FGF19/FGFR4-mediated elevation of ETV4 facilitates hepatocellular carcinoma metastasis by upregulating PD-L1 and CCL2.

BACKGROUND & AIMS: Metastasis remains the major reason for the high mortality of patients with hepatocellular carcinoma (HCC). This study was designed to investigate the role of E-twenty-six-specific sequence variant 4 (ETV4) in promoting HCC metastasis and to explore a new combination therapy strategy for ETV4-mediated HCC metastasis. METHODS: PLC/PRF/5, MHCC97H, Hepa1-6, and H22 cells were used to establish orthotopic HCC models. Clodronate liposomes were used to clear macrophages in C57BL/6 mice. Gr-1 monoclonal antibody was used to clear myeloid-derived suppressor cells (MDSCs) in C57BL/6 mice. Flow cytometry and immunofluorescence were used to detect the changes of key immune cells in the tumour microenvironment. RESULTS: ETV4 expression was positively related to higher tumour-node-metastasis (TNM) stage, poor tumour differentiation, microvascular invasion, and poor prognosis in human HCC. Overexpression of ETV4 in HCC cells transactivated PD-L1 and CCL2 expression, which increased tumour-associated macrophage (TAM) and MDSC infiltration and inhibited CD8+ T-cell accumulation. Knockdown of CCL2 by lentivirus or CCR2 inhibitor CCX872 treatment impaired ETV4-induced TAM and MDSC infiltration and HCC metastasis. Furthermore, FGF19/FGFR4 and HGF/c-MET jointly upregulated ETV4 expression through the ERK1/2 pathway. Additionally, ETV4 upregulated FGFR4 expression, and downregulation of FGFR4 decreased ETV4-enhanced HCC metastasis, which created a FGF19-ETV4-FGFR4 positive feedback loop. Finally, anti-PD-L1 combined with FGFR4 inhibitor BLU-554 or MAPK inhibitor trametinib prominently inhibited FGF19-ETV4 signalling-induced HCC metastasis. CONCLUSIONS: ETV4 is a prognostic biomarker, and anti-PD-L1 combined with FGFR4 inhibitor BLU-554 or MAPK inhibitor trametinib may be effective strategies to inhibit HCC metastasis. IMPACT AND IMPLICATIONS: Here, we reported that ETV4 increased PD-L1 and chemokine CCL2 expression in HCC cells, which resulted in TAM and MDSC accumulation and CD8+ T-cell inhibition to facilitate HCC metastasis. More importantly, we found that anti-PD-L1 combined with FGFR4 inhibitor BLU-554 or MAPK inhibitor trametinib markedly inhibited FGF19-ETV4 signalling-mediated HCC metastasis. This preclinical study will provide a theoretical basis for the development of new combination immunotherapy strategies for patients with HCC.

Comprehensive analysis of partial epithelial mesenchymal transition-related genes in hepatocellular carcinoma.

Increasing evidence has revealed that cancer cells undergoing an intermediate state, partial epithelial mesenchymal transition (p-EMT), tend to metastasize rather than complete EMT. We performed a comprehensive analysis of E-cadherin and 25 p-EMT-related genes in HCC to explore the roles and regulatory mechanisms of them in HCC. We analysed E-cadherin and 25 p-EMT-related genes in HCC and constructed an mRNA-miRNA-lncRNA ceRNA subnetwork containing p-EMT-related genes by bioinformatic approaches. IHC was used to identify the protein expression of key p-EMT-related genes, P4HA2, ITGA5, MMP9, MT1X and SPP1. Complete EMT is not necessary for HCC progression. Overexpression of P4HA2, ITGA5, MMP9, SPP1 and down-regulation of MT1X were found in HCC tissues, which were significantly associated with poor prognosis of HCC patients. By means of stepwise reverse prediction and validation from mRNA to lncRNA, an mRNA-miRNA-lncRNA ceRNA subnetwork correlated with HCC prognosis was identified by expression and survival analysis. This study implied that key p-EMT-related genes P4HA2, ITGA5, MMP9, MT1X, SPP1 could be prognostic biomarkers and potential targets of therapy for HCC patients. We constructed an mRNA-miRNA-lncRNA subnetwork containing p-EMT-related genes successfully, among which each component might be utilized as a prognostic biomarker of HCC.

SPOCK1 overexpression induced by platelet-derived growth factor-BB promotes hepatic stellate cell activation and liver fibrosis through the integrin α5ß1/PI3K/Akt signaling pathway.

Sparc/osteonectin, cwcv, and kazal-like domain proteoglycan 1 (SPOCK1) is a matricellular protein which regulates cell proliferation, invasion, and survival but the function of SPOCK1 in liver fibrosis is obscure. In this study, we found that SPOCK1 expression increased significantly in fibrotic liver tissues and activated primary rat hepatic stellate cells (R-HSCs). SPOCK1 co-localized with α-smooth muscle actin (α-SMA) in the cytoplasm. Mechanistically, we found platelet-derived growth factor-BB (PDGF-BB) induced SPOCK1 expression by activating the PI3K/Akt/forkhead box M1 (FoxM1) signaling pathway. Intracellular SPOCK1 downregulation decreased the HSC activation, proliferation, and migration induced by PDGF-BB. Furthermore, intracellular SPOCK1 overexpression or recombinant SPOCK1 treatment promoted HSC activation, proliferation, and migration by activating the PI3K/Akt signaling pathway. Co-immunoprecipitation, double immunofluorescence staining indicated that SPOCK1 interacted with integrin α5ß1, and neutralization of integrin α5ß1 significantly reduced the role of recombinant SPOCK1 in HSCs. In vivo HSC-specific SPOCK1 knockdown following lentivirus administration dramatically ameliorated thioacetamide (TAA)-induced collagen deposition in rat livers. Collectively, our study indicates that SPOCK1 is crucial for hepatic fibrosis and it might be a promising therapeutic target.

Overexpression of KLF14 protects against immune-mediated hepatic injury in mice.

The underlying immunopathogenic mechanisms of autoimmune hepatitis (AIH) have not yet been well elucidated. An impairment in regulatory T cells (Tregs) is key to the development of AIH. Krüppel-like factors (KLFs) regulate a broad of cellular processes including immunocyte maturation. KLF14 may regulate Treg differentiation, but the biological functions remain far from elucidated. In this study, we identified the hepatic expression of KLF14 in human and murine liver diseases. Immune-mediated hepatitis was induced by concanavalin A (Con A). A KLF14 recombinant adenoviruses plasmid (Ad-KLF14) was constructed and injected into mice. Tregs were assessed by flow cytometry analysis; inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), were tested by enzyme-linked immunosorbent assay (ELISA). The expression of KLF14 was suppressed in a time-and dose-dependent manner. Changes in cytokine levels were consistent with the degree of hepatic injury. Overexpression of KLF14 protected the liver from immune-mediated damage in vivo. Ad-KLF14 transfection before Con A challenge increased the frequency of Tregs in liver mononuclear cells (MNCs), and suppressed the expression of cytokines. All of these improvements were completely abrogated after Treg deletion in vivo by intraperitoneal injection of a CD25 antibody. In conclusion, these data suggest that KLF14 plays an as-yet unrecognized role in immune-mediated hepatitis mainly via induced Treg differentiation. Our findings extend the knowledge of the biological function of KLF14 to the autoimmune disease field, and indicate the possibility of KLF14 as a therapeutic target in AIH patients.

L-lysine supplementation attenuates experimental autoimmune hepatitis in a chronic murine model.

The incidence of autoimmune hepatitis (AIH) has increased significantly worldwide. The present study aims to explore the protective effect of L-lysine supplementation against AIH and to investigate its potential underlying mechanisms. A chronic experimental AIH mouse model was established by repeated tail vein injection of human cytochrome P450 2D6 (CYP2D6) plasmid. Starting from day 14 of the modeling, mice in the CYP2D6-AIH +L-lysine group were given 200 µl of purified water containing 10 mg/kg L-lysine by gavage until day27, once a day, and mice in the healthy control group and model group were given an equal volume of purified water by gavage. Our results showed that L-lysine supplementation partially reversed the liver injury mediated by CYP2D6 overexpression. These effects were consistent with the restraining impacts of L-lysine supplementation on decreasing pro-inflammatory cytokines expression level and CD4+ and CD8+ T lymphocytes infiltration, as well as curbing hepatic oxidative stress. Furthermore, L-lysine supplement relieved liver fibrosis in the context of AIH. In conclusion, L-lysine supplementation attenuates CYP2D6-induced immune liver injury in mice, which may serve as a novel nutrition support approach for AIH.

MTAP-ANRIL gene fusion promotes melanoma epithelial-mesenchymal transition-like process by activating the JNK and p38 signaling pathways.

Gene fusions caused by cytogenetic aberrations play important roles in the initiation and progression of cancers. The recurrent MTAP-ANRIL fusion gene was reported to have a frequency of greater than 7% in melanoma in our previous study. However, its functions remain unclear. Truncated MTAP proteins resulting from point mutations in the last three exons of MTAP can physically interact with the wild-type MTAP protein, a tumor suppressor in several human cancers. Similarly, MTAP-ANRIL, which is translated into a truncated MTAP protein, would influence wild-type MTAP to act as an oncogene. Here, we found that MTAP-ANRIL gene fusion downregulated the expression of wild-type MTAP and promoted epithelial-mesenchymal transition-like process through the activation of JNK and p38 MAPKs in vitro and in vivo. Our results suggest that MTAP-ANRIL is a potential molecular prognostic biomarker and therapeutic target for melanoma.

Bowel preparation efficacy and safety of compound polyethylene glycol electrolyte powder combined with linaclotide for colonoscopy: A randomized controlled trial.

Background and Aim: Adequate bowel preparation is essential for colonoscopy, which is important for detecting colon polyps and preventing colorectal cancer. Linaclotide is approved for irritable bowel syndrome with predominant constipation (IBS-C) symptoms. The main objective of this study was to explore the quality of bowel preparation by low-volume compound polyethylene glycol (PEG) combined with linaclotide. Methods: A total of 266 patients who underwent colonoscopy in Shangrao People's Hospital from June 2021 to June 2022 were randomized to 1 of 3 split PEG regimens: 4LPEG, 2LPEG, and 2LPEG + L (linaclotide). The primary end point was adequate bowel preparation (Boston Bowel Preparation Scale [BBPS] total score of ≥6, with each of three colonic segments subscores ≥2). Secondary outcomes were polyp detection rates and the incidence of adverse reactions. Results: Over 12 months, 266 subjects were randomized into 2LPEG (n = 12), 4LPEG (n = 112), or 2LPEG + L (n = 142). There were no significant differences between the 4LPEG and 2LPEG + L groups in achieving adequate bowel preparation (P > 0.05). The mean BBPS score of the total colon, left hemi-colon, right hemi-colon, and transverse in the 2LPEG + L group was higher than that in the 2LPEG group (P < 0.001). Patient's sleeping quality and the incidence of adverse reactions of 2LPEG + L group were compatible with 2LPEG group, but it was significantly lower than that in 4LPEG group. There was no statistically significant difference in the detection rate of colon polyps between each group. Conclusion: The quality of bowel preparation of the compound polyethylene glycol electrolyte powder combined with linaclotide is approximately the same as that of 4LPEG, and it can reduce the adverse reactions in the process of bowel preparation, and its intestinal cleansing effect is also better than that of 2LPEG.

EZH2-mediated inhibition of KLF14 expression promotes HSCs activation and liver fibrosis by downregulating PPARγ.

OBJECTIVES: Induction of deactivation and apoptosis of hepatic stellate cells (HSCs) are principal therapeutic strategies for liver fibrosis. Krüppel-like factor 14 (KLF14) regulates various biological processes, however, roles, mechanisms and implications of KLF14 in liver fibrosis are unknown. MATERIALS AND METHODS: KLF14 expression was detected in human, rat and mouse fibrotic models, and its effects on HSCs were assessed. Chromatin immunoprecipitation assays were utilized to investigate the binding of KLF14 to peroxisome proliferator-activated receptor γ (PPARγ) promoter, and the binding of enhancer of zeste homolog 2 (EZH2) to KLF14 promoter. In vivo, KLF14-overexpressing adenovirus was injected via tail vein to thioacetamide (TAA)-treated rats to investigate the role of KLF14 in liver fibrosis progression. EZH2 inhibitor EPZ-6438 was utilized to treat TAA-induced rat liver fibrosis. RESULTS: KLF14 expression was remarkably decreased in human, rat and mouse fibrotic liver tissues. Overexpression of KLF14 increased LD accumulation, inhibited HSCs activation, proliferation, migration and induced G2/M arrest and apoptosis. Mechanistically, KLF14 transactivated PPARγ promoter activity. Inhibition of PPARγ blocked the suppressive role of KLF14 overexpression in HSCs. Downregulation of KLF14 in activated HSCs was mediated by EZH2-regulated histone H3 lysine 27 trimethylation. Adenovirus-mediated KLF14 overexpression ameliorated TAA-induced rat liver fibrosis in PPARγ-dependent manner. Furthermore, EPZ-6438 dramatically alleviated TAA-induced rat liver fibrosis. Importantly, KLF14 expression was decreased in human with liver fibrosis, which was significantly correlated with EZH2 upregulation and PPARγ downregulation. CONCLUSIONS: KLF14 exerts a critical anti-fibrotic role in liver fibrosis, and targeting the EZH2/KLF14/PPARγ axis might be a novel therapeutic strategy for liver fibrosis.

FOXC1 promotes HCC proliferation and metastasis by Upregulating DNMT3B to induce DNA Hypermethylation of CTH promoter.

BACKGROUND: Forkhead box C1 (FOXC1), as a member of the FOX family, is important for promote HCC invasion and metastasis. FOX family protein lays a pivotal role in metabolism. ROS is involved in tumor progression and is associated with the expression of lots of transcription factors. We next explored the mechanism underlying FOXC1 modulating the metabolism and ROS hemostasis in HCC. METHODS: We used amino acids arrays to verify which metabolism is involved in FOXC1-induced HCC. The kits were used to detect the ROS levels in HCC cells with over-expression or down-expression of FOXC1. After identified the downstream target genes and candidate pathway which regulated by FOXC1 during HCC progression in vitro and in vivo, we used western blot, immunohistochemistry, bisulfite genomic sequencing, methylation-specific PCR, chromatin immunoprecipitation analysis and luciferase reporter assays to explore the relationship of FOXC1 and downstream genes. Moreover, the correlation between FOXC1 and target genes and the correlation between target genes and the recurrence and overall survival were analyzed in two independent human HCC cohorts. RESULTS: Here, we reported that FOXC1 could inhibit the cysteine metabolism and increase reactive oxygen species (ROS) levels by regulating cysteine metabolism-related genes, cystathionine γ-lyase (CTH). Overexpression of CTH significantly suppressed FOXC1-induced HCC proliferation, invasion and metastasis, while the reduction in cell proliferation, invasion and metastasis caused by the inhibition of FOXC1 could be reversed by knockdown of CTH. Meanwhile, FOXC1 upregulated de novo DNA methylase 3B (DNMT3B) expression to induce DNA hypermethylation of CTH promoter, which resulted in low expression of CTH in HCC cells. Moreover, low levels of ROS induced by N-acetylcysteine (NAC) which is an antioxidant inhibited the cell proliferation, migration, and invasion abilities mediated by FOXC1 overexpression, whereas high levels of ROS induced by L-Buthionine-sulfoximine (BSO) rescued the suppression results mediated by FOXC1 knockdown. Our study demonstrated that the overexpression of FOXC1 that was induced by the ROS dependent on the extracellular regulated protein kinases 1 and 2 (ERK1/2)- phospho-ETS Transcription Factor 1 (p-ELK1) pathway. In human HCC tissues, FOXC1 expression was positively correlated with oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), p-ELK1 and DNMT3B expression, but negatively correlated with CTH expression. HCC patients with positive co-expression of 8-OHdG/FOXC1 or p-ELK1/FOXC1 or FOXC1/DNMT3B had the worst prognosis, whereas HCC patients who had positive FOXC1 and negative CTH expression exhibited the worst prognosis. CONCLUSION: In a word, we clarify that the positive feedback loop of ROS-FOXC1-cysteine metabolism-ROS is important for promoting liver cancer proliferation and metastasis, and this pathway may provide a prospective clinical treatment approach for HCC.

Comprehensive analysis of key biomarkers, immune infiltration and potential therapeutic agents for ulcerative colitis.

AIMS: There has been an increasing trend towards the ulcerative colitis (UC) incidence worldwide. The present study aimed to explore novel biomarkers and potential therapeutic agents for UC. MATERIALS AND METHODS: Differentially expressed genes (DEGs) among UC and healthy control samples were identified by GEO2R online tool. Functional analysis was performed and protein-protein interaction networks were constructed. The hub genes were explored by Cytoscape, and quantitative real-time-PCR (qRT-PCR) was used to valid their expression in clinical samples. ImmuCellAI was utilized to analyze the fraction of 24 types of immune cells. The L1000 platform was applied to determine potential agents for UC treatment. The dextran sulfate sodium (DSS)-induced colitis model was used to identify the therapeutic effect of meclofenamic acid. KEY FINDINGS: A total of 270 DEGs were identified among UC and healthy control samples. Functional analysis indicated that the DEGs were primarily enriched in several immune response and digestion pathways. A proportion of 18 immune-cell types was found to be significantly altered between UC and healthy control samples. 10 compounds were predicted to have therapeutic potentials for treating UC. Among them, we selected meclofenamic acid to identify its therapeutic effect on UC treatment by animal experiments. SIGNIFICANCE: The current study comprehensively analyzed the DEGs and immune infiltration in UC, as well as screened for potential agents for UC treatment.

SIX4 promotes hepatocellular carcinoma metastasis through upregulating YAP1 and c-MET.

Metastasis is the main reason for high mortality in hepatocellular carcinoma (HCC) patients and the molecular mechanism remains unclear. Therefore, it is important to elucidate the mechanism underlying HCC metastasis. Here, we report a novel role of SIX homeobox 4 (SIX4), one of the SIX gene family, in promoting HCC metastasis. The elevated expression of SIX4 was positively correlated with loss of tumor encapsulation, microvascular invasion, higher TNM stage, and poor prognosis in human HCC. SIX4 expression was an independent and significant risk factor for the recurrence and survival in HCC patients. Upregulation of SIX4 promoted HCC invasion and metastasis, whereas downregulation of SIX4 decreased HCC invasion and metastasis. SIX4 transactivated Yes1 associated transcriptional regulator (YAP1) and MET proto-oncogene, receptor tyrosine kinase (MET) expression through directly binding to their promoters. Knockdown of YAP1 and c-MET inhibited SIX4-medicated HCC metastasis, while the stable overexpression of YAP1 and c-MET reversed the decreased metastasis induced by SIX4 knockdown. Hepatocyte growth factor (HGF), the specific ligand of c-MET, upregulated SIX4 expression through ERK/NF-κB pathway. Knockdown of SIX4 significantly decreased HGF-enhanced HCC metastasis. In human HCC tissues, SIX4 expression was positively correlated with nuclear YAP1, c-MET and HGF expression. Patients with positive coexpression of SIX4/ nuclear YAP1, SIX4/c-MET or HGF/SIX4 had the poorest prognosis. Moreover, the combination treatment of YAP1 inhibitor Verteporfin and c-MET inhibitor Capmatinib significantly suppressed SIX4-mediated HCC metastasis. In conclusion, SIX4 is a prognostic biomarker in HCC patients and targeting the HGF-SIX4-c-MET positive feedback loop may provide a promising strategy for the treatment of SIX4-driven HCC metastasis.

Netrin-1 promotes the collective cell migration of liver cancer cells in a 3D cell culture model.

Collective cell migration plays an important role in embryonic development, wound healing, and cancer metastasis. We aimed to investigate the expression, role, and mechanism of Netrin-1 in collective cell migration using a3D culture model. An immunohistochemical study showed that certain cells invaded surrounding tissue by collective migration and that Netrin-1 expression in these cells was increased, especially at the invasive front. In the 3D culture model, collective cell migration was clearly observed, as leader cells were followed by cells migrating along a canal. N-cadherin-mediated cell junctions were observed in collective cell migration, and Netrin-1 expression was elevated in these cells. Netrin-1 did not affect the expression of N-cadherin in 2D-cultured cells; however, in 3D culture, the overexpression of Netrin-1 increased N-cadherin and promoted the collective migration of Huh7 cells, while the knockdown of Netrin-1 decreased N-cadherin and inhibited collective migration in SK-Hep-1 cells. Interestingly, N-cadherin knockdown in Huh7 cells significantly diminished Netrin-1-promoted collective cell migration, while the overexpression of N-cadherin restored collective migration in Netrin-1-knockdown SK-Hep1 cells. These results suggest that Netrin-1 enhances N-cadherin junctions to promote liver cancer cell collective migration in 3D cell culture and may subsequently increase liver cancer metastasis.

A Potential Model for Detecting Crowding-induced Epithelial Cell and Cancer Cell Extrusion.

Overcrowding and cell deformation lead to the shedding of apoptotic and live cells to maintain homeostasis in the epithelium. Recent studies have attempted to explain the effect of extrusion on epithelial homeostasis and tumor metastasis, but lack the requisite quantitative models for testing extrusion. Here, we designed a petri dish inversion model to detect the extrusion ability of both normal epithelial cells and epithelial cancer cells. Firstly, we found cell extrusion was observed in both normal epithelial cells (LO2 cells) and cancer cells; in confluent LO2 cell culture, certain cells were surrounded by their neighbors, suffered "collective attack", and were then made round in shape. Green fluorescent protein (GFP)-labeled cancer cells were also found to be squeezed by normal LO2 cells. Using the petri dish inversion model, we quantified the number of extrusion cells, and demonstrated that the ability of cancer cell extrusion was related to the metastatic potential of cancer cell lines. Our findings provide a novel model to detect crowding-induced epithelial cell and cancer cell extrusion. This novel model provides a quantitative method for research into apoptotic and cancer cell extrusion, particularly in human hepatocellular carcinoma.

CFIm25 inhibits hepatocellular carcinoma metastasis by suppressing the p38 and JNK/c-Jun signaling pathways.

Alternative polyadenylation (APA), a post-transcriptional modification, has been implicated in many diseases, but especially in tumor proliferation. CFIm25, the 25 kDa subunit of human cleavage factor Im (CFIm), is a key factor in APA. We show that CFIm25 expression is reduced in human hepatocellular carcinoma (HCC), and its expression correlates with metastasis. Kaplan-Meier analysis indicated that CFIm25 is related to overall survival in HCC. Moreover, CFIm25 expression is negatively related to the metastatic potential of HCC cell lines. CFIm25 knockdown promotes cell invasion and migration in vitro, while overexpression of CFIm25 inhibits cell invasion and migration in vitro and inhibits intrahepatic and lung metastasis in vivo. Additional studies showed that CFIm25 disrupts epithelial-mesenchymal transition by increasing E-cadherin, that it inhibits HCC cell migration and invasion by blocking the p38 and JNK/c-Jun signaling pathways, and that CFIm25 knockdown increases the transcriptional activity of activating protein-1 (AP-1). These findings indicate that therapy directed at increasing CFIm25 expression is a potential HCC treatment.