DEAD-Box Helicase 17 exacerbates non-alcoholic steatohepatitis via transcriptional repression of cyp2c29, inducing hepatic lipid metabolism disorder and eliciting the activation of M1 macrophages.

OBJECTIVE: Our study was to elucidate the role of RNA helicase DEAD-Box Helicase 17 (DDX17) in NAFLD and to explore its underlying mechanisms. METHODS: We created hepatocyte-specific Ddx17-deficient mice aim to investigate the impact of Ddx17 on NAFLD induced by a high-fat diet (HFD) as well as methionine and choline-deficient l-amino acid diet (MCD) in adult male mice. RNA-seq and lipidomic analyses were conducted to depict the metabolic landscape, and CUT&Tag combined with chromatin immunoprecipitation (ChIP) and luciferase reporter assays were conducted. RESULTS: In this work, we observed a notable increase in DDX17 expression in the livers of patients with NASH and in murine models of NASH induced by HFD or MCD. After introducing lentiviruses into hepatocyte L02 for DDX17 knockdown or overexpression, we found that lipid accumulation induced by palmitic acid/oleic acid (PAOA) in L02 cells was noticeably weakened by DDX17 knockdown but augmented by DDX17 overexpression. Furthermore, hepatocyte-specific DDX17 knockout significantly alleviated hepatic steatosis, inflammatory response and fibrosis in mice after the administration of MCD and HFD. Mechanistically, our analysis of RNA-seq and CUT&Tag results combined with ChIP and luciferase reporter assays indicated that DDX17 transcriptionally represses Cyp2c29 gene expression by cooperating with CCCTC binding factor (CTCF) and DEAD-Box Helicase 5 (DDX5). Using absolute quantitative lipidomics analysis, we identified a hepatocyte-specific DDX17 deficiency that decreased lipid accumulation and altered lipid composition in the livers of mice after MCD administration. Based on the RNA-seq analysis, our findings suggest that DDX17 could potentially have an impact on the modulation of lipid metabolism and the activation of M1 macrophages in murine NASH models. CONCLUSION: These results imply that DDX17 is involved in NASH development by promoting lipid accumulation in hepatocytes, inducing the activation of M1 macrophages, subsequent inflammatory responses and fibrosis through the transcriptional repression of Cyp2c29 in mice. Therefore, DDX17 holds promise as a potential drug target for the treatment of NASH.

Tubular cell transcriptional intermediary factor 1γ deficiency exacerbates kidney injury-induced tubular cell polyploidy and fibrosis.

Tubulointerstitial fibrosis is considered the final convergent pathway of progressive chronic kidney diseases (CKD) regardless of etiology. However, mechanisms underlying kidney injury-induced fibrosis largely remain unknown. Recent studies have indicated that transcriptional intermediary factor 1γ (TIF1γ) inhibits the progression of fibrosis in other organs. Here, we found that TIF1γ was highly expressed in the cytoplasm and nucleus of the kidney proximal tubule. Interestingly, we found tubular TIF1γ expression was decreased in patients with CKD, including those with diabetes, hypertension, and IgA nephropathy, and in mouse models with experimental kidney fibrosis (unilateral ureteral obstruction [UUO], folic acid nephropathy [FAN], and aristolochic acid-induced nephrotoxicity). Tubule-specific knock out of TIF1γ in mice exacerbated UUO- and FAN-induced tubular cell polyploidy and subsequent fibrosis, whereas overexpression of kidney TIF1γ protected mice against kidney fibrosis. Mechanistically, in tubular epithelial cells, TIF1γ exerted an antifibrotic role via transforming growth factor-ß (TGF-ß)-dependent and -independent signaling. TIF1γ hindered TGF-ß signaling directly by inhibiting the formation and activity of the transcription factor Smad complex in tubular cells, and we discovered that TIF1γ suppressed epidermal growth factor receptor (EGFR) signaling upstream of TGF-ß signaling in tubular cells by ubiquitylating EGFR at its lysine 851/905 sites thereby promoting EGFR internalization and lysosomal degradation. Pharmacological inhibition of EGFR signaling attenuated exacerbated polyploidization and the fibrotic phenotype in mice with tubule deletion of TIF1γ. Thus, tubular TIF1γ plays an important role in kidney fibrosis by suppressing profibrotic EGFR and TGF-ß signaling. Hence, our findings suggest that maintaining homeostasis of tubular TIF1γ may be a new therapeutic option for treating tubulointerstitial fibrosis and subsequent CKD.

Discoidin domain receptor 1 promotes hepatocellular carcinoma progression through modulation of SLC1A5 and the mTORC1 signaling pathway.

PURPOSE: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with a high mortality rate. Receptor tyrosine kinases play important roles in the occurrence and development of various cancers. Discoid protein domain receptor 1 (DDR1) is a special type of transmembrane receptor tyrosine kinase. Here, we show that the expression of DDR1 is significantly increased in HCC and is related to a poor clinical prognosis. METHODS: The expression of DDR1 in HCC cell lines and primary HCC specimens was evaluated using Western blotting and immunohistochemistry. A correlation between DDR1 and SLC1A5 expression was also investigated in primary HCC specimens. Cell proliferation was evaluated using in vitro CCK8 and colony formation assays. Gene knock-down and overexpression assays, CHX, NH4CL and Mg132 interference tests and immunoprecipitation, as well as nude mouse xenograft models were used to assess the mechanism by which DDR1 promotes tumorigenesis in vitro and in vivo. RESULTS: We found that DDR1 promotes the proliferation of HCC cells and accelerates the growth of HCC tumor xenografts, while DDR1 downregulation had the opposite effect. We also found that loss or gain of DDR1 expression affected HCC cell cycle progression. Mechanistically, we found that DDR1 interacts with SLC1A5, which belongs to the solute carrier (SLC) family of transporters, and regulates its stability, thereby affecting the mTORC1 signaling pathway. In addition, we found that SLC1A5 regulation by DDR1 can be restored by lysosome inhibitors. We also found that DDR1 is highly expressed in HCC tissues and that increased DDR1 expression predicts a shorter overall survival (OS) time. We additionally found that the expression of SLC1A5 was positively correlated with that of DDR1. Together, our data indicate that DDR1 acts as a tumor-promoting factor that can control HCC cell proliferation and cell cycle progression by stabilizing SLC1A5 in a lysosome-dependent way. CONCLUSIONS: Our study reveals a new mechanism by which DDR1 plays a liver cancer-promoting role. We also found that DDR1 expression serves as an independent prognostic marker, and that DDR1 and SLC1A5 expression levels are positively correlated in clinical samples. Our findings provide a new perspective for understanding HCC development and offers new targets for the treatment and management of HCC.

The 14-3-3σ protein promotes HCC anoikis resistance by inhibiting EGFR degradation and thereby activating the EGFR-dependent ERK1/2 signaling pathway.

Resistance to anoikis, cell death due to matrix detachment, is acquired during tumor progression. The 14-3-3σ protein is implicated in the development of chemo- and radiation resistance, indicating a poor prognosis in multiple human cancers. However, its function in anoikis resistance and metastasis in hepatocellular carcinoma (HCC) is currently unknown. Methods: Protein expression levels of 14-3-3σ were measured in paired HCC and normal tissue samples using western blot and immunohistochemical (IHC) staining. Statistical analysis was performed to evaluate the clinical correlation between 14-3-3σ expression, clinicopathological features, and overall survival. Artificial modulation of 14-3-3σ (downregulation and overexpression) was performed to explore the role of 14-3-3σ in HCC anoikis resistance and tumor metastasis in vitro and in vivo. Association of 14-3-3σ with epidermal growth factor receptor (EGFR) was assayed by co-immunoprecipitation. Effects of ectopic 14-3-3σ expression or knockdown on EGFR signaling, ligand-induced EGFR degradation and ubiquitination were examined using immunoblotting and co-immunoprecipitation, immunofluorescence staining, and flow cytometry analysis. The levels of EGFR ubiquitination, the interaction between EGFR and 14-3-3σ, and the association of EGFR with c-Cbl after EGF stimulation, in 14-3-3σ overexpressing or knockdown cells were examined to elucidate the mechanism by which 14-3-3σ inhibits EGFR degradation. Using gain-of-function or loss-of-function strategies, we further investigated the role of the EGFR signaling pathway and its downstream target machinery in 14-3-3σ-mediated anoikis resistance of HCC cells. Results: We demonstrated that 14-3-3σ was upregulated in HCC tissues, whereby its overexpression was correlated with aggressive clinicopathological features and a poor prognosis. In vitro and in vivo experiments indicated that 14-3-3σ promoted anoikis resistance and metastasis of HCC cells. Mechanistically, we show that 14-3-3σ can interact with EGFR and significantly inhibit EGF-induced degradation of EGFR, stabilizing the activated receptor, and therefore prolong the activation of EGFR signaling. We demonstrated that 14-3-3σ downregulated ligand-induced EGFR degradation by inhibiting EGFR-c-Cbl association and subsequent c-Cbl-mediated EGFR ubiquitination. We further verified that activation of the ERK1/2 pathway was responsible for 14-3-3σ-mediated anoikis resistance of HCC cells. Moreover, EGFR inactivation could reverse the 14-3-3σ-mediated effects on ERK1/2 phosphorylation and anoikis resistance. Expression of 14-3-3σ and EGFR were found to be positively correlated in human HCC tissues. Conclusions: Our results indicate that 14-3-3σ plays a pivotal role in the anoikis resistance and metastasis of HCC cells, presumably by inhibiting EGFR degradation and regulating the activation of the EGFR-dependent ERK1/2 pathway. To our best knowledge, this is the first report of the role of 14-3-3σ in the anoikis resistance of HCC cells, offering new research directions for the treatment of metastatic cancer by targeting 14-3-3σ.

Microenvironment characterization and multi-omics signatures related to prognosis and immunotherapy response of hepatocellular carcinoma.

BACKGROUND: Immune cell infiltration in the tumor microenvironment (TME) affects tumor initiation, patients' prognosis and immunotherapy strategies. However, their roles and interactions with genomics and molecular processes in hepatocellular carcinoma (HCC) still have not been systematically evaluated. METHODS: We performed unsupervised clustering of total 1000 HCC samples including discovery and validation group from available public datasets. Immune heterogeneity of each subtype was explored by multi-dimension analysis. And a support vector machine (SVM) model based on multi-omics signatures was trained and tested. Finally, we performed immunohistochemistry to verify the immune role of signatures. RESULTS: We defined three immune subtypes in HCC, with diverse clinical, molecular, and genomic characteristics. Cluster1 had worse prognosis, better anti-tumor characteristics and highest immune scores, but also accompanied by immunosuppression and T cell dysfunction. Meanwhile, a better anti-PD1/CTLA4 immunotherapeutic response was predicted in cluster1. Cluster2 was enriched in TAM-M2 and stromal cells, indicating immunosuppression. Cluster3, with better prognosis, had lowest CD8 T cell but highest immune resting cells. Further, based on genomic signatures, we developed an SVM classifier to identify the patient's immunological status, which was divided into Type A and Type B, in which Type A had poorer prognosis, higher T cell dysfunction despite higher T cell infiltration, and had better immunotherapeutic response. At the same time, MMP9 may be a potential predictor of the immune characteristics and immunotherapeutic response in HCC. CONCLUSIONS: Our work demonstrated 3 immune clusters with different features. More importantly, multi-omics signatures, such as MMP9 was identified based on three clusters to help us recognize patients with different prognosis and responses to immunotherapy in HCC. This study could further reveal the immune status of HCC and provide potential predictors for immune checkpoint treatment response.

Genome-wide screening diagnostic biomarkers and the construction of prognostic model of hepatocellular carcinoma.

Although methods in diagnosis and therapy of hepatocellular carcinoma (HCC) have made significant progress in decades, the overall survival (OS) of HCC remains dissatisfactory, so it is particularly important to find better diagnostic and prognostic biomarkers. In this study, we found a more reliable potential diagnostic biomarkers and constructed a more accurate prognostic evaluation model based on integrated transcriptome sequencing analysis of multiple independent data sets. First, we performed quality evaluation and differential analysis on seven Gene Expression Omnibus (GEO) data sets, and then comprehensively analyzed the differentially expressed genes with a robust rank aggregation algorithm. Next, Least absolute shrinkage and selection operator (LASSO) regression was used to establish an 8-gene prognostic risk score (RS) model. Finally, the prognostic model was further validated in the GEO data set. Also, RS has independence on other clinicopathological characteristics but has similarities in prognostic assessment compared with the T stage. Moreover, the combination of T stage and prognostic RS model based on the 8-gene had a better prognostic evaluation effect. In brief, our research suggest that the prognostic risk model of 8 genes has important clinical significance in HCC patients, and can further enrich the prognostic guidance value of the traditional T stage.

CRISPR/Cas9-mediated knockout of HBsAg inhibits proliferation and tumorigenicity of HBV-positive hepatocellular carcinoma cells.

Chronic hepatitis B virus (HBV) infection remains the most common risk factor for hepatocellular carcinoma (HCC). High HBV surface antigen (HBsAg) levels are highly correlated with hepatocarcinogenesis and HBV-associated HCC development. However, the role and detailed mechanisms associated with HBsAg in HCC development remain elusive. In this study, we designed specific single guide RNAs (sgRNAs) targeting the open reading frames, preS1/preS2/S, of the HBV genome and established HBsAg knockout HCC cell lines using the CRISPR/Cas9 system. We showed that knockout of HBsAg in HCC cell lines decreased HBsAg expression and significantly attenuated HCC proliferation in vitro, as well as tumorigenicity in vivo. We also found that overexpression of HBsAg, including the large (LHBs), middle (MHBs), and small (SHBs) surface proteins promoted proliferation and tumor formation in HCC cells. Moreover, we demonstrated that knockout of HBsAg in HCC cells decreased interleukin (IL)-6 production and inhibited signal transducer and activator of transcription 3 (STAT3) signaling, while overexpression of HBsAg induced a substantial accumulation of pY-STAT3. Collectively, these results highlighted the tumorigenic role of HBsAg and implied that the IL-6-STAT3 pathway may be implicated in the HBsAg-mediated malignant potential of HBV-associated HCC.