Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS: Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS: SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS: This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

The intrahepatic signalling niche of hedgehog is defined by primary cilia positive cells during chronic liver injury.

BACKGROUND & AIMS: In vertebrates, canonical Hedgehog (Hh) pathway activation requires Smoothened (SMO) translocation to the primary cilium (Pc), followed by a GLI-mediated transcriptional response. In addition, a similar gene regulation occurs in response to growth factors/cytokines, although independently of SMO signalling. The Hh pathway plays a critical role in liver fibrosis/regeneration, however, the mechanism of activation in chronic liver injury is poorly understood. This study aimed to characterise Hh pathway activation upon thioacetamide (TAA)-induced chronic liver injury in vivo by defining Hh-responsive cells, namely cells harbouring Pc and Pc-localised SMO. METHODS: C57BL/6 mice (wild-type or Ptc1(+/-)) were TAA-treated. Liver injury and Hh ligand/pathway mRNA and protein expression were assessed in vivo. SMO/GLI manipulation and SMO-dependent/independent activation of GLI-mediated transcriptional response in Pc-positive (Pc(+)) cells were studied in vitro. RESULTS: In vivo, Hh activation was progressively induced following TAA. At the epithelial-mesenchymal interface, injured hepatocytes produced Hh ligands. Progenitors, myofibroblasts, leukocytes and hepatocytes were GLI2(+). Pc(+) cells increased following TAA, but only EpCAM(+)/GLI2(+) progenitors were Pc(+)/SMO(+). In vitro, SMO knockdown/hGli3-R overexpression reduced proliferation/viability in Pc(+) progenitors, whilst increased proliferation occurred with hGli1 overexpression. HGF induced GLI transcriptional activity independently of Pc/SMO. Ptc1(+/-) mice exhibited increased progenitor, myofibroblast and fibrosis responses. CONCLUSIONS: In chronic liver injury, Pc(+) progenitors receive Hh ligand signals and process it through Pc/SMO-dependent activation of GLI-mediated transcriptional response. Pc/SMO-independent GLI activation likely occurs in Pc(-)/GLI2(+) cells. Increased fibrosis in Hh gain-of-function mice likely occurs by primary progenitor expansion/proliferation and secondary fibrotic myofibroblast expansion, in close contact with progenitors.

Impact of Intensive Care Unit Discharge Delay on Liver Transplantation Outcomes.

Background: For general intensive care unit (ICU) patients, ICU discharge delay (ICUDD) has been associated with an increased hospital length of stay (LOS) and the acquisition of multi-resistant organism (MRO) infections. The impact of ICUDD on liver transplant (LT) recipients is unknown. Methods: We retrospectively studied consecutive adult LT between 2011 and 2019. ICUDD was defined as >8 h between a patient being cleared for discharge to ward and the patient leaving the ICU. Results: 550 patients received LT and the majority (68.5%) experienced ICUDD. The median time between clearance for ward and the patient leaving the ICU was 25.6 h. No donor or recipient variables were associated with ICUDD. Patients cleared for discharge early in the week (Sunday-Tuesday) and those discharged outside routine work hours were more likely to experience ICUDD (p = 0.001 and p < 0.001, respectively). The median hospital LOS was identical (18 days, p = 0.96) and there were no differences in other patient outcomes. Patients who became colonized with MRO in the ICU spent a longer time there compared to those who remained MRO-free (9 vs. 6 days, p < 0.001); however, this was not due to ICUDD. Conclusion: ICUDD post-LT is common and does not prolong hospital LOS. ICUDD is not associated with adverse patient outcomes or MRO colonization.

Lenvatinib for the treatment of hepatocellular carcinoma-a real-world multicenter Australian cohort study.

INTRODUCTION: Hepatocellular carcinoma (HCC) is a serious complication of chronic liver disease. Lenvatinib is an oral multikinase inhibitor registered to treat advanced HCC. This study evaluates the real-world experience with lenvatinib in Australia. METHODS: We conducted a retrospective cohort study of patients treated with lenvatinib for advanced HCC between July 2018 and November 2020 at 11 Australian tertiary care hospitals. Baseline demographic data, tumor characteristics, lenvatinib dosing, adverse events (AEs) and clinical outcomes were collected. Overall survival (OS) was the primary outcome. Progression free survival (PFS) and AEs were secondary outcomes. RESULTS: A total of 155 patients were included and were predominantly male (90.7%) with a median age of 65 years (interquartile range [IQR]: 59-75). The main causes of chronic liver disease were hepatitis C infection (40.0%) and alcohol-related liver disease (34.2). Median OS and PFS were 7.7 (95% confidence interval [CI]: 5.8-14.0) and 5.3 months (95% CI: 2.8-9.2) respectively. Multivariate predictors of mortality were the need for dose reduction due to AEs (Hazard ratio [HR] 0.41, p < 0.01), new or worsening hypertension (HR 0.42, p < 0.01), diarrhoea (HR 0.47, p = 0.04) and more advanced BCLC stage (HR 2.50, p = 0.04). Multivariable predictors of disease progression were higher Child-Pugh score (HR 1.25, p = 0.04), the need for a dose reduction (HR 0.45, p < 0.01) and age (HR 0.96, p < 0.001). AEs occurred in 83.9% of patients with most being mild (71.6%). CONCLUSIONS: Lenvatinib remains safe and effective in real-world use. Treatment emergent diarrhoea and hypertension, and the need for dose reduction appear to predict better OS.

Reply to "Comments on the Editor Re: The Relationship of Obesity, Nutritional Status and Muscle Wasting in Patients Assessed for Liver Transplantation, Nutrients 2019, 11, 2097."

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Fibroblast activation protein in liver fibrosis.

Fibroblast activation protein (FAP) belongs to the dipeptidyl peptidase IV (DPP4; CD26) gene family. Other related genes in this family of enzyme include DPP4, 8 and 9. The FAP serine protease has the rare property of both dipeptidyl peptidase and endopeptidase activities capable of cleaving the post-proline bond at two or more residues from the N-terminus. FAP is involved in a variety of biological processes but its expression in healthy tissues is low. In contrast, FAP is significantly elevated in pathological conditions such as at sites of tissue remodelling and repair. Its differential pattern of expression in diseases supports the emerging concept for FAP as a potential disease biomarker as well as a useful therapeutic target for drug intervention. This review summarizes the current knowledge of FAP, particularly its diagnostic and pathological significance in liver fibrosis.

The Relationship of Obesity, Nutritional Status and Muscle Wasting in Patients Assessed for Liver Transplantation.

INTRODUCTION: Obesity co-exists with malnutrition and muscle atrophy in patients with cirrhosis. Muscle wasting is a feature of sarcopenia, a known determinant of patient outcomes. This is the first description of a relationship between obesity, subjective global assessment (SGA) of nutritional status and muscle wasting in patients with cirrhosis. METHODS: The relationship between body mass index (BMI with obesity defined as ≥ 30 kg/m2), nutritional status (assessed by liver-specific subjective global assessment-SGA) and muscle wasting (assessed by corrected total cross-sectional psoas muscle area-cTPA) was analysed in patients with cirrhosis considered for liver transplantation between 1 January 2012 and 31 December 2014. RESULTS: There were 205 patients, of whom 70% were males. The mean age was 52 ± 0.7 years and the Model for End-Stage Liver Disease (MELD) score was 16.8 ± 0.5. Overall, 31% of patients were obese and 56% of well-nourished (SGA A) individuals were obese. Muscle wasting was identified in 86% of all patients, irrespective of their nutritional status (A, B, C). All obese males classified as well-nourished (SGA A) were sarcopenic and 62% of obese females classified as SGA A were sarcopenic. Muscle wasting was worse in obese individuals (cTPA 230.9 mm2/m2 ± 12.9, p < 0.0001) and more likely to be associated with hepatic encephalopathy (p = 0.03). Univariate and multivariate analysis demonstrated testosterone deficiency was significantly associated with muscle wasting (p = 0.007) but not obesity (p = 0.8). CONCLUSION: Obesity combined with muscle wasting is common in patients with cirrhosis. Muscle wasting is common in well-nourished (SGA A) obese patients. Consequently, all patients assessed for liver transplantation should undergo additional screening for malnutrition and muscle wasting irrespective of BMI.

Randomised clinical trial: oral taurine supplementation versus placebo reduces muscle cramps in patients with chronic liver disease.

BACKGROUND: Painful muscle cramps occur in the majority of patients with cirrhosis impacting significantly on quality of life and sleep patterns. They are frequently unrecognised or overlooked. Current management is based on anecdotal evidence or case study reports. AIM: To investigate the effect of oral taurine supplementation on frequency, duration, and intensity of muscle cramps in patients with chronic liver disease. METHODS: Patients with chronic liver disease who experienced three or more muscle cramps/week were enrolled in a double-blinded, randomised control, crossover, taurine dose-variable study. Each participant received either taurine supplementation or placebo for 4 weeks then crossed to the alternative arm. Primary outcome data for frequency, duration, and intensity of muscle cramps was recorded by participants. Participants recorded frequency, duration, and location of muscle cramps. Biochemical parameters, including serum taurine and methionine levels, were measured at each time point. Linear mixed models were used to analyse outcomes. RESULTS: Forty-nine patients were enrolled in the study and 30 patients completed the protocol. Participants who were unable to complete the protocol were not included in the final analysis due to the absence of outcome data. The mean age of participants was 54.7 years and 70% were males. Oral taurine supplementation increased serum taurine levels (P < 0.001). There were no adverse side effects associated with taurine supplementation. Participants receiving 2 g taurine/d experienced a reduction in cramp frequency (seven cramps fewer/fortnight, P = 0.03), duration (89 minutes less/fortnight P = 0.03), and severity (1.4 units less on a Likert scale P < 0.004) compared to placebo. CONCLUSIONS: Oral supplementation with 2 g taurine/d results in a clinically significant reduction in the frequency, duration, and intensity of muscle cramps in patients with chronic liver disease. Taurine should be considered as a safe and effective intervention in the management of muscle cramps in individuals with chronic liver disease. This study was registered with the Australian New Zealand Clinical Trials Register: ACTRN12612000289819.

Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease.

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (>99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMO-dependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness.

Rapid improvement of autonomic and peripheral neuropathy after liver transplantation: a single case report.

Peripheral and autonomic neuropathies are known but often unrecognized associations of cirrhosis from any cause. The pathogenesis of these effects are ill understood. Liver transplantation has been shown to reverse autonomic manifestations, but little evidence exists for an effect on peripheral neuropathy. This case report documents improvement in peripheral and autonomic neuropathy in a 40-year-old man with hepatitis B virus--related cirrhosis. A return to normal neurophysiological function was seen within 9 months of successful liver transplantation, suggesting a metabolic, rather than a structural, cause of such changes in the peripheral nervous system.