Distinct single-cell immune ecosystems distinguish true and de novo HBV-related hepatocellular carcinoma recurrences.

OBJECTIVE: Revealing the single-cell immune ecosystems in true versus de novo hepatocellular carcinoma (HCC) recurrences could help the optimal development of immunotherapies. DESIGN: We performed 5'and VDJ single-cell RNA-sequencing on 34 samples from 20 recurrent HCC patients. Bulk RNA-sequencing, flow cytometry, multiplexed immunofluorescence, and in vitro functional analyses were performed on samples from two validation cohorts. RESULTS: Analyses of mutational profiles and evolutionary trajectories in paired primary and recurrent HCC samples using whole-exome sequencing identified de novo versus true recurrences, some of which occurred before clinical diagnosis. The tumour immune microenvironment (TIME) of truly recurrent HCCs was characterised by an increased abundance in KLRB1+CD8+ T cells with memory phenotype and low cytotoxicity. In contrast, we found an enrichment in cytotoxic and exhausted CD8+ T cells in the TIME of de novo recurrent HCCs. Transcriptomic and interaction analyses showed elevated GDF15 expression on HCC cells in proximity to dendritic cells, which may have dampened antigen presentation and inhibited antitumour immunity in truly recurrent lesions. In contrast, myeloid cells' cross talk with T cells-mediated T cell exhaustion and immunosuppression in the TIME of de novo recurrent HCCs. Consistent with these findings, a phase 2 trial of neoadjuvant anti-PD-1 immunotherapy showed more responses in de novo recurrent HCC patients. CONCLUSION: True and de novo HCC recurrences occur early, have distinct TIME and may require different immunotherapy strategies. Our study provides a source for genomic diagnosis and immune profiling for guiding immunotherapy based on the type of HCC recurrence and the specific TIME.

Cripto-1 promotes tumor invasion and predicts poor outcomes in hepatocellular carcinoma.

Cripto-1 (CR1), an oncofetal protein, had been implied to reactivate in some cancers. However, the relationship between CR1 expression and patient outcomes and the tumor biological function of CR1 contributing to invasion and metastasis in hepatocellular carcinoma (HCC) is poorly defined. In this study, we demonstrated that CR1 was expressed in over 80% of HCCs in a training cohort (n = 242) and a validation cohort (n = 159). High CR1 expression was significantly correlated with aggressive HCC phenotypes (i.e. portal vein tumor thrombus, microscopic vascular invasion, multiple tumors and poor tumor differentiation). In both the training and validation cohorts, patients with high CR1 expression had remarkably shorter disease-free survival and overall survival rates than those with low CR1 expression. A series in vitro and in vivo assays showed that CR1 substantially promoted HCC cell migration, invasion and metastasis. Mechanistically, we demonstrated that CR1 induced HCC cells to undergo epithelial-mesenchymal transition through activating the Akt/NFκB/p65 signaling. Chromatin immunoprecipitation assay showed that NFκB/p65 enhanced CR1 expression by binding its promoter. Thus, CR1 and NFκB/p65 form a positive feedback loop that sustained the process of migration and invasion of HCC. Therefore, CR1 plays an important role in HCC invasion and metastasis and may be an effective and reliable prognostic biomarker for HCC recurrence after resection. Targeting CR1 may be a promising treatment for HCC.

Cripto-1 promotes tumor invasion and predicts poor outcomes in hepatocellular carcinoma.

Cripto-1 (CR1), an oncofetal protein, had been implied to reactivate in some cancers. However, the relationship between CR1 expression and patient outcomes and the tumor biological function of CR1 contributing to invasion and metastasis in hepatocellular carcinoma (HCC) is poorly defined. In this study, we demonstrated that CR1 was expressed in over 80% of HCCs in a training cohort (n = 242) and a validation cohort (n = 159). High CR1 expression was significantly correlated with aggressive HCC phenotypes (i.e. portal vein tumor thrombus, microscopic vascular invasion, multiple tumors and poor tumor differentiation). In both the training and validation cohorts, patients with high CR1 expression had remarkably shorter disease-free survival and overall survival rates than those with low CR1 expression. A series in vitro and in vivo assays showed that CR1 substantially promoted HCC cell migration, invasion and metastasis. Mechanistically, we demonstrated that CR1 induced HCC cells to undergo epithelial-mesenchymal transition through activating the Akt/NFκB/p65 signaling. Chromatin immunoprecipitation assay showed that NFκB/p65 enhanced CR1 expression by binding its promoter. Thus, CR1 and NFκB/p65 form a positive feedback loop that sustained the process of migration and invasion of HCC. Therefore, CR1 plays an important role in HCC invasion and metastasis and may be an effective and reliable prognostic biomarker for HCC recurrence after resection. Targeting CR1 may be a promising treatment for HCC.

Kinesin family member 2C aggravates the progression of hepatocellular carcinoma and interacts with competing endogenous RNA.

Kinesin family member 2C (KIF2C), a substantial mitotic regulator, has been verified to exert a malignant function in several cancers. However, its function in hepatocellular carcinoma (HCC) remains unclear. In this study, the expression profile of KIF2C in HCC was characterized through the dataset from the TCGA and clinical tissue microarrays containing 220 pairs of resected HCC tissues and adjacent nontumor tissues in our hospital. The results indicated that KIF2C was substantially higher expression in tumor tissues than adjacent nontumor tissues. High expression of KIF2C significantly correlated with large tumor (>5.0 cm) (P = .001) and implied a dismal postoperative overall survival (OS) (hazard ratio [HR] = 1.729; P = .002) in our cohort of patients. Gain and loss of function assays displayed that KIF2C promoted HCC cell proliferation, accelerated cell cycle progression, and impeded apoptosis. By bioinformatic tools and mechanistic investigation, we found that KIF2C interacted with various cell-cycle-related proteins and was significantly involved in growth-promoting pathways. KIF2C upregulated PCNA and CDC20 expression. Subsequently, we investigated the regulation of KIF2C by competing endogenous RNA and elucidated that has-miR-6715a-3p was directly bond to the 3'-untranslated region of KIF2C through dual luciferase assays, thereby inhibiting KIF2C expression. Furthermore, the long noncoding RNA GS1-358P8.4 was found to be a candidate of KIF2C for has-miR-6715a-3p binding. HCC patients with high lncRNA-GS1-358P8.4 expression had shorter OS and relapse-free survival compared to those with low expression, which was accordance with the KIF2C. Taken together, KIC2C aggravated HCC progression, it could serve as a prognostic indicator and confer a novel target for clinical treatment.

Mechanical confinement promotes heat resistance of hepatocellular carcinoma via SP1/IL4I1/AHR axis.

Mechanical stress can modulate the fate of cells in both physiological and extreme conditions. Recurrence of tumors after thermal ablation, a radical therapy for many cancers, indicates that some tumor cells can endure temperatures far beyond physiological ones. This unusual heat resistance with unknown mechanisms remains a key obstacle to fully realizing the clinical potential of thermal ablation. By developing a 3D bioprinting-based thermal ablation system, we demonstrate that hepatocellular carcinoma (HCC) cells in this 3D model exhibit enhanced heat resistance as compared with cells on plates. Mechanistically, the activation of transcription factor SP1 under mechanical confinement enhances the transcription of Interleukin-4-Induced-1, which catalyzes tryptophan metabolites to activate the aryl hydrocarbon receptor (AHR), leading to heat resistance. Encouragingly, the AHR inhibitor prevents HCC recurrence after thermal ablation. These findings reveal a previously unknown role of mechanical confinement in heat resistance and provide a rationale for AHR inhibitors as neoadjuvant therapy.

Genomic Instability of Mutation-Derived Gene Prognostic Signatures for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the major cancer-related deaths worldwide. Genomic instability is correlated with the prognosis of cancers. A biomarker associated with genomic instability might be effective to predict the prognosis of HCC. In the present study, data of HCC patients from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases were used. A total of 370 HCC patients from the TCGA database were randomly classified into a training set and a test set. A prognostic signature of the training set based on nine overall survival (OS)-related genomic instability-derived genes (SLCO2A1, RPS6KA2, EPHB6, SLC2A5, PDZD4, CST2, MARVELD1, MAGEA6, and SEMA6A) was constructed, which was validated in the test and TCGA and ICGC sets. This prognostic signature showed more accurate prediction for prognosis of HCC compared with tumor grade, pathological stage, and four published signatures. Cox multivariate analysis revealed that the risk score could be an independent prognostic factor of HCC. A nomogram that combines pathological stage and risk score performed well compared with an ideal model. Ultimately, paired differential expression profiles of genes in the prognostic signature were validated at mRNA and protein level using HCC and paratumor tissues obtained from our institute. Taken together, we constructed and validated a genomic instability-derived gene prognostic signature, which can help to predict the OS of HCC and help us to explore the potential therapeutic targets of HCC.

Cathepsin C Interacts with TNF-α/p38 MAPK Signaling Pathway to Promote Proliferation and Metastasis in Hepatocellular Carcinoma.

PURPOSE: Although cathepsin C (CTSC) has been reported to maintain malignant biological properties in various cancers, its functions in hepatocellular carcinoma (HCC) remain obscure. We aimed to investigate the potential role of CTSC in HCC. MATERIALS AND METHODS: HCC tissue microarrays (n=122) were employed to analyze the correlation between CTSC expression and clinicopathological characteristics through immunohistochemistry staining. Quantitative real-time polymerase chain reaction, western blot assay, Cell Counting Kit-8 assay, colony formation, cell migration, and invasion assays, xenograft mice model were adopted to validate what had been indicated by the bioinformatic web tools. RESULTS: By bioinformatic tools and tissue microarrays, CTSC was found upregulated in HCC compared with normal liver tissues, and its higher expression was correlated with poor prognosis of HCC patients (hazard ratio, 2.402; 95% confidence interval, 1.493 to 3.865; p < 0.001). By gain/loss-of-function assays, we implicated that CTSC functioned as an oncogene to promote the proliferation and metastasis of HCC cells. Mechanistically, we revealed that CTSC was involved in several cancer-related signaling pathways by Gene Set Enrichment Analysis, among which tumor necrosis factor α (TNF-α)/p38 pathway was verified to be activated by CTSC. Furthermore, we found that TNF-α could activate CTSC expression in a concentration- dependent manner. Ralimetinib, an oral p38 mitogen-activated protein kinase (MAPK) inhibitor could inhibit CTSC expression. These indicated a potential positive feedback loop between CTSC and TNF-α/MAPK (p38) signaling. CONCLUSION: Taken together, CTSC plays an important role in the growth and metastasis of HCC and may be a promising therapeutic target upon HCC.

Transcatheter closure with use of the SHSMA occluder in 180 patients with congenital heart defects: preliminary results.

Transcatheter closure of congenital heart defects with the use of septal occluders has been widely accepted as a preferred treatment; however, the high cost of these devices limits their clinical application in some countries. Few clinical data are available regarding lower-cost products. Accordingly, we evaluated the efficacy and safety of the Chinese-made Shanghai Shape Memory Alloy (SHSMA) occluder in patients with congenital heart defects. From December 2001 through December 2008, a total of 180 patients with congenital heart defects (ages, 3-68 yr; mean age, 17.35 ± 13.22 yr) underwent transcatheter closure with use of the SHSMA occluder: 73 had atrial septal defects; 64, ventricular septal defects; 40, patent ductus arteriosus; and 3, complex congenital defects. The mean diameters of the defects were 20 ± 7.6 mm (atrial septal), 4.9 ± 2.1 mm (ventricular septal), and 5.6 ± 2.2 mm (patent ductus arteriosus). The procedural success rates were 98.6% for atrial defects, 98.4% for ventricular defects, and 100% for patent ductus arteriosus and for complex defects. The overall incidences of sequelae were 5.5%, 9.4%, 2.5%, and 0, respectively. Six months postprocedurally, complete occlusion was associated with a significant decrease in the right ventricular Tei index in atrial septal defect patients (P < 0.05) and with improvement of body mass index in 11 children. These results suggest that the SHSMA occluder is a safe, effective device for the transcatheter closure of congenital heart defects. For confirmation, a randomized controlled trial with more patients and a longer follow-up period is warranted.