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Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested that these 24 mutants affect 24 different genes. We applied unsupervised clustering algorithms to classify the recovered mutants into three classes unbiasedly. Further computational analyses revealed that each of the recovered mutations in these three classes shows a unique effect on node subtype composition and connection property distribution of the intrahepatic biliary network. Besides, we found that most recovered mutations are viable. In those mutant fish, biliary network phenotypes persist into adulthood, which themselves are good animal models to study chronic cholestatic liver diseases. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

Hepatocellular carcinoma (HCC) is a complex biological process and is often diagnosed at advanced stages with no effective treatment options. With advances in tumor biology and molecular genetic profiling, several different signaling pathways and molecular mechanisms have been identified as responsible for initiating and promoting HCC. Targeting these critical pathways, which include the receptor tyrosine kinase pathways, the Ras mitogen-activated protein kinase (Ras/Raf/MAPK), the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), the Wnt/β-catenin signaling pathway, the ubiquitin/proteasome degradation and the hedgehog signaling pathway has led to the identification of novel therapeutics for HCC treatment. In this review, we elaborated on our current understanding of the signaling pathways involved in the development and initiation of HCC and anticipate the potential targets for therapeutic drug development.

The intrahepatic biliary network is a highly branched three-dimensional network lined by biliary epithelial cells, but how its branching patterns are precisely established is not clear. We designed a new computer-based algorithm that quantitatively computes the structural differences of the three-dimensional networks. Utilizing the algorithm, we showed that inhibition of Cyclin-dependent kinase 5 (Cdk5) led to reduced branching in the intrahepatic biliary network in zebrafish. Further, we identified a previously unappreciated downstream kinase cascade regulated by Cdk5. Pharmacological manipulations of this downstream kinase cascade produced a crowded branching defect in the intrahepatic biliary network and influenced actin dynamics in biliary epithelial cells. We generated larvae carrying a mutation in cdk5 regulatory subunit 1a (cdk5r1a), an essential activator of Cdk5. cdk5r1a mutant larvae show similar branching defects as those observed in Cdk5 inhibitor-treated larvae. A small-molecule compound that interferes with the downstream kinase cascade rescued the mutant phenotype. These results provide new insights into branching morphogenesis of the intrahepatic biliary network.

Cancer cells undergo metabolic adaptation to sustain uncontrolled proliferation. Aerobic glycolysis and glutaminolysis are two of the most essential characteristics of cancer metabolic reprogramming. Hyperactivated phosphoinositide 3-kinase (PI3K)/Akt serine/threonine kinase (Akt) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways play central roles in cancer cell metabolic adaptation given that their downstream effectors, such as Akt and c-Myc, control most of the glycolytic and glutaminolysis genes. Here, we report that the cytosolic flavoprotein, NAD(P)H quinone dehydrogenase 1 (Nqo1), is strongly overexpressed in mouse and human hepatocellular carcinoma (HCC). Knockdown of Nqo1 enhanced activity of the serine/threonine phosphatase, protein phosphatase 2A, which operates at the intersection of the PI3K/Akt and MAPK/ERK pathways and dephosphorylates and inactivates pyruvate dehydrogenase kinase 1, Akt, Raf, mitogen-activated protein kinase kinase, and ERK1/2. Nqo1 ablation also induced the expression of phosphatase and tensin homolog, a dual protein/lipid phosphatase that blocks PI3K/Akt signaling, through the ERK/cAMP-responsive element-binding protein/c-Jun pathway. Together, Nqo1 ablation triggered simultaneous inhibition of the PI3K/Akt and MAPK/ERK pathways, suppressed the expression of glycolysis and glutaminolysis genes and blocked metabolic adaptation in liver cancer cells. Conversely, Nqo1 overexpression caused hyperactivation of the PI3K/Akt and MAPK/ERK pathways and promoted metabolic adaptation. Conclusion: In conclusion, Nqo1 functions as an upstream activator of both the PI3K/Akt and MAPK/ERK pathways in liver cancer cells, and Nqo1 ablation blocked metabolic adaptation and inhibited liver cancer cell proliferation and HCC growth in mice. Therefore, our results suggest that Nqo1 may function as a therapeutic target to inhibit liver cancer cell proliferation and inhibit HCC.

Cachexia is a complex tissue-wasting syndrome characterized by inflammation, hypermetabolism, increased energy expenditure, and anorexia. Browning of white adipose tissue (WAT) is one of the significant factors that contribute to energy wasting in cachexia. By utilizing a cell implantation model, we demonstrate here that the lipid mobilizing factor zinc-α(2)-glycoprotein (ZAG) induces WAT browning in mice. Increased circulating levels of ZAG not only induced lipolysis in adipose tissues but also caused robust browning in WAT. Stimulating WAT progenitors with ZAG recombinant protein or expression of ZAG in mouse embryonic fibroblasts (MEFs) strongly enhanced brown-like differentiation. At the molecular level, ZAG stimulated peroxisome proliferator–activated receptor γ (PPARγ) and early B cell factor 2 expression and promoted their recruitment to the PR/SET domain 16 (Prdm16) promoter, leading to enhanced expression of Prdm16, which determines brown cell fate. In brown adipose tissue, ZAG stimulated the expression of PPARγ and PPARγ coactivator 1α and promoted recruitment of PPARγ to the uncoupling protein 1 (Ucp1) promoter, leading to increased expression of Ucp1. Overall, our results reveal a novel function of ZAG in WAT browning and highlight the targeting of ZAG as a potential therapeutic application in humans with cachexia.—Elattar, S., Dimri, M., Satyanarayana, A. The tumor secretory factor ZAG promotes white adipose tissue browning and energy wasting.

Ligand bound beta 2 adrenergic receptor (β2AR) crystal structures are in use for screening of compound libraries for identifying inducers and blockers. However, in case of G protein coupled receptors (GPCR), docking and binding energy (BE) calculations are not enough to discriminate agonist and antagonists. Absence of a reliable model for discriminating β2AR antagonist is still a major hurdle. Docking of known antagonists and agonists into activated and ground state β2AR revealed several key intermolecular interactions and H-bonding patterns, which in combination, emerged as a model for prediction of antagonists. Present study identifies an alternative binding orientation, within the binding pocket, for blockers and a minimum grid size to lessen the false positive predictions. Cluster analysis revealed structural variability among the antagonists and a conserved pattern in case of agonists. A combination of docking and structure activity relationship (SAR) model reliably discriminated antagonists. Based on key intermolecular interactions, a set of agonists and antagonists useful to SAR, quantitative structure activity relationship (QSAR) and pharmacophore modeling, has also been proposed for identifying antagonists.

The Johns Hopkins Clinical Compound Library (JHCCL), a collection of Food and Drug Administration (FDA)-approved small molecules (1400), was screened in silico for identification of novel β2AR blockers and tested for hematopoietic stem cell (HSC) expansion and radioprotection in zebrafish embryos. Docking studies, followed by the capacity to hasten erythropoiesis, identified todralazine (Binding energy, -8.4 kcal/mol) as a potential HSC-modulating agent. Todralazine (5 μM) significantly increased erythropoiesis in caudal hematopoietic tissue (CHT) in wild-type and anemic zebrafish embryos (2.33- and 1.44-folds, respectively) when compared with untreated and anemic control groups. Todralazine (5 μM) treatment also led to an increased number of erythroid progenitors, as revealed from the increased expression of erythroid progenitor-specific genes in the CHT region. Consistent with these effects, zebrafish embryos, Tg(cmyb:gfp), treated with 5 μM todralazine from 24 to 36 hours post fertilization (hpf) showed increased (approximately two-folds) number of HSCs at the aorta-gonad-mesonephros region (AGM). Similarly, expression of HSC marker genes, runx1 (3.3-folds), and cMyb (1.41-folds) also increased in case of todralazine-treated embryos, further supporting its HSC expansion potential. Metoprolol, a known beta blocker, also induced HSC expansion (1.36- and 1.48-fold increase in runx1 and cMyb, respectively). Todralazine (5 μM) when added 30 min before 20 Gy gamma radiation, protected zebrafish from radiation-induced organ toxicity, apoptosis, and improved survival (80% survival advantage over 6 days). The 2-deoxyribose degradation test further suggested hydroxyl (OH) radical scavenging potential of todralazine, and the same is recapitulated in vivo. These results suggest that todralazine is a potential HSC expanding agent, which might be acting along with important functions, such as antioxidant and free radical scavenging, in manifesting radioprotection.

According to the World Health Organization, hepatocellular carcinoma (HCC) is the 2nd leading cause of cancer related deaths worldwide. HCC is highly resistant to conventional chemotherapies; therefore, identification of specific molecular targets for the development of targeted therapies is indispensable. Cancer cells undergo metabolic adaptation to sustain uncontrolled proliferation and depends on the high rate of glycolysis and glutaminolysis. Hence, identifying and targeting proteins that assist cancer cells in metabolic reprogramming is an effective strategy to block their proliferation. We detected a strong upregulation of NAD(P)H Quinone Dehydrogenase 1 (Nqo1), a cytosolic flavoprotein, in vivo in mouse and human liver tumors as well as in liver cancer cell lines in vitro. Knockdown of Nqo1 in liver cancer cells by shRNA caused a significant downregulation of both glycolytic and glutaminolysis genes, impaired cell proliferation, colony formation and in vivo xenograft tumor growth. Nqo1-/- mice with HCC displayed significant reduction in tumor number, size and growth. In Nqo1+/+ mice, histological analysis of liver tumors revealed large tumor cells with hyperchromatic nuclei in a compact growth pattern, whereas Nqo1-/- livers appeared normal. Hyper-activated PI3K/Akt and MAPK/ERK signaling pathways play central role in cancer cell metabolic adaptation since their downstream effectors such as Akt and c-Myc control most of the glycolytic and glutaminolysis genes. Interestingly, at the molecular level, Nqo1 knockdown caused strong upregulation of the tumor suppressor PTEN, and enhanced activation of the serine/threonine phosphatase PP2A, leading to impaired activation of the PI3K/Akt and MAPK/ERK/c-Myc pathways. Our findings indicate that, by targeting PP2A and PTEN, Nqo1 enhances the expression and/or activity of PDK1, Akt, ERK1/2 and c-Myc and promotes cancer cell metabolic reprogramming and hence may function as a key therapeutic target for HCC inhibition. Citation Format: Manali Dimri, Ashley Humphries, Archana Laknaur, Sawsan Elattar, Ashok Sharma, Ravindra Kolhe, Ande Satyanarayana. Nqo1 ablation inhibits activation of the PI3K/Akt and MAPK/ERK pathways and blocks metabolic adaptation in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1835.

Drug repositioning is an approach of significant translatability, and the present study was undertaken to screen a collection of FDA approved small-molecule clinical compounds for identification of novel radioprotective agents. Screening of JHCCL (Johns Hopkins Clinical Compound Library), a collection of 1,400 FDA approved small molecules, lead to identification of prilocaine hydrochloride, a local anesthetic used widely during dental procedures, as a potential radioprotector. Prilocaine, at a concentration of 20 µM, protected zebrafish from radiation induced (20 Gy) pericardial edema (PE), microphthalmia and rendered 60 % survival advantage over radiation exposed controls. While 40 % survival advantage over radiation exposed controls was achieved with 10 µM prilocaine. Prilocaine, in a dose-dependent manner, scavenged, radiation-induced hydroxyl radicals and maximally (43 %) at the highest concentration (1 mM) tried in this study. However, prilocaine exerted a mild superoxide anion scavenging potential (around 5 %) at all the concentrations used within this study. Prilocaine, at 20 µM concentration, significantly increased erythropoiesis, a marker for HSC function, in caudal hematopoietic tissue (CHT) in wild type and anemic zebrafish embryos (1.48 and 0.85 folds respectively) when compared to untreated (1) and phenylhydrazine (PHZ) (0.41 fold) treated control groups respectively. These results suggest that prilocaine is a radioprotective agent and free radical scavenging and HSC expanding potential seems to be contributing towards its radioprotective action.

Cyclooxygenase-2 (COX-2) is well established for its role in inflammation, cancer and has also been reported to play a significant role in radiation induced inflammation and bystander effect. It has already been reported to have a role in protection against radiation induced damage, suggesting it to be an important target for identifying novel radiation countermeasure agents. Present study aims at identifying novel small molecules from pharmacopeia using COX-2 as target in silico. Systematic search of the molecules that are reported to exhibit radiation protection revealed that around 30% (40 in 130) of them have a role in inflammation and a small percentage of these molecules (20%; 8 in 40) are reported to act as non-steroidal anti-inflammatory drugs (NSAIDS). Docking studies further clarified that antiinflammatory compounds exhibited higher binding energy (BE). Out of 15 top hits, 14 molecules are reported to have anti-inflammatory property, suggesting the significant role of COX-2 in radiation protection. Further, Johns Hopkins Clinical Compound Library (JHCCL), a collection of small molecule clinical compounds, was screened virtually for COX-2 inhibition by docking approach. Docking of around 1400 small molecules against COX-2, leads to identification of a number of previously unreported molecules, which are likely to act as radioprotectors.