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

Upper GI involvement in Crohn’s disease (CD) is thought to occur in 13% to 15% of cases. However, despite expansion of the therapeutic armamentarium for CD over the last 30 years, there continues to be minimal data on optimal therapeutic strategies for upper GI CD. This knowledge gap presents major clinical challenges in managing this subset of patients.

Aims & Methods

We conducted an extensive systematic review using multiple databases investigating pharmacological or dietary interventions for patients with upper GI CD. We included fully published, English-language studies that compared at least two active interventions with a placebo from 1990 to 2025. Due to the lack of a standardized definition for “upper GI” CD, we included studies that reported data on CD affecting the esophagus to the jejunum. We excluded studies that included <10 upper GI CD patients, reported data for “small bowel CD” in general or limited to ileal CD, or did not report data separately for upper GI CD. There is no age limitation.

Results

Among 1206 citations, 14 observational studies (11 retrospective, 3 prospective) and 1 post hoc of an RCT met our inclusion criteria, amounting to 487 patients with UGI CD. Majority of patients had concomitant ileocolonic involvement. Five studies with 137 patients had esophageal CD. Follow-up ranged from 1 to 28 years. Interventions and reported outcomes varied. Steroids (mainly prednisone) and anti-TNF, either alone or in combination, showed the highest rates of success for clinical improvement, endoscopic healing and histologic remission for upper GI CD compared to other therapies. Other therapies used, alone or in combination, were EEN, PPI, H2RA, immunomodulators (azathioprine, 6-mercaptopurine), 5-ASA, anti-integrins, and anti-IL 12/23, showing moderate to minimal improvement in clinical or endoscopic response and remission. (Table)

Conclusion

Our comprehensive literature search of comparative studies highlights an almost void of evidence to inform therapeutic strategies in upper GI CD. Steroids and anti-TNF showed the best outcomes, but only from small observational, uncontrolled studies. Data for biologics and advanced therapies remain limited highlighting a large unmet need.

Disclosure

Financial Disclosures:
MC: none.
DA: none.
YY: has received consulting fees from Alimentiv.
VJ: has received consulting/advisory board fees from AbbVie, Alimentiv, Anaptyis Bio, Arena pharmaceuticals, Asahi Kasei Pharma, Asieris, Astra Zeneca, Avoro Capital, Bristol Myers Squibb, Celltrion, Eli Lilly, Endpoint Health, Enthera, Ensho, Ferring, Flagship Pioneering, Fresenius Kabi, Galapagos, Gilde Healthcare, GlaxoSmithKline, Genentech, Gilead, Innomar, JAMP, Janssen, Merck, Metacrine, Mylan, MRM Health, Pandion, Pendopharm, Pfizer, Protagonist, Prometheus Biosciences, Reistone Biopharma, Roche, Roivant, Sandoz, Second Genome, Sorriso, Synedgen, Takeda, TD Securities, Teva, Topivert, Ventyx, Vividion; speaker’s fees from, Abbvie, Ferring, Bristol Myers Squibb, Galapagos, Janssen Pfizer Shire, Takeda, Tillotts, Fresenius Kabi
RS: has received consulting fees from AbbVie, Alimentiv and Takeda.

Introduction

Recently, a Japanese expert consensus introduced oncological resectability criteria for hepatocellular carcinoma (HCC) [1]. Notably, these criteria do not incorporate the concept of unresectable tumors, but rather classify borderline resectability into two distinct subgroups, defined from the therapeutic perspective of hepatic resection. Based on these classifications, resectability is categorized into three groups: resectable (R), borderline resectable 1 (BR1), and borderline resectable 2 (BR2).

Aims & Methods

In this study, we aimed to assess the clinical outcomes of patients with HCC who were treated with atezolizumab plus bevacizumab (Atezo/Bev) in a real-world context, categorized by oncological resectability criteria. Between May 2018 and May 2024, a total of 1,055 patients with unresectable HCC were treated with Atezo/Bev across 26 institutions in Japan. Of these, 467 met the following inclusion criteria: (1) Child–Pugh class A, (2) Atezo/Bev administered as the first-line systemic therapy, (3) Eastern Cooperative Oncology Group performance status (ECOG-PS) of 0 or 1, (4) BR1 or BR2 classification, (5) treatment conducted within a clinical trial setting, and (6) availability of complete clinical data.

Results

Of 467 patients, 102 (21.8%) were women, and 365 (78.2%) were men. The median age was 74.0 (69.0–79.0) years. Patients were categorized into BR1 (n=153 [32.8%]) and BR2 (n=314 [67.2%]) groups. The median follow-up period was 14.3 (7.5–24.0) months. The median progression-free survival (PFS) was 9.0 months in the BR1 group and 6.8 months in the BR2 group (p = 0.014). Multivariable analysis identified the following independent prognostic factors for PFS: age ≥75 years (hazard ratio [HR], 1.309; 95% confidence interval [CI], 1.057–1.622; p=0.014), albumin–bilirubin (ALBI) grade 2 or 3 (HR, 1.494; 95%CI, 1.201–1.859; p<0.001), neutrophil-to-lymphocyte ratio (NLR) ≥3 (HR, 1.289; 95%CI, 1.030–1.613; p=027), α-fetoprotein (AFP) ≥100 ng/mL (HR, 1.523; 95%CI, 1.214–1.912; p<0.001), and BR2 classification (HR, 1.360; 95%CI, 1.076–1.718; p=0.010). The median overall survival (OS) was 25.3 months in the BR1 group and 22.3 months in the BR2 group (p = 0.048). Similarly, multivariable analysis identified the following independent prognostic factors for OS: age ≥75 years (HR, 1.522; 95%CI, 1.155–2.007; p=0.033), ALBI grade 2 or 3 (HR, 2.411; 95%CI, 1.802–3.224; p<0.011), NLR ≥3 (HR, 1.635; 95%CI, 1.233–2.169; p<0.011), AFP ≥100 ng/mL (HR, 1.530; 95%CI, 1.152–2.032; p=0.003), and BR2 classification (HR, 1.421; 95%CI, 1.048–1.927; p=0.024). When oncological resectability factors (tumor number and size, vascular invasion, and extrahepatic spread) were incorporated into the multivariable analysis, major vascular invasion emerged as a significant predictor of both PFS (HR, 3.188; 95%CI, 2.051–4.953; p<0.011) and OS (HR, 2.650; 95%CI, 1.541–4.556; p<0.001). No significant differences were observed between the BR1 and BR2 groups regarding concomitant or subsequent treatments following Atezo/Bev therapy, as indicated by the Sankey diagram (p = 0.391).

Conclusion

Among HCC patients with a low probability of surgical resection (i.e., those classified as BR1 or BR2) undergoing Atezo/Bev treatment, vascular invasion serves as a critical prognostic factor for tumor progression. Moreover, beyond tumor progression, preserved liver function also plays a pivotal role in determining the prognosis of patients treated with Atezo/Bev.

References

1. Akahoshi K, Shindoh J, Tanabe M, Ariizumi S, Eguchi S, Okamura Y, et al. Oncological Resectability Criteria for Hepatocellular Carcinoma in the Era of Novel Systemic Therapies: The Japan Liver Cancer Association and Japanese Society of Hepato-Biliary-Pancreatic Surgery Expert Consensus Statement 2023. Liver Cancer 2024; 13: 1–11.

Disclosure

Toshifumi Tada: lecture fees from AbbVie, AstraZeneca, Eisai, and Chugai
Atsushi Hiraoka: lecture fees from Eisai, Bayer, Eli Lilly, and Otsuka
Hidenori Toyoda: lecture fees from AbbVie, Eisai, Gilead, Terumo, and Bayer

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.