Background: Colorectal cancer (CRC) is the leading cause of cancer-related death worldwide. Exosome shave emerged as crucial regulators of intercellular communication and that abundant Circular RNAs (circRNAs) are enriched within exosomes. CircRNAs are novel members of noncoding RNAs regulating cancer proliferation and progression. However, the function and regulatory mechanism of cancer-derived exosomal circRNAs in CRC remains unclear. Methods: CRC cells-derived exosomes were characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blot. CCK-8, wound healing and transwell assays, and flow cytometry assays were conducted to assess whether exosomes would affect the proliferation, metastasis, and apoptosis of CRC cells, respectively. Moreover, we performed the RNA sequencing and RT-qPCR to identify circRNAs in exosome-stimulated CRC cells. Fluorescence in situ hybridization (FISH) assay was used to detect the cellular distribution of circPACRGL. Bioinformatic analyses (StarBase 2.0) were used to pool the miRNA targets of circPACRGL. Luciferase assays were performed to verify the direct interaction. Finally, flow cytometry was used to detect the differentiation of N1-N2 neutrophils. Results: Our study identified a novel CRC-derived exosomal circRNA, circPACRGL. We found circPACRGL was significantly upregulated in CRC cells after tumor-derived exosomes addition. Moreover, circPACRGL serves as a sponge for miR-142-3p/miR-506-3p to facilitate the transforming growth factor-β1 (TGF-β1) expression. As a result, circPACRGL promoted CRC cell proliferation, migration and invasion, as well as differentiation of N1 to N2 neutrophils via miR-142-3p/miR-506-3p-TGF-β1 axis. Conclusion: Our study, the first to reveal that cancer-derived exosomal circPACRGL plays an oncogenic role in CRC proliferation and metastasis, providing mechanistic insights into the roles of circRNAs in CRC progression and a valuable marker for CRC treatment.

Background: An in-depth understanding of immune evasion mechanisms in tumors is crucial to overcome resistance and enable innovative advances in immunotherapy. Circular RNAs (circRNAs) have been implicated in cancer progression. However, much remains unknown regarding whether circRNAs impact immune escape in non-small-cell lung carcinoma (NSCLC). Methods: We performed bioinformatics analysis to profile and identify the circRNAs mediating immune evasion in NSCLC. A luciferase reporter assay, RNA immunoprecipitation (RIP), RNA pulldown assays and fluorescence in situ hybridization were performed to identify the interactions among circIGF2BP3, miR-328-3p, miR-3173-5p and plakophilin 3 (PKP3). In vitro T cell-mediated killing assays and in vivo syngeneic mouse models were used to investigate the functional roles of circIGF2BP3 and its downstream target PKP3 in antitumor immunity in NSCLC. The molecular mechanism of PKP3-induced PD-L1 upregulation was explored by immunoprecipitation, RIP, and ubiquitination assays. Results: We demonstrated that circIGF2BP3 (hsa_circ_0079587) expression was increased in NSCLC and negatively correlated with CD8⁺ T cell infiltration. Functionally, elevated circIGF2BP3 inactivated cocultured T cells in vitro and compromised antitumor immunity in an immunocompetent mouse model, and this effect was dependent on CD8⁺ T cells. Mechanistically, METTL3 mediates the N⁶-methyladenosine (m⁶A) modification of circIGF2BP3 and promotes its circularization in a manner dependent on the m⁶A reader protein YTHDC1. circIGF2BP3 competitively upregulates PKP3 expression by sponging miR-328-3p and miR-3173-5p to compromise the cancer immune response. Furthermore, PKP3 engages with the RNA-binding protein FXR1 to stabilize OTUB1 mRNA, and OTUB1 elevates PD-L1 abundance by facilitating its deubiquitination. Tumor PD-L1 deletion completely blocked the impact of the circIGF2BP3/PKP3 axis on the CD8⁺ T cell response. The inhibition of circIGF2BP3/PKP3 enhanced the treatment efficacy of anti-PD-1 therapy in a Lewis lung carcinoma mouse model. Collectively, the PKP3/PD-L1 signature and the infiltrating CD8⁺ T cell status stratified NSCLC patients into different risk groups. Conclusion: Our results reveal the function of circIGF2BP3 in causing immune escape from CD8⁺ T cell-mediated killing through a decrease in PD-L1 ubiquitination and subsequent proteasomal degradation by stabilizing OTUB1 mRNA in a PKP3-dependent manner. This work sheds light on a novel mechanism of PD-L1 regulation in NSCLC and provides a rationale to enhance the efficacy of anti-PD-1 treatment in NSCLC.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal forms of adult cancer with poor prognosis. Substantial evidence indicates that reactive oxygen species (ROS) are important modulators of aggressive cancer behavior. However, the mechanism by which ESCC cells integrate redox signals to modulate carcinoma progression remains elusive. Methods: The expression of interferon alpha inducible protein 6 (IFI6) in clinical ESCC tissues and cell lines was detected by RT-PCR and Western blotting. The correlation between IFI6 expression levels and aggressive ESCC disease stage was examined by immunohistochemistry. Bioinformatic analysis was conducted to explore the potential function of IFI6 in ESCC. ESCC cell lines stably depleted of IFI6 and ectopically expressing IFI6 were established using lentiviruses expressing shRNAs and an IFI6 expression plasmid, respectively. The effects of IFI6 on ESCC cells were determined by cell-based analyses, including EdU assay, apoptotic assay, cellular and mitochondria-specific ROS detection, seahorse extracellular flux, and mitochondrial calcium flux assays. Blue native-polyacrylamide gel electrophoresis was used to determine mitochondrial supercomplex assembly. Transcriptional activation of NADPH oxidase 4 (NOX4) via ATF3 was confirmed by dual luciferase assay. In vivo tumor growth was determined in mouse xenograft models. Results: We find that the expression of IFI6, an IFN-stimulated gene localized in the inner mitochondrial membrane, is markedly elevated in ESCC patients and a panel of ESCC cell lines. High IFI6 expression correlates with aggressive disease phenotype and poor prognosis in ESCC patients. IFI6 depletion suppresses proliferation and induces apoptosis by increasing ROS accumulation. Mechanistically, IFI6 ablation induces mitochondrial calcium overload by activating mitochondrial Ca²⁺ uniporter and subsequently ROS production. Following IFI6 ablation, mitochondrial ROS accumulation is also induced by mitochondrial supercomplex assembly suppression and oxidative phosphorylation dysfunction, while IFI6 overexpression produces the opposite effects. Furthermore, energy starvation induced by IFI6 inhibition drives endoplasmic reticulum stress through disrupting endoplasmic reticulum calcium uptake, which upregulates NOX4-derived ROS production in an ATF3-dependent manner. Finally, the results in xenograft models of ESCC further corroborate the in vitro findings. Conclusion: Our study unveils a novel redox homeostasis signaling pathway that regulates ESCC pathobiology and identifies IFI6 as a potential druggable target in ESCC.

Background: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive and treatment‐resistant tumor. The biological implications and molecular mechanism of cancer stem‐like cells (CSCs) in ESCC, which contribute to therapeutic resistance such as radioresistance, remain elusive.Methods: Quantitative real‐time polymerase chain reaction, western blotting, immunohistochemistry, and in situ hybridization assays were used to detect methyltransferase‐like 14 miR‐99a‐5p tribble 2 (METTL14/miR‐99a‐5p/TRIB2) expression in ESCC. The biological functions of METTL14/miR‐99a‐5p/TRIB2 were demonstrated in vitro and in vivo. Mass spectrum analysis was used to identify the downstream proteins regulated by TRIB2. Chromatin immunoprecipitation (IP), IP, N⁶‐methyladenosine (m⁶A)‐RNA IP, luciferase reporter, and ubiquitination assays were employed to explore the molecular mechanisms underlying this feedback circuit and its downstream pathways.Results: We found that miR‐99a‐5p was significantly decreased in ESCC. miR‐99a‐5p inhibited CSCs persistence and the radioresistance of ESCC cells, and miR‐99a‐5p downregulation predicted an unfavorable prognosis of ESCC patients. Mechanically, we unveiled a METTL14‐miR‐99a‐5p‐TRIB2 positive feedback loop that enhances CSC properties and radioresistance of ESCC cells. METTL14, an m⁶A RNA methyltransferase downregulated in ESCC, suppresses TRIB2 expression via miR‐99a‐5p‐mediated degradation of TRIB2 mRNA by targeting its 3′ untranslated region, whereas TRIB2 induces ubiquitin‐mediated proteasomal degradation of METTL14 in a COP1‐dependent manner. METTL14 upregulates miR‐99a‐5p by modulating m⁶A‐mediated, DiGeorge critical region 8‐dependent pri‐mir‐99a processing. Hyperactivation of TRIB2 resulting from this positive circuit was closely correlated with radioresistance and CSC characteristics. Furthermore, TRIB2 activates HDAC2 and subsequently induces p21 epigenetic repression through Akt/mTOR/S6K1 signaling pathway activation. Pharmacologic inhibition of HDAC2 effectively attenuates the TRIB2‐mediated effect both in vitro and in patient‐derived xenograft models.Conclusion: Our data highlight the presence of the METTL14/miR‐99a‐5p/TRIB2 axis and show that it is positively associated with CSC characteristics and radioresistance of ESCC, suggesting potential therapeutic targets for ESCC treatment.

Background: Cisplatin-based chemotherapy is a mainstay systematic therapy for advanced esophageal squamous cell carcinoma (ESCC), and cisplatin resistance, which is not uncommon, is the major barrier to improving patient outcomes. Circular RNAs (circRNAs) are novel noncoding RNAs that are implicated in cancer progression, but their involvement in modulating cisplatin responsiveness in ESCC remains unknown. Methods: Bioinformatics analysis was used to profile and identify the circRNAs involved in cisplatin responsiveness in ESCC. The chemosensitive role of cDOPEY2 was confirmed both in vitro and in vivo. The molecular mechanism of cDOPEY2 was investigated by mass spectrometry, immunoprecipitation, and ubiquitination analyses. Results: We report that a novel circRNA (cDOPYE2, hsa_circ_0008078) was markedly downregulated in cisplatin-resistant ESCC cells (ESCC-CR) compared with parental chemosensitive cells. Re-expression of cDOPEY2 substantially enhanced the cell-killing ability of cisplatin by augmenting the apoptotic process in ESCC-CR cells, which was achieved by decreasing the abundance of the antiapoptotic protein Mcl-1. Mechanistically, we showed that cDOPEY2 acted as a protein scaffold to enhance the interaction between the cytoplasmic polyadenylation element binding protein (CPEB4) and the E3 ligase TRIM25, which in turn facilitated the ubiquitination and degradation of CPEB4. The increased Mcl-1 expression in ESCC-CR cells was dependent on the binding of CPEB4 to its untranslated mRNA, and depletion of CPEB4 mediated by cDOPEY2 reversed this effect. Rescue experiments confirmed that the critical role of cDOPEY2 in maintaining cisplatin sensitivity was dependent on the depletion of CEPB4 and its downstream target Mcl-1. Clinical and in vivo data further corroborated the significant relevance of cDOPEY2 to cisplatin responsiveness in ESCC. Conclusions: We provide evidence that cDOPEY2 inhibits CPEB4-mediated Mcl-1 translation by promoting the ubiquitination and degradation of CPEB4 to alleviate cisplatin resistance, indicating that cDOPEY2 may serve as a valuable biomarker and potential therapeutic target in ESCC.

Background Transcatheter mitral valve‐in‐valve (TMVIV) and valve‐in‐ring (TMVIR) implantation for degenerated mitral bioprostheses and failed annuloplasty rings have recently emerged as treatment options for patients deemed unsuitable for repeat surgery. Methods A systematic literature review was conducted to summarize the data regarding the baseline characteristics and clinical outcomes of patients undergoing TMVIV and TMVIR procedures. Results A total of 245 patients (172 patients who underwent TMVIV surgery and 73 patients who underwent TMVIR surgery) were included in the study; 93.5% of patients experienced successful TMVIV or TMVIR implantation. The mortality rates at discharge, 30 days, and 6 months were 5.7%, 8.1%, and 23.4%, respectively. The transapical (TA) access route was used in most procedures (55.2%). The TA and transseptal (TS) access routes resulted in similar outcomes. No significant differences were observed in the short‐term outcomes between the patients who developed mitral stenosis versus mitral regurgitation as the mode of failure. Conclusions TMVIV and TMVIR implantation for degenerated mitral bioprostheses and failed annuloplasty rings are safe and effective. Both procedures, via TA or TS access, can result in excellent short‐term clinical outcomes in patients with mitral stenosis or regurgitation, but long‐term follow‐up data are currently lacking to determine the durability of these procedures.