Identification of a novel GSK3ß inhibitor involved in abrogating KRas dependent pancreatic tumors in Wnt/beta-catenin and NF-kB dependent manner.

Pancreatic cancer is an aggressive malignancy with a poor survival rate because it is difficult to diagnose the disease during its early stages. The currently available treatments, which include surgery, chemotherapy and radiation therapy, offer only limited survival benefit. Pharmacological interventions to inhibit Glycogen Synthase Kinase-3beta (GSK3ß) activity is an important therapeutic strategy for the treatment of pancreatic cancer because GSK3ß is one of the key factors involved in the onset, progression as well as in the acquisition of chemoresistance in pancreatic cancer. Here, we report the identification of MJ34 as a potent GSK3ß inhibitor that significantly reduced growth and survival of human mutant KRas dependent pancreatic tumors. MJ34 mediated GSK3ß inhibition was seen to induce apoptosis in a ß-catenin dependent manner and downregulate NF-kB activity in MiaPaCa-2 cells thereby impeding cell survival and anti-apoptotic processes in these cells as well as in the xenograft model of pancreatic cancer. In vivo acute toxicity and in vitro cardiotoxicity studies indicate that MJ34 is well tolerated without any adverse effects. Taken together, we report the discovery of MJ34 as a potential drug candidate for the therapeutic treatment of mutant KRas-dependent human cancers through pharmacological inhibition of GSK3ß.

Impaired Bile Secretion Promotes Hepatobiliary Injury in Sickle Cell Disease.

BACKGROUND AND AIMS: Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS: SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS: These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.

Blood-Bile Barrier: Morphology, Regulation, and Pathophysiology.

The term blood-bile barrier (BBlB) refers to the physical structure within a hepatic lobule that compartmentalizes and hence segregates sinusoidal blood from canalicular bile. Thus, this barrier provides physiological protection in the liver, shielding the hepatocytes from bile toxicity and restricting the mixing of blood and bile. BBlB is primarily composed of tight junctions; however, adherens junction, desmosomes, gap junctions, and hepatocyte bile transporters also contribute to the barrier function of the BBlB. Recent findings also suggest that disruption of BBlB is associated with major hepatic diseases characterized by cholestasis and aberrations in BBlB thus may be a hallmark of many chronic liver diseases. Several molecular signaling pathways have now been shown to play a role in regulating the structure and function and eventually contribute to regulation of the BBlB function within the liver. In this review, we will discuss the structure and function of the BBlB, summarize the methods to assess the integrity and function of BBlB, discuss the role of BBlB in liver pathophysiology, and finally, discuss the mechanisms of BBlB regulation. Collectively, this review will demonstrate the significance of the BBlB in both liver homeostasis and hepatic dysfunction.

Dual catenin loss in murine liver causes tight junctional deregulation and progressive intrahepatic cholestasis.

ß-Catenin, the downstream effector of the Wnt signaling, plays important roles in hepatic development, regeneration, and tumorigenesis. However, its role at hepatocyte adherens junctions (AJ) is relatively poorly understood, chiefly due to spontaneous compensation by γ-catenin. We simultaneously ablated ß- and γ-catenin expression in mouse liver by interbreeding ß-catenin-γ-catenin double-floxed mice and Alb-Cre transgenic mice. Double knockout mice show failure to thrive, impaired hepatocyte differentiation, cholemia, ductular reaction, progressive cholestasis, inflammation, fibrosis, and tumorigenesis, which was associated with deregulation of tight junctions (TJ) and bile acid transporters, leading to early morbidity and mortality, a phenotype reminiscent of progressive familial intrahepatic cholestasis (PFIC). To address the mechanism, we specifically and temporally eliminated both catenins from hepatocytes using adeno-associated virus 8 carrying Cre-recombinase under the thyroid-binding globulin promoter (AAV8-TBG-Cre). This led to a time-dependent breach of the blood-biliary barrier associated with sequential disruption of AJ and TJ verified by ultrastructural imaging and intravital microscopy, which revealed unique paracellular leaks around individual hepatocytes, allowing mixing of blood and bile and leakage of blood from one sinusoid to another. Molecular analysis identified sequential losses of E-cadherin, occludin, claudin-3, and claudin-5 due to enhanced proteasomal degradation, and of claudin-2, a ß-catenin transcriptional target, which was also validated in vitro. CONCLUSION: We report partially redundant function of catenins at AJ in regulating TJ and contributing to the blood-biliary barrier. Furthermore, concomitant hepatic loss of ß- and γ-catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Mice with dual catenin loss develop progressive intrahepatic cholestasis, providing a unique model to study diseases such as PFIC. (Hepatology 2018;67:2320-2337).

ß-Catenin Signaling and Roles in Liver Homeostasis, Injury, and Tumorigenesis.

ß-catenin (encoded by CTNNB1) is a subunit of the cell surface cadherin protein complex that acts as an intracellular signal transducer in the WNT signaling pathway; alterations in its activity have been associated with the development of hepatocellular carcinoma and other liver diseases. Other than WNT, additional signaling pathways also can converge at ß-catenin. ß-catenin also interacts with transcription factors such as T-cell factor, forkhead box protein O, and hypoxia inducible factor 1α to regulate the expression of target genes. We discuss the role of ß-catenin in metabolic zonation of the adult liver. ß-catenin also regulates the expression of genes that control metabolism of glucose, nutrients, and xenobiotics; alterations in its activity may contribute to the pathogenesis of nonalcoholic steatohepatitis. Alterations in ß-catenin signaling may lead to activation of hepatic stellate cells, which is required for fibrosis. Many hepatic tumors such as hepatocellular adenomas, hepatocellular cancers, and hepatoblastomas have mutations in CTNNB1 that result in constitutive activation of ß-catenin, so this molecule could be a therapeutic target. We discuss how alterations in ß-catenin activity contribute to liver disease and how these might be used in diagnosis and prognosis, as well as in the development of therapeutics.

PDGFRα in liver pathophysiology: emerging roles in development, regeneration, fibrosis, and cancer.

Platelet-derived growth factor receptor α (PDGFRα) is an isoform of the PDGFR family of tyrosine kinase receptors involved in cell proliferation, survival, differentiation, and growth. In this review, we highlight the role of PDGFRα and the current evidence of its expression and activities in liver development, regeneration, and pathology-including fibrosis, cirrhosis, and liver cancer. Studies elucidating PDGFRα signaling in processes ranging from profibrotic signaling, angiogenesis, and oxidative stress to epithelial-to-mesenchymal transition point toward PDGFRα as a potential therapeutic target in various hepatic pathologies, including hepatic fibrosis and liver cancer. Furthermore, PDGFRα localization and modulation during liver development and regeneration may lend insight into its potential roles in various pathologic states. We will also briefly discuss some of the current targeted treatments for PDGFRα, including multi receptor tyrosine kinase inhibitors and PDGFRα-specific inhibitors.

Cellular and molecular basis of liver development.

Liver is a prime organ responsible for synthesis, metabolism, and detoxification. The organ is endodermal in origin and its development is regulated by temporal, complex, and finely balanced cellular and molecular interactions that dictate its origin, growth, and maturation. We discuss the relevance of endoderm patterning, which truly is the first step toward mapping of domains that will give rise to specific organs. Once foregut patterning is completed, certain cells within the foregut endoderm gain competence in the form of expression of certain transcription factors that allow them to respond to certain inductive signals. Hepatic specification is then a result of such inductive signals, which often emanate from the surrounding mesenchyme. During hepatic specification bipotential hepatic stem cells or hepatoblasts become apparent and undergo expansion, which results in a visible liver primordium during the stage of hepatic morphogenesis. Hepatoblasts next differentiate into either hepatocytes or cholangiocytes. The expansion and differentiation is regulated by cellular and molecular interactions between hepatoblasts and mesenchymal cells including sinusoidal endothelial cells, stellate cells, and also innate hematopoietic elements. Further maturation of hepatocytes and cholangiocytes continues during late hepatic development as a function of various growth factors. At this time, liver gains architectural novelty in the form of zonality and at cellular level acquires polarity. A comprehensive elucidation of such finely tuned developmental cues have been the basis of transdifferentiation of various types of stem cells to hepatocyte-like cells for purposes of understanding health and disease and for therapeutic applications.

Role of Wnt/ß-catenin signaling in liver metabolism and cancer.

Wnt/ß-catenin signaling is known for its role in embryogenesis as well as carcinogenesis. In the liver, it plays many critical roles during hepatic development and regeneration, and its dysregulation is evident in aberrant hepatic growth during various liver tumors. Its chief cellular roles in the liver include regulation of processes of cell proliferation, apoptosis, oxidative stress and differentiation, which in turn contributes to hepatic growth, zonation, xenobiotic metabolism and other metabolic processes inherent to the liver. Most of these functions of the Wnt/ß-catenin signaling are dictated through the highly temporal and tissue-specific or non-specific transcriptional targets of the pathway. In addition, some of the critical functions such as cell-cell adhesion and perhaps maintenance of various junctions that are critical from an epithelial cell biology perspective are also a function of ß-catenin, which is the central component of the canonical Wnt pathway. Various animal models and clinical studies have demonstrated the spectra of Wnt/ß-catenin signaling in liver health and disease. Thus therapeutic modulation of this pathway for improved hepatic health is inevitable in the future. The current review discusses the advances in our understanding of the Wnt/ß-catenin signaling in liver physiology and pathology especially in hepatic metabolism and various tumors in adult liver and goes on to extrapolate the pre-clinical significance and possible translational implications of such findings.

The nitric oxide donor S-nitrosoglutathione reduces apoptotic primary liver cell loss in a three-dimensional perfusion bioreactor culture model developed for liver support.

INTRODUCTION: Artificial extracorporeal support for hepatic failure has met with limited clinical success. In hepatocytes, nitric oxide (NO) functions as an antiapoptotic modulator in response to a variety of stresses. We hypothesized that NO administration would yield improved viability and hepatocellular restructuring in a four-compartment, hollow fiber-based bioreactor with integral oxygenation for dynamic three-dimensional perfusion of hepatic cells in bioartificial liver support systems. METHODS: Isolated adult rat liver cells were placed in culture medium alone (control) or medium supplemented with various concentrations of an NO donor (S-nitrosoglutathione [GSNO]) in the bioreactors. Media samples were obtained from the cell perfusion circuit to monitor cellular response. After 24 and 72 h, histology biopsies were taken to investigate spontaneous restructuring of the cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to quantify apoptotic nuclei. RESULTS: Control bioreactors exhibited 47.9 +/- 2.9% (mean +/- standard error of the mean) apoptotic nuclei. In contrast, NO-treated bioreactors exhibited a biphasic response. Fewer apoptotic nuclei were seen in the 200 and 500 microM GSNO groups (14.4 +/- 0.4%). No effect was observed in the 10 microM GSNO group (47.3%), and increased TUNEL staining was observed in the 1000 microM GSNO group (82.6%). Media lactate dehydrogenase levels were lower in bioreactor groups treated with 200 or 500 microM GSNO (310 +/- 38 IU/L) compared with the control group (919 +/- 188 IU/L; p < 0.05). Protein synthesis was not affected, as measured by albumin levels in the media (115 +/- 19 microg/day/cell inoculum in GSNO-treated bioreactors at 24 h vs. 110 +/- 13 in controls; p = 0.851). Histologically, all of the bioreactor groups exhibited liver cell aggregates with some attached to the bioreactor capillaries. Increased numbers of cells in the aggregates and superior spontaneous restructuring of the cells were seen at 24 and 72 h in the bioreactor groups treated with either 200 or 500 microM GSNO compared with the control groups. CONCLUSION: Addition of an NO donor reduces adult rat liver cell apoptosis during the initial 24 h after cell inoculation within a three-dimensional perfusion bioreactor system for liver support and promotes liver cell aggregation and spontaneous restructuring of the cells at 24 and 72 h. GSNO-treated bioreactors remain metabolically active and show significantly lower levels of cellular injury as compared with controls. Further studies will be required to evaluate the impact of NO treatment of liver support bioreactors for clinical studies.