supplementary materials and methods, Tables 1-3: Supplementary Table 1: Clinical Pathological Factors and CYP3A5 Expression Level Based on Immunohistochemisry in HCC Tissue Microarray. Supplementary Table 2: The summary demographics of patients participated in overall survival and tumor free survival analysis. Supplementary Table 3: Univariate Analysis of Clinical Variables Associated With Recurrence and Survival.

Lung adenocarcinoma (LUAD) is a leading cause of cancer-related mortality worldwide, with limited treatment options due to inherent chemotherapy resistance. Ferroptosis, an iron-dependent form of regulated cell death, has recently emerged as a promising strategy to overcome this resistance. Inducing ferroptosis may sensitize LUAD cells to chemotherapy, but its molecular regulators and therapeutic potential remain largely unexplored. This study aims to investigate the role of Distal-less homeobox 6 (DLX6) in regulating ferroptosis and its contribution to LUAD progression. To elucidate the function of DLX6 in LUAD, we employed a multi-omics approach, integrating TCGA data analysis, in vitro cell culture models, and in vivo xenograft experiments. Transcriptome sequencing, chromatin immunoprecipitation (ChIP) assays, and dual-luciferase reporter assays were utilized to uncover the underlying molecular mechanisms. We found that DLX6 is significantly upregulated in LUAD and associated with poor patient prognosis. Functional studies showed that silencing DLX6 suppressed cell proliferation, triggered ferroptosis, and sensitized LUAD cells to chemotherapy. Mechanistically, DLX6 acted as a transcriptional activator of Glutathione Peroxidase 4 (GPX4), a central regulator of ferroptosis, by directly binding to its promoter region. Inhibition of DLX6 triggered ferroptosis in LUAD cells, enhancing treatment sensitivity to cisplatin. Our findings highlight DLX6 as a key regulator of ferroptosis and cisplatin sensitivity in LUAD. By promoting GPX4 expression and inhibiting ferroptosis, DLX6 plays a crucial role in LUAD progression. This study provides valuable insights into the molecular mechanisms driving LUAD and offers a potential therapeutic target for sensitizing cisplatin treatment and improving chemotherapy resistance.

Supplementary Figure S7: ROS enrichment and the dissociation of mTOR-Ribosome are necessary for the function of CYP3A5

Supplementary Fig. 1. Filter ICD related circRNAs.

Supplementary Fig. 8. cEMSY activates antiviral immune signaling.

Supplementary Fig. 6. In vitro and in vivo characterization of designed circRNA.

Background: Non-small cell lung cancer (NSCLC) is a type of lung cancer with high mortality. Mesenchymal-to-epithelial transition (MET) fusions are present in NSCLC and are associated with tumorigenesis. The Ephrin type-B receptor 4 (EPHB4)-MET fusion gene is a recently discovered gene, and there is a lack of research on its oncogenicity and driving mechanisms. In this study, we focused on the oncogenic properties and mechanism of the EPHB4-MET fusion gene. Methods: To create EPHB4-MET gene fusion cells, BEAS-2B cells were infected with lentivirus harboring EPHB4-MET. The expression efficiency was measured using reverse-transcriptase polymerase chain reaction (RT-PCR). Subsequently, cell counting kit-8 (CCK-8), crystal violet staining, and transwell assays were performed to investigate the oncogenicity of EPHB4-MET gene fusion. Finally, dual luciferase assay and chromatin immunoprecipitation (ChIP) were used to probe the binding of homeobox domain transcription factor A9 (HOXA9) to the EPHB4 promoter. Results: EPHB4-MET expression increased after infection with lentivirus, indicating that the gene fusion cell construction was successful. In addition, EPHB4-MET promoted MET activation of the downstream AKT pathway. Genes harboring EPHB4-MET significantly promoted cell proliferation, cell migration, and invasion. Subsequent research showed that via binding to the EPHB4 promoter region, HOXA9 induced the expression of EPHB4-MET, and knockdown of HOXA9 inhibited EPHB4-MET-induced oncogenic properties. Conclusion: In this study, we demonstrated that HOXA9 drove the expression of the fusion gene EPHB4-MET and exerted a tumorigenesis signature. HOXA9 may be a useful target for both diagnosis and treatment in patients with EPHB4-MET fusion genes in lung cancer.

RATIONALE:During tumorigenesis, approximately 20% of EGFR-mutant lung adenocarcinomas (LUADs) progress rapidly to aggressive subtypes. Multi-omics analyses of stage I LUAD cohorts have revealed that centrally located lesions exhibit enhanced tumorigenic potential compared with peripheral counterparts, whereas the underlying mechanisms remain elusive. OBJECTIVES:To define the spatial-clinical determinants of early aggressive progression in EGFR-mutant LUAD and develop a lineage-based mechanistic framework connecting regional microenvironmental constraints with epithelial cell-state remodeling and invasive acquisition. METHODS:We conducted an integrated multi-omics analysis combining clinical cohort data (n = 277), single-cell and spatial transcriptomics, and functional studies in genetically engineered mouse models to identify spatial-clinical patterns. Mechanistic studies were carried out using mouse models, 3D organoids, and controlled oxygen interventions to investigate the effects of hypoxia on cellular transformation. MEASUREMENTS AND MAIN RESULTS:Our analysis revealed that centrally located lesions display enhanced tumorigenic potential compared to peripheral counterparts, driven by hypoxic niche. Hypoxic preconditioning (10% O2) induced ribosome collisions in EGFR-driven mouse models and organoids, activating the ZAKα-MAPK-c-Fos axis to disrupt alveolar lineage imbalance, characterizing as suppressing alveolar epithelial factor NKX2-1 while elevating stem-like progenitor FOXD1. Therapeutic hyperoxia (60% O2) restored lineage balance and attenuated tumorigenesis. CONCLUSIONS:A preexisting hypoxic niche is a key spatial determinant of early malignant progression in EGFR-mutant LUAD, engaging ribosome-collision signaling through the ZAKα-MAPK-c-Fos axis and promoting alveolar lineage imbalance. Restoring oxygenation, either through controlled hyperoxia or pharmacologic inhibition of this pathway, may help curb tumorigenesis and rapid progression of centrally located lesions.

Objectives: Circular RNA (circRNA) is linked to the pathogenesis of various malignancies mediated by cancer-associated fibroblasts (CAFs). However, their role and molecular mechanisms in lung adenocarcinoma (LUAD) remain largely unexplored. Methods: CAFs and normal fibroblasts (NFs) isolated from LUAD patients were subjected to RNA sequencing to identify differentially expressed circRNAs. Candidate circRNAs with differential expression were quantitatively validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in CAFs and plasma from LUAD patients. Their prognostic value was assessed by survival analysis using the Kaplan-Meier method. To validate the function of SPECC1 circular RNA (circSPECC1) in CAFs, knockdown and overexpression vectors were constructed. In vitro co-culture systems and functional assays were employed to evaluate the impact of circSPECC1 on the growth and metastatic potential of LUAD cells. Furthermore, RNA pull-down, RNA immunoprecipitation, mass spectrometry, and qRT-PCR were used to investigate the underlying mechanism of circSPECC1. Results: Several differentially expressed circRNAs were identified between CAFs and NFs, from which six core circRNAs were selected based on fold-change criteria. The expression patterns of these core circRNAs, as determined by RNA sequencing, were confirmed by qRT-PCR in CAFs (vs. NFs) and in plasma from LUAD patients (vs. normal). Among them, circSPECC1, which we are interested in, showed significant differential expression and was significantly correlated with advanced pathological stages and LUAD cohorts with poor clinical outcomes. Additionally, circSPECC1 is a cytoplasm-localized circRNA generated by back-splicing of exon 4 of the SPECC1 gene. Overexpression of circSPECC1 in CAFs promoted LUAD cell migration and invasion without affecting LUAD cell proliferation. Mechanistically, circSPECC1 was found to bind to heterogeneous nuclear ribonucleoprotein K (hnRNPK) and to enhance the stability of chemokine (C-C motif) ligand 2 (CCL2) mRNA. Conclusion: CircSPECC1 is an emerging prognostic indicator in LUAD and is associated with the migration and invasion of LUAD cells induced by CAFs, potentially via its interaction with hnRNPK and modulation of CCL2 mRNA stability. Our study provides novel insights into developing CAF-targeted therapies in LUAD.

Background: Lung adenocarcinoma (LUAD) remains a leading cause of cancer-related mortality worldwide, and effective biomarkers for prognosis and therapeutic guidance are still lacking. TRPM4, a sodium-related ion channel, has been implicated in tumor progression; however, its role in LUAD, particularly at the single-cell level, remains unclear. Methods: Single-cell RNA sequencing data were used to analyze the cellular distribution of TRPM4 in LUAD. TRPM4-related genes were identified through correlation analysis in the TCGA cohort, followed by construction of a prognostic model using Cox and LASSO regression analyses. The model was validated in an independent GEO dataset. Functional enrichment and drug sensitivity analyses were performed to explore the underlying mechanisms and therapeutic implications. Random forest analysis was applied to identify key genes, and in vitro experiments were conducted to validate their biological functions. Results: A TRPM4-related prognostic signature was established, demonstrating robust predictive performance in both training and validation cohorts. High-risk patients were characterized by activation of cell cycle and DNA replication pathways, showing differences in computationally predicted sensitivity to multiple chemotherapeutic and targeted agents. Furthermore, RNPEPL1 was identified as a key gene and experimentally validated to promote LUAD cell proliferation, migration, and invasion. Conclusions: The TRPM4-related signature and RNPEPL1 may provide novel insights for risk stratification and personalized therapeutic strategies.

BACKGROUND:With the rising incidence of colon cancer, several studies have indicated that aquaporin 1 (AQP1) expression is associated with the development of colon cancer. This study aims to elucidate the potential molecular mechanisms between them. METHODS:We screened data from The Cancer Genome Atlas (TCGA) database and retrospectively examined AQP1 protein expression in 127 colon cancer patients to analyze the relationship between AQP1 expression and pathological stages, prognosis. We created stable colon cancer cell lines with differential AQP1 expression, the effect of AQP1 expression on the proliferation and migration of colon cancer cells was assessed by in vitro and in vivo studies, and explored potential molecular mechanisms through Western blotting. RESULTS:High AQP1 expression was associated with poorer survival (overall survival [OS], p = .028) in colon cancer patients from the TCGA database. Similarly, retrospective clinical data indicated that high AQP1 expression was associated with reduced disease-free survival and OS (p = .036 and p = .017, respectively). The low-expressing AQP1 colon cancer cells exhibited a decrease in proliferation and migration ability of colon cancer cells compared to the overexpressing AQP1 group (p < .05) in vitro and in vivo. Immunohistochemistry and western blotting experiments validated heightened expression of N-cadherin, vimentin, and claudin- 1 in the tumor tissues of the overexpressing AQP1 group. Conversely, reduced AQP1 expression resulted in decreased expression of claudin- 1. CONCLUSIONS:AQP1 correlates with unfavorable prognosis in colon cancer and potentially enhances the proliferation and migration of colon cancer by up-regulating claudin-1 expression.

Ubiquitin-specific protease 7 (USP7), a deubiquitinase, is involved in tumor progression. However, its roles in pancreatic neuroendocrine neoplasms (pNENs) remain unclear. The main objective of this study was therefore to investigate the molecular mechanism of how USP7 promoted pNEN progression. Proteomics and ubiquitin-omics were used to identify the substrates for USP7. We investigated the roles of USP7 and histone H1.2 in DNA repair in pNEN cells using comet assays and γ-H2AX immunofluorescence. The synergistic effects of cisplatin and the USP7 inhibitor, P005091, were evaluated using CCK-8, colony formation, and EdU assays. Western blot analysis was conducted to characterize the PI3K/AKT/mTOR signaling pathway. In vivo, the efficacy of the combination therapy was tested in xenograft models. The results showed a significant increase in USP7 levels in the tissues and cells of pNENs. Furthermore, USP7 was found to promote the proliferation, migration, and invasion of pNENs both in vitro and in vivo. Mechanistically, USP7 facilitated DNA repair through its interaction with histone H1.2 and the activation of the PI3K/AKT/mTOR pathway. The combination of cisplatin and P005091, a USP7 inhibitor, synergistically inhibited tumor growth and DNA repair in both in vitro and in vivo models, without exhibiting significant toxicity. In conclusion, USP7 was a key regulator of DNA repair in pNENs. The combination of cisplatin and P005091 therefore holds promise as a therapeutic strategy for pNENs.

Esophageal cancer is a major contributor to the global cancer burden, yet comprehensive global assessments of its burden across subtypes are lacking. This study examines the incidence, risk factors, and trends of esophageal cancer by histological and anatomical subtypes, with stratification by sex and age across different countries, using GLOBOCAN 2022 estimates, Cancer Incidence in Five Continents data, and country-level socioeconomic, lifestyle, and metabolic risk-factor indicators. Here we show that Eastern Asia has the highest age-standardized rate (ASR) of incidence of esophageal squamous cell carcinoma (ESCC) (7.2 per 100,000 population) and upper- and middle-third esophageal cancer (6.0), whereas Northern Europe exhibits the highest ASR of incidence of esophageal adenocarcinoma (EAC) (3.2) and lower‑third esophageal cancer (3.6). EAC and lower‑third esophageal cancer generally share risk factors and exhibit increasing trends; in contrast, ESCC and the upper- and middle-third esophageal cancer share a different risk profile and show an overall decreasing trend. In developed regions, EAC and lower‑third esophageal cancer are more common, whereas in developing regions, ESCC and upper- and middle-third esophageal cancer predominate. Our findings may assist the development of tailored prevention strategies based on locally emerging patterns of esophageal cancer subtypes and relevant risk factors. There is a need for more insights into subtype differences, temporal trends and related risk factors of oesophageal cancer. Here, the authors find that the incidence, risk factors, and trends of oesophageal cancer vary significantly by histological and anatomical subtypes in different populations globally.

Supplementary Fig. 7. In vitro-transcribed cEMSY combined with anti-PD-1 promotes antitumor cure.

Supplementary Fig. 2. Characteristics of cEMSY circRNA and expression profiles in LUAD.

Supplementary Fig. 3. cEMSY induces ICD in vitro.

Vascular invasion is associated with metastasis and poor prognosis in patients with lung adenocarcinoma (LUAD). Recent studies have highlighted the role of intercellular mitochondrial transfer, mediated by both contact-dependent and contact-independent mechanisms, in tumor progression. Here, we reveal an abnormal accumulation of mitochondria in vascular endothelial cells invaded by tumor cells. Further in vivo and in vitro experiments demonstrated that tumor cells can transfer mitochondria with highly expressed mitochondrial tRNA-derived small RNAs (tsRNAs), mt-5'tiRNA-34-GlnTTG, to endothelial cells. Mechanistic investigations indicated that mt-5'tiRNA-34-GlnTTG binds to FUBP3 and facilitates its translocation, ultimately enhancing ribosomal assembly efficiency and translation rates in endothelial cells. This molecular cascade leads to increased endothelial cell proliferation and migration, thereby promoting LUAD metastasis. Moreover, we demonstrated the potential clinical translational value of lipid nanoparticles(LNPs) encapsulating mt-5'tiRNA-34-GlnTTG inhibitors in animal experiments. Utilizing LNPs encapsulating mt-5'tiRNA-34-GlnTTG inhibitors effectively suppressed lung cancer metastasis in in vivo models. These findings reveal a novel mechanism of LUAD progression mediated by mitochondrial tsRNA transfer and highlight a promising therapeutic strategy for limiting metastatic spread.

Depression imposes substantial individual and societal burdens, yet more than 80