Introduction

Colorectal cancer ranks second in cancer-related deaths worldwide. O-GlcNAc glycosylation, as a crucial post-translational modification, is overexpressed in various cancer. Hippo pathway effector YAP can be O-GlcNAc glycosylated to promote development of some types of cancer. However, the effects of YAP's O-GlcNAc glycosylation on colorectal cancer growth are poorly understood, and the development of targeted drugs is still blank.

Aims & Methods

We aim to develop a novel anti-colorectal cancer drug targeting OGT and investigate its anti-tumor mechanism. The expression level of OGT, YAP and YAP's phosphorylation were examined through WB, IHC and bioinformatics analysis. YAP's O-GlcNAc glycosylation and OGT/YAP interaction were assessed by molecular dynamics simulations and Co-IP. OSMI-1, an OGT inhibitor, was used to construct an OSMI-1's folate-targeted diselenide-responsive liposome. Basic characteristics of liposome were identified. Next, the effect of OSMI-1's liposome on colon cancer development and invasiveness was examined, and the change of Hippo-YAP pathway as well as downstream gene expression were assessed. Finally, the metabolism, drug distribution, toxicity, and therapeutic effect of OSMI-1's liposome were investigated in subcutaneous tumor mice model.

Results

Based on TCGA and GTEx database, gene of YAP (p<0.05) and OGT (p<0.01) were remarkably overexpressed in colon cancer. High level OGT gene expression was linked with poor OS (p=0.016) and DFS (p=0.02). OGT (p=0.05) and YAP (P<0.01) expression raised with stage. WB and IHC revealed an over-expression of YAP and OGT in both colon cancer tissue and cell lines. Co-IP verified the YAP/OGT interaction and molecular dynamics simulations predicted glycosylation sites of serine 103, 366, and 216, and threonine 213 and 41, which might affect the neighboring amino acids phosphorylation. OSMI-1's folate-targeted diselenide-responsive liposome appeared as stable particles of average 205.5 nm in size. Drug loading amount was 280ug/ml. Mass spectrum indicated a standard (Cholesterol-Se)₂ in liposome and it stably released over 90% of OMSI-1 within 16 hours in GSH/H2O2 environment. IC50 of the drug for colon cancer was 1240ug/ml, while it presented low toxicity to normal colon cell. OSMI-1's liposome effectively inhibited the proliferation and invasion ability of colon cancer cell CACO2 and HCT116. It suppressed glycosylation of YAP but increased its phosphorylation level relatively and YAP presented more cytoplasmic translocation. The downstream target genes CTGF, Bcl-2, E-cadherin, and Vitemin were all significantly downregulated. In nude mice, the drug was significant accumulated at the subcutaneous tumor site within 12 hours and its half-life time was 20 hours. After treated with OSMI-1's liposome for 15 days, the average subcutaneous tumor volume of mice was significantly smaller than OSMI-1 group and the control group (16.9mm³ vs 27.6mm³ vs 46.6mm³, p<0.05, n=15), and there was no significant change in the body weight of the mice.

Conclusion

In summary, we successfully constructed an OSMI-1's folate-targeted diselenide-responsive liposome, which exhibited well targetability and efficacy against colon cancer tissue with low toxicity to normal tissue. Furthermore, we demonstrating that OGT inhibition can reduce the YAP's O-GlcNAcylation and enhance its phosphorylation thus suppress YAP nuclear translocation, to suppress the development of colon cancer. Our research provides a brand new targeted therapy for colon cancer, with significant application value and broad development prospects.

References

1. Ferlay J, Colombet M, Soerjomataram I, et al. Cancer statistics for the year 2020: An overview [published online ahead of print, 2021 Apr 5]. Int J Cancer. 2021;10.1002/ijc.33588. doi:10.1002/ijc.33588
2. Ohta Y, Fujii M, Takahashi S, et al. Cell-matrix interface regulates dormancy in human colon cancer stem cells. Nature. 2022;608(7924):784-794. doi:10.1038/s41586-022-05043-y
3. Yang Y, Yan Y, Yin J, et al. O-GlcNAcylation of YTHDF2 promotes HBV-related hepatocellular carcinoma progression in an N6-methyladenosine-dependent manner. Signal Transduct Target Ther. 2023;8(1):63. Published 2023 Feb 10. doi:10.1038/s41392-023-01316-8
4. Li X, Wu Z, He J, et al. OGT regulated O-GlcNAcylation promotes papillary thyroid cancer malignancy via activating YAP. Oncogene. 2021;40(30):4859-4871. doi:10.1038/s41388-021-01901-7

Introduction

Colorectal carcinogenesis is intricately associated with dynamic interactions between gut microbiota and immune microenvironment remodeling(1). While microbial colonization within tumors creates complex pathophysiological conditions, emerging evidence paradoxically indicates suppressed tumor proliferation in bacteria-rich regions compared to microbe-deficient areas(2), suggesting indirect oncogenic mechanisms. This study investigates the immunomodulatory axis of microbiota-CRC interactions, particularly focusing on bacterial translocation-triggered immune reprogramming. Our previous findings showed that gallic acid ameliorates colitis through barrier enhancement and bacterial containment, we now extended it to CRC models to delineate how microbial translocation modulates tumor-associated immune landscapes.

Aims & Methods

1) To elucidate the effect of interaction between immune cells and gut bacteria in CRC progression and the potential mechanism;
2) find novel therapeutic targets at the microbiota-immunity interface for precision CRC intervention.
We used AOM/DSS and APCmin/+model to evaluate the effect of Gallic acid on colonic carcinogenesis. Single cell sequencing was used to deep investigate the effect of bacteria translocation on immune landscape. Conditional gene edited animals were used for mechanistic study.

Results

GA suppresses CRC progression by reinforcing barrier function and reducing microbial translocation. Mechanistically, bacterial invasion drives neutrophil infiltration, whose-derived reactive oxygen species (ROS) promote tumor oxidative stress and establish an immunosuppressive niche (elevated Tregs, reduced and impaired CD8+T cells). Further investigations identified tumor necrosis factor receptor 2 (TNFR2) as a pivotal regulator of this process: TNFR2 supports Treg expansion under oxidative stress but exacerbates CD8+ T cell dysfunction. Genetic ablation of TNFR2 in Tregs or CD8+ T cells attenuates CRC progression, and therapeutic targeting of TNFR2 with neutralizing antibodies achieves comparable efficacy.

Conclusion

Our study establishes a “gut microbiota-neutrophil-TNFR2” immunosuppressive axis that promotes CRC development.

References

(1) C. C. Wong, J. Yu, Gut microbiota in colorectal cancer development and therapy. Nat Rev Clin Oncol, (2023).
(2) J. L. Galeano Nino et al., Effect of the intratumoral microbiota on spatial and cellular heterogeneity in cancer. Nature 611, 810-817 (2022).

Introduction

Lipid metabolism dysregulation plays a crucial role in colorectal cancer (CRC) progression. Our perivious study found that upregulation of Slc26a9 promotes colorectal tumorigenesis (Zhang M, et al. Cell Death Discov. 2024). This study investigates the role of Slc26a9 in regulating lipid metabolism through the RRBP1-HSPA5 axis in CRC.

Aims & Methods

In this study, we assessed the expression of Slc26a9 in mouse colorectal cancer tissues using immunohistochemistry and immunofluorescence (IF). Slc26a9-interacting proteins in Colo201 cells, such as RRBP1, were identified through immunoprecipitation followed by mass spectrometry (IP-MS). Bioinformatic analysis of the TCGA and GEO databases was performed to evaluate the correlation between Slc26a9 and RRBP1 expression and their clinical significance. Protein interaction sites were predicted using Protein-Protein Docking. Furthermore, the effects of Slc26a9 overexpression on mitochondrial metabolism, lipid metabolism, the RRBP1-HSPA5 pathway, and endoplasmic reticulum (ER) stress were investigated using Oil Red O staining, Nile Red staining, qRT-PCR, Western blotting (WB), co-immunoprecipitation (co-IP), and IF.

Results

In this study, we employed the widely recognized AOM/DSS protocol to establish a colorectal cancer model in C57BL/6J mice. We observed a significant upregulation of Slc26a9 in colorectal cancer tissues compared to adjacent non-cancerous tissues, consistent with our previous findings in human colorectal cancer samples (Zhang M, et al. Cell Death Discov. 2024). To investigate the precise molecular mechanisms by which Slc26a9 regulates the development of CRC, we performed IP-MS to identify Slc26a9-interacting proteins in Colo201 cells. RRBP1 was found to have the highest binding affinity with Slc26a9 and has been shown to localize at mitochondria–ER contact sites, where it plays a role in regulating organelle interactions. Moreover, GO and KEGG enrichment analyses indicated that Slc26a9 is associated with lipid metabolism in CRC cells. Overexpression of Slc26a9 also promoted endoplasmic reticulum stress, mitochondrial energy metabolism, and lipid accumulation in Colo201 cells. Moreover, Slc26a9 enhanced the expression of markers related to endoplasmic reticulum stress (PERK-ATF4, IRE1α-XBP1, and ATF6), mitochondrial metabolism (Mitofilin, MFN1, and COXIV), and lipid metabolism (FASN, ACC, and SCD1). Moreover, Slc26a9 upregulation also enhanced the expression of the endoplasmic reticulum stress–related signaling pathway RRBP1/HSPA5. Protein-protein docking analysis revealed the formation of multiple hydrogen bonds between Slc26a9 and RRBP1, indicating a high degree of structural complementarity and strong binding affinity between the two proteins. Co-IP and IF experiments demonstrated that Slc26a9 interacts with RRBP1 and co-localizes with it. In addition, bioinformatics analysis of GEO dataset (GSE39582) showed a positive correlation between Slc26a9 and RRBP1 expression in human CRC tissues, with their co-high expression predicting poor overall survival.

Conclusion

These findings highlight that Slc26a9 interacts with RRBP1 to activate the HSPA5-mediated ER stress response, driving mitochondrial metabolism-lipid metabolic reprogramming and promoting CRC progression. Slc26a9 and RRBP1 may serve as potential prognostic biomarkers, and their axis represents a novel therapeutic target for CRC.

Introduction

Chemoresistance is common in colorectal cancer (CRC) and it contributes to treatment failure and relapse. Cancer stem cells (CSCs) is a sub-population of tumor-initiating cells that drive chemoresistance in CRC. However, the interaction between gut microbiome and CSCs remains unclear.

Aims & Methods

In this study, we investigated the effect of a CRC pathogen, Peptostreptococcus anaerobius, in promoting colorectal CSC stemness and chemoresistance, and elucidated its molecular mechanism of action. The effect of P. anaerobius on CRC stemness and chemoresistance were investigated in human CSCs, and validated in Apc^Min/+ and AOM/DSS induced CRC tumorigenesis mouse models. Attachment of P. anaerobius on human CSCs was evaluated by transmission electron microscopy and scanning electron microscopy. P. anaerobius proteins interacting with CSC receptors were identified by GST pulldown and surface plasmon resonance (SPR) assays. The protein binding sites were determined by crosslinking mass spectrometry (MS) and docking.

Results

Intratumoral P. anaerobius abundance was positively correlated with stemness-related genes, including CD133 and LGR5 in CRC patients (n=73). P. anaerobius gavage promoted CRC formation and elevated Cd133⁺ and Lgr5⁺ cell populations in CRC tissues from Apc^Min/+ mice and AOM/DSS-induced CRC models. Consistently, P. anaerobius co-culture increased the self-renewal capacity and sphere formation in human colorectal CSCs, along with increased expression of CD133 and LGR5. In line with its stemness boosting effect, P. anaerobius promoted resistance to oxaliplatin and 5-fluorouracil treatment in human CSCs in vitro, and in AOM/DSS-induced CRC tumorigenesis model. Mechanistically, we revealed that the outer membrane protein NlpC/P60 from P. anaerobius interacts with the Annexin A2 receptor on colorectal CSCs. Co-immunoprecipitation, SPR assays, and crosslinking MS revealed that NlpC/P60 competed with Yes-associated protein 1 (YAP) for binding to Annexin A2 (ANXA2). This interaction disrupted the intracellular binding between ANXA2 and YAP, leading to the release of YAP from the cell membrane, promoting YAP translocation into nucleus to activate Hippo signaling pathway. Anti-ANXA2 neutralizing antibody blocks the binding between P. anaerobius and colorectal CSCs and abrogated the effect of P. anaerobius on promoting stemness and chemoresistance. Corrobrating these pre-clinical observations, Levels of P. anaerobius were found to be increased in chemotherapy non-responders as compared to responders in CRC patients.

Conclusion

P. anaerobius drives CRC stemness and chemoresistance via NlpC/P60- Annexin A2 interaction, leading to YAP1-driven activation of Hippo signaling pathway. Blocking NlpC/P60-Annexin interaction using Anti-ANXA2 antibody could overcome P. anaerobius-mediated chemoresistance in CRC.

Introduction

Colorectal cancer, with an estimated two million new cases each year worldwide, poses a significant socioeconomic burden on modern society. It is the second leading cause of cancer-related deaths, accounting for nearly one million premature deaths annually.

Aims & Methods

The role of Signal Transducer and Activator of Transcription 2 (STAT2) in this context is not yet understood, and no therapies targeting this transcription factor are currently being pursued. We explored the function of STAT2 in colorectal cancer by employing experimental mouse models, as well as conducting functional assays with three-dimensional tumoroids in the context of anticancer therapy.

Results

In vivo studies showed that Stat2KO mice were more resistant to developing tumors in both inflammation-driven and inflammation-independent models of experimental colorectal cancer. This unexpected outcome could not be reconciled with the known role of STAT2 in inducing cell death downstream of type I interferon signaling. We consequently performed ex vivo experiments with tumoroids derived from Stat2KO mice carrying the multiple intestinal neoplasia (Min) mutant allele of the adenomatous polyposis coli (Apc) gene. These tumoroids exhibited slower growth, were generally smaller in size, and showed a higher degree of differentiation compared to those derived from ApcMin/+ wildtype mice. Remarkably, when treated with anti-tumor agents that induce cell death through various mechanisms, tumoroids derived from ApcMin/+ Stat2KO mice were found to be highly sensitive. Additional functional studies indicated that the underlying mechanism might involve a STAT2-dependent disruption of innate immune DNA sensing.

Conclusion

Our results establish STAT2 as a promoter of colorectal cancer and a contributor to resistance against anti-tumor therapy. These findings suggest that targeting STAT2-dependent signaling could be a promising therapeutic strategy for patients with colorectal cancer.

Introduction

Both gut microbiome dysbiosis and dysregulated RNA N6-methyladenosine (m6A) modification have been independently linked to colorectal cancer (CRC) pathogenesis. Yet, how these two key factors interact during early CRC development remains unclear.

Aims & Methods

​​​​​​Aims: This study investigates YTHDF1 as a novel link between m6A epitranscriptomics and gut microbiome dysbiosis in CRC development.
Methods: We performed comparative transcriptomic analysis of YTHDF1 expression in normal colon tissue (n = 65), adenoma (n = 69), and carcinoma (n = 74) specimens from human cohorts. Intestine-specific Ythdf1 knock-in (Ythdf1^cki) mice were generated to assess the effects of YTHDF1 on gut microbiota and metabolites by shotgun metagenomic sequencing and LC-MS/MS metabolomics, respectively. Fecal microbiota transplantation (FMT) from Ythdf1^cki mice into germ-free and antibiotic-treated SPF mice was conducted to evaluate microbiota-driven tumorigenesis. Two independent human CRC cohorts, a publicly available dataset (PRJNA941834, n = 110) and a local Hong Kong cohort (n = 208), were analyzed to correlate YTHDF1 expression with microbial shifts, particularly taxa A, a CRC-associated bacterium. MTT assays assessed the effects of taxa A and its metabolites on CRC cell proliferation in vitro. The Apc^Min/+model was used to test the tumor-promoting role of taxa A in vivo.

Results

Among m⁶A regulators, YTHDF1 exhibited progressive upregulation from adenoma to CRC, suggesting an early role in tumorigenesis. Compared to WT littermates, Ythdf1^cki mice displayed gut dysbiosis and metabolic alteration prior to tumor development, characterized by enrichment of 18 bacterial species and 21 metabolites alongside depletion of 8 bacterial species and 49 metabolites. FMT from Ythdf1^cki mice accelerated tumorigenesis in both Apc^Min/+ mice and the AOM/DSS model, including antibiotic-treated SPF or germ-free recipients. Notably, Ythdf1^cki recipients showed elevated serum levels of FITC-Dextran, LPS, and LBP, coupled with reduced expression of E-cadherin, indicating compromised gut barrier integrity. Furthermore, Ythdf1^cki FMT enhanced colonic epithelial proliferation (indicated by elevated Ki67⁺ cells per crypt) and amplified pro-inflammatory responses, including marked activation of NF-κB, IL-17, and TNF-α signalings. Similarly, fecal metabolites derived from Ythdf1^cki mice significantly enhanced CRC cell proliferation compared to WT metabolites. Together, these findings establish that intestinal YTHDF1 upregulation promotes CRC initiation by remodeling the gut microbiota and metabolome. Notably, taxa A, a CRC-enriched bacterium, showed significant expansion in both Ythdf1^cki mice and recipient mice following FMT from Ythdf1^cki donors. Consistently, we observed a significant positive correlation between both YTHDF1 mRNA (PRJNA941834 dataset, p < 0.01, Spearman' s ρ = 0.34) and protein expression (Hong Kong cohort, p < 0.05, Spearman' s ρ = 0.1705) with taxa A abundance in human CRC specimens, suggesting evolutionary conservation of YTHDF1's regulation over this bacterium from mice to humans. Further functional validation demonstrated that taxa A and its metabolites promoted CRC cell proliferation in vitro. These findings were corroborated in vivo, where taxa A significantly increased intestinal tumor burden in Apc^Min/+mice.

Conclusion

Our study identifies YTHDF1 as a novel regulator of the gut microbiota, specifically enriching taxa A, which drives CRC initiation. These findings reveal a m⁶A-microbiota axis in early colorectal tumorigenesis, offering potential therapeutic targets.