Introduction

We previously demonstrated that SCNN1B is a potential tumor suppressor in colorectal cancer (CRC), however, its role in the tumor microenvironment is largely unknown. In this study, we have constructed a conditional, colon specific SCNN1B knockout mouse model, and investigated the effects of SCNN1B knockout on colorectal tumorigenesis, and the associated alterations in the gut microbiome and tumor immune microenvironment.

Aims & Methods

Conditional SCNN1B knockout mouse was established, and then crossed to CDX2-CreERT2 mouse to generate colon specific SCNN1B knockout mouse model. To knockout SCNN1B, mice were injected with tamoxifen (100mg/kg, i.p., 6 times). After SCNN1B knockout, the mice were subjected to the Azoxymethane (AOM)/Dextran Sodium Sulfate (DSS)-induced colorectal tumorigenesis. Gut barrier function was determined by FITC-dextran permeability assay. Mucosal and fecal microbiota in SCNN1B knockout mice and control mice were determined by 16S rRNA sequencing and shot gun metagenomic sequencing, respectively. The tumor immune cell infiltration was determined by flow cytometry.

Results

Colon-specific SCNN1B knockout mice were successfully constructed. Compared to wildtype controls, SCNN1B knockout significantly increased tumor number (P<0.05) and load (P<0.05) in colon. Notably, gut permeability was increased in SCNN1B knockout mice, as evidenced by increased levels of circulation FITC-dextran in the gut permeability assays. Analysis of the mucosal microbiota revealed that knockout of SCNN1B led to decreased microbial diversity according to alpha- and beta-diversities as compared to wildtype counterparts, suggesting that knockout of SCNN1B promoted gut dysbiosis. SCNN1B knockout mice were enriched in the bacterial genera of Lawsonibacter, Duncaniella, and Desulfovibrionaceae, together with the depletion of Geosporobacter. In terms of microbial function, SCNN1B knockout mice microbiota was enriched in dark sulphur oxidation activity, which is frequently associated with pathogenic processes. Meanwhile, flow cytometry demonstrated the loss of SCNN1B promoted immunosuppression, characterized by increased infiltration of myeloid-derived suppressor cells (MDSCs) and decreased CD8+ T cell activation.

Conclusion

SCNN1B functions as a tumor suppressor in CRC. Colon specific knockout of SCNN1B in mice dysregulated gut microbiota and impaired antitumor immune response, which might contribute to the pathogenesis of 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

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.

Introduction

Immune checkpoint blockade (ICB) for treatment of colorectal cancer (CRC) has been approved for clinical use since 2017. However, the approval of PD-L1 inhibitor for CRC has been encumbered by the lack of durable clinical benefits. Increasing evidence from other cancer types support that PD-L1 expression correlates to the efficacy of ICB and tumor PD-L1 is essential for ICB-mediated tumor cytotoxicity [1-3]. Concomitantly, many studies have also demonstrated the infrequent expression of PD-L1 in CRC cells [4, 5], thereby alluding to the possibility of low/absent PD-L1 expression as a primary obstacle to ICB efficacy.

Aims & Methods

Given the integral role of tumour microenvironment (TME) in shaping the tumour’s natural history, we sought to elucidate the role of tumour stroma in regulating CRC PD-L1 expression. To this end, we aim to rescue tumor PD-L1 and assess its utility in ICB-mediated anti-tumour immunity.
​To explore the role of different cell types in the TME on CRC PD-L1 expression, cocultures between major cell compartments of the TME and CRC cell lines were performed. Further various omics approaches were utilised to characterise the crosstalk between CRC and stromal cells that saw significant suppression of CRC PD-L1 expression.

Results

Myeloid cells are able to exert a significant suppression of PD-L1 expression in CRC and this suppression is regulated at a post-transcriptional level. The underlying mechanism is orchestrated by CRC metabolic shift from glycolysis to oxidative phosphorylation in response to harsh metabolic competition within the TME.

Conclusion

We have identified potential cellular compartment of the TME and mechanisms which may explain, in part, low/absent CRC PD-L1 expression. This can thus serve to evaluate appropriate external manipulations that can alleviate CRC PD-L1 suppression and explore the possibility of combinatory therapy with anti-PD-L1 ICB to improve its clinical efficacy and durability.

References

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4. Llosa, N.J., et al., The Vigorous Immune Microenvironment of Microsatellite Instable Colon Cancer Is Balanced by Multiple Counter-Inhibitory Checkpoints. Cancer Discovery, 2015. 5(1): p. 43-51.
5. Taube, J.M., et al., Association of PD-1, PD-1 Ligands, and Other Features of the Tumor Immune Microenvironment with Response to Anti–PD-1 Therapy. Clinical Cancer Research, 2014. 20(19): p. 5064-5074.