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BACKGROUND & AIMS: Despite remarkable advances in treatment, most patients with hepatocellular carcinoma (HCC) respond poorly to anti-programmed cell death 1 (anti-PD1) therapy. A deeper insight into the tolerance mechanism of HCC against this therapy is urgently needed. METHODS: We performed next-generation sequencing, multiplex immunofluorescence, and dual-color immunohistochemistry and constructed an orthotopic HCC xenograft tumor model to identify the key gene associated with anti-PD1 tolerance. A spontaneously tumorigenic transgenic mouse model, an in vitro coculture system, mass cytometry, and multiplex immunofluorescence were used to explore the biological function of zinc finger protein 64 (ZFP64) on tumor progression and immune escape. Molecular and biochemical strategies like RNA-sequencing, chromatin immunoprecipitation-sequencing and mass spectrometry were used to gain insight into the underlying mechanisms of ZFP64. RESULTS: We showed that ZFP64 is frequently upregulated in tumor tissues from patients with anti-PD1-resistant HCC. Elevated ZFP64 drives anti-PD1 resistance by shifting macrophage polarization toward an alternative activation phenotype (M2) and fostering an inhibitory tumor microenvironment. Mechanistically, we primarily demonstrated that protein kinase C alpha (PKCα) directly phosphorylates ZFP64 at S226, leading to its nuclear translocation and the transcriptional activation of macrophage colony-stimulating factor (CSF1). HCC-derived CSF1 transforms macrophages to the M2 phenotype to drive immune escape and anti-PD1 tolerance. Notably, Gö6976, a protein kinase inhibitor, and lenvatinib, a multi-kinase inhibitor, reset the tumor microenvironment and restore sensitivity to anti-PD1 by blocking the PKCα/ZFP64/CSF1 axis. CONCLUSIONS: We propose that the PKCα/ZFP64/CSF1 axis is critical for triggering immune evasion and anti-PD1 tolerance. Inhibiting this axis with Gö6976 or lenvatinib overcomes anti-PD1 resistance in HCC. LAY SUMMARY: Despite remarkable treatment progress, most patients with hepatocellular carcinoma respond poorly to anti-PD1 therapy (a type of immunotherapy). A deeper insight into the tolerance mechanisms to this therapy is urgently needed. Herein, we unravel a previously unexplored mechanism linking tumor progression, macrophage polarization, and anti-PD1 resistance, and offer an attractive novel target for anti-PD1 combination therapy, which may benefit patients with hepatocellular carcinoma.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Colony-Stimulating Factors , DNA-Binding Proteins , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Mice , Protein Kinase C-alpha/genetics , Protein Kinase Inhibitors , Transcription Factors , Tumor MicroenvironmentABSTRACT
BACKGROUND: Circular RNAs (circRNAs) have been reported to have critical regulatory roles in tumor biology. However, their contribution to melanoma remains largely unknown. METHODS: CircRNAs derived from oncogene CD151 were detected and verified by analyzing a large number of melanoma samples through quantitative real-time polymerase chain reaction (qRT-PCR). Melanoma cells were stably transfected with lentiviruses using circ_0020710 interference or overexpression plasmid, and then CCK-8, colony formation, wound healing, transwell invasion assays, and mouse xenograft models were employed to assess the potential role of circ_0020710. RNA immunoprecipitation, luciferase reporter assay and fluorescence in situ hybridization were used to evaluate the underlying mechanism of circ_0020710. RESULTS: Our findings indicated that circ_0020710 was generally overexpressed in melanoma tissues, and high level of circ_0020710 was positively correlated with malignant phenotype and poor prognosis of melanoma patients. Elevated circ_0020710 promoted melanoma cell proliferation, migration and invasion in vitro as well as tumor growth in vivo. Mechanistically, we found that high level of circ_0020710 could upregulate the CXCL12 expression via sponging miR-370-3p. CXCL12 downregulation could reverse the malignant behavior of melanoma cells conferred by circ_0020710 over expression. Moreover, we also found that elevated circ_0020710 was correlated with cytotoxic lymphocyte exhaustion, and a combination of AMD3100 (the CXCL12/CXCR4 axis inhibitor) and anti-PD-1 significantly attenuated tumor growth. CONCLUSIONS: Elevated circ_0020710 drives tumor progression via the miR-370-3p/CXCL12 axis, and circ_0020710 is a potential target for melanoma treatment.
Subject(s)
Biomarkers, Tumor/metabolism , Chemokine CXCL12/metabolism , Gene Expression Regulation, Neoplastic , Melanoma/pathology , MicroRNAs/genetics , RNA, Circular/genetics , Tetraspanin 24/genetics , Animals , Apoptosis , Biomarkers, Tumor/genetics , Case-Control Studies , Cell Movement , Cell Proliferation , Chemokine CXCL12/genetics , Disease Progression , Female , Humans , Immune Evasion , Male , Melanoma/genetics , Melanoma/immunology , Melanoma/metabolism , Mice , Mice, Inbred C57BL , Middle Aged , Neoplasm Invasiveness , Prognosis , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Intrahepatic cholangiocarcinoma (iCCA) is an aggressive liver malignancy with limited treatment options and a dismal prognosis. The tumor immune microenvironment (TIME) is crucial for iCCA progression, yet its comprehensive characterization remains incomplete. This study utilized mass cytometry by time of flight (CyTOF) to comprehensively analyze immune cell populations in fresh iCCA tumor samples and adjacent peritumor liver tissues. Notably, NK cell percentages significantly decreased in iCCA lesions compared to peritumor liver tissues. Conversely, an enrichment of immunosuppressive CD39+Foxp3+CD4+ regulatory T cells (CD39+T-regs) and exhausted-like CD8+T cells (with pronounced CD39 and PD-1 expression) within TIME was identified and confirmed by multiplex immunofluorescence staining in an independent patient cohort (n = 140). Crucially, tumor-infiltrating CD39+T-regs and CD39+PD-1+CD8+T cells emerged as independent prognostic indicators associated with an unfavorable prognosis in iCCA. These findings unveil the intricate immune landscape within iCCA, offering valuable insights for disease management and novel cancer immunotherapies.
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Background: Trastuzumab (TRA) shows significant efficacy in patients with human epidermal growth factor receptor 2 (HER2)-positive gastric cancer (GC). While TRA can help treat HER2-positive breast cancer, TRA resistance is a key clinical challenge. Nestin reportedly regulates the cellular redox homeostasis in lung cancer. This study aimed at identifying the functions of Nestin on the TRA sensitivity of HER2-positive GC cells. Methods: Real-time polymerase chain reaction (PCR) and Western blotting (WB) were performed to explore the association between the mRNA and protein expression profiles, respectively, of Nestin and the Keap1-Nrf2 pathway. The influence of Nestin overexpression on the in vitro sensitivity of GC cells to TRA was explored by Cell Counting Kit-8 (CCK-8) assay, colony formation assay, reactive oxygen species (ROS) detection, and flow cytometry. Results: TRA treatment caused Nestin downregulation in two HER2-positive GC cell lines (MKN45 and NCI-N87). Nestin overexpression reduced the sensitivity of GC cells to TRA. The expression and activity of Nrf2 and relevant downstream antioxidant genes were increased by Nestin overexpression. Nestin overexpression also significantly suppressed TRA-induced apoptosis and ROS generation. In vivo tumor growth experiment with female BALB/c nude mice indicated that Nestin upregulation restored the tumor growth rate which was inhibited by TRA treatment. Conclusions: Collectively, the inhibitory effect of Nestin on the TRA sensitivity of cells to TRA was confirmed in this study. These results imply that the antioxidant Nestin-Nrf2 axis may play a role in the mechanism underlying the resistance of GC cells to TRA.
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Aim: Circular RNAs are widely and abnormally expressed in human cancer cells, and they participate in cancer progression. However, they have rarely been investigated in the immune evasion of non-small cell lung cancer (NSCLC). Here, we elucidated the function and molecular mechanism of hsa_circ_0020714 in promoting the resistance to anti-PD-1 immunotherapy of NSCLC. Methods: The expression of hsa_circ_0020714 were examined by qRT-PCR. In vivo experiments were executed to investigate the biological function of hsa_circ_0020714 in the sensitivity of NSCLC to anti-PD-1 immunotherapy. The qRT-PCR, fluorescence in situ hybridization, RNA pulldown, RNA immunoprecipitation, and western blot were carried out to investigate the potential regulatory mechanisms of hsa_circ_0020714 in NSCLC immune evasion. Results: The expression of hsa_circ_0020714 was upregulated in NSCLC tissues compared to the paired adjacent non-tumor tissues, and an increased expression of hsa_circ_0020714 was significantly associated with a bad prognosis and resistance to anti-PD-1 immunotherapy in patients with NSCLC. Mechanistically, hsa_circ_0020714 functions as an endogenous miR-30a-5p sponge to enhance SOX4 expression, thereby promoting immune evasion and anti-PD-1 resistance in NSCLC patients. Conclusion: Hsa_circ_0020714 induces the immune evasion and resistance to anti-PD-1 immunotherapy of NSCLC via the miR-30a-5p/SOX4 axis, and may be an promising immunotherapeutic target in NSCLC.
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BACKGROUND: The immunosuppressive microenvironment is closely related to tumorigenesis and cancer development, including colorectal cancer (CRC). The aim of the current study was to identify new immune biomarkers for the diagnosis and treatment of CRC. MATERIALS AND METHODS: CRC data were downloaded from the Gene Expression Omnibus and The Cancer Genome Atlas databases. Sequences of immune-related genes (IRGs) were obtained from the ImmPort and InnateDB databases. Gene set enrichment analysis (GSEA) and transcription factor regulation analysis were used to explore potential mechanisms. An immune-related classifier for CRC prognosis was conducted using weighted gene co-expression network analysis (WGCNA), Cox regression analysis, and least absolute shrinkage and selection operator (LASSO) analysis. ESTIMATE and CIBERSORT algorithms were used to explore the tumor microenvironment and immune infiltration in the high-risk CRC group and the low-risk CRC group. RESULTS: By analyzing the IRGs that were significantly associated with CRC in the module, a set of 13 genes (CXCL1, F2RL1, LTB4R, GPR44, ANGPTL5, BMP5, RETNLB, MC1R, PPARGC1A, PRKDC, CEBPB, SYP, and GAB1) related to the prognosis of CRC were identified. An IRG-based prognostic signature that can be used as an independent potentially prognostic indicator was generated. The ROC curve analysis showed acceptable discrimination with AUCs of 0.68, 0.68, and 0.74 at 1-, 3-, and 5- year follow-up respectively. The predictive performance was validated in the train set. The potential mechanisms and functions of prognostic IRGs were analyzed, i.e., NOD-like receptor signaling, and transforming growth factor beta (TGFß) signaling. Besides, the stromal score and immune score were significantly different in high-risk group and low-risk group (p=4.6982e-07, p=0.0107). Besides, the proportions of resting memory CD4+ T cells was significantly higher in the high-risk groups. CONCLUSIONS: The IRG-based classifier exhibited strong predictive capacity with regard to CRC. The survival difference between the high-risk and low-risk groups was associated with tumor microenvironment and immune infiltration of CRC. Innovative biomarkers for the prediction of CRC prognosis and response to immunological therapy were identified in the present study.
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In the original publication of this article [1], the author would like to revise Figure 4.
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Although recent therapeutic advances based on our understanding of molecular phenomena have prolonged the survival of melanoma patients, the prognosis of melanoma remains dismal and further understanding of the underlying mechanism of melanoma progression is needed. In this study, differential expression analyses revealed that many genes, including AKT1 and CDK2, play important roles in melanoma. Functional analyses of differentially expressed genes (DEGs), obtained from the GEO (Gene Expression Omnibus) database, indicated that high proliferative and metastatic abilities are the main characteristics of melanoma and that the PI3K and MAPK pathways play essential roles in melanoma progression. Among these DEGs, major facilitator superfamily domain-containing 12 (MFSD12) was found to have significantly and specifically upregulated expression in melanoma, and elevated MFSD12 level promoted cell proliferation by promoting cell cycle progression. Mechanistically, MFSD12 upregulation was found to activate PI3K signaling, and a PI3K inhibitor reversed the increase in cell proliferation endowed by MFSD12 upregulation. Clinically, high MFSD12 expression was positively associated with shorter overall survival (OS) and disease-free survival (DFS) in melanoma patients, and MFSD12 was an independent prognostic factor for OS and DFS in melanoma patients. Therapeutically, in vivo assays further confirmed that MFSD12 interference inhibited tumor growth and lung metastasis in melanoma. In conclusion, elevated MFSD12 expression promotes melanoma cell proliferation, and MFSD12 is a valuable prognostic biomarker and promising therapeutic target in melanoma.
Subject(s)
Cell Proliferation/genetics , Cell Transformation, Neoplastic/genetics , Melanoma/genetics , Membrane Proteins/genetics , Skin Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/genetics , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Computational Biology/methods , Disease Progression , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Male , Melanoma/mortality , Melanoma/pathology , Melanoma/therapy , Membrane Proteins/metabolism , Middle Aged , Molecular Targeted Therapy/methods , Molecular Targeted Therapy/trends , Prognosis , Skin Neoplasms/mortality , Skin Neoplasms/pathology , Skin Neoplasms/therapy , Survival Analysis , Up-Regulation/geneticsABSTRACT
BACKGROUND: ATPase associated with a variety of cellular activities (AAA ATPase) family members are closely linked to tumor formation and progression. However, their roles in hepatocellular carcinoma (HCC) largely remain unclear. METHODS: Bioinformatic analyses of public databases were used to excavate the potential AAA ATPases that may contribute to HCC, and thyroid hormone receptor interactor 13 (TRIP13) was selected to following researches because of its most prominently differential expression. Western blot, qRT-PCR and immunohistochemistry were used to detect the expression of TRIP13 in HCC tissues, and then the relationship between TRIP13 expression and clinicopathological parameters were evaluated. Finally, its functions and potential mechanisms were investigated through a series gain- and loss-of-function strategies both in vitro and in vivo. RESULTS: TRIP13 was significantly overexpressed in HCC tissues and high level of TRIP13 was closely correlated with a worse clinical outcome. Functionally, elevated TRIP13 facilitated cell proliferation, migration, invasion, and promoted cellular epithelial-mesenchymal transition (EMT) in vitro, while promote tumor growth and lung metastasis in vivo. Mechanistically, TRIP13 interacted with ACTN4 and positively regulated its expression, thus activating the AKT/mTOR pathway to drive tumor progression. Moreover, miR-192-5p served as an upstream regulator of TRIP13 by directly binding to TRIP13 mRNA 3' UTR, which may partially explain the high expression of TRIP13 in HCC. CONCLUSION: Our findings identified TRIP13 as a promising candidate oncogene in HCC, and TRIP13 induced cell migration, invasion and metastasis of HCC through the AKT/mTOR signaling via interacting with ACTN4.
Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle Proteins/metabolism , Liver Neoplasms/pathology , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Actinin/metabolism , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/mortality , Case-Control Studies , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Movement , Disease Progression , Female , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/mortality , Male , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/metabolism , Middle Aged , Signal Transduction , Survival Rate , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Ring finger proteins (RNFs) were involved in carcinogenesis. Here, we aimed to explore the detailed mechanism of RNF128 in the progression of melanoma. METHODS: We reanalyzed several gene expression profiles from the Gene Expression Omnibus (GEO) database and obtained the overlapped differential expressed RNF genes. Among them, RNF128 was selected to further explore its expression, the biological significance, and the underlying molecular mechanism, as well as the clinical relevance in melanoma patients. RESULTS: RNF128 was found to be significantly downregulated in the selected datasets, which was further verified in our melanoma tissues. Moreover, RNF128 downregulation was shown to correlate with the malignant phenotype of melanoma, and further functional assays demonstrated that low levels of RNF128 promoted melanoma progression via inducing cell epithelial-mesenchymal transition (EMT) and the acquisition of stemness. Mechanistically, RNF128 interference activated the Wnt pathway via simultaneously ubiquitinating CD44/cortactin (CTTN), resulting in CD44 and c-Myc transcription, thus revealed that RNF128 participated in a positive feedback of the Wnt pathway-CD44 loop. Clinically, we found that patients expressing low RNF128 and high CD44/CTTN levels had a poor prognosis. CONCLUSION: Downregulated RNF128 activates Wnt signaling to induce cellular EMT and stemness by ubiquitinating and degrading CD44/CTTN, and RNF128 is a reliable diagnostic and prognostic biomarker, and a deeper understanding of RNF128 may contribute to the treatment of melanoma.
Subject(s)
Cortactin/metabolism , Hyaluronan Receptors/metabolism , Melanoma/metabolism , Ubiquitin-Protein Ligases/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Cell Line, Tumor , Down-Regulation , Epithelial-Mesenchymal Transition , Female , Humans , Male , Melanoma/genetics , Melanoma/pathology , Mice , Mice, Nude , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Survival Rate , Transcriptome , Transfection , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
BACKGROUND: There is growing evidence that tripartite motif-containing protein 44 (TRIM44) plays crucial role in tumor development. However, the underlying mechanism of this deubiquitinating enzyme remains unclear. METHODS: Large clinical samples were used to detect TRIM44 expression and its associations with clinicopathological features and prognosis. Gain- and loss-of-function experiments in cell lines and mouse xenograft models were performed to elucidate the function and underlying mechanisms of TRIM44 induced tumor progression. Co-immunoprecipitation (Co-IP) assays and mass spectrometric analyses were applied to verify the interacting proteins of TRIM44. RESULTS: We found that TRIM44 was commonly amplified in melanoma tissues compared with paratumoral tissues. TRIM44 expression also positively correlated with more aggressive clinicopathological features, such as Breslow depth (p = 0.025), distant metastasis (p = 0.012), and TNM stage (p = 0.002). Importantly, we found that TRIM44 was an independent indicator of prognosis for melanoma patients. Functionally, overexpression of TRIM44 facilitated cell invasion, migration, apoptosis resistance and proliferation in vitro, and promoted lung metastasis and tumorigenic ability in vivo. Importantly, high level of TRIM44 induced melanoma cell epithelial-mesenchymal transition (EMT), which is one of the most important mechanisms for the promotion of tumor metastasis. Mechanistically, high levels of TRIM44 increased the levels of p-AKT (T308) and p-mTOR (S2448), and a specific AKT inhibitor inhibited TRIM44-induced tumor progression. Co-IP assays and mass spectrometric analyses indicated that TRIM44 overexpression induces cell EMT through activating AKT/mTOR pathway via directly binding and stabilizing TOLL-like receptor 4 (TLR4), and TLR4 interference impeded TRIM44 induced tumor progression. Moreover, we demonstrated that TRIM44 is the target of miR-26b-5p, which is significantly downregulated in melanoma tissues and may be responsible for the overexpression of TRIM44. CONCLUSIONS: TRIM44, regulated by miR-26b-5p, promotes melanoma progression by stabilizing TLR4, which then activates the AKT/mTOR pathway. TRIM44 shows promise as a prognostic predictor and a therapeutic target for melanoma patients.
Subject(s)
Carrier Proteins/metabolism , Melanoma/pathology , MicroRNAs/genetics , Signal Transduction , Toll-Like Receptor 4/metabolism , Up-Regulation , Animals , Carrier Proteins/genetics , Cell Line, Tumor , Cell Movement , Disease Progression , Female , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins , Male , Melanoma/genetics , Melanoma/metabolism , Mice , Neoplasm Staging , Neoplasm Transplantation , Prognosis , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , Toll-Like Receptor 4/genetics , Tripartite Motif ProteinsABSTRACT
BACKGROUND: In our previous study, kindlin-2 promoted skin wound healing and decreased the permeability of neovascularization during angiogenesis. Herein, we explored the biological function and underlying mechanism of kindlin-2 in cutaneous melanoma. METHODS AND RESULTS: Through a series of in vitro assays, we found that high levels of kindlin-2 promoted migration and invasion of melanoma cells without influencing cell proliferation. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses showed that upregulated kindlin-2 promoted the cellular epithelial-mesenchymal transition (EMT). Importantly, we found that melanoma cells overexpressing kindlin-2 promoted angiogenesis and VEGFA secretion in vitro and facilitated tumour growth and lung metastasis in vivo. To unveil the underlying mechanism, we conducted Next-generation sequencing (NGS) and differential expression analyses. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that overlapping differentially expressed genes (DEGs) were primarily enriched in the TGF-ß, mTOR and VEGF signalling pathways. Then, we confirmed that the mTOR/VEGFA pathway was activated during the process of kindlin-2-induced melanoma progression and angiogenesis. Moreover, we demonstrated that kindlin-2 was significantly overexpressed in clinical melanoma samples and that a high level of kindlin-2 predicted a poor prognosis. CONCLUSIONS: Taken together, these findings showed that kindlin-2 promotes angiogenesis and tumour progression via the mTOR/VEGFA pathway.