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 ApcMin/+ 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
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