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).

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

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

The adhesion G protein-coupled receptor F5 (ADGRF5) belongs to seven-transmembrane receptor family which action seems to be crucial for various cancers and immune response [1-3]. Nevertheless, limited efforts have been made to explore ADGRF5 significance in gastrointestinal cancers.

Aims & Methods

The aim of the study was to assess the significance of ADGRF5 for macrophages function and its impact on gastrointestinal cancer progression. Bone marrow of wild type (WT) and ADGRF5 knockout (KO) mice was used to isolate macrophage population employing differentiation medium. M1 and M2 macrophage phenotype was obtained by polarization of naïve macrophages with a mixture of lipopolysaccharide (LPS) with interferon γ (IFN-γ) and interleukin (IL) 4, respectively. Macrophage phenotype was confirmed using real-time PCR. In vivo studies employing MC38 and PK5L1940 cell-derived allograft murine model of colon and pancreas cancers, respectively, were performed. Multiplex analysis was used to explore secretome in media obtained from tumors.

Results

In murine model of colon cancer, animals with ADGRF5 KO developed significantly smaller tumors which was accompanied by enhanced secretion of macrophage-related cytokines (such as macrophage colony-stimulating factor) in colon tumors compared to WT animals. Ex vivo analysis revealed overexpression of IL-12, nitric oxidase synthase 2 and tumor necrosis factor α in M1 macrophages with ADGRF5 KO compared with WT macrophages under LPS with IFN-γ treatment. In contrast, lower expression of arginase 1 was noted in M2 macrophages with ADGRF5 KO compared with WT macrophages treated with IL-4. In vivo studies employing allograft model of colon and pancreas cancers revealed that the administration of ADGRF5 KO macrophages led to a reduction in tumor size when compared to WT macrophages.

Conclusion

Collectively, ADGRF5 appears to be crucial regulator of gastrointestinal cancer progression through regulating macrophage function. Our study also noted that ADGRF5 depletion reprograms macrophages into M1 phenotype which is responsible for anti-tumor activity.

References

[1] Jacenik D., Hikisz P., Beswick E.J., Fichna J. The clinical relevance of the adhesion G protein-coupled receptor F5 for human diseases and cancers. Biochim Biophys Acta Mol Basis Dis. 2023; 1869: 166683. doi:10.1016/j.bbadis.2023.166683
[2] Kubo F., Ariestanti D.M., Oki S., Fukuzawa T., Demizu R., Sato T., Sabirin R. M., Hirose S., Nakamura N. Loss of the adhesion G-protein coupled receptor ADGRF5 in mice induces airway inflammation and the expression of CCL2 in lung endothelial cells. Respir Res. 2019; 20: 11. doi:10.1186/s12931-019-0973-6
[3] Tang X., Jin R., Qu G., Wang X., Li Z., Yuan Z., Zhao C., Siwko S., Shi T., Wang P., Xiao J., Liu M., Luo J. GPR116, an adhesion G-protein-coupled receptor, promotes breast cancer metastasis via the Gαq-p63RhoGEF-Rho GTPase pathway. Cancer Res, 2013; 73: 6206–6218. doi:10.1158/0008-5472.CAN-13-1049

Introduction

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome caused by mutations in the APC gene, a negative regulator of the Wnt signaling pathway. It is characterized by the development of hundreds to thousands of Wnt-driven adenomatous polyps in the colon. To prevent the inevitable progression to colorectal cancer, patients with FAP typically undergo total proctocolectomy in early adulthood. Currently, no effective medical therapy exists for FAP. Casein kinase I alpha (CKIα), another negative regulator of Wnt signaling, has emerged as a potential therapeutic target. Prior work in our lab demonstrated that, unlike APC loss, epithelial deletion of Csnk1a1 (CKIα) in the gut does not induce tumorigenesis. Instead, it activates a non-canonical DNA damage response, leading to robust p53 stabilization and suppression of tumorigenic changes. Given that TP53 mutations are typically late events in the adenoma–carcinoma sequence, early-stage lesions in FAP often retain wild-type p53. Unlike sporadic adenomas, which can be resected during colonoscopy, the high polyp burden in FAP precludes endoscopic management, underscoring the need for effective chemoprevention.

Aims & Methods

Our aim in this work was to investigated CKIα as a therapeutic target in preclinical models of FAP.
We employed Csnk1a1^fl/fl, APC^fl/fl, Villin-CreERT2, and CDX2-CreERT2 transgenic mice for in vivo studies, with tamoxifen-inducible gene ablation. Intestinal organoids derived from normal and tumor tissues were used for ex vivo experiments.

Results

In vivo, simultaneous deletion of APC and CKIα in intestinal epithelium did not further enhance Wnt target gene expression compared to APC loss alone. Instead, dual knockout (DKO) stabilized p53, promoted apoptosis, and blocked the dysplastic progression normally observed in APC-deficient gut tissue. In organoid models, treatment with the novel CKIα degrader BTX1050 selectively inhibited tumor-derived organoid proliferation, but did not fully eliminate tumor-initiating cells. We therefore tested A51, a dual CKIα and CDK7/9 inhibitor developed in our lab, which both stabilized and activated p53 and suppressed transcription of super-enhancer-driven oncogenes. A51 treatment significantly downregulated Myc and Mcl1, increased apoptosis in tumor organoids, but only partially suppressed tumor growth in vivo. Notably, A51 induced MAPK pathway activation, as indicated by increased pERK levels. This prompted combination therapy with A51 and the MEK inhibitor trametinib, which demonstrated potent synergistic anti-tumor activity in both organoid and mouse models.

Conclusion

CKIα depletion exerts anti-tumor effects in intestinal tumor models with wild-type p53. Our findings support the combination of A51 and MEK inhibition as a promising "chemical polypectomy" approach for FAP patients.

Disclosure

n/a

Introduction

Gastric cancer (GC) is a multifactorial disease, with a significant microbial contribution. Beyond Helicobacter pylori, the bacterium most commonly linked to GC, lactic acid-producing bacteria (LAB) have been frequently reported to be more abundant in later stages of the gastric carcinogenesis cascade. LAB are known for producing considerably large amounts of the oncometabolite lactate, which is strongly linked to the main hallmarks of cancer (i.e., inflammation, immune escape, angiogenesis, invasion, and metastasis).

Aims & Methods

We aimed to understand the potential association of LAB with gastric precancerous lesions (GPL) and GC in two high-risk populations (Colombian Mestizo and Han Chinese). DNA and RNA were simultaneously extracted from snap frozen gastric biopsies from subjects presenting with functional dyspepsia, GPL (atrophic gastritis, intestinal metaplasia, and dysplasia), and GC. 16S gene (DNA, n=257) and transcript (cDNA, n=129) amplicons (V4 region) were sequenced with Illumina MiSeq 2x250 bp. Alpha, beta, and per-taxa analyses were performed.

Results

H. pylori significantly impacted the diversity and composition of the gastric microbiota. Gastric active microbiota (cDNA) assessment showed that there were significant differences in beta diversity between FD controls and GPL and GC patients. Further, there were strong positive associations between LAB, including Otu00023.Lactobacillus (100% similarity to Lactobacillus gasseri), Otu00045.Limosilactobacillus (100% similarity to Limosilactobacillus fermentum), Otu00046.Oribacterium (100% similarity to Oribacterium sinus), Otu00120.Streptococcus (100% similarity to Streptococcus anginosus), and Otu00232.Limosilactobacillus (100% similarity to Lactobacillus timonensis) and disease status (GPL and GC). These findings were maintained even when Otu00001.Helicobacter (100% similarity to H. pylori) was removed from the analysis, evidencing a strong signature of LAB regardless of the presence of H. pylori in the active gastric microbiota. Enrichment of Otu00046.Oribacterium and Otu00023.Lactobacillus in GPL and GC patients, was validated when the gastric resident/transient microbiota was assessed (DNA).

Conclusion

The gastric active microbiota of GPL and GC patients from two high-risk populations geographically and ethnically diverse is significantly dominated by LAB (Otu00046.Oribacterium and Otu00023.Lactobacillus), demonstrating a potential role for these bacteria in the pathogenesis of GC. Assessment of the viable gastric microbiota appears to be more informative compared to resident/transient microbiota assessment.