Heterozygous midnolin knockout attenuates severity of nonalcoholic fatty liver disease in mice fed a Western-style diet high in fat, cholesterol, and fructose.

Although midnolin has been studied for over 20 years, its biological roles in vivo remain largely unknown, especially due to the lack of a functional animal model. Indeed, given our recent discovery that the knockdown of midnolin suppresses liver cancer cell tumorigenicity and that this antitumorigenic effect is associated with modulation of lipid metabolism, we hypothesized that knockout of midnolin in vivo could potentially protect from nonalcoholic fatty liver disease (NAFLD) which has become the most common cause of chronic liver disease in the Western world. Accordingly, in the present study, we have developed and now report on the first functional global midnolin knockout mouse model. Although the overwhelming majority of global homozygous midnolin knockout mice demonstrated embryonic lethality, heterozygous knockout mice were observed to be similar to wild-type mice in their viability and were used to determine the effect of reduced midnolin expression on NAFLD. We found that global heterozygous midnolin knockout attenuated the severity of NAFLD in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism. Collectively, our results support a role for midnolin in regulating cholesterol/lipid metabolism in the liver. Thus, midnolin may represent a novel therapeutic target for NAFLD. Finally, our observation that midnolin was essential for survival underscores the broad importance of this gene beyond its role in liver biology.NEW & NOTEWORTHY We have developed and now report on the first functional global midnolin knockout mouse model. We found that global heterozygous midnolin knockout attenuated the severity of nonalcoholic fatty liver disease (NAFLD) in mice fed a Western-style diet, high in fat, cholesterol, and fructose, and this attenuation in disease was associated with significantly reduced levels of large lipid droplets, hepatic free cholesterol, and serum LDL, with significantly differential gene expression involved in cholesterol/lipid metabolism.

Pharmacologically Targeting the WNT/ß-Catenin Signaling Cascade: Avoiding the Sword of Damocles.

WNT/ß-catenin signaling plays fundamental roles in numerous developmental processes and in adult tissue homeostasis and repair after injury, by controlling cellular self-renewal, activation, division, differentiation, movement, genetic stability, and apoptosis. As such, it comes as no surprise that dysregulation of WNT/ß-catenin signaling is associated with various diseases, including cancer, fibrosis, neurodegeneration, etc. Although multiple agents that specifically target the WNT/ß-catenin signaling pathway have been studied preclinically and a number have entered clinical trials, none has been approved by the FDA to date. In this chapter, we provide our insights as to the reason(s) it has been so difficult to safely pharmacologically target the WNT/ß-catenin signaling pathway and discuss the significant efforts undertaken towards this goal.

Targeted genomic profiling identifies frequent deleterious mutations in FAT4 and TP53 genes in HBV-associated hepatocellular carcinoma.

BACKGROUND: Hepatitis B virus (HBV) is the major risk factor for hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV-associated HCC pathogenesis is still unclear. Genetic alterations in cancer-related genes have been linked to many human cancers. Here, we aimed to explore genetic alterations in selected cancer-related genes in patients with HBV-associated HCC. METHODS: Targeted sequencing was used to analyze six cancer-related genes (PIK3CA, TP53, FAT4, IRF2, HNF4α and ARID1A) in eight pairs of HBV-associated HCC tumors and their adjacent non-tumor tissues. Sanger sequencing, quantitative PCR, Western-blotting and RNAi-mediated gene knockdown were used to further validate findings. RESULTS: Targeted sequencing revealed thirteen non-synonymous mutations, of which 9 (69%) were found in FAT4 and 4 (31%) were found in TP53 genes. Non-synonymous mutations were not found in PIK3CA, IRF2, HNF4α and ARID1A. Among these 13 non-synonymous mutations, 12 (8 in FAT4 and 4 in TP53) were predicted to have deleterious effect on protein function by in silico analysis. For TP53, Y220S, R249S and P250R non-synonymous mutations were solely identified in tumor tissues. Further expression profiling of FAT4 and TP53 on twenty-eight pairs of HCC tumor and non-tumor tissues confirmed significant downregulation of both genes in HCC tumors compared with their non-tumor counterparts (P < 0.001 and P < 0.01, respectively). Functional analysis using RNAi-mediated knockdown of FAT4 revealed an increased cancer cell growth and proliferation, suggesting the putative tumor suppressor role of FAT4 in HCC. CONCLUSIONS: This study highlights the importance of FAT4 and TP53 in HCC pathogenesis and identifies new genetic variants that may have potentials for development of precise therapy for HCC.

Stearoyl-CoA Desaturase Promotes Liver Fibrosis and Tumor Development in Mice via a Wnt Positive-Signaling Loop by Stabilization of Low-Density Lipoprotein-Receptor-Related Proteins 5 and 6.

BACKGROUND & AIMS: Stearoyl-CoA desaturase (SCD) synthesizes monounsaturated fatty acids (MUFAs) and has been associated with the development of metabolic syndrome, tumorigenesis, and stem cell characteristics. We investigated whether and how SCD promotes liver fibrosis and tumor development in mice. METHODS: Rodent primary hepatic stellate cells (HSCs), mouse liver tumor-initiating stem cell-like cells (TICs), and human hepatocellular carcinoma (HCC) cell lines were exposed to Wnt signaling inhibitors and changes in gene expression patterns were analyzed. We assessed the functions of SCD by pharmacologic and conditional genetic manipulation in mice with hepatotoxic or cholestatic induction of liver fibrosis, orthotopic transplants of TICs, or liver tumors induced by administration of diethyl nitrosamine. We performed bioinformatic analyses of SCD expression in HCC vs nontumor liver samples collected from patients, and correlated levels with HCC stage and patient mortality. We performed nano-bead pull-down assays, liquid chromatography-mass spectrometry, computational modeling, and ribonucleoprotein immunoprecipitation analyses to identify MUFA-interacting proteins. We examined the effects of SCD inhibition on Wnt signaling, including the expression and stability of low-density lipoprotein-receptor-related proteins 5 and 6 (LRP5 and LRP6), by immunoblot and quantitative polymerase chain reaction analyses. RESULTS: SCD was overexpressed in activated HSC and HCC cells from patients; levels of SCD messenger RNA (mRNA) correlated with HCC stage and patient survival time. In rodent HSCs and TICs, the Wnt effector ß-catenin increased sterol regulatory element binding protein 1-dependent transcription of Scd, and ß-catenin in return was stabilized by MUFAs generated by SCD. This loop required MUFA inhibition of binding of Ras-related nuclear protein 1 (Ran1) to transportin 1 and reduced nuclear import of elav-like protein 1 (HuR), increasing cytosolic levels of HuR and HuR-mediated stabilization of mRNAs encoding LRP5 and LRP6. Genetic disruption of Scd and pharmacologic inhibitors of SCD reduced HSC activation and TIC self-renewal and attenuated liver fibrosis and tumorigenesis in mice. Conditional disruption of Scd2 in activated HSCs prevented growth of tumors from TICs and reduced the formation of diethyl nitrosamine-induced liver tumors in mice. CONCLUSIONS: In rodent HSCs and TICs, we found SCD expression to be regulated by Wnt-ß-catenin signaling, and MUFAs produced by SCD provided a forward loop to amplify Wnt signaling via stabilization of Lrp5 and Lrp6 mRNAs, contributing to liver fibrosis and tumor growth. SCD expressed by HSCs promoted liver tumor development in mice. Components of the identified loop linking HSCs and TICs might be therapeutic targets for liver fibrosis and tumors.

Extracellular matrix dynamics in hepatocarcinogenesis: a comparative proteomics study of PDGFC transgenic and Pten null mouse models.

We are reporting qualitative and quantitative changes of the extracellular matrix (ECM) and associated receptor proteomes, occurring during the transition from liver fibrosis and steatohepatitis to hepatocellular carcinoma (HCC). We compared two mouse models relevant to human HCC: PDGFC transgenic (Tg) and Pten null mice, models of disease progression from fibrosis and steatohepatitis to HCC. Using mass spectrometry, we identified in the liver of both models proteins for 26 collagen-encoding genes, providing the first evidence of expression at the protein level for 16 collagens. We also identified post-transcriptional protein variants for six collagens and lysine hydroxylation modifications for 14 collagens. Tumor-associated collagen proteomes were similar in both models with increased expression of collagens type IV, VI, VII, X, XIV, XV, XVI, and XVIII. Splice variants for Col4a2, Col6a2, Col6a3 were co-upregulated while only the short form of Col18a1 increased in the tumors. We also identified tumor specific increases of nidogen 1, decorin, perlecan, and of six laminin subunits. The changes in these non-collagenous ECM proteins were similar in both models with the exception of laminin ß3, detected specifically in the Pten null tumors. Pdgfa and Pdgfc mRNA expression was increased in the Pten null liver, a possible mechanism for the similarity in ECM composition observed in the tumors of both models. In contrast and besides the strong up-regulation of integrin α5 protein observed in the liver tumors of both models, the expression of the six other integrins identified was specific to each model, with integrins α2b, α3, α6, and ß1 up-regulated in Pten null tumors and integrins α8 and ß5 up-regulated in the PDGFC Tg tumors. In conclusion, HCC-associated ECM proteins and ECM-integrin networks, common or specific to HCC subtypes, were identified, providing a unique foundation to using ECM composition for HCC classification, diagnosis, prevention, or treatment.

Midnolin Regulates Liver Cancer Cell Growth In Vitro and In Vivo.

Hepatocellular carcinoma (HCC) ranks worldwide as one of the most lethal cancers. In spite of the vast existing knowledge about HCC, the pathogenesis of HCC is not completely understood. Discovery of novel genes that contribute to HCC pathogenesis will provide new insights for better understanding and treating HCC. The relatively obscure gene midnolin has been studied for over two decades; however, its biological roles are largely unknown. Our study is the first to demonstrate the functional significance of midnolin in HCC/cancer: Midnolin expression correlates with poor prognosis in HCC patients, and suppression of midnolin severely inhibits tumorigenicity of HCC cells in vitro and in mice and disrupts retinoic acid/lipid metabolism in these cells.

Thioacetamide-Induced Norepinephrine Production by Hepatocytes is Associated with Hepatic Stellate Cell Activation and Liver Fibrosis.

BACKGROUND: Collagen production by activated hepatic stellate cells (HSCs) to encapsulate injury is part of the natural wound-healing response in injured liver. However, persistent activation of HSCs can lead to pathological fibrogenesis. Such persistent HSC activation could be mediated by norepinephrine (NE), a reaction product of dopamine beta-hydroxylase (DBH). OBJECTIVE: To investigate the potential paracrine role of NE in hepatotoxin thioacetamide (TAA)-induced liver fibrosis. METHODS: In TAA-treated mice, fibrotic liver tissue showed significant increases in the mRNA expression of DBH up to 14-fold and collagen up to 7-fold. Immunohistochemical staining showed increased DBH protein expression in fibrotic liver tissue. Parenchymal hepatocyte cell line HepG2 expressed DBH and secreted NE, and the conditioned medium of HepG2 cells promoted collagenesis in nonparenchymal HSC cell line LX-2. TAA treatment increased DBH expression by 170% in HepG2 cells, as well as increased NE by 120% in the conditioned medium of HepG2 cells. The conditioned medium of TAA-treated HepG2 cells was used to culture LX-2 cells, and was found to increase collagen expression by 80% in LX-2 cells. Collagen expression was reduced by pre-treating HepG2 cells with siRNA targeting DBH or by adding NE antagonists to the conditioned medium. RESULTS: Finally, TAA-induced oxidative stress in HepG2 cells was associated with induction of DBH expression. Collectively, our results suggest a potential role for DBH/NE-mediated crosstalk between hepatocytes and HSCs in fibrogenesis. CONCLUSION: From a therapeutic standpoint, antagonism of DBH/NE induction in hepatocytes might be a useful strategy to suppress pathological fibrogenesis.

Magnetic resonance imaging for characterization of hepatocellular carcinoma metabolism.

With a better understanding of the pathophysiological and metabolic changes in hepatocellular carcinoma (HCC), multiparametric and novel functional magnetic resonance (MR) and positron emission tomography (PET) techniques have received wide interest and are increasingly being applied in preclinical and clinical research. These techniques not only allow for non-invasive detection of structural, functional, and metabolic changes in malignant tumor cells but also characterize the tumor microenvironment (TME) and the interactions of malignant tumor cells with the TME, which has hypoxia and low pH, resulting from the Warburg effect and accumulation of metabolites produced by tumor cells and other cellular components. The heterogeneity and complexity of the TME require a combination of images with various parameters and modalities to characterize tumors and guide therapy. This review focuses on the value of multiparametric magnetic resonance imaging and PET/MR in evaluating the structural and functional changes of HCC and in detecting metabolites formed owing to HCC and the TME.

Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK.

BACKGROUND & AIMS: Mechanisms underlying synergistic liver injury caused by alcohol and obesity are not clear. We have produced a mouse model of synergistic steatohepatitis by recapitulating the natural history of the synergism seen in patients for mechanistic studies. METHODS: Moderate obesity was induced in mice by 170% overnutrition in calories using intragastric overfeeding of high fat diet. Alcohol (low or high dose) was then co-administrated to determine its effects. RESULTS: Moderate obesity plus alcohol intake causes synergistic steatohepatitis in an alcohol dose-dependent manner. A heightened synergism is observed when a high alcohol dose (32g/kg/d) is used, resulting in plasma ALT reaching 392±28U/L, severe steatohepatitis with pericellular fibrosis, marked M1 macrophage activation, a 40-fold induction of iNos, and intensified nitrosative stress in the liver. Hepatic expression of genes for mitochondrial biogenesis and metabolism are significantly downregulated, and hepatic ATP level is decreased. Synergistic ER stress evident by elevated XBP-1, GRP78 and CHOP is accompanied by hyperhomocysteinemia. Despite increased caspase 3/7 cleavage, their activities are decreased in a redox-dependent manner. Neither increased PARP cleavage nor TUNEL positive hepatocytes are found, suggesting a shift of apoptosis to necrosis. Surprisingly, the synergism mice have increased plasma adiponectin and hepatic p-AMPK, but adiponectin resistance is shown downstream of p-AMPK. CONCLUSIONS: Nitrosative stress mediated by M1 macrophage activation, adiponectin resistance, and accentuated ER and mitochondrial stress underlie potential mechanisms for synergistic steatohepatitis caused by moderate obesity and alcohol.

Improved reflexive testing algorithm for hepatitis C infection using signal-to-cutoff ratios of a hepatitis C virus antibody assay.

BACKGROUND: Chemiluminescence immunoassay (CIA) is used to detect hepatitis C virus (HCV) antibody status on the basis of signal-to-cutoff (S/Co) ratios. Positive results of antibody to HCV (anti-HCV) are followed by either recombinant immunoblot assay (RIBA) to confirm anti-HCV positivity or reverse transcription (RT)-PCR to detect viremia. We hypothesized that by analyzing S/Co ratios, we could determine a strategy to reduce unnecessary supplementary testing in our population. METHODS: CIA was performed to screen for anti-HCV, and positive results were followed up with RT-PCR testing. Negative RT-PCR results were followed up with RIBA, whereas positive RT-PCR results were assumed to be RIBA positive. ROC curves were analyzed to determine the optimal S/Co ratios to predict HCV infection. RESULTS: We determined the S/Co ratios on 34 243 veteran patient samples. We found that with the CIA method 9.0% of patients had positive test results for anti-HCV. An S/Co ratio <3.0 ruled out active HCV infection and exposure with 100% negative predictive value. When the S/Co ratio was ≥20.0, positive predictive values were 98.5% compared with RIBA results, and 81.0% compared with RT-PCR results. CONCLUSIONS: RIBA is not necessary to confirm negative or positive CIA anti-HCV if the S/Co ratio is <3.0 or ≥20.0, respectively. To confirm HCV exposure, samples with an S/Co ratio between 3.0 and 19.9 should be followed up with RIBA unless PCR testing has been performed and the result is positive. Samples with an S/Co ratio ≥20.0 or positive RIBA results should be further tested by RT-PCR to determine HCV viremia status.

Differential Kat3 Usage Orchestrates the Integration of Cellular Metabolism with Differentiation.

The integration of cellular status with metabolism is critically important and the coupling of energy production and cellular function is highly evolutionarily conserved. This has been demonstrated in stem cell biology, organismal, cellular and tissue differentiation and in immune cell biology. However, a molecular mechanism delineating how cells coordinate and couple metabolism with transcription as they navigate quiescence, growth, proliferation, differentiation and migration remains in its infancy. The extreme N-termini of the Kat3 coactivator family members, CBP and p300, by far the least homologous regions with only 66% identity, interact with members of the nuclear receptor family, interferon activated Stat1 and transcriptionally competent ß-catenin, a critical component of the Wnt signaling pathway. We now wish to report based on multiomic and functional investigations, utilizing p300 knockdown, N-terminal p300 edited and p300 S89A edited cell lines and p300 S89A knockin mice, that the N-termini of the Kat3 coactivators provide a highly evolutionarily conserved hub to integrate multiple signaling cascades to coordinate cellular metabolism with the regulation of cellular status and function.

p300 Serine 89: A Critical Signaling Integrator and Its Effects on Intestinal Homeostasis and Repair.

Differential usage of Kat3 coactivators, CBP and p300, by ß-catenin is a fundamental regulatory mechanism in stem cell maintenance and initiation of differentiation and repair. Based upon our earlier pharmacologic studies, p300 serine 89 (S89) is critical for controlling differential coactivator usage by ß-catenin via post-translational phosphorylation in stem/progenitor populations, and appears to be a target for a number of kinase cascades. To further investigate mechanisms of signal integration effected by this domain, we generated p300 S89A knock-in mice. We show that S89A mice are extremely sensitive to intestinal insult resulting in colitis, which is known to significantly increase the risk of developing colorectal cancer. We demonstrate cell intrinsic differences, and microbiome compositional differences and differential immune responses, in intestine of S89A versus wild type mice. Genomic and proteomic analyses reveal pathway differences, including lipid metabolism, oxidative stress response, mitochondrial function and oxidative phosphorylation. The diverse effects on fundamental processes including epithelial differentiation, metabolism, immune response and microbiome colonization, all brought about by a single amino acid modification S89A, highlights the critical role of this region in p300 as a signaling nexus and the rationale for conservation of this residue and surrounding region for hundreds of million years of vertebrate evolution.

Targeting the CBP/ß-Catenin Interaction to Suppress Activation of Cancer-Promoting Pancreatic Stellate Cells.

BACKGROUND: Although cyclic AMP-response element binding protein-binding protein (CBP)/ß-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. METHODS: To investigate the role of the CBP/ß-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/ß-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. RESULTS: We report for the first time that CBP/ß-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of "activation" markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/ß-catenin antagonism also suppresses PSC-induced migration of cancer cells. CONCLUSION: CBP/ß-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.

HBV X protein mutations affect HBV transcription and association of histone-modifying enzymes with covalently closed circular DNA.

The hepatitis B X protein (HBx) plays a role in the epigenetic regulation of hepatitis B virus (HBV) replication. This study investigated the effects of HBx mutations on HBV transcription and the recruitment of HBx, histone acetyl-transferase P300 and histone deacetylase 1 (HDAC1) to circularized HBV DNA (which resembles covalently closed circular DNA [cccDNA]). Compared with wild type, majority of mutants had lower levels of intracellular HBV RNA (44-77% reduction) and secretory HBsAg (25-81% reduction), and 12 mutants had a reduction in intracellular encapsidated HBV DNA (33-64% reduction). Eight mutants with >70% reduction in HBV RNA and/or HBsAg were selected for chromatin immunoprecipitation analysis. Four HBx mutants with mutations in amino acid residues 55-60 and 121-126 had a lower degree of HBx-cccDNA association than wild type HBx (mean % input: 0.02-0.64% vs. 3.08% in wild type). A reduced association between cccDNA and P300 (mean % input: 0.69-1.81% vs. 3.48% in wild type) and an augmented association with HDAC1 (mean % input: 4.01-14.0% vs. 1.53% in wild type) were detected. HBx amino acid residues 55-60 and 121-126 may play an important role in HBV transcription regulation, via their impeded interaction with cccDNA and altered recruitment of histone modifying enzymes to cccDNA.

Comprehensive and quantitative proteome profiling of the mouse liver and plasma.

UNLABELLED: We report a comprehensive and quantitative analysis of the mouse liver and plasma proteomes. The method used is based on extensive fractionation of intact proteins, further separation of proteins based on their abundance and size, and high-accuracy mass spectrometry. This analysis reached a depth in proteomic profiling not reported to date for a mammalian tissue or a biological fluid, with 7099 and 4727 proteins identified with high confidence in the liver and in the corresponding plasma, respectively. This method allowed for the identification in both compartments of low-abundance proteins such as cytokines, chemokines, and receptors and for the detection in plasma of proteins in the pg/mL concentration range. This method also allowed for semiquantitation of all identified proteins. The calculated abundance scores correlated with the abundance of the corresponding transcripts for the large majority of the proteins identified in the liver. Finally, comparison of the liver and plasma datasets demonstrated that a significant number of proteins identified in the liver can be detected in plasma. These included proteins involved in complement and coagulation, in fatty acid, purine and pyruvate metabolism, in gluconeogenesis and glycolysis, in protein ubiquitination, and in insulin, interleukin-4, epidermal growth factor, and platelet-derived growth factor signaling. CONCLUSION: This in-depth analysis of the mouse liver and corresponding plasma proteomes provides a strong basis for investigations of liver pathobiology and biology that employ mouse models of hepatic diseases in an effort to better understand, diagnose, treat, and prevent human hepatic diseases.

Convergence of Canonical and Non-Canonical Wnt Signal: Differential Kat3 Coactivator Usage.

BACKGROUND: The ancient and highly evolutionarily conserved Wnt signaling pathway is critical in nearly all tissues and organs for an organism to develop normally from embryo through adult. Wnt signaling is generally parsed into "canonical" or Wnt-ß-catenin-dependent or "non-canonical" ß-catenin-independent signaling. Even though designating Wnt signaling as either canonical or noncanonical allows for easier conceptual discourse about this signaling pathway, in fact canonical and non-canonical Wnt crosstalk regulates complex nonlinear networks. OBJECTIVE: In this perspective, we discuss the integration of canonical and non-canonical Wnt signaling via differential Kat3 (CBP and p300) coactivator usage, thereby regulating and coordinating gene expression programs associated with both proliferation and cellular differentiation and morphogenesis. METHODS: Pharmacologic inhibitors, cell culture, real-time PCR, chromatin immunoprecipitation, protein immunoprecipitation, Western blotting, reporter-luciferase, protein purification, site-directed mutagenesis, in vitro phosphorylation and binding assays, and immunofluorescence were utilized. CONCLUSION: Coordinated integration between both canonical and non-canonical Wnt pathways appears to be crucial not only in the control of fundamental morphologic processes but also in the regulation of normal as well as pathologic events. Such integration between both canonical and non-canonical Wnt signaling is presumably effected via reversible phosphorylation mechanism (e.g., protein kinase C) to regulate differential ß -catenin/Kat3 coactivator usage in order to coordinate proliferation with differentiation and adhesion.

The Mode of Stem Cell Division Is Dependent on the Differential Interaction of ß-Catenin with the Kat3 Coactivators CBP or p300.

Normal long-term repopulating somatic stem cells (SSCs) preferentially divide asymmetrically, with one daughter cell remaining in the niche and the other going on to be a transient amplifying cell required for generating new tissue in homeostatic maintenance and repair processes, whereas cancer stem cells (CSCs) favor symmetric divisions. We have previously proposed that differential ß-catenin modulation of transcriptional activity via selective interaction with either the Kat3 coactivator CBP or its closely related paralog p300, regulates symmetric versus asymmetric division in SSCs and CSCs. We have previously demonstrated that SSCs that divide asymmetrically per force retain one of the dividing daughter cells in the stem cell niche, even when treated with specific CBP/ß-catenin antagonists, whereas CSCs can be removed from their niche via forced stochastic symmetric differentiative divisions. We now demonstrate that loss of p73 in early corticogenesis biases ß-catenin Kat3 coactivator usage and enhances ß-catenin/CBP transcription at the expense of ß-catenin/p300 transcription. Biased ß-catenin coactivator usage has dramatic consequences on the mode of division of neural stem cells (NSCs), but not neurogenic progenitors. The observed increase in symmetric divisions due to enhanced ß-catenin/CBP interaction and transcription leads to an immediate increase in NSC symmetric differentiative divisions. Moreover, we demonstrate for the first time that the complex phenotype caused by the loss of p73 can be rescued in utero by treatment with the small-molecule-specific CBP/ß-catenin antagonist ICG-001. Taken together, our results demonstrate the causal relationship between the choice of ß-catenin Kat3 coactivator and the mode of stem cell division.

Animal Models: Challenges and Opportunities to Determine Optimal Experimental Models of Pancreatitis and Pancreatic Cancer.

At the 2018 PancreasFest meeting, experts participating in basic research met to discuss the plethora of available animal models for studying exocrine pancreatic disease. In particular, the discussion focused on the challenges currently facing the field and potential solutions. That meeting culminated in this review, which describes the advantages and limitations of both common and infrequently used models of exocrine pancreatic disease, namely, pancreatitis and exocrine pancreatic cancer. The objective is to provide a comprehensive description of the available models but also to provide investigators with guidance in the application of these models to investigate both environmental and genetic contributions to exocrine pancreatic disease. The content covers both nongenic and genetically engineered models across multiple species (large and small). Recommendations for choosing the appropriate model as well as how to conduct and present results are provided.

Nuclear receptor/Wnt beta-catenin interactions are regulated via differential CBP/p300 coactivator usage.

Over 400 million years ago, the evolution of vertebrates gave rise to a life cycle in which the organism began to live longer particularly as an adult. To accommodate such a longer lifespan, the organism underwent adaptation, developing a mechanism for long-lived cellular homeostasis. This adaptation required a population of long-lived relatively quiescent somatic stem cells (SSCs) along with a more proliferative differentiated daughter cell population, and was necessary to safeguard the genetic attributes with which SSCs were endowed. Intriguingly, cAMP response element binding protein (CREB)-binding protein (CBP) and E1A-binding protein, 300 kDa (p300), the highly homologous Kat3 coactivators had diverged, through duplication of ancestral Kat3, immediately preceding the evolution of vertebrates, given that both CBP and p300 have been detected in nearly all vertebrates versus non-vertebrates. We now demonstrate that a relatively small, highly evolutionarily conserved, amino terminal 9 amino acid deletion in CBP versus p300, plays a critical role in allowing for both robust maintenance of genomic integrity in stem cells and the initiation of a feed-forward differentiation mechanism by tightly controlling the interaction of the nuclear receptor family with the Wnt signaling cascade in either an antagonistic or synergistic manner.

Differentiation Therapy Targeting the ß-Catenin/CBP Interaction in Pancreatic Cancer.

BACKGROUND: Although canonical Wnt signaling is known to promote tumorigenesis in pancreatic ductal adenocarcinoma (PDAC), a cancer driven principally by mutant K-Ras, the detailed molecular mechanisms by which the Wnt effector ß-catenin regulates such tumorigenesis are largely unknown. We have previously demonstrated that ß-catenin's differential usage of the Kat3 transcriptional coactivator cyclic AMP-response element binding protein-binding protein (CBP) over its highly homologous coactivator p300 increases self-renewal and suppresses differentiation in other types of cancer. AIM/METHODS: To investigate Wnt-mediated carcinogenesis in PDAC, we have used the specific small molecule CBP/ß-catenin antagonist, ICG-001, which our lab identified and has extensively characterized, to examine its effects in human pancreatic cancer cells and in both an orthotopic mouse model and a human patient-derived xenograft (PDX) model of PDAC. RESULTS/CONCLUSION: We report for the first time that K-Ras activation increases the CBP/ß-catenin interaction in pancreatic cancer; and that ICG-001 specific antagonism of the CBP/ß-catenin interaction sensitizes pancreatic cancer cells and tumors to gemcitabine treatment. These effects were associated with increases in the expression of let-7a microRNA; suppression of K-Ras and survivin; and the elimination of drug-resistant cancer stem/tumor-initiating cells.