DNA of Neutrophil Extracellular Traps Binds TMCO6 to Impair CD8+ T-cell Immunity in Hepatocellular Carcinoma.

Neutrophil extracellular traps (NET), formed by the extracellular release of decondensed chromatin and granules, have been shown to promote tumor progression and metastasis. Tumor-associated neutrophils in hepatocellular carcinoma (HCC) are prone to NET formation, highlighting the need for a more comprehensive understanding of the mechanisms of action of NETs in liver cancer. Here, we showed that DNA of NETs (NET-DNA) binds transmembrane and coiled-coil domains 6 (TMCO6) on CD8+ T cells to impair antitumor immunity and thereby promote HCC progression. TGFß1 induced NET formation, which recruited CD8+ T cells. Binding to NET-DNA inhibited CD8+ T cells function while increasing apoptosis and TGFß1 secretion, forming a positive feedback loop to further stimulate NET formation and immunosuppression. Mechanistically, the N-terminus of TMCO6 interacted with NET-DNA and suppressed T-cell receptor signaling and NFκB p65 nuclear translocation. Blocking NET formation by inhibiting PAD4 induced potent antitumor effects in wild-type mice but not TMCO6-/- mice. In clinical samples, CD8+ T cells expressing TMCO6 had an exhausted phenotype. TGFß1 signaling inhibition or TMCO6 deficiency combined with anti-PD-1 abolished NET-driven HCC progression in vivo. Collectively, this study unveils the role of NET-DNA in impairing CD8+ T-cell immunity by binding TMCO6 and identifies targeting this axis as an immunotherapeutic strategy for blocking HCC progression. SIGNIFICANCE: TMCO6 is a receptor for DNA of NETs that mediates CD8+ T-cell dysfunction in HCC, indicating that the NET-TMCO6 axis is a promising target for overcoming immunosuppression in liver cancer.

Sex Differences in Genomic Features of Hepatitis B-Associated Hepatocellular Carcinoma With Distinct Antitumor Immunity.

BACKGROUND & AIMS: Aflatoxin exposure increases the risk for hepatocellular carcinoma (HCC) in hepatitis B virus (HBV)-infected individuals, particularly males. We investigated sex-based differences in the HCC genome and antitumor immunity. METHODS: Whole-genome, whole-exome, and RNA sequencing were performed on 101 HCC patient samples (47 males, 54 females) that resulted from HBV infection and aflatoxin exposure from Qidong. Androgen on the expression of aflatoxin metabolism-related genes and nonhomologous DNA end joining (NHEJ) factors were examined in HBV-positive HCC cell lines, and further tested in tumor-bearing syngeneic mice. RESULTS: Qidong HCC differed between males and females in genomic landscape and transcriptional dysfunction pathways. Compared with females, males expressed higher levels of aflatoxin metabolism-related genes, such as AHR and CYP1A1, and lower levels of NHEJ factors, such as XRCC4, LIG4, and MRE11, showed a signature of up-regulated type I interferon signaling/response and repressed antitumor immunity. Treatment with AFB1 in HBV-positive cells, the addition of 2 nmol/L testosterone to cultures significantly increased the expression of aflatoxin metabolism-related genes, but reduced NHEJ factors, resulting in more nuclear DNA leakage into cytosol to activate cGAS-STING. In syngeneic tumor-bearing mice that were administrated tamoxifen daily via oral gavage, favorable androgen signaling repressed NHEJ factor expression and activated cGAS-STING in tumors, increasing T-cell infiltration and improving anti-programmed cell death protein 1 treatment effect. CONCLUSIONS: Androgen signaling in the context of genotoxic stress repressed DNA damage repair. The alteration caused more nuclear DNA leakage into cytosol to activate the cGAS-STING pathway, which increased T-cell infiltration into tumor mass and improved anti-programmed cell death protein 1 immunotherapy in HCCs.

High Mobility Group Protein B1 Decreases Surface Localization of PD-1 to Augment T-cell Activation.

High-mobility group protein B1 (HMGB1) is a danger signaling molecule that has been found to trigger an effective antitumor immune response. However, the mechanisms underlying its antitumor effects are not fully understood. Here, we found that HMGB1 release induced by chemotherapy in patients with non-small cell lung cancer was negatively correlated with PD-1 expression on CD8+ T cells. In vitro analysis indicated that treatment with HMGB1 led to a significant decrease in the level of expression of PD-1 on CD8+ T cells. Further analysis demonstrated that HMGB1 reduced PD-1 expression by inducing dynamin-mediated internalization of the protein, leading to early endocytosis in the cytoplasm, and subsequently degradation in the lysosomes. In a xenograft model, HER2-targeted chimeric antigen receptor (CAR) T cells had enhanced function in the presence of HMGB1. These data identify a role for HMGB1 as a negative regulator of PD-1 signaling in lung cancer and the observed antitumor effect of HMGB1 on CAR T cells may provide a theoretical foundation for a new immunotherapy combination.

Long Noncoding RNA lncNDEPD1 Regulates PD-1 Expression via miR-3619-5p in CD8+ T Cells.

Therapies targeting programmed cell death protein 1 (PD-1) have gained great success in patients with multiple types of cancer. The regulatory mechanisms underlying PD-1 expression have been extensively explored. However, the impact of long noncoding RNAs on PD-1 expression remains elusive. In this study, we identified the Notch1/lncNDEPD1 axis, which plays a critical role in PD-1 expression in human CD8+ T cells. RNA sequencing and quantitative reverse transcription PCR data showed that lncNDEPD1 was upregulated in activated T cells, especially in PD-1high subsets. Fluorescence in situ hybridization demonstrated that lncNDEPD1 was localized in the cytoplasm. A mechanistic study showed that lncNDEPD1 could bind with miR-3619-5p and PDCD1 mRNA to prevent PDCD1 mRNA degradation and then upregulate PD-1 expression. A chromatin immunoprecipitation assay showed that Notch1 directly binds to the promoter of lncNDEPD1 instead of PDCD1 Furthermore, chimeric Ag receptor T cells expressing lncNDEPD1-specific short hairpin RNAs were generated. Chimeric Ag receptor T cells with decreased lncNDEPD1 expression showed enhanced tumoricidal effects when PD-L1 was present. Our work uncovered a new regulatory mechanism of PD-1 expression and thus provided a potential target to decrease PD-1 without affecting T cell function.

Galectin-3 favours tumour metastasis via the activation of ß-catenin signalling in hepatocellular carcinoma.

BACKGROUND: High probability of metastasis limited the long-term survival of patients with hepatocellular carcinoma (HCC). Our previous study revealed that Galectin-3 was closely associated with poor prognosis in HCC patients. METHODS: The effects of Galectin-3 on tumour metastasis were investigated in vitro and in vivo, and the underlying biological and molecular mechanisms involved in this process were evaluated. RESULTS: Galectin-3 showed a close correlation with vascular invasion and poor survival in a large-scale study in HCC patients from multiple sets. Galectin-3 was significantly involved in diverse metastasis-related processes in HCC cells, such as angiogenesis and epithelial-to-mesenchymal transition (EMT). Mechanistically, Galectin-3 activated the PI3K-Akt-GSK-3ß-ß-catenin signalling cascade; the ß-catenin/TCF4 transcriptional complex directly targeted IGFBP3 and vimentin to regulate angiogenesis and EMT, respectively. In animal models, Galectin-3 enhanced the tumorigenesis and metastasis of HCC cells via ß-catenin signalling. Moreover, molecular deletion of Galectin-3-ß-catenin signalling synergistically improved the antitumour effect of sorafenib. CONCLUSIONS: The Galectin-3-ß-catenin-IGFBP3/vimentin signalling cascade was determined as a central mechanism controlling HCC metastasis, providing possible biomarkers for predicating vascular metastasis and sorafenib resistance, as well as potential therapeutic targets for the treatment of HCC patients.

Large-scale analyses identify a cluster of novel long noncoding RNAs as potential competitive endogenous RNAs in progression of hepatocellular carcinoma.

The abnormal expression of noncoding RNAs has attracted increasing interest in the field of hepatocellular carcinoma progression. However, the underlying molecular mechanisms mediated by noncoding RNAs in these processes are unclear. Here, we obtained the expression profiles of long noncoding RNAs, microRNAs, and mRNAs from the Gene Expression Omnibus database and identified hepatocarcinogenesis-specific differentially expressed transcripts. Next, we identified significant Gene Ontology and pathway terms that the differentially expressed transcripts involved in. Using functional analysis and target prediction, we constructed a hepatocellular carcinoma-associated deregulated competitive endogenous RNA network to reveal the potential mechanisms underlying tumor progression. By analyzing The Cancer Genome Atlas dataset, six key long noncoding RNAs showed significant association with overall survival as well as strong correlation with some microRNAs and mRNAs in the competitive endogenous RNA network. We further validated the above results and determined their diagnostic and prognostic value in clinical samples. Importantly, by large-scale analyses, we identified a cluster of long noncoding RNAs, GBAP1, MCM3AP-AS1, SLC16A1-AS1, C3P1, DIO3OS, and HNF4A-AS1 as candidate biomarkers for the diagnosis and prognosis of hepatocellular carcinoma, which will improve our understanding of competitive endogenous RNA-mediated regulatory mechanisms underlying hepatocellular carcinoma development and will provide novel therapeutic targets in the future.

Low-Dose IFNγ Induces Tumor Cell Stemness in Tumor Microenvironment of Non-Small Cell Lung Cancer.

IFNγ is conventionally recognized as an inflammatory cytokine that plays a central role in antitumor immunity. Although it has been used clinically to treat a variety of malignancies, low levels of IFNγ in the tumor microenvironment (TME) increase the risk of tumor metastasis during immunotherapy. Accumulating evidence suggests that IFNγ can induce cancer progression, yet the mechanisms underlying the controversial role of IFNγ in tumor development remain unclear. Here, we reveal a dose-dependent effect of IFNγ in inducing tumor stemness to accelerate cancer progression in patients with a variety of cancer types. Low levels of IFNγ endowed cancer stem-like properties via the intercellular adhesion molecule-1 (ICAM1)-PI3K-Akt-Notch1 axis, whereas high levels of IFNγ activated the JAK1-STAT1-caspase pathway to induce apoptosis in non-small cell lung cancer (NSCLC). Inhibition of ICAM1 abrogated the stem-like properties of NSCLC cells induced by the low dose of IFNγ both in vitro and in vivo. This study unveils the role of low levels of IFNγ in conferring tumor stemness and elucidates the distinct signaling pathways activated by IFNγ in a dose-dependent manner, thus providing new insights into cancer treatment, particularly for patients with low expression of IFNγ in the TME. SIGNIFICANCE: These findings reveal the dose-dependent effect of IFNγ in inducing tumor stemness and elucidate the distinct molecular mechanisms activated by IFNγ in a dose-dependent manner.

Cancer-cell-secreted CXCL11 promoted CD8+ T cells infiltration through docetaxel-induced-release of HMGB1 in NSCLC.

BACKGROUND: Chemotherapy combined with immunotherapy becomes the main trend in lung cancer intervention; however, how chemotherapy promotes the immune function remains elusive. Therefore, we sought to determine how chemotherapy promotes the immune function. METHODS: We determined in 100 NSCLC patients the expression of CD8, functional markers (IFN-γ, Granzyme B, and Perforin) and specific chemokines by quantitative real-time reverse transcriptase-PCR. Functional experiments were carried out to check whether docetaxel (DOC), a chemotherapeutic agent, modifies the expression of HMGB1 and CXCL11, and influences the infiltration properties of CD8+ T cells to the tumor microenvironment. The mechanism of the release of HMGB1 and CXCL11 was determined by flow cytometry, immunofluorescence and western blotting. In in vivo experiment, we confirmed how DOC enhanced the recruitment of HER2-CAR T cells to tumor sites. RESULTS: We found that DOC upregulated the expression of chemokine receptor ligand CXCL11 in tumor microenvironment and subsequently enhanced CD8+ T cell recruitment. DOC treatment significantly increased HMGB1 release in an ROS-dependent manner. Recombinant protein HMGB1 stimulated the secretion of CXCL11 via NF-κB activation in vitro. Tumors from DOC-treated mice exhibited higher expression of HMGB1 and CXCL11, more HER2-CAR T cell infiltration, and reduced progression, relative to control. Increased HMGB1 and CXCL11 expressions were positively correlated with prolonged overall survival of lung cancer patients. CONCLUSIONS: Our results demonstrate that DOC induces CD8+ T cell recruitment to the tumor microenvironment by enhancing the secretion of HMGB1 and CXCL11, thus improving the anti-tumor efficacy, indicating that modulating the HMGB1-CXCL11 axis might be helpful for NSCLC treatment.

A cycle involving HMGB1, IFN-γ and dendritic cells plays a putative role in anti-tumor immunity.

An important subset in regulating antitumor immunity is the maturation and accumulation of intratumor dendritic cells (DCs), inducing potent T cell cytotoxicity. In this study, we explored how the soluble abundant high-mobility group box 1 protein (HMGB1) affected DC activation and retention within lung cancers, and in which way the resultant interferon-γ (IFN-γ) further enhanced DC maturation and accumulation. It was discovered that HMGB1 was correlated with DC markers HLA-DR and CD86 in lung cancers at both mRNA and protein level. Further analyses showed HMGB1 enhanced the maturation of DCs, indicated by upregulated IFN-γ in CD8+ T cells. Additionally, HMGB1 increased the accumulation of DCs by promoting CCR5 and CXCR3 production. Moreover, the resultant IFN-γ elevated the levels of HMGB1 and DC-associated chemokines, CCL5, CXCL10 and CXCL11 in tumor cells. Hence, the HMGB1-IFN-γ cycle may represent an important mechanism underlying DC-mediated anti-tumor immune response.