Circular RNA circFCHO2(hsa_circ_0002490) promotes the proliferation of melanoma by directly binding to DND1.

Circular RNAs (circRNAs) have been documented to play crucial roles in the biology of various cancers. However, their investigation in melanoma is still at an early stage, particularly as a broader mechanism beyond acting as miRNA sponges needs to be explored. We report here that circFCHO2(hsa_circ_0002490), a circRNA encompassing exons 19 and 20 of the FCHO2 gene, exhibited a consistent overexpression in melanoma tissues. Furthermore, elevated circFCHO2 levels demonstrated a positive correlation with the malignant phenotype and poor prognosis among the 158 melanoma patients studied. Besides, we observed that heightened levels of circFCHO2 promoted melanoma cell proliferation, migration, and invasion in vitro, along with contributing to tumor growth in vivo. Furthermore, we found differences in the secondary structure of circFCHO2 compared to most other circular RNA structures. It has fewer miRNA binding sites, while it has more RNA binding protein binding sites. We therefore speculate that circFCHO2 may have a function of interacting with RNA binding proteins. Mechanistically, it was confirmed by fluorescence in situ hybridization (FISH), RNA-pull down, RNA immunoprecipitation (RIP), and western blotting assays that circFCHO2 interacts with dead end protein homolog 1 (DND1) and reverses the inhibition of the PI3K/AKT signaling pathway by binding to DND1. Our findings reveal that circFCHO2 drives melanoma progression by regulating the PI3K/AKT signaling pathway through direct binding to DND1 and may serve as a potential diagnostic biomarker and therapeutic target for the treatment of melanoma.

Melanoma Derived Exosomes Amplify Radiotherapy Induced Abscopal Effect via IRF7/I-IFN Axis in Macrophages.

Radiotherapy (RT) can induce tumor regression outside the irradiation field, known as the abscopal effect. However, the detailed underlying mechanisms remain largely unknown. A tumor-bearing mouse model is successfully constructed by inducing both subcutaneous tumors and lung metastases. Single-cell RNA sequencing, immunofluorescence, and flow cytometry are performed to explore the regulation of tumor microenvironment (TME) by RT. A series of in vitro assays, including luciferase reporter, RNA Pulldown, and fluorescent in situ hybridization (FISH) assays, are performed to evaluate the detailed mechanism of the abscopal effect. In addition, in vivo assays are performed to investigate combination therapy strategies for enhancing the abscopal effect. The results showed that RT significantly inhibited localized tumor and lung metastasis progression and improved the TME. Mechanistically, RT promoted the release of tumor-derived exosomes carrying circPIK3R3, which is taken up by macrophages. circPIK3R3 promoted Type I interferon (I-IFN) secretion and M1 polarization via the miR-872-3p/IRF7 axis. Secreted I-IFN activated the JAK/STAT signaling pathway in CD8+ T cells, and promoted IFN-γ and GZMB secretion. Together, the study shows that tumor-derived exosomes promote I-IFN secretion via the circPIK3R3/miR-872-3p/IRF7 axis in macrophages and enhance the anti-tumor immune response of CD8+ T cells.

Prediction of survival and immunotherapy response by the combined classifier of G protein-coupled receptors and tumor microenvironment in melanoma.

BACKGROUND: Melanoma is the deadliest form of skin tumor, and G protein-coupled receptors (GPCRs) play crucial roles in its carcinogenesis. Furthermore, the tumor microenvironment (TME) affects the overall survival (OS) and the response to immunotherapy. The combination of GPCRs and TME from a multi-omics perspective may help to predict the survival of the melanoma patients and their response to immunotherapy. METHODS: Bulk-seq, single-cell RNA sequencing (scRNA-seq), gene mutations, immunotherapy responses, and clinicopathologic feature data were downloaded from public databases, and prognostic GPCRs and immune cells were screened using multiple machine learning algorithms. The expression levels of GPCRs were detected using real-time quantitative polymerase chain reaction (qPCR) in A375 and HaCaT cell lines. The GPCR-TME classifier was constructed and verified using different cohorts and multi-omics. Gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), and tracking tumor immunophenotype (TIP) were used to identify the key biological pathways among the GPCR-TME subgroups. Then, tumor mutational burden (TMB), vital mutant genes, antigen presentation genes, and immune checkpoints were compared among the subgroups. Finally, the differences in immunotherapy response rates among the GPCR-TME subgroups were investigated. RESULTS: A total of 12 GPCRs and five immune cell types were screened to establish the GPCR-TME classifier. No significant differences in the expression levels of the 12 GPCRs were found in the two cell lines. Patients with high GPCR score or low TME score had a poor OS; thus, the GPCRlow/TMEhigh subgroup had the most favorable OS. The scRNA-seq result revealed that immune cells had a higher GPCR score than tumor and stromal cells. The GPCR-TME classifier acted as an independent prognostic factor for melanoma. GSEA, WGCNA, and TIP demonstrated that the GPCRlow/TMEhigh subgroup was related to the activation and recruitment of anti-tumor immune cells and the positive regulation of the immune response. From a genomic perspective, the GPCRlow/TMEhigh subgroup had higher TMB, and different mutant genes. Ultimately, higher expression levels of antigen presentation genes and immune checkpoints were observed in the GPCRlow/TMEhigh subgroup, and the melanoma immunotherapy cohorts confirmed that the response rate was highest in the GPCRlow/TMEhigh cohort. CONCLUSIONS: We have developed a GPCR-TME classifier that could predict the OS and immunotherapy response of patients with melanoma highly effectively based on multi-omics analysis.

Redox-Reversible Near-Infrared Fluorescent Probe for Imaging of Acute Kidney Oxidative Injury and Remedy.

Drug-induced acute kidney injury (DIAKI) is associated with high morbidity and mortality. It remains a diagnostic and therapeutic dilemma due to failure of providing unambiguous real-time feedback on nephrotoxicity, which is regarded as a serious problem in clinics. Herein, we report a reversible fluorescence probe, NRN, to monitor the ONOO-/GSH in an acute kidney injury model. The NRN near-infrared fluorescent probe features a big Stokes shift (83 nm), which was oxidized by ONOO- and reduced by succussive glutathione (GSH) with excellent selectivity and good sensitivity (detection limit: 418 nM and 0.28 mM, respectively). Taking the reversibility of NRN toward ONOO- and GSH, real-time evaluations in vivo with cisplatin (CP) alone and CP combined with acetaminophen-stimulated acute kidney injury and the following remedy process with l-carnitine were realized for the first time. The experiments revealed that acute kidney injury caused by combined drugs might be more serious and irreversible under certain conditions. Therefore, NRN could act as a potential tool for understanding oxidative stress-related DIAKI disease processes.

Intratumoral CD73: An immune checkpoint shaping an inhibitory tumor microenvironment and implicating poor prognosis in Chinese melanoma cohorts.

Background: As a novel immune checkpoint, CD73 has been reported to play prominent roles in several malignancies. However, the significance of CD73 in melanoma remains ambiguous. This study sought to reveal the impact of CD73 on the tumor microenvironment (TME) and patients' prognosis, and to investigate whether CD73 could be a therapeutic target in Chinese melanomas, which were dominated by acral and mucosal subtypes. Methods: Two independent Chinese cohorts of 194 patients with melanoma were enrolled. CD73 and PD-L1 expression as well as CD8+ and CD56+ cell infiltrations were evaluated by immunohistochemistry in 194 resected melanoma samples. Clinical outcomes of patients were assessed utilizing the Kaplan-Meier plotter and Cox proportional hazard analysis. RNA-seq data was obtained from TCGA database. Gene set functional annotations were performed based on GO, KEGG and GSEA analysis. CIBERSORT, ssGSEA and TIMER were used to explore the association between CD73 and immune infiltration. These findings were validated by establishing tumor xenograft model, and functions of tumor-infiltrating immune cells were examined by flow cytometry and immunofluorescence. Results: High CD73 expression showed poorer clinical outcomes and was identified as an independent prognostic indicator for survival in two cohorts. Expression of CD73 was more prevalent than PD-L1 in Chinese melanoma cohorts (54.6% vs 23.2%). Co-expression of both immune checkpoints was infrequent (12.9%) in melanoma, and 54.4% of PD-L1 negative cases showed elevated expression of CD73. CD73high tumors showed a microenvironment with fewer CD8+ T cells and CD56+ NK cells infiltration, which displayed a dysfunctional phenotype. With the treatment of CD73 inhibitor APCP, the amount of CD8+ T cells and CD56+ NK cells infiltrated in tumors was elevated and the immunosuppressive effect of CD73 was eliminated. Conclusions: High CD73 expression was associated with an inhibitory TME and adverse clinical outcomes of melanoma. In comparison to PD-L1, CD73 was more prevalent and possessed more definite prognostic significance. Therefore, it may serve as a prognostic indicator and immunotherapeutic target next to PD-L1 in melanoma for Chinese population.

Golgi-Targeting Fluorescent Probe for Monitoring CO-Releasing Molecule-3 In Vitro and In Vivo.

CO-releasing molecule-3 (CORM-3), mainly metal carbonyl compounds, is widely used as experimental tools to deliver CO, a biological "gasotransmitter", in mammalian systems. CORM-3 is also proposed as a potential new antimicrobial agent, which kills bacteria effectively and rapidly in vitro and in animal models. Organelle-targeting therapy, as a highly effective therapeutic strategy with little toxic and side effects, has important research significance and development prospects. Therefore, the development of effective methods for detecting and tracking CORM-3 at the subcellular level has important implications. In this paper, an easily available Golgi-targetable fluorescent probe (Golgi-Nap-CORM-3) was proposed for CORM-3 detection. In the probe Golgi-Nap-CORM-3, the phenyl sulfonamide group was selected as the Golgi-targetable unit, naphthalimide dye was chosen as a fluorophore, and the nitro group was selected as a CORM-3-responsive unit. Golgi-Nap-CORM-3 shows a CORM-3-reponsive increase of fluorescence emission at 520 nm. Using the excellent probe, the change of CORM-3 in HeLa cells, HepG2 cells, and zebrafish is successfully monitored. This study demonstrates very important information for the study of CORM-3 in vivo systems.

Overcoming chemoresistance to b-raf inhibitor in melanoma via targeted inhibition of phosphoenolpyruvate carboxykinase1 using 3-mercaptopropionic acid.

The resistance of melanoma to BRAF inhibitors remains a tough clinical challenge. In order to explore the underlying mechanism of drug resistance in melanoma, we established the resistant cell line to vemurafenib, and assessed the changes of drug-resistant cells on proliferation, apoptosis, oxidative stress and tumor stemness. Our results suggest that phosphoenolpyruvate carboxykinase1 (PCK1) is activated and inhibits the oxidative stress caused by vemurafenib in drug-resistant cells. Long term treatment of vemurafenib increases the expression of PCK1 which reduces the production of reactive oxygen species (ROS) by activating PI3K/Akt pathway. After the inhibition of PCK1 by 3-mercaptopropionic acid (3-MPA), the therapeutic sensitivity of vemurafenib is restored. In conclusion, this study disclosed that drug-resistant cells appeared to regulate their own proliferation, oxidative stress and tumor dryness by activating Akt/PCK1/ROS pathway, and shed new insights into acquiring drug resistance in melanoma.