Background: The role of N1-methyladenosine (m1A) in cancer is poorly understood. Here we explored the function of RNA methyltransferase TRNA methyltransferase 61A (TRMT61A) in colorectal cancer (CRC) and its potential as a therapeutic target.
Methods: RNA m1A levels were assessed through liquid chromatography-mass spectrometry. The expression and clinical significance of TRMT61A were investigated across five human CRC cohorts. The function of TRMT61A was elucidated using CRC cell lines, patient-derived organoids, xenografts, and transgenic mouse models. Integrated analyses of m1A-sequencing and RNA-sequencing revealed the underlying mechanisms of TRMT61A. A nanoparticle-based small interfering RNA (siRNA) delivery system and a specific inhibitor were developed to target TRMT61A. The efficacy and safety of targeting TRMT61A were assessed.
Results: Our research revealed a consistent increase in TRMT61A expression and total RNA m1A levels within primary CRCs. High TRMT61A expression was associated with poor prognosis of CRC patients. Through CRISPR/Cas9 screenings, we identified TRMT61A as the most essential gene among m1A regulators. Furthermore, we established that TRMT61A promoted CRC tumorigenesis and progression by enhancing the mRNA stability of critical targets in an m1A-dependent manner. In particular, TRMT61A boosted the mRNA stability of one cut homeobox 2 (ONECUT2), which in turn triggered son of sevenless homolog 1 (SOS1) transcription, leading to the induction of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling in CRC. Notably, our study underscored the safety and substantial anti-CRC effects achievable by inhibiting TRMT61A using nanoparticle-encapsulated siTRMT61A or our newly discovered small molecule compound, pentagalloylglucose.
Conclusions: Our study unveiled the tumor-promoting role of TRMT61A in CRC via the m1A-ONECUT2-SOS1-MAPK/ERK pathway. Targeting TRMT61A showed promise as a therapeutic strategy for treating CRC.
Background: Blood-based cell-free DNA (cfDNA) methylation testing has emerged as a promising approach for multi-cancer early detection (MCED), holding the potential to improve cancer survival rates. However, traditional bisulfite-based methods often encounter sensitivity limitations in detecting early-stage malignancies or certain cancer types. In the INSPECTOR study, we developed a MCED and cancer signal origin (CSO) system specifically designed for early-stage or hard-to-detect cancers, including those of the lung, breast, colorectum, liver, esophagus, stomach, pancreas, and ovary.
Methods: We established a comprehensive methylation marker discovery database (n = 6,342) by integrating public datasets (n = 4,699) and in-house samples (n = 1,643), all processed using human TET (hTET) enzyme-assisted whole-methylome sequencing (GM-seq). This enabled the design of a targeted panel encompassing 155,362 methylated CpG sites. Leveraging hTET-assisted high-depth next-generation sequencing (NGS), our blood test achieved a median unique depth of 1,093×. Multicenter case-control cohorts, including various pathological subtypes, were used for training, validation, and independent validation of MCED and CSO models, and to verify the clinical feasibility.
Results: Clinical validation was conducted across multi-center case-control cohorts, including 1,071 participants in the training set, 581 in the validation set, and 824 in the independent validation set. The MCED assay demonstrated robust performance with a specificity of 99.1% and sensitivity of 83.2% in the training set, 99.0% and 81.8% in the validation set, and comparable results in the independent validation set (99.0% specificity, 81.9% sensitivity). Notably, sensitivity reached 65.5% for stage I cancers, 79.7% for stage II, and 71.3% for stages I-II combined. The sensitivities for different cancer types were as follows: esophageal (79.2%), gastric (76.1%), colorectal (86.2%), pancreatic (66.7%), liver (100.0%), lung (72.9%), breast (88.9%), and ovarian (87.9%). The CSO model exhibited strong accuracy, with top-1 cancer origin prediction rates of 87.9% (validation) and 87.4% (independent validation), rising to 95.1% and 94.5% for top-2 predictions, respectively. For stage I cancers specifically, the top-1 accuracy was 85.5%.
Conclusions: These findings underscore the efficacy of the hTET-assisted cfDNA methylation sequencing system across diverse cancer types, particularly in early stages. Enzyme-assisted NGS test of methylated cfDNA thus enhances the clinical utility of non-invasive blood-based screening.
Background: Brain metastasis, a leading cause of death in patients with lung adenocarcinoma (LUAD), arises from tumor cells adapting to the unique microenvironment of the brain through metabolic remodeling regulated by key oncogenes. Here, we aimed to determine the role of high mobility group protein box 3 (HMGB3) in regulating tumor cell metabolism to promote the progression and brain metastasis of LUAD.
Methods: A LUAD cell model predisposed to brain metastasis was established, followed by differential gene expression analysis. HMGB3 expression was quantified via single-cell RNA sequencing (scRNA-seq) and immunohistochemistry, with clinical relevance assessed in two retrospective cohorts: the primary LUAD and the LUAD brain metastasis cohorts. Gene enrichment analysis of scRNA-seq and bulk RNA-seq data, along with Western blotting, were performed to identify HMGB3-associated pathways. Co-immunoprecipitation combined with mass spectrometry was used to detect HMGB3-interacting proteins. Gain-of-function, loss-of-function and rescue experiments targeting HMGB3 downstream pathways were conducted in vitro and in vivo.
Results: HMGB3 expression was significantly elevated in both primary LUAD lesions and brain metastatic foci, and its upregulation was strongly associated with poor prognosis in LUAD patients, as well as in those with concomitant brain metastasis. HMGB3 enhanced the migration, invasion, and epithelial-mesenchymal transition (EMT) capabilities of LUAD cells in vitro and promoted the development of brain metastasis in vivo. Mechanistically, HMGB3 recruited and interacted with single-stranded DNA-binding protein 1 (SSBP1), inducing its nuclear translocation and reprogramming mitochondrial metabolism. This process elevated cytoplasmic reactive oxygen species levels, which subsequently activated the phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathway through downregulating phosphatase and tensin homolog (PTEN), ultimately promoting tumor cell proliferation, migration, invasion, and EMT.
Conclusions: This study demonstrated HMGB3 as a key regulator of the brain metastasis of LUAD, orchestrating tumor cells’ metabolic adaptation to the brain microenvironment through modulation of mitochondrial metabolism, thereby offering potential therapeutic targets for LUAD brain metastases.
Background: Metastasis is the leading cause of cancer-related mortality, with circulating tumor cell (CTC) clusters serving as highly efficient precursors of distant metastasis. Survival of CTC clusters in the bloodstream is the primary contributor to tumor metastasis. However, the underlying mechanisms of how CTC clusters respond to the blood environment and drive metastasis remain elusive. This study aimed to elucidate the potential mechanisms that enable CTC clusters to adapt and survive in the bloodstream.
Methods: CTC clusters were detected using a microfluidic system in cancer patients, as well as in patient-derived xenograft (PDX), cell line-derived xenograft, and syngeneic models. The key molecules responsible for the adaptive survival of CTC clusters were characterized using RNA-sequencing (RNA-seq), gene interference, and flow cytometry. To investigate the underlying mechanisms of adaptive survival, RNA-seq, targeted metabolomics, isotope tracing experiments, chromatin immunoprecipitation (ChIP) sequencing, and immunofluorescence (IF) staining were employed. The therapeutic potential of survival pathway inhibitor combined with chemotherapy drug was evaluated in patient-derived CTCs and the PDX model.
Results: CTC clusters exhibited superior survival and metastatic capacity compared to single CTCs and were associated with adverse clinical outcomes. The unfolded protein response mediator protein kinase R-like endoplasmic reticulum kinase (PERK) was activated in CTC clusters and maintained S-adenosylmethionine (SAM) availability, facilitating their adaptive survival in the bloodstream. Mechanistically, PERK mediated the upregulation of activating transcription factor 4 (ATF4), which enhanced methionine adenosyltransferase 2A (MAT2A) expression, contributing to SAM synthesis. Increased SAM enhanced H3K4me3 modification of the platelet-derived growth factor B (PDGFB) promoter, leading to elevated PDGFB secretion and its accumulation in the intercellular region within CTC clusters. PDGFB functioned as a shared survival signal, triggering the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway via platelet-derived growth factor receptor beta (PDGFRβ), supporting CTC cluster survival in the bloodstream. Inhibition of PERK and PDGFRβ profoundly impaired the survival signaling and suppressed the metastatic dissemination of CTC clusters.
Conclusions: Our findings revealed a PERK/MAT2A/PDGFB axis that confers adaptive survival capabilities to CTC clusters in the bloodstream. Targeting this survival signaling pathway represents a promising therapeutic strategy for metastatic cancer.
Background: The prognosis for non-small cell lung cancer (NSCLC) patients with extensive brain metastases (BMs) treated with radiotherapy alone remains poor. Based on the synergistic potential of radiotherapy and angiogenesis inhibitors, we initiated this phase II study to assess the efficacy and safety of combining bevacizumab (Bev) with fractionated stereotactic radiotherapy (FSRT) in managing extensive BMs in NSCLC patients who had stable extracranial disease.
Methods: Patients with extensive BMs from NSCLC, deemed unsuitable for stereotactic radiosurgery, were prospectively enrolled following multidisciplinary tumor board evaluation. Patients received FSRT (40 Gy in 10 fractions or 30 Gy in 5 fractions) in combination with Bev (7.5 mg/kg on day 1 prior to FSRT and on day 21 post-FSRT). The primary endpoint was intracranial progression-free survival (IPFS). Secondary endpoints included overall survival, progression-free survival, quality of life (QOL), and toxicities. For comparison, NSCLC patients with extensive BMs treated with whole-brain radiotherapy (WBRT) plus FSRT or FSRT alone were matched 1:1 with the study group (Bev + FSRT) using the propensity score matching.
Results: One hundred and six patients were included in the Bev + FSRT group, with a median follow-up duration of 35.8 months. The median IPFS was 18.3 months (95% confidence interval, 15.2-23.3 months). The Bev + FSRT group showed a significant improvement in IPFS compared to both the WBRT + FSRT group (9.6 months, P < 0.001) and the FSRT alone group (8.9 months, P < 0.001). Treatment was well tolerated, with grade 1 radiation necrosis in 1 patient. Bev + FSRT treatment significantly reduced tumor volume (P < 0.001), peritumoral edema volume (P = 0.004), and vascular leakage (P < 0.001). Furthermore, QOL was significantly improved after Bev + FSRT treatment, particularly in patients with symptomatic extensive BMs.
Conclusion: These findings support the combination of Bev and FSRT as a safe and effective treatment strategy for extensive BMs in NSCLC patients, offering improved intracranial disease control and symptom relief while avoiding the neurotoxicity associated with WBRT. A randomized trial is warranted to validate the findings of the current study.
Trial registration: ClinicalTrials.gov, NCT04345146. Registration date: 2020-02-22.