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1.
Oncogene ; 33(24): 3151-60, 2014 Jun 12.
Article in English | MEDLINE | ID: mdl-23851502

ABSTRACT

Mesothelioma is diagnosed in ∼2500 patients in the United States every year, most often arising in the pleural space, but also occurring as primary peritoneal mesothelioma. The vast majority of patients with mesothelioma die of their disease within 3 years. We developed a new mouse model of mesothelioma by bladder or intraperitoneal injection of adenovirus Cre into mice with conditional alleles of each of Tp53 and Tsc1. Such mice began to develop malignant ascites about 6 months after injection, which was due to peritoneal mesothelioma, on the basis of tumor morphology and immunohistochemical staining. Mesothelioma cell lines were established, which showed loss of both Tsc1 and Tp53, with mammalian target of rapamycin complex (mTORC)1 activation. Treatment of mice with malignant ascites due to mesothelioma with rapamycin led to a marked reduction in ascites, extended survival and a 95-99% reduction in the mesothelioma tumor volume, in comparison with vehicle-treated mice. To see whether TSC1/TSC2 loss was a common genetic event in human mesothelioma, we examined nine human mesothelioma cell lines, and found that four of nine showed persistent activation of mTORC1, although none had loss of TSC1 or TSC2. A tissue microarray analysis of 198 human mesothelioma specimens showed that 33% of cases had reduced TSC2 expression and 60% showed activation of mTOR, indicating that mTOR activation is common in human mesothelioma, suggesting that it is a potential therapeutic target.


Subject(s)
Mesothelioma/pathology , Peritoneal Neoplasms/pathology , Tumor Suppressor Protein p53/physiology , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/physiology , Animals , Blotting, Western , Humans , Immunoenzyme Techniques , Male , Mesothelioma/genetics , Mesothelioma/mortality , Mice , Mice, Knockout , Peritoneal Neoplasms/genetics , Peritoneal Neoplasms/mortality , Survival Rate , Tissue Array Analysis , Tuberous Sclerosis Complex 1 Protein , Tumor Cells, Cultured
2.
Oncogene ; 32(13): 1660-9, 2013 Mar 28.
Article in English | MEDLINE | ID: mdl-22710717

ABSTRACT

Ubiquitination of epidermal growth factor receptor (EGFR) is required for downregulation of the receptor by endocytosis. Impairment of this pathway results in constitutively active EGFR, which is associated with carcinogenesis, particularly in lung cancer. We previously demonstrated that the deubiquitinating enzyme ubiquitin-specific protease 2a (USP2a) has oncogenic properties. Here, we show a new role for USP2a as a regulator of EGFR endocytosis. USP2a localizes to early endosomes and associates with EGFR, stabilizing the receptor, which retains active downstream signaling. HeLa cells transiently expressing catalytically active, but not mutant (MUT), USP2a show increased plasma membrane-localized EGFR, as well as decreased internalized and ubiquitinated EGFR. Conversely, USP2a silencing reverses this phenotype. Importantly, USP2a prevents the degradation of MUT in addition to wild-type EGFR. Finally, we observed that USP2a and EGFR proteins are coordinately overexpressed in non-small cell lung cancers. Taken together, our data indicate that USP2a antagonizes EGFR endocytosis and thus amplifies signaling activity from the receptor. Our findings suggest that regulation of deubiquitination could be exploited therapeutically in cancers overexpressing EGFR.


Subject(s)
Endocytosis/physiology , Endopeptidases/physiology , ErbB Receptors/metabolism , Proteolysis , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Down-Regulation/genetics , Endocytosis/genetics , Endopeptidases/genetics , Endopeptidases/metabolism , ErbB Receptors/genetics , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Protein Stability , Ubiquitin Thiolesterase , Ubiquitin-Specific Proteases , Ubiquitination/genetics
3.
Ann Oncol ; 21(4): 884-894, 2010 Apr.
Article in English | MEDLINE | ID: mdl-19825886

ABSTRACT

BACKGROUND: AZD5438 is an orally bioavailable inhibitor of cyclin E-cdk2, cyclin A-cdk2 and cyclin B-cdk1 complexes. Three phase I studies assessed the clinical safety, tolerability, pharmacokinetics and pharmacodynamics of AZD5438 when administered in different dosing schedules. PATIENTS AND METHODS: AZD5438 was administered four times daily, once every 7 days (study 1), for 14 consecutive days followed by 7 days of rest (study 2), or continuously (study 3), to patients with advanced solid tumours. Dose escalation proceeded until the emergence of dose-limiting toxic effects. RESULTS: Sixty-four patients were included across the three studies (19, 17 and 28, respectively). Nausea and vomiting were the most common adverse events. When dosed continuously, 40 mg four times daily was considered intolerable, and due to safety issues, all studies were terminated prematurely. Consequently, no intolerable dose was identified during the weekly schedule. Pharmacokinetics demonstrated dose-proportional exposure, high interpatient variability and accumulation after multiple doses. Skin biopsies indicated reduced retinoblastoma protein phosphorylation at cdk2 phospho-sites; other pharmacodynamic assessments did not reveal consistent trends. CONCLUSIONS: AZD5438 was generally well tolerated in a weekly dosing schedule, but not in continuous schedules. The clinical development programme for AZD5438 was discontinued owing to tolerability and exposure data from these studies.


Subject(s)
Imidazoles/administration & dosage , Imidazoles/adverse effects , Imidazoles/pharmacokinetics , Neoplasms/drug therapy , Pyrimidines/administration & dosage , Pyrimidines/adverse effects , Pyrimidines/pharmacokinetics , Administration, Oral , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Biomarkers, Pharmacological/analysis , Cohort Studies , Cyclin-Dependent Kinases/antagonists & inhibitors , Disease Progression , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Humans , Male , Neoplasms/pathology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/pharmacokinetics , Treatment Outcome
4.
Oncogene ; 27(34): 4702-11, 2008 Aug 07.
Article in English | MEDLINE | ID: mdl-18408761

ABSTRACT

Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/drug therapy , Quinazolines/therapeutic use , Receptor, ErbB-2/antagonists & inhibitors , Afatinib , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/pathology , Cell Survival/drug effects , Disease Models, Animal , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Humans , Lung Neoplasms/pathology , Mice , Mice, Nude , Mice, Transgenic , NIH 3T3 Cells , Phosphorylation/drug effects , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Treatment Outcome , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
5.
Biotech Histochem ; 82(4-5): 189-97, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17917854

ABSTRACT

Knowledge of the exact cell content of frozen tissue samples is of growing importance in genomic research. We developed a microaliquoting technique to measure and optimize the cell composition of frozen tumor specimens for molecular studies. Frozen samples of 31 mesothelioma cases were cut in alternating thin and thick sections. Thin sections were stained and evaluated visually. Thick sections, i.e., microaliquots, were annotated using bordering stained sections. A range of cellular heterogeneity was observed among and within samples. Precise annotation of samples was obtained by integration and compared to conventional single face and "front and back"' section estimates of cell content. Front and back estimates were more highly correlated with block annotation by microaliquoting than were single face estimates. Both methods yielded discrepant estimates, however, and for some studies may not adequately account for the heterogeneity of mesothelioma or other malignancies with variable cellular composition. High yield and quality RNA was extracted from precision annotated, tumor-enriched subsamples prepared by combining individual microaliquots with the highest tumor cellularity estimates. Microaliquoting provides accurate cell content annotation and permits genomic analysis of enriched subpopulations of cells without fixation or amplification.


Subject(s)
Frozen Sections , Tissue Fixation , Adult , Aged , Aged, 80 and over , Female , Frozen Sections/methods , Humans , Male , Mesothelioma , Middle Aged , Pathology, Molecular , RNA/analysis , Tissue Fixation/methods
6.
Am J Physiol Endocrinol Metab ; 279(5): E1003-11, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11052954

ABSTRACT

The current study assessed in vivo the effect of insulin on triglyceride-rich lipoprotein (TRL) production by rat liver. Hepatic triglyceride and apolipoprotein B (apoB) production were measured in anesthetized, fasted rats injected intravenously with Triton WR-1339 (400 mg/kg). After intravascular catabolism was blocked by detergent treatment, glucose (500 mg/kg) was injected to elicit insulin secretion, and serum triglyceride and apoB accumulation were monitored over the next 3 h. In glucose-injected rats, triglyceride secretion averaged 22.5 +/- 2.1 microg.ml(-1).min(-1), which was significantly less by 30% than that observed in saline-injected rats, which averaged 32.1 +/- 1.4 microg.ml(-1).min(-1). ApoB secretion was also significantly reduced by 66% in glucose-injected rats. ApoB immunoblotting indicated that both B100 and B48 production were significantly reduced after glucose injection. Results support the conclusion that insulin acts in vivo to suppress hepatic very low density lipoprotein (VLDL) triglyceride and apoB secretion and strengthen the concept of a regulatory role for insulin in VLDL metabolism postprandially.


Subject(s)
Apolipoproteins B/biosynthesis , Glucose/pharmacology , Insulin/metabolism , Lipoproteins/biosynthesis , Liver/metabolism , Triglycerides/biosynthesis , Animals , Apolipoproteins B/metabolism , Blood Glucose/metabolism , Cells, Cultured , Dexamethasone/pharmacology , Fasting , Glucagon/pharmacology , Glucocorticoids/pharmacology , Glucose Clamp Technique , Insulin/pharmacology , Insulin Secretion , Kinetics , Lipoproteins/blood , Liver/drug effects , Male , Oleic Acid/pharmacology , Rats , Rats, Sprague-Dawley , Triglycerides/blood
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