Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Serine-arginine rich splicing factor 3 (SRSF3) plays a critical role in hepatocyte function and its loss in mice promotes chronic liver damage and leads to HCC. Hepatocyte-specific SRSF3 knockout mice (SKO mice) also overexpress insulin-like growth factor 2 (IGF2). In the present study, double deletion of Igf2 and Srsf3 (DKO mice) prevents hepatic fibrosis and inflammation, and completely prevents tumor formation, and is associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. This is confirmed in vitro, where IGF2 treatment of HepG2 hepatoma cells decreases DNA repair enzyme expression and causes DNA damage. Tumors from the SKO mice also show mutational signatures consistent with homologous recombination and mismatch repair defects. Analysis of frozen human samples shows that SRSF3 protein is decreased sixfold in HCC compared to normal liver tissue but SRSF3 mRNA is increased. Looking at public TCGA data, HCC patients having high SRSF3 mRNA expression show poor survival, as do patients with alterations in known SRSF3-dependent splicing events. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer.

Exercise Improves Outcomes of Surgery on Fatty Liver in Mice: A Novel Effect Mediated by the AMPK Pathway.

OBJECTIVE: To investigate whether exercise improves outcomes of surgery on fatty liver, and whether pharmacological approaches can substitute exercising programs. SUMMARY OF BACKGROUND DATA: Steatosis is the hepatic manifestation of the metabolic syndrome, and decreases the liver's ability to handle inflammatory stress or to regenerate after tissue loss. Exercise activates adenosine monophosphate-activated kinase (AMPK) and mitigates steatosis; however, its impact on ischemia-reperfusion injury and regeneration is unknown. METHODS: We used a mouse model of simple, diet-induced steatosis and assessed the impact of exercise on metabolic parameters, ischemia-reperfusion injury and regeneration after hepatectomy. The same parameters were evaluated after treatment of mice with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Mice on a control diet served as age-matched controls. RESULTS: A 4-week-exercising program reversed steatosis, lowered insulin levels, and improved glucose tolerance. Exercise markedly enhanced the ischemic tolerance and the regenerative capacity of fatty liver. Replacing exercise with AICAR was sufficient to replicate the above benefits. Both exercise and AICAR improved survival after extended hepatectomy in mice challenged with a Western diet, indicating protection from resection-induced liver failure. CONCLUSIONS: Exercise efficiently counteracts the metabolic, ischemic, and regenerative deficits of fatty liver. AICAR acts as an exercise mimetic in settings of fatty liver disease, an important finding given the compliance issues associated with exercise. Exercising, or its substitution through AICAR, may provide a feasible strategy to negate the hepatic consequences of energy-rich diet, and has the potential to extend the application of liver surgery if confirmed in humans.

Exploration of the Transcriptional Landscape of ALPPS Reveals the Pathways of Accelerated Liver Regeneration.

Background and Aims: ALPPS (associating liver partition and portal vein ligation for staged hepatectomy), a novel 2-staged hepatectomy, dramatically accelerates liver regeneration and thus enables extensive liver tumor resection. The signaling networks underlying the ALPPS-induced accelerated regeneration process are largely unknown. Methods: We performed transcriptome profiling (TP) of liver tissue obtained from a mouse model of ALPPS, standard hepatectomy (68% model), and additional control surgeries (sham, PVL and Tx). We also performed TP using human liver biopsies (n = 5) taken from the occluded lobe and the future liver remnant (FLR) during the first step of ALPPS surgery (4-5 h apart). We used Oncofinder computational tools, which covers 378 ISPs, for unsupervised, unbiased quantification of ISP activity. Results: Gene expression cluster analysis revealed an ALPPS specific signature: the IGF1R Signaling Pathway (Cell survival), the ILK Pathway (Induced cell proliferation), and the IL-10 Pathway (Stability determination) were significantly enriched, whereas the activity of the Interferon Pathway (Transcription) was reduced (p < 0.05). Further, the PAK- and ILK-associated ISPs were activated at an earlier time point, reflecting significant acceleration of liver regeneration (p < 0.001). These pathways, which were also recovered in human liver biopsies, control cell growth and proliferation, inflammatory response, and hypoxia-related processes. Conclusions: ALPPS is not a straightforward addition of portal vein ligation (PVL) plus transection-it is more. The early stages of normal and accelerated liver regeneration are clearly discernible by a significantly increased and earlier activation of a small number of signaling pathways. Compounds mimicking these responses may help to improve the ALPPS method and further reduce the hospitalization time of the patient.

JNK1 induces hedgehog signaling from stellate cells to accelerate liver regeneration in mice.

BACKGROUND & AIMS: To improve outcomes of two-staged hepatectomies for large/multiple liver tumors, portal vein ligation (PVL) has been combined with parenchymal transection (associating liver partition and portal vein ligation for staged hepatectomy [coined ALPPS]) to greatly accelerate liver regeneration. In a novel ALPPS mouse model, we have reported paracrine Indian hedgehog (IHH) signaling from stellate cells as an early contributor to augmented regeneration. Here, we sought to identify upstream regulators of IHH. METHODS: ALPPS in mice was compared against PVL and additional control surgeries. Potential IHH regulators were identified through in silico mining of transcriptomic data. c-Jun N-terminal kinase (JNK1 [Mapk8]) activity was reduced through SP600125 to evaluate its effects on IHH signaling. Recombinant IHH was injected after JNK1 diminution to substantiate their relationship during accelerated liver regeneration. RESULTS: Transcriptomic analysis linked Ihh to Mapk8. JNK1 upregulation after ALPPS was validated and preceded the IHH peak. On immunofluorescence, JNK1 and IHH co-localized in alpha-smooth muscle actin-positive non-parenchymal cells. Inhibition of JNK1 prior to ALPPS surgery reduced liver weight gain to PVL levels and was accompanied by downregulation of hepatocellular proliferation and the IHH-GLI1-CCND1 axis. In JNK1-inhibited mice, recombinant IHH restored ALPPS-like acceleration of regeneration and re-elevated JNK1 activity, suggesting the presence of a positive IHH-JNK1 feedback loop. CONCLUSIONS: JNK1-mediated induction of IHH paracrine signaling from hepatic stellate cells is essential for accelerated regeneration of parenchymal mass. The JNK1-IHH axis is a mechanism unique to ALPPS surgery and may point to therapeutic alternatives for patients with insufficient regenerative capacity. LAY SUMMARY: Associating liver partition and portal vein ligation for staged hepatectomy (so called ALPPS), is a new two-staged approach to hepatectomy, which induces an unprecedented acceleration of liver regeneration, enabling treatment of patients with liver tumors that would otherwise be considered unresectable. Herein, we demonstrate that JNK1-IHH signaling from stellate cells is a key mechanism underlying the regenerative acceleration that is induced by ALPPS.

Early career researchers want Open Science.

Open Science is encouraged by the European Union and many other political and scientific institutions. However, scientific practice is proving slow to change. We propose, as early career researchers, that it is our task to change scientific research into open scientific research and commit to Open Science principles.

Hedgehog pathway mediates early acceleration of liver regeneration induced by a novel two-staged hepatectomy in mice.

BACKGROUND & AIMS: ALPPS, a novel two-staged approach for the surgical removal of large/multiple liver tumors, combines portal vein ligation (PVL) with parenchymal transection. This causes acceleration of compensatory liver growth, enabling faster and more extensive tumor removal. We sought to identify the plasma factors thought to mediate the regenerative acceleration following ALPPS. METHODS: We compared a mouse model of ALPPS against PVL and additional control surgeries (n=6 per group). RNA deep sequencing was performed to identify candidate molecules unique to ALPPS liver (n=3 per group). Recombinant protein and a neutralizing antibody combined with appropriate surgeries were used to explore candidate functions in ALPPS (n=6 per group). Indian hedgehog (IHH/Ihh) levels were assessed in human ALPPS patient plasma (n=6). RESULTS: ALPPS in mouse confirmed significant acceleration of liver regeneration relative to PVL (p<0.001). Ihh mRNA, coding for a secreted ligand inducing hedgehog signaling, was uniquely upregulated in ALPPS liver (p<0.001). Ihh plasma levels rose 4h after surgery (p<0.01), along with hedgehog pathway activation and subsequent cyclin D1 induction in the liver. When combined with PVL, Ihh alone was sufficient to induce ALPPS-like acceleration of liver growth. Conversely, blocking Ihh markedly inhibited the accelerating effects of ALPPS. In the small cohort of ALPPS patients, IHH tended to be elevated early after surgery. CONCLUSIONS: Ihh and hedgehog pathway activation provide the first mechanistic insight into the acceleration of liver regeneration triggered by ALPPS surgery. The accelerating potency of recombinant Ihh, and its potential effect in human ALPPS may lead to a clinical role for this protein. LAY SUMMARY: ALPPS, a novel two-staged hepatectomy, accelerates liver regeneration, thereby helping to treat patients with otherwise unresectable liver tumors. The molecular mechanisms behind this accelerated regeneration are unknown. Here, we elucidate that Indian hedgehog, a secreted ligand important for fetal development, is a crucial mediator of the regenerative acceleration triggered by ALPPS surgery.

Deletion of serine/arginine-rich splicing factor 3 in hepatocytes predisposes to hepatocellular carcinoma in mice.

UNLABELLED: Alterations in RNA splicing are associated with cancer, but it is not clear whether they result from malignant transformation or have a causative role. We show here that hepatocyte-specific deletion of serine/arginine-rich splicing factor 3 (SRSF3) impairs hepatocyte maturation and metabolism in early adult life, and mice develop spontaneous hepatocellular carcinoma (HCC) with aging. Tumor development is preceded by chronic liver disease with progressive steatosis and fibrosis. SRSF3 protects mice against CCl4 -induced fibrosis and carcinogenesis and suppresses inclusion of the profibrogenic EDA exon in fibronectin 1. Loss of SRSF3 increases expression of insulin-like growth factor 2 and the A-isoform of the insulin receptor, allowing aberrant activation of mitogenic signaling, promotes aberrant splicing and expression of epithelial to mesenchymal transition (EMT) genes, and activates Wnt/ß-catenin signaling leading to c-Myc induction. Finally, SRSF3 expression is either decreased or the protein mislocalized in human HCC. CONCLUSION: Our data suggest a potential role for SRSF3 in preventing hepatic carcinogenesis by regulating splicing to suppress fibrosis, mitogenic splicing, and EMT. Thus, these mice may provide an attractive model to discover the pathogenic mechanisms linking aberrant pre-messenger RNA splicing with liver damage, fibrosis, and HCC.