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Background: The incidence of cutaneous melanoma continues to rise rapidly and has an extremely poor prognosis. Immunotherapy strategies are the most effective approach for patients who have developed metastases, but not all cases have been successful due to the complex and variable mechanisms of melanoma response to immune checkpoint inhibition., Methods: We synthesized collagen-coding gene expression data (second-generation and single-cell sequencing) from public Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Bioinformatics analysis was performed using R software and several database resources such as Metascape database, Gene Set Cancer Analysis (GSCA) database, and Cytoscape software, etc., to investigate the biological mechanisms that may be related with collagens. Immunofluorescence and immunohistochemical staining were used to validate the expression and localization of Nidogen-2 (NID2)., Results: Melanoma patients can be divided into two collagen clusters. Patients with high collagen levels (C1) had a shorter survival than those with low collagen levels (C2) and were less likely to benefit from immunotherapy. We demonstrated that NID2 is a potential key factor in the collagen phenotype, is involved in fibroblast activation in melanoma, and forms a barrier to limit the proximity of CD8+ T cells to tumor cells., Conclusion: We clarified the adverse effects of collagen on melanoma patients and identified NID2 as a potential therapeutic target., Competing Interests: The authors declare no conflicts of interest in this work., (© 2023 Sha et al.)

Background: As one of the most immunogenic malignancies, skin cutaneous melanoma (SKCM) is mainly characterized by a high prevalence in immune-compromised patients and a brisk lymphocyte infiltration in the tumor microenvironment (TME). However, to date, studies on deubiquitination in SKCM are still very limited., Methods: Public data with regard to this study in SKCM patients were acquired from The Cancer Genome Atlas (TCGA) and the Gene-Expression Omnibus (GEO) databases. We stratified TCGA-SKCM cases using consensus clustering and identified independent prognostic factors in deubiquitinating enzymes encoding genes (DECGs) by LASSO-Cox analysis. USP35 transcriptome level was examined using public data and validated by Immunohistochemical (IHC) staining at the protein level. Enrichment analysis was used to explore the potential functions of USP35 , and the TISCH database, providing further evidence at the single-cell level. The CIBERSORT algorithm was used to assess the relationship between USP35 and the immune microenvironment, and IHC was used to further evaluate the relationship between USP35 and immunotherapy response. Finally, we used the cBioPortal and the Methsurv database to analyze the significance of genomic alterations of USP35 in melanoma., Results: Our results showed that DECGs can be effectively used to stratify SKCM patients, suggesting their potential significance in the development of SKCM. Furthermore, USP35 overexpression was significantly associated with an unfavorable prognosis. We further revealed that USP35 may be involved in the activation of TORC1 signaling. Most importantly, USP35 was found to be significantly associated with an immunosuppressive TME, both in terms of negative correlation with the abundance of infiltrating CD8+ T cells and in terms of the fact that patients with high USP35 expression may benefit less from immunotherapy than those with low USP35 expression., Conclusion: Deubiquitinating enzymes are of great importance in the diagnosis and treatment of SKCM, and USP35 is an extremely promising target for immunotherapy., Competing Interests: The authors have declared no conflicts of interest., (© 2022 Zhang et al.)

Background: Gap junctions, as one of the major ways to maintain social connections between cells, are now considered as one of the potential regulators of tumor metastasis. However, to date, studies on the relationship between gap junctions and colorectal cancer (CRC) are limited., Methods: We synthesized connexins-coding gene expression data from public Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Bioinformatics analysis was performed using R software and several database resources such as MEXPRESS database, Gene Set Cancer Analysis (GSCA) database, Human Protein Atlas (HPA) database, Tumor Immune Single Cell Hub (TISCH) database, Search Tool for Retrieval of Gene Interaction Relationships (STRING), and Cytoscape software, etc., to investigate the biological mechanisms that may be involved in connexins. Immunofluorescence and immunohistochemical staining were used to validate the expression and localization of GJA4., Results: We found that CRC patients can be divided into two connexin clusters and that patients in cluster C1 had shorter survival than in cluster C2. The infiltration of M1 macrophages and NK cells was lower in cluster C1, while the levels of M2 macrophages and immune checkpoints were higher, indicating an immunosuppressed state in cluster C1. In addition, the epithelial-mesenchymal transition (EMT) phenotype was significantly activated in cluster C1. We observed that GJA4 was up-regulated in colorectal cancer tissues, which was related to poor prognosis. It was mainly expressed in fibroblasts, but the expression levels in normal intestinal epithelial cells were low. Finally, we found that GJA4 was associated with M2 macrophages and may be a potential immunosuppressive factor., Conclusion: We found that there is a significant correlation between abnormal connexins expression and patients' prognosis, and connexins play an important role in stromal-tumor interactions. Connexins, especially GJA4, can help enhance our understanding of tumor microenvironment (TME) and may guide more effective immunotherapeutic strategies., Competing Interests: The authors declare no conflicts of interest., (© 2022 Liu et al.)

Purpose: Low-grade gliomas (LGG) are primary brain tumors that often affect predominantly young adults, which usually have a painless course, and have a longer survival period compared to patients with high-grade gliomas. Relatively established treatment options include surgery, radiotherapy, chemotherapy or combination therapy, as well as individualized management based on tumor location, histology, molecular features and patient characteristics. Due to the rapid development of targeted therapies, the development of new molecular targets is now a very promising research direction., Methods: We explored the diagnostic value, clinical relevance, and molecular function of deoxynucleotidyl transferase terminal-interacting proteins 2 ( DNTTIP2 ) in LGG using MethSurv, MEXPRESS, STRING, cBioPortal, Tumor Immunity Estimation Resource (TIMER) database, Gene Expression Profiling Interactive Analysis (GEPIA) databases. Besides, the "CIBERSORT" algorithm was conducted to estimate immune cells infiltration abundance, with "ggplot2" package visualizing the results. In vivo and vitro experiments were used to verify the speculations of bioinformatics analysis., Results: In LGG patients, DNTTIP1/2 were over-expressed at mRNA levels and high DNTTIP1/2 levels correlated with poor survival in LGG patients. We confirmed that DNTTIP2 significantly promotes M2 macrophage activation and angiogenesis, which may be related to the IL6/JAK/STAT3 signaling pathway. In addition, we found that DNTTIP2 amplification was associated with an unfavorable prognosis in LGG patients. We demonstrated, finally, a correlation between DNTTIP2 gene hypermethylation and a poor prognosis in LGG., Conclusion: This study demonstrated that DNTTIP1/2 had diagnostic and prognostic value in LGG patients. The biological mechanisms of DNTTIP2 regarding angiogenesis and macrophage activation may provide new insights into the treatment of glioma., Competing Interests: The authors have declared no conflicts of interest in this work., (© 2022 Liu et al.)

Purpose: The identification of biomarkers and effective therapeutic targets for gastric cancer (GC), the most common cause of cancer-related deaths around the world, is currently a major focus in research. Here, we examined the utility of LHFPL6 as a prognostic biomarker and therapeutic target for GC., Methods: We explored the clinical relevance, function, and molecular role of LHFPL6 in GC using the MethSurv, cBioPortal, TIMER, Gene Expression Profiling Interactive Analysis, ONCOMINE, MEXPRESS, and EWAS Atlas databases. The GSE118919, GSE29272, and GSE13861 datasets were used for differential expression analysis. Using The Cancer Genome Atlas, we developed a Cox regression model and assessed the clinical significance of LHFPLs. In addition, we used the "CIBERSORT" algorithm to make reliable immune infiltration estimations. Western blot and immunohistochemistry were used to examine protein expression. Cell migration and invasion were assessed using transwell experiments. THP-1-derived macrophages and GC cells were co-cultured in order to model tumor-macrophage interactions in vitro. The levels of CD206 and CD163 were measured using immunofluorescence assays. The results were visualized with the "ggplot2" and "circlize" packages., Results: Our results showed that in GC, LHFPL6 overexpression was significantly associated with a poor prognosis. Our findings also suggested that LHFPL6 may be involved in the activation of the epithelial-mesenchymal transition. Furthermore, LHFPL6 expression showed a positive correlation with the abundance of M2 macrophages, which are potent immunosuppressors., Conclusion: LHFPL6 could be a prognostic biomarker and therapeutic target for GC., Competing Interests: The authors have declared no conflicts of interest., (© 2021 Liu et al.)

Purpose: Gastric cancer (GC) is a common type of cancer worldwide. It can relapse and metastasize even after standard treatment; therefore, it has a poor prognosis. Moreover, sensitive biomarkers for prognosis prediction in GC are lacking. In this study, using a bioinformatics approach, we aimed to examine the value of DAZ Interacting Protein 1 ( DZIP1 ) as a prognostic predictor and therapeutic target in GC., Methods: We explored the clinical relevance, function, and molecular role of DZIP1 in GC using MethSurv, cBioPortal, TIMER, Gene Expression Profiling Interactive Analysis, IMEx, ONCOMINE, MEXPRESS, and EWAS Atlas databases. The GSE118919 dataset was used to plot receiver operating characteristic curves. Using The Cancer Genome Atlas, we developed a Cox regression model and assessed the clinical significance of DZIPs . In addition, we used the "xCELL" algorithm to make reliable immune infiltration estimations. Western blot and immunohistochemistry were used to examine protein expression. The results were visualized with the 'ggplot2' and "circlize" packages., Results: In GC patients, DZIP1 was over-expressed at both the mRNA and protein levels. High levels of DZIP1 were found to be associated with poor survival in patients with GC. Our results indicated that DZIP1 could be involved in multiple cancer-related pathways such as the PI3K-Akt signaling pathway, WNT signaling pathway, and RAS signaling pathway, and its expression was correlated with the infiltration of activated myeloid dendritic cells, naive CD4+ T cells, and naive CD8+ T cells. Furthermore, we found that mutations in DZIP1 were correlated with a good prognosis in GC patients. Finally, we demonstrated a correlation between hypomethylation of the DZIP1 gene promoter and a poor prognosis in GC., Conclusion: This study is the first to demonstrate a significant correlation between high levels of DZIP1 and a poor prognosis in GC patients. Our results clarify multiple potential mechanisms that could contribute to this correlation and may thus provide novel insights into the clinical diagnosis and treatment of GC., Competing Interests: The authors declare no conflicts of interest., (© 2021 Liu et al.)

Purpose: Follistatin-related gene 3 ( FSTL3 ), an established oncogene, can modulate target gene expression via members of the transforming growth factor β (TGF-β) superfamily. The present study was conducted to evaluate the expression of FSTL3 in gastric cancer (GC) and to determine its prognostic significance. We also evaluated the possible mechanisms involved in the oncogenic role of FSTL3 in gastric carcinogenesis and development., Methods: We obtained data from the Human Protein Atlas, MethSurv, cBioPortal, UALCAN, TIMER, GEPIA, STRING, GeneMANIA, ONCOMINE, and MEXPRESS databases and examined it using R software. RNAi was used to establish stable FSTL3 -knockdown (shFSTL3) and overexpression (OE) cell strains. Western blot; enzyme-linked immunosorbent (ELISA); and immunohistochemical (ICH), immunofluorescence, and phalloidin staining were used for examining protein expression. Cell invasion and migration were determined using transwell and scratch-wound assays. After tumor-associated macrophage (TAM) generation, co-culturing of cancer cells with TAMs was performed to confirm the relationship between FSTL3 and TAMs., Results: In GC patients, FSTL3 mRNA and protein levels were upregulated. FSTL3 expression was significantly linked to cancer stage as well as to pathological tumor grade in GC. Moreover, a high expression of FSTL3 was associated with a dismal survival duration in patients with GC. Furthermore, functional enrichment analysis demonstrated that FSTL3 overexpression could activate epithelial-mesenchymal transition (EMT) by promoting F-actin expression and BMP/SMAD signaling. Finally, immunofluorescence staining confirmed that the overexpression of FSTL3 promoted the proliferation of M2 TAMs., Conclusion: Taken together, our findings suggest that FSTL3 may be involved in GC progression via the promotion of BMP/SMAD signaling-mediated EMT and M2 macrophage activation., Competing Interests: The authors have declared no conflicts of interest., (© 2021 Liu et al.)