Gp78 deficiency in hepatocytes alleviates hepatic ischemia-reperfusion injury via suppressing ACSL4-mediated ferroptosis.

Ferroptosis, which is driven by iron-dependent lipid peroxidation, plays an essential role in liver ischemia-reperfusion injury (IRI) during liver transplantation (LT). Gp78, an E3 ligase, has been implicated in lipid metabolism and inflammation. However, its role in liver IRI and ferroptosis remains unknown. Here, hepatocyte-specific gp78 knockout (HKO) or overexpressed (OE) mice were generated to examine the effect of gp78 on liver IRI, and a multi-omics approach (transcriptomics, proteomics, and metabolomics) was performed to explore the potential mechanism. Gp78 expression decreased after reperfusion in LT patients and mice with IRI, and gp78 expression was positively correlated with liver damage. Gp78 absence from hepatocytes alleviated liver damage in mice with IRI, ameliorating inflammation. However, mice with hepatic gp78 overexpression showed the opposite phenotype. Mechanistically, gp78 overexpression disturbed lipid homeostasis, remodeling polyunsaturated fatty acid (PUFA) metabolism, causing oxidized lipids accumulation and ferroptosis, partly by promoting ACSL4 expression. Chemical inhibition of ferroptosis or ACSL4 abrogated the effects of gp78 on ferroptosis and liver IRI. Our findings reveal a role of gp78 in liver IRI pathogenesis and uncover a mechanism by which gp78 promotes hepatocyte ferroptosis by ACSL4, suggesting the gp78-ACSL4 axis as a feasible target for the treatment of IRI-associated liver damage.

TP53/mTORC1-mediated bidirectional regulation of PD-L1 modulates immune evasion in hepatocellular carcinoma.

BACKGROUND: Immunotherapy has facilitated great breakthroughs in the treatment of hepatocellular carcinoma (HCC). However, the efficacy and response rate of immunotherapy are limited and vary among different patients with HCC. TP53 mutation substantially affects the expression of immune checkpoint molecules in multiple cancers. However, the regulatory relationship between programmed death ligand 1 (PD-L1) and TP53 is poorly studied in HCC. We aimed to elucidate the regulatory mechanism of PD-L1 in HCC with different TP53 statuses and to assess its role in modulating immune evasion in HCC. METHODS: HCC mouse models and cell lines with different TP53 statuses were constructed. PD-L1 levels were detected by PCR, western blotting and flow cytometry. RNA-seqencing, immunoprecipitation, chromatin immunoprecipitation and transmission electron microscopy were used to elucidate the regulatory mechanism in HCC with different TP53 status. HCC mouse models and patient with HCC samples were analyzed to demonstrate the preclinical and clinical significance of the findings. RESULTS: We report that loss of p53 promoted PD-L1 expression and reduced CD8+ T-cell infiltration in patient with HCC samples and mouse models. Mammalian target of rapamycin (mTOR) pathway was activated in p53-loss-of-function HCC or after knocking down TP53. The transcription factor E2F1 was found to bind to the p53 protein in TP53 wild-type HCC cells, and inhibiting mammalian target of rapamycin complex 1 (mTORC1) disrupted this binding and enhanced E2F1 translocation to the nucleus, where it bound to the PD-L1 promoter and transcriptionally upregulated PD-L1. In p53-loss-of-function HCC cells, autophagosomes were activated after mTORC1 suppression, promoting the degradation of PD-L1 protein. The combination of mTOR inhibitor and anti-PD-L1 antibody enhanced CD8+ T-cell infiltration and tumor suppression in TP53 wild-type HCC mouse models, but no benefit was observed in p53-loss-of-function HCC mouse models. In patients with TP53 wild-type HCC, PD-L1 levels were significantly higher in the high E2F1 group than in the low E2F1 group, and the low E2F1 level group had significantly superior survival. CONCLUSION: We revealed the bidirectional regulatory mechanism of PD-L1 mediated by TP53/mTORC1 in HCC. The combination of mTOR inhibitor and anti-PD-L1 antibody could be a novel precise immunotherapy scheme for TP53 wild-type HCC.

Insulin-induced gene 2 protects against hepatic ischemia-reperfusion injury via metabolic remodeling.

BACKGROUND: Hepatic ischemia-reperfusion (IR) injury is the primary reason for complications following hepatectomy and liver transplantation (LT). Insulin-induced gene 2 (Insig2) is one of several proteins that anchor the reticulum in the cytoplasm and is essential for metabolism and inflammatory responses. However, its function in IR injury remains ambiguous. METHODS: Insig2 global knock-out (KO) mice and mice with adeno-associated-virus8 (AAV8)-delivered Insig2 hepatocyte-specific overexpression were subjected to a 70% hepatic IR model. Liver injury was assessed by monitoring hepatic histology, inflammatory responses, and apoptosis. Hypoxia/reoxygenation stimulation (H/R) of primary hepatocytes and hypoxia model induced by cobalt chloride (CoCl2) were used for in vitro experiments. Multi-omics analysis of transcriptomics, proteomics, and metabolomics was used to investigate the molecular mechanisms underlying Insig2. RESULTS: Hepatic Insig2 expression was significantly reduced in clinical samples undergoing LT and the mouse IR model. Our findings showed that Insig2 depletion significantly aggravated IR-induced hepatic inflammation, cell death and injury, whereas Insig2 overexpression caused the opposite phenotypes. The results of in vitro H/R experiments were consistent with those in vivo. Mechanistically, multi-omics analysis revealed that Insig2 is associated with increased antioxidant pentose phosphate pathway (PPP) activity. The inhibition of glucose-6-phosphate-dehydrogenase (G6PD), a rate-limiting enzyme of PPP, rescued the protective effect of Insig2 overexpression, exacerbating liver injury. Finally, our findings indicated that mouse IR injury could be attenuated by developing a nanoparticle delivery system that enables liver-targeted delivery of substrate of PPP (glucose 6-phosphate). CONCLUSIONS: Insig2 has a protective function in liver IR by upregulating the PPP activity and remodeling glucose metabolism. The supplementary glucose 6-phosphate (G6P) salt may serve as a viable therapeutic target for alleviating hepatic IR.

Prognostic implication of early posttransplant hypercholesterolemia in liver transplantation for patients with hepatocellular carcinoma.

BACKGROUND: Hyperlipidemia is a common complication after liver transplantation (LT) and develops mostly in the early posttransplant period. Recently, some studies have reported a positive correlation between hyperlipidemia and favorable prognosis in patients with hepatocellular carcinoma (HCC) undergoing hepatectomy. This study aimed to evaluate the possibility of predicting prognosis in HCC patients receiving LT by early posttransplant dyslipidemia. METHODS: From January 2015 to December 2017, a total of 806 HCC patients from China Liver Transplant Registry database were retrospectively enrolled. The prognostic relevance of early posttransplant hypertriglyceridemia or hypercholesterolemia was examined using survival analysis, and subgroup analysis was implemented based on LT criteria. RESULTS: Early posttransplant hypercholesterolemia (EPHC) was independently inversely associated with the risk of recurrence [hazard ratio (HR) = 0.630; P = 0.022], but was not significantly correlated with the mortality. However, early posttransplant hypertriglyceridemia was not related to prognosis. Intriguingly, with further classification, we found that borderline EPHC (B-EPHC), instead of significant EPHC, was a predictor of lower risk for both recurrence (HR = 0.504; P = 0.006) and mortality (HR = 0.511; P = 0.023). Compared with non-EPHC patients, B-EPHC patients achieved significantly superior 1-year and 3-year tumor-free survival (89.6% and 83.7% vs. 83.8% and 72.7% respectively; P = 0.023), and 1-year and 3-year overall survival (95.8% and 84.8% vs. 94.6% and 77.6% respectively; P = 0.039). In the subgroup analysis, B-EPHC remained an independent predictor of better prognosis in patients beyond Milan criteria and those within Hangzhou criteria; whereas there was no significant relationship between B-EPHC and prognosis in patients within Milan criteria and those beyond Hangzhou criteria. More interestingly, patients beyond Milan criteria but within Hangzhou criteria were identified as the crucial subpopulation who benefited from B-EPHC (recurrence HR = 0.306, P = 0.011; mortality HR = 0.325, P = 0.031). CONCLUSIONS: B-EPHC could assist transplant teams in dynamically evaluating prognosis after LT for HCC as a postoperative non-oncological biomarker, especially in patients beyond Milan criteria but within Hangzhou criteria.

E2F7 promotes mammalian target of rapamycin inhibitor resistance in hepatocellular carcinoma after liver transplantation.

The mammalian target of rapamycin (mTOR) pathway is frequently deregulated and has critical roles in cancer progression. mTOR inhibitor has been widely used in several kinds of cancers and is strongly recommended in patients with hepatocellular carcinoma (HCC) after liver transplantation (LT). However, the poor response to mTOR inhibitors due to resistance remains a challenge. Hypoxia-associated resistance limits the therapeutic efficacy of targeted drugs. The present study established models of HCC clinical samples and cell lines resistance to mTOR inhibitor sirolimus and screened out E2F7 as a candidate gene induced by hypoxia and promoting sirolimus resistance. E2F7 suppressed mTOR complex 1 via directly binding to the promoter of the TSC1 gene and stabilizes hypoxia-inducible factor-1α activating its downstream genes, which are responsible for E2F7-dependent mTOR inhibitor resistance. Clinically, low E2F7 expression could be an effective biomarker for recommending patients with HCC for anti-mTOR-based therapies after LT. Targeting E2F7 synergistically inhibited HCC growth with sirolimus in vivo. E2F7 is a promising target to reverse mTOR inhibition resistance. Collectively, our study points to a role for E2F7 in promoting mTOR inhibitor resistance in HCC and emphasizes its potential clinical significance in patients with HCC after LT.

The metabolic flexibility of quiescent CSC: implications for chemotherapy resistance.

Quiescence has been observed in stem cells (SCs), including adult SCs and cancer SCs (CSCs). Conventional chemotherapies mostly target proliferating cancer cells, while the quiescent state favors CSCs escape to chemotherapeutic drugs, leaving risks for tumor recurrence or metastasis. The tumor microenvironment (TME) provides various signals that maintain resident quiescent CSCs, protect them from immune surveillance, and facilitates their recurrence potential. Since the TME has the potential to support and initiate stem cell-like programs in cancer cells, targeting the TME components may prove to be a powerful modality for the treatment of chemotherapy resistance. In addition, an increasing number of studies have discovered that CSCs exhibit the potential of metabolic flexibility when metabolic substrates are limited, and display increased robustness in response to stress. Accompanied by chemotherapy that targets proliferative cancer cells, treatments that modulate CSC quiescence through the regulation of metabolic pathways also show promise. In this review, we focus on the roles of metabolic flexibility and the TME on CSCs quiescence and further discuss potential treatments of targeting CSCs and the TME to limit chemotherapy resistance.

NEAT1/hsa-miR-372-3p axis participates in rapamycin-induced lipid metabolic disorder.

Rapamycin is a crucial immunosuppressive regimen for patients that have undergone liver transplantation (LT). However, one of the major side effects of rapamycin include metabolic disorders such as dyslipidemia, and the mechanism remains unknown. This study aims to explore the biomolecules that are responsible for rapamycin-induced dyslipidemia and the control strategies that can reverse the lipid metabolism disorder. In this study, data collected from LT patients, cell and mouse models treated with rapamycin were analyzed. Results showed an increase of triglycerides (TGs) induced by rapamycin. MicroRNAs (miRNAs) play important roles in many vital biological processes including TG metabolism. hsa-miR-372-3p was filtered using RNA sequencing and identified as a key regulator in rapamycin-induced TGs accumulation. Using bioinformatics and experimental analyses, target genes of hsa-miR-372-3p were predicted. These genes were alkylglycerone phosphate synthase (AGPS) and apolipoprotein C4 (APOC4), which are reported to be involved in TG metabolism. LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) was also identified as an upstream regulatory factor of hsa-miR-372-3p. From the results of this study, NEAT1/hsa-miR-372-3p/AGPS/APOC4 axis plays a vital role in rapamycin-disruption of lipid homeostasis. Therefore, targeting this axis is a potential therapeutic target combating rapamycin-induced dyslipidemia after LT.

Delivery of microRNA-33 Antagomirs by Mesoporous Silica Nanoparticles to Ameliorate Lipid Metabolic Disorders.

Lipid metabolic disorders have become a major global public health concern. Fatty liver and dyslipidemia are major manifestations of these disorders. Recently, MicroRNA-33 (miR-33), a post-transcriptional regulator of genes involved in cholesterol efflux and fatty acid oxidation, has been considered as a good therapeutic target for these disorders. However, the traditional methods of gene therapy impede their further clinical transformation into a mature treatment system. To counter this problem, in this study we used mesoporous silica nanoparticles (MSNs) as nanocarriers to deliver miR-33 antagomirs developing nanocomposites miR-MSNs. We observed that the hepatocellular uptake of miR-33 antagomirs increased by ∼5 times when they were delivered using miR-MSNs. The regulation effects of miR-MSNs on miR-33 and several genes involved in lipid metabolism were confirmed in L02 cells. In a high-fat diet fed mice, miR-33 intervention via miR-MSNs lowered the serum triglyceride levels remarkably by 18.9% and reduced hepatic steatosis. Thus, our results provide a proof-of-concept for a potential strategy to ameliorate lipid metabolic disorders.

The circFASN/miR-33a pathway participates in tacrolimus-induced dysregulation of hepatic triglyceride homeostasis.

Dyslipidemia exhibits a high incidence after liver transplantation, in which tacrolimus, a widely used immunosuppressant, plays a fundamental role. MicroRNAs and related circRNAs represent a class of noncoding RNAs that have been recognized as important regulators of genes associated with lipid metabolism. However, their transcriptional activities and functional mechanisms in tacrolimus-related dyslipidemia remain unclear. In this study, we observed that tacrolimus could induce triglyceride accumulation in hepatocytes by stimulating sterol response element-binding proteins (SREBPs) and miR-33a. Our in silico and experimental analyses identified miR-33a as a direct target of circFASN. Tacrolimus could downregulate circFASN and result in elevated miR-33a in vivo and in vitro. Overexpression of circFASN or silencing of miR-33a decreased the promoting effects of tacrolimus on triglyceride accumulation. Clinically, the incidence of dyslipidemia in liver transplant recipients with elevated serum miR-33a after liver transplantation was higher than that in patients without elevated serum miR-33a (46.3% vs. 18.8% p = 0.012, n = 73). Our results showed that the circFASN/miR-33a regulatory system plays a distinct role in tacrolimus-induced disruption of lipid homeostasis. MiR-33a is likely a risk factor for tacrolimus-related dyslipidemia, providing a potential therapeutic target to combat tacrolimus-induced dyslipidemia after liver transplantation.

The tacrolimus-induced glucose homeostasis imbalance in terms of the liver: From bench to bedside.

Tacrolimus (TAC), the mainstay of maintenance immunosuppressive agents, plays a crucial role in new-onset diabetes after transplant (NODAT). Previous studies investigating the diabetogenic effects of TAC have focused on the ß cells of islets. In this study, we found that TAC contributed to NODAT through directly affecting hepatic metabolic homeostasis. In mice, TAC-induced hypoglycemia rather than hyperglycemia during starvation via suppressing gluconeogenetic genes, suggesting the limitation of fasting blood glucose in the diagnosis of NODAT. In addition, TAC caused hepatic insulin resistance and triglyceride accumulation through insulin receptor substrate (IRS)2/AKT and sterol regulatory element binding protein (SREBP1) signaling, respectively. Furthermore, we found a pivotal role of CREB-regulated transcription coactivator 2 (CRTC2) in TAC-induced metabolic disorders. The restoration of hepatic CRTC2 alleviated the metabolic disorders through its downstream molecules (eg, PCK1, IRS2, and SREBP1). Consistent with the findings from bench, low CRTC2 expression in graft hepatocytes was an independent risk factor for NODAT (odds ratio = 2.692, P = .023, n = 135). Integrating grafts' CRTC2 score into the clinical model could significantly increase the predictive capacity (areas under the receiver operating characteristic curve: 0.71 vs 0.79, P = .048). Taken together, in addition to its impact on pancreatic cells, TAC induces "hematogenous diabetes" via CRTC2 signaling. Liver-targeted management may be of help to prevent or heal TAC-associated diabetes.

USP22 promotes hypoxia-induced hepatocellular carcinoma stemness by a HIF1α/USP22 positive feedback loop upon TP53 inactivation.

OBJECTIVE: We aimed to elucidate the mutual regulation mechanism of ubiquitin-specific protease 22 (USP22) and hypoxia inducible factor-1α (HIF1α), and the mechanism they promote the stemness of hepatocellular carcinoma (HCC) cells under hypoxic conditions. DESIGN: Cell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were established to detect the stemness of HCC cells. Immunoprecipitation, ubiquitination assay and chromatin immunoprecipitation assay were used to elucidate the mutual regulation mechanism of USP22 and HIF1α. HCC patient samples and The Cancer Genome Atlas data were used to demonstrate the clinical significance. In vivo USP22-targeting experiment was performed in mice bearing HCC. RESULTS: USP22 promotes hypoxia-induced HCC stemness and glycolysis by deubiquitinating and stabilising HIF1α. As direct target genes of HIF1α, USP22 and TP53 can be transcriptionally upregulated by HIF1α under hypoxic conditions. In TP53 wild-type HCC cells, HIF1α induced TP53-mediated inhibition of HIF1α-induced USP22 upregulation. In TP53-mutant HCC cells, USP22 and HIF1α formed a positive feedback loop and promote the stemness of HCC. HCC patients with a loss-of-function mutation at TP53 and high USP22 and/or HIF1α expression tend to have a worse prognosis. The USP22-targeting lipopolyplexes caused high tumour inhibition and high sorafenib sensitivity in mice bearing HCC. CONCLUSION: USP22 promotes hypoxia-induced HCC stemness by a HIF1α/USP22 positive feedback loop on TP53 inactivation. USP22 is a promising target for the HCC therapy.

High Expression of Human AugminComplex Submit 3 Indicates Poor Prognosis and Associates with Tumor Progression in Hepatocellular Carcinoma.

The function of human augmin complex unit 3(Haus3), a component of the HAU augmin-like complex, in various cancers is not clear. This study aims to elucidate the clinical significance and the role of Haus3 in tumor progression of hepatocellular carcinoma (HCC). We analyzed the expression of Haus3 in 50 HCC patients from The Cancer Genome Atlas and 137 HCC patients in our hospital. Compared with adjacent normal tissue, Haus3 expression assessed by immunohistochemical staining was dramatically increased in tumor tissues. A high level of Haus3 expression was significantly correlated with large tumor size (p=0.025) and tumor multiplicity (p=0.004). Univariate and multivariate survival analysis showed thatexpression of Haus3 was an independent prognostic factor for overall survival ofHCCpatients. Western blot analysis showed that Haus3 regulated the phosphorylation of PLK1-T210 and activity of the Cdk1/cyclin B1 complex, indicating that Haus3 disrupted G2/M phase arrest. In immunofluorescence studies, expression of Haus3 correlated with the level ofα-tubulin and γ-tubulin. In summary, Haus3 plays a vital role in regulatingtheactivityof PLK2-T210 and Cdk1/cyclin B1 complex in G2/M phasetransition and the expression of tubulins to ensure normal mitotic progression. Our data suggest that Haus3 might be a promising prognostic biomarker and molecular target of HCC.

Middle hepatic vein reconstruction in adult right lobe living donor liver transplantation improves recipient survival.

BACKGROUND: The efficacy and necessity of middle hepatic vein (MHV) reconstruction in adult-to-adult right lobe living donor liver transplantation (LDLT) remain controversial. The present study aimed to evaluate the survival beneficiary of MHV reconstructions in LDLT. METHODS: We compared the clinical outcomes of liver recipients with MHV reconstruction (n = 101) and without MHV reconstruction (n = 43) who underwent LDLT using right lobe grafts at our institution from January 2006 to May 2017. RESULTS: The overall survival (OS) rate of recipients with MHV reconstruction was significantly higher than that of those without MHV reconstruction in liver transplantation (P = 0.022; 5-yr OS: 76.2% vs 58.1%). The survival of two segments (segments 5 and 8) hepatic vein reconstruction was better than that of the only one segment (segment 5 or segment 8) hepatic vein reconstruction (P = 0.034; 5-yr OS: 83.6% vs 67.4%). The survival of using two straight vascular reconstructions was better than that using Y-shaped vascular reconstruction in liver transplantation with two segments hepatic vein reconstruction (P = 0.020; 5-yr OS: 100% vs 75.0%). The multivariate analysis demonstrated that MHV tributary reconstructions were an independent beneficiary prognostic factor for OS (hazard ratio=0.519, 95% CI: 0.282-0.954, P = 0.035). Biliary complications were significantly increased in recipients with MHV reconstruction (28.7% vs 11.6%, P = 0.027). CONCLUSIONS: MHV reconstruction ensured excellent outflow drainage and favored recipient outcome. The MHV tributaries (segments 5 and 8) should be reconstructed as much as possible to enlarge the hepatic vein anastomosis and reduce congestion.

Exclusive enteral nutrition versus corticosteroids for treatment of pediatric Crohn's disease: a meta-analysis.

BACKGROUND: Many studies have examined the effects of exclusive enteral nutrition (EEN) in children with Crohn's disease (CD), but corticosteroids are considered a superior therapy and are frequently used in China. This meta-analysis aims to compare the efficacy of EEN with corticosteroids in treating pediatric CD. METHODS: A comprehensive retrieval from medical databases, including PubMed, EMBASE, MEDLINE, Web of Science, Wanfang data, VIP and CNKI, was performed using the search terms "diet therapy", "exclusive enteral nutrition", "Crohn's disease", "inflammatory bowel diseases", "child" and "pediatrics" from January 1990 to April 2017. RESULTS: We included 18 studies from 1329 identified sources in this meta-analysis. EEN was as effective as corticosteroids in inducing remission rate of children suffering from CD (OR = 1.35; 95% CI 0.90, 2.10; P = 0.14). Nevertheless, patients who received EEN were more likely to achieve both endoscopic mucosal healing (OR = 5.24; 95% CI 2.06, 13.37; P = 0.0005) and histological mucosal healing (OR = 4.78; 95% CI 1.89, 12.08; P = 0.0009) than those who received corticosteroids; the Pediatric Crohn's Disease Activity Index was lower [mean difference (MD) = - 3.67; 95% CI - 4.91, - 2.43] and weight gain was higher (MD = 1.92; 95% CI 0.02, 3.83; P = 0.05) in those patients who received EEN than in those who received corticosteroids. No difference was found in relapse rate (OR = 0.57; 95% CI 0.25, 1.29; P = 0.18), height for age or body mass index between the patients treated with EEN and corticosteroids at the 1-year end point. CONCLUSIONS: This meta-analysis reveals that there is no significant difference between EEN and corticosteroids in the efficacy of inducing remission rate of CD in a pediatric population, but EEN is superior to corticosteroids in improving short-term mucosal inflammation and reducing the PCDAI index.

Metformin ameliorates arsenic trioxide hepatotoxicity via inhibiting mitochondrial complex I.

Arsenic trioxide (ATO) is a well-accepted chemotherapy agent in managing promyelocytic leukemia. ATO often causes severe health hazards such as hepatotoxicity, dermatosis, neurotoxicity, nephrotoxicity and cardiotoxicity. The production of reactive oxygen species, (ROS) play a significant role in ATO-induced hepatotoxicity. The oral hypoglycemic drug, metformin, is considered to be a potential novel agent for chemoprevention in the treatment of cancer. Moreover, metformin has also been shown to have hepatoprotective effects. In the present study, we demonstrated that metformin protected normal hepatocytes from ATO-induced apoptotic cell death in vitro and in vivo. Gene expression screening revealed that glucose metabolism might be related to the metformin-induced protective effect on ATO-treated AML12 cells. The metformin-promoted or induced glycolysis was not responsible for the protection of AML12 cells from ATO-induced apoptotic cell death. Instead, metformin increased the intracellular NADH/NAD+ ratio by inhibiting mitochondrial respiratory chain complex I, further decreasing the intracellular ROS induced by ATO. Treatment with low glucose or rotenone, a mitochondrial respiratory chain complex I inhibitor, also protected AML12 cells from ATO-induced apoptotic cell death. We show for the first time that metformin protects the hepatocyte from ATO by regulating the mitochondrial function. With its properties of chemoprevention, chemosensitization and the amelioration of liver damage, metformin has great prospects for clinical application other than type 2 diabetes mellitus (T2DM).