Introduction

The widespread presence of N1-methyladenosine (m¹A) modifications controls RNA metabolism and is pivotal to fundamental biological processes. However, the understanding of RNA m¹A involved in cancer is still minimal. Here we reported for the first time the role and underlying mechanism of RNA m¹A methyltransferase TRMT61A in colorectal cancer (CRC), one of the most common causes of cancer-related deaths, and assessed its clinical significance.

Aims & Methods

RNA m¹A level was detected by liquid chromatography-mass spectrometry. Targeted CRISPR/Cas9 dropout screening was conducted to explore essential m¹A regulators. The expression and clinical implication of TRMT61A were investigated in three different human CRC cohorts (n=379). The role of TRMT61A was elucidated via in vitro CRC cell lines, patient-derived CRC organoids models, and in vivo CRC xenograft mouse models. The underlying mechanisms of TRMT61A were studied by integrative m¹A sequencing and RNA sequencing analyses. Nanoparticle-mediated siRNA delivery system was developed to deplete TRMT61A expression. Meanwhile, a potent TRMT61A inhibitor was identified by structure-based virtual screening. The therapeutic potential of targeting TRMT61A in vivo was assessed through different CRC mouse models.

Results

Higher RNA m¹A levels were observed in primary CRCs compared to paired adjacent normal tissues (n=24, P<.05), inferring the abnormal change of RNA m¹A profile during CRC development. Among different m¹A regulators, TRMT61A was identified as the top essential gene in different CRC cell lines using CRISPR/Cas9 screening. TRMT61A catalyzed RNA m¹A modification and was overexpressed in 81.3% (122/150) and 84.0% (42/50) of primary CRCs from two independent cohorts (both P<.001). Accordingly, TRMT61A mRNA expression was positively correlated with global RNA m¹A levels of CRCs (P<.05). Of note, high TRMT61A protein expression predicted poor survival of CRC patients (n=179, P<.05). Functionally, TRMT61A overexpression increased CRC cells proliferation, migration, and invasion, but suppressed apoptosis; while knockout of TRMT61A exerted opposite effects in vitro and significantly inhibited CRC xenograft growth, and lung and liver metastasis in vivo. Mechanistically, ONECUT2 was identified as a downstream target of TRMT61A by m¹A sequencing and RNA sequencing. Wild-type but not catalytically inactive TRMT61A increased the m¹A level of ONECUT2 to stabilize its mRNA, leading to elevated ONECUT2 expression that activated MAPK/ERK pathway. Notably, depletion of ONECUT2 or inhibition of MAPK/ERK abrogated the tumor-promoting role of TRMT61A, implying that TRMT61A induced ONECUT2-MAPK/ERK axis to facilitate CRC. Finally, we explored the therapeutic potential of targeting TRMT61A via genetic depletion or pharmacological inhibition of TRMT61A. We generated PEG-PLGA-based nanoparticles for delivery of TRMT61A-siRNA which posed a strong inhibitory effect against CRC growth in vivo. Meanwhile, our structure-based virtual screening of 100,100 compounds identified a highly potent and selective TRMT61A inhibitor PGG. PGG could directly bind to TRMT61A protein, as is demonstrated by thermal shift assay and drug affinity responsive targets stability assay. Treatment with PGG suppressed CRC xenograft growth and intestinal tumorigenesis of Apc^Min/+ mice, accompanied by decreased RNA m¹A levels.

Conclusion

We unravel for the first time the connections between TRMT61A-induced m¹A modification and CRC development. TRMT61A could serve as a prognostic marker and therapeutic target in CRC.

References

Zhang C, Jia G. Reversible RNA Modification N(1)-methyladenosine (m¹A) in mRNA and tRNA. Genomics Proteomics Bioinformatics 2018;16:155-161.
Shi H, Chai P, Jia R, et al. Novel insight into the regulatory roles of diverse RNA modifications: Re-defining the bridge between transcription and translation. Mol Cancer 2020;19:78.
Siegel RL, Miller KD, Goding Sauer A, et al. Colorectal cancer statistics, 2020. CA Cancer J Clin 2020;70:145-164.

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

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

In recent years, emphasis has been shifted on to understanding the role of N1-methyladenosine (m1A) in tumor progression. While little is known about its regulatory effect on mRNA and its role in metastatic colorectal cancer (CRC).

Aims & Methods

We performed methylated RNA immunoprecipitation sequencing with tumor tissues and tumor-adjacent normal tissues from three CRC patients to determine the profile of m1A methylation in CRC. Expression of Diaphanous-related formin 3 (DIAPH3) and its correlation with clinicopathological characteristics of CRC were evaluated using immunohistochemistry and online datasets. The role of DIAPH3 in the migration and invasion of CRC cells was evaluated by wound healing and transwell assays. Besides, the downstream targets of DIAPH3 were screened by mass spectrometry analysis. By co-transfecting DIAPH3 siRNA and Keratin 19 (KRT19) ectopic plasmid in CRC cells, the role of DIAPH3-KRT19 signaling axis was confirmed.

Results

The mRNA level of DIAHP3 and its m1A modification were increased simultaneously in CRC. Besides, high expression of DIAPH3 in CRC tissues was significantly associated with metastasis, and tended to progress to an advanced stage. After knocking down the expression of DIAPH3, the migration and invasion capabilities of CRC cells suffered notable decline, which can be rescued by overexpressing KRT19. Besides, the degradation of KRT19 might be regulated by DIAPH3 in a proteasome-dependent pathway.

Conclusion

DIAPH3 mRNA was upregulated in CRC cells through m1A methylation. Targeting DIAPH3 results in suppressed migration and invasion of CRC cells, potentially through proteasome-dependent degradation of downstream KRT19.

Introduction

Colorectal cancer (CRC) is a leading global malignancy and a major cause of cancer-related mortality. Exosomes—small extracellular vesicles—mediate intercellular communication by transferring microRNAs, which regulate gene expression and influence tumor behavior. Elevated levels of miR-1290 have been observed in CRC patient blood and tumor tissues. However, the biological function of exosomal miR-1290 in CRC progression remains poorly understood.

Aims & Methods

Plasma and tissue samples from CRC patients and healthy individuals were collected with ethical approval. Exosomes were isolated from patient plasma and CRC cell line-conditioned media using ultracentrifugation and membrane affinity methods. In vitro, CRC cells were transfected with miR-1290 mimics or inhibitors to assess effects on proliferation (CCK-8, EdU assays), clonogenicity (colony formation), apoptosis (flow cytometry), and migration (transwell assay). Candidate miR-1290 targets were predicted bioinformatically, and CBFA2T3 was validated by dual-luciferase reporter assay. Stable CBFA2T3-overexpressing CRC cell lines were established to evaluate phenotypic rescue. In vivo, CRC xenografts in nude mice were treated with exosomes derived from miR-1290–overexpressing or control cells. Tumor growth and expression of Ki-67 and PCNA were analyzed.

Results

Plasma exosomal miR-1290 levels were lowest in healthy controls and progressively increased across advanced adenoma, early-stage CRC, and advanced CRC groups. Notably, miR-1290 distinguished advanced CRC from controls with an AUC of 0.77 and moderately distinguished early CRC (AUC = 0.69). Consistent with clinical findings, miR-1290 expression was significantly elevated in CRC tissues versus adjacent normal mucosa and in CRC cell lines compared to normal colonic epithelial cells. CRC cells also secreted exosomes enriched in miR-1290. In vitro, miR-1290 mimics enhanced CRC cell proliferation, DNA synthesis, and clonogenic potential, without significantly affecting apoptosis or migration. Bioinformatic analysis identified CBFA2T3 as a key target of miR-1290. Dual-luciferase assays confirmed direct binding of miR-1290 to the 3′-UTR of CBFA2T3, resulting in its downregulation. In CRC cells, miR-1290 upregulation—via mimics or exosomal transfer—decreased CBFA2T3 mRNA and protein levels. Restoration of CBFA2T3 expression significantly counteracted miR-1290-induced proliferation and colony formation. In vivo, nude mice injected with CRC cells and treated with miR-1290–enriched exosomes developed significantly larger tumors compared to controls. Tumors exhibited reduced CBFA2T3 levels and elevated expression of proliferation markers Ki-67 and PCNA, supporting the tumor-promoting role of exosomal miR-1290.

Conclusion

Exosomal miR-1290 correlates with CRC progression and shows promise as a non-invasive diagnostic biomarker. Functionally, it promotes CRC proliferation and tumor growth by targeting and downregulating the tumor suppressor CBFA2T3, highlighting its potential as a therapeutic target.