6. A model with all 13 markers gave an AUC of 0.71 for CD vs SC. Only C4M showed capacity of differentiating between UC and CD with
an AUC 0.66. Lastly, differentiation of UC from SC using only C6M had an AUC of 0.62, increasing to 0.64 when adding C7M and PRO-C3 to the model.

Conclusion

This study revealed a moderate diagnostic accuracy of predicting IBD versus SC. However, the ability to separate patients with CD from SC was promising, motivating further exploration of biomarker utility for CD subgroup distinctions.

Disclosure

JH has received consulting and/or advisory board fees from: AbbVie, Alfasigma, Aqilion, Bristol Myers Squibb, Celgene, Celltrion, Eli Lilly, Ferring, Galapagos, Gilead, Hospira, Index Pharma, Janssen, Johnson & Johnson, MEDA, Medivir, Medtronic, Merck, Merck Sharp & Dohme, Novartis, Pfizer, Prometheus Laboratories Inc., Sandoz, Shire, STADA, Takeda, Thermo Fisher Scientific, Tillotts Pharma, Vifor Pharma, UCB; and speaker’s fees from: AbbVie, Alfasigma, Bristol Myers Squibb, Celgene, Eli Lilly, Ferring, Galapagos, Gilead, Hospira, Janssen, Johnson & Johnson, Merck Sharp & Dohme, Novartis, Pfizer, Shire, Takeda, Thermo Fisher Scientific, Tillotts Pharma; and research grant support from Janssen, Merck Sharp & Dohme and Takeda.
AWM has received and researcher-initiated grant from Takeda
VS has received research funding from Takeda
VA is a member of the Merck advisory board and has consulted for Janssen
JDF reports travel grants from Tillotts Pharma
VAK has received consulting and/or advisory board fees from Takeda, Tillotts Pharma, Janssen-Cilag, and speaker fees from Thermo Fisher Scientific.
MLH has received investigator-initiated research grants from Takeda, Pfizer, Tillotts, Ferring and Janssen, speaker fees from Takeda, Tillotts, Ferring, AbbVie, Pfizer, Galapagos, MSD and Meda, and advisory board fees from Takeda, Galapagos, MSD, Lilly, Janssen, Pfizer and AbbVie

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