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.

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

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

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[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
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