William S. Twaddell

Comparative Genomic Analysis of Esophageal Adenocarcinoma and Squamous Cell Carcinoma

Esophageal cancer ranks sixth in cancer death. To explore its genetic origins, we conducted exomic sequencing on 11 esophageal adenocarcinomas (EAC) and 12 esophageal squamous cell carcinomas (ESCC) from the United States. Interestingly, inactivating mutations of NOTCH1 were identified in 21% of ESCCs but not in EACs. There was a substantial disparity in the spectrum of mutations, with more indels in ESCCs, A:T>C:G transversions in EACs, and C:G>G:C transversions in ESCCs (P < 0.0001). Notably, NOTCH1 mutations were more frequent in North American ESCCs (11 of 53 cases) than in ESCCs from China (1 of 48 cases). A parallel analysis found that most mutations in EACs were already present in matched Barrett esophagus. These discoveries highlight key genetic differences between EACs and ESCCs and between American and Chinese ESCCs, and suggest that NOTCH1 is a tumor suppressor gene in the esophagus. Finally, we provide a genetic basis for the evolution of EACs from Barrett esophagus.

Targeting abnormal DNA repair in therapy-resistant breast cancers

Although hereditary breast cancers have defects in the DNA damage response that result in genomic instability, DNA repair abnormalities in sporadic breast cancers have not been extensively characterized. Recently, we showed that, relative to nontumorigenic breast epithelial MCF10A cells, estrogen receptor–positive (ER+) MCF7 breast cancer cells and progesterone receptor–positive (PR+) MCF7 breast cancer cells have reduced steady-state levels of DNA ligase IV, a component of the major DNA–protein kinase (PK)-dependent nonhomologous end joining (NHEJ) pathway, whereas the steady-state level of DNA ligase IIIα, a component of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, is increased. Here, we show that tamoxifen- and aromatase-resistant derivatives of MCF7 cells and ER−/PR− cells have even higher steady-state levels of DNA ligase IIIα and increased levels of PARP1, another ALT NHEJ component. This results in increased dependence upon microhomology-mediated ALT NHEJ to repair DNA double-strand breaks (DSB) and the accumulation of chromosomal deletions. Notably, therapy-resistant derivatives of MCF7 cells and ER−/PR− cells exhibited significantly increased sensitivity to a combination of PARP and DNA ligase III inhibitors that increased the number of DSBs. Biopsies from ER−/PR− tumors had elevated levels of ALT NHEJ and reduced levels of DNA–PK-dependent NHEJ factors. Thus, our results show that ALT NHEJ is a novel therapeutic target in breast cancers that are resistant to frontline therapies and suggest that changes in NHEJ protein levels may serve as biomarkers to identify tumors that are candidates for this therapeutic approach. Mol Cancer Res; 10(1); 96–107. ©2011 AACR.

NEU1 Sialidase Expressed in Human Airway Epithelia Regulates Epidermal Growth Factor Receptor (EGFR) and MUC1 Protein Signaling

Background: Airway epithelia express sialoglycoproteins that respond to danger signals and initiate repair programs. Results: NEU1 sialidase desialylates EGFR and MUC1 in airway epithelia to regulate their responsiveness to ligands and adhesiveness to P. aeruginosa. Conclusion: NEU1 provides an additional level of regulation over airway epithelial responsiveness to ligands and pathogens. Significance: The downstream effects of EGFR desialylation require further investigation. Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6–1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7–1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38–56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR- and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli.

NEU1 and NEU3 Sialidase Activity Expressed in Human Lung Microvascular Endothelia: NEU1 RESTRAINS ENDOTHELIAL CELL MIGRATION, WHEREAS NEU3 DOES NOT*

Background: The vascular endothelial surface is highly sialylated. Results: Vascular endothelia express catalytically active NEU1 and NEU3 sialidases, and NEU1 restrains the endothelial migratory response to wounding. Conclusion: NEU1 regulates endothelial remodeling in response to injury. Significance: Learning how NEU1 and NEU3 regulate sialylated molecules on the endothelial surface is key to understanding endothelial receptor-ligand, cell-cell, and host-pathogen interactions. The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.

SlimQuick™ - associated hepatotoxicity in a woman with alpha-1 antitrypsin heterozygosity

Green tea (Camellia sinensis)-associated hepatotoxicity is reported. However, the presence of alpha-1 antitrypsin MZ phenotype as a predisposing factor to green tea-associated drug-induced liver injury (DILI) is unknown. A previously healthy woman with alpha-1 antitrypsin MZ phenotype who took SlimQuick™, an herbal supplement containing green tea extract, developed severe hepatotoxicity requiring corticosteroid treatment. Green tea-associated hepatotoxicity is reviewed and alpha-1 antitrypsin MZ phenotype as a predisposing factor to green tea-associated DILI is discussed. Liver biopsy demonstrated marked inflammation with necrosis suggestive of toxic injury with diffuse alpha-1 antitrypsin globule deposition on immunostaining. Corticosteroid therapy resulted in rapid clinical improvement. Alpha-1 antitrypsin MZ phenotype may increase vulnerability to herbal hepatotoxicity.

M1 Muscarinic Receptors Modify Oxidative Stress Response to Acetaminophen-Induced Acute Liver Injury

The role of muscarinic receptor subtypes in modulating acute liver injury is unknown. We detected M1 muscarinic receptor (M1R) expression in human and murine hepatocytes, and investigated the consequences of M1R deficiency on acute liver injury in vivo and inhibiting M1R activation on hepatocyte injury in vitro. Age-matched wild-type (WT) and M1R-deficient (Chrm1(-/-)) male mice were injected intraperitoneally with 200mg/kg acetaminophen (APAP) and euthanized 0, 2, 4, 16, 24, and 36h later. Biochemical and histological parameters indicated that liver injury peaked within 16h after APAP treatment and resolved by 24h. Compared to WT, M1R-deficient mice had reduced intrahepatic hemorrhage and hepatocyte necrosis, reflected by an attenuated rise in serum alanine aminotransferase levels. Livers of M1R-deficient mice showed reduced hepatocyte DNA fragmentation and attenuated expression of injury cytokines (Il-1α, Il-1β, Il-6, and Fasl). In all mice hepatic glutathione levels decreased after APAP injection, but they recovered more quickly in M1R-deficient mice. During the course of APAP-induced liver injury in M1R-deficient compared to WT mice, hepatic Nrf-2, Gclc, and Nqo1 expressions increased and nitrotyrosine generation decreased. APAP metabolic pathways were not altered by M1R deficiency; expression of hepatic Cyp2e1, Cyp1a2, Cyp3a11, Cyp3a13, Car, and Pxr was similar in Chrm1(-/-) and WT mice. Finally, treatment of murine AML12 hepatocytes with a novel M1R antagonist, VU0255035, attenuated H2O2-induced oxidative stress, prevented GSH depletion, and enhanced viability. We conclude that M1R modify hepatocyte responses to oxidative stress and that targeting M1R has therapeutic potential for toxic liver injury.

Molecular Interactions between MUC1 Epithelial Mucin, β-Catenin, and CagA Proteins

Interleukin (IL)-8-driven neutrophil infiltration of the gastric mucosa is pathognomonic of persistent Helicobacter pylori infection. Our prior study showed that ectopic over-expression of MUC1 in human AGS gastric epithelial cells reduced H. pylori-stimulated IL-8 production compared with cells expressing MUC1 endogenously. Conversely, Muc1 knockout (Muc1−/−) mice displayed an increased level of transcripts encoding the keratinocyte chemoattractant (KC), the murine equivalent of human IL-8, in gastric mucosa compared with Muc1+/+ mice during experimental H. pylori infection. The current study tested the hypothesis that a decreased IL-8 level observed following MUC1 over-expression is mediated through the ability of MUC1 to associate with β-catenin, thereby inhibiting H. pylori-induced β-catenin nuclear translocation. Increased neutrophil infiltration of the gastric mucosa of H. pylori-infected Muc1−/− mice was observed compared with Muc1+/+ wild type littermates, thus defining the functional consequences of increased KC expression in the Muc1-null animals. Protein co-immunoprecipitation (co-IP) studies using lysates of untreated or H. pylori-treated AGS cells demonstrated that (a) MUC1 formed a co-IP complex with β-catenin and CagA, (b) MUC1 over-expression reduced CagA/β-catenin co-IP, and (c) in the absence of MUC1 over-expression, H. pylori infection increased the nuclear level of β-catenin, (d) whereas MUC1 over-expression decreased bacteria-driven β-catenin nuclear localization. These results suggest that manipulation of MUC1 expression in gastric epithelia may be an effective therapeutic strategy to inhibit H. pylori-dependent IL-8 production, neutrophil infiltration, and stomach inflammation.

Effects of modulating M3 muscarinic receptor activity on azoxymethane-induced liver injury in mice

Previously, we reported that azoxymethane (AOM)-induced liver injury is robustly exacerbated in M3 muscarinic receptor (M3R)-deficient mice. We used the same mouse model to test the hypothesis that selective pharmacological modulation of M3R activity regulates the liver injury response. Initial experiments confirmed that giving a selective M3R antagonist, darifenacin, to AOM-treated mice mimicked M3R gene ablation. Compared to vehicle controls, mice treated with the M3R antagonist had reduced survival and increased liver nodularity and fibrosis. We next assessed AOM-induced liver injury in mice treated with a selective M3R agonist, pilocarpine. After pilocarpine treatment, stimulation of post-M3R signaling in the liver was evidenced by ERK and AKT activation. In contrast to the damaging effects of the M3R antagonist, administering pilocarpine to AOM-treated mice significantly attenuated hepatic stellate cell activation, collagen deposition, bile ductule proliferation, and liver fibrosis and nodularity. As anticipated from these findings, livers from pilocarpine-treated mice exhibited reduced expression of key players in fibrosis (α1 collagen, α-smooth muscle actin, TGF-β1, PGDF, TGF-β1R, PGDFR) and decreased mRNA levels for molecules that regulate extracellular matrix formation (TIMP-1, TIMP-2, MMP-2, MMP-13). Cleaved caspase-3, nitrotyrosine and BrdU immunostaining provided evidence that pilocarpine treatment reduced hepatocyte apoptosis and oxidative stress, while increasing hepatocyte proliferation. Collectively, these findings identify several downstream mechanisms whereby M3R activation ameliorates toxic liver injury. These novel observations provide a proof-of-principle that selectively stimulating M3R activation to prevent or diminish liver injury is a therapeutic strategy worthy of further investigation.

Adenomatoid tumor of the testis with intratesticular growth: a case report and review of the literature.

Adenomatoid tumor of the male genitourinary tract is a rare benign neoplasm thought to be of mesothelial origin. In exceptional cases, these lesions may involve the testicular parenchyma, of which there are only 9 published cases in the literature. The authors describe a rare case of a testicular tumor in a 41-year-old male with normal tumor markers. Histopathology and immunohistochemical studies revealed an adenomatoid tumor with intratesticular growth. No involvement of the epididymis or testicular membranes was identified. The morphological clues leading to the correct diagnosis of adenomatoid tumor and the possible histogenesis and differential diagnosis are discussed.

Human airway epithelia express catalytically active NEU3 sialidase

Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.