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BACKGROUND: USP18 (ubiquitin-specific protease 18) removes ubiquitin-like modifier interferon stimulated gene 15 (ISG15) from conjugated proteins. USP18 null mice in a FVB/N background develop tumors as early as 2 months of age. These tumors are leiomyosarcomas and thus represent a new murine model for this disease. METHODS: Heterozygous USP18 +/- FVB/N mice were bred to generate wild-type, heterozygous and homozygous cohorts. Tumors were characterized immunohistochemically and two cell lines were derived from independent tumors. Cell lines were karyotyped and their responses to restoration of USP18 activity assessed. Drug testing and tumorigenic assays were also performed. USP18 immunohistochemical staining in a large series of human leiomyosacomas was examined. RESULTS: USP18 -/- FVB/N mice spontaneously develop tumors predominantly on the back of the neck with most tumors evident between 6-12 months (80 % penetrance). Immunohistochemical characterization of the tumors confirmed they were leiomyosarcomas, which originate from smooth muscle. Restoration of USP18 activity in sarcoma-derived cell lines did not reduce anchorage dependent or independent growth or xenograft tumor formation demonstrating that these cells no longer require USP18 suppression for tumorigenesis. Karyotyping revealed that both tumor-derived cell lines were aneuploid with extra copies of chromosomes 3 and 15. Chromosome 15 contains the Myc locus and MYC is also amplified in human leiomyosarcomas. MYC protein levels were elevated in both murine leiomyosarcoma cell lines. Stabilized P53 protein was detected in a subset of these murine tumors, another feature of human leiomyosarcomas. Immunohistochemical analyses of USP18 in human leiomyosarcomas revealed a range of staining intensities with the highest USP18 expression in normal vascular smooth muscle. USP18 tissue array analysis of primary leiomyosarcomas from 89 patients with a clinical database revealed cases with reduced USP18 levels had a significantly decreased time to metastasis (P = 0.0441). CONCLUSIONS: USP18 null mice develop leiomyosarcoma recapitulating key features of clinical leiomyosarcomas and patients with reduced-USP18 tumor levels have an unfavorable outcome. USP18 null mice and the derived cell lines represent clinically-relevant models of leiomyosarcoma and can provide insights into both leiomyosarcoma biology and therapy.
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Carcinogénesis/genética , Leiomiosarcoma/genética , Ubiquitina Tiolesterasa/genética , Neoplasias Uterinas/genética , Animales , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leiomiosarcoma/patología , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Proteína p53 Supresora de Tumor/genética , Ubiquitina Tiolesterasa/biosíntesis , Neoplasias Uterinas/patologíaRESUMEN
BACKGROUND: Essential tremor (ET) is one of the most common movement disorders worldwide. In medically refractory ET, deep brain stimulation (DBS) of the ventral intermediate nucleus of the thalamus is the current standard of care. However, DBS carries an inherent 2% to 3% risk of hemorrhage, a risk that can be much higher in patients with concomitant coagulopathy. Magnetic resonance imaging-guided focused ultrasound (MRgFUS) thalamotomy is a surgical alternative that is highly effective in treating ET, with no reports of intracranial hemorrhage to date. OBSERVATIONS: This is the first documented case of successful MRgFUS thalamotomy in a patient with von Willebrand disease (VWD). A 60-year-old left-handed male had medically refractory ET, VWD type 2B, and a family history of clinically significant hemorrhage after DBS. He underwent right-sided MRgFUS thalamotomy and received a perioperative course of VONVENDI (recombinant von Willebrand factor) to ensure appropriate hemostasis. Postprocedure imaging confirmed a focal lesion in the right thalamus without evidence of hemorrhage. The patient reported 90% improvement of his left-hand tremor and significant improvement in his quality of life without obvious side effects. LESSONS: MRgFUS thalamotomy with peri- and postoperative hematological management is a promising alternative to DBS for patients with underlying coagulopathies.
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KRAS is frequently mutated in lung cancers and is associated with aggressive biology and chemotherapy resistance. Therefore, innovative approaches are needed to treat these lung cancers. Prior work implicated the IFN-stimulated gene 15 (ISG15) deubiquitinase (DUB) USP18 as having antineoplastic activity by regulating lung cancer growth and oncoprotein stability. This study demonstrates that USP18 affects the stability of the KRAS oncoprotein. Interestingly, loss of USP18 reduced KRAS expression, and engineered gain of USP18 expression increased KRAS protein levels in lung cancer cells. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability. Intriguingly, loss of USP18 altered KRAS subcellular localization by mislocalizing KRAS from the plasma membrane. To explore the biologic consequences, immunohistochemical (IHC) expression profiles of USP18 were compared in lung cancers of KrasLA2/+ versus cyclin E engineered mouse models. USP18 expression was higher in Kras-driven murine lung cancers, indicating a link between KRAS and USP18 expression in vivo To solidify this association, loss of Usp18 in KrasLA2/+ /Usp18-/- mice was found to significantly reduce lung cancers as compared with parental KrasLA2/+ mice. Finally, translational relevance was confirmed in a human lung cancer panel by showing that USP18 IHC expression was significantly higher in KRAS-mutant versus wild-type lung adenocarcinomas.Implications: Taken together, this study highlights a new way to combat the oncogenic consequences of activated KRAS in lung cancer by inhibiting the DUB USP18. Mol Cancer Res; 15(7); 905-14. ©2017 AACR.
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Adenocarcinoma/genética , Endopeptidasas/genética , Neoplasias Pulmonares/genética , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Membrana Celular/genética , Ciclina E/genética , Cicloheximida/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Ratones Noqueados , Mutación , Ubiquitina TiolesterasaRESUMEN
Histone deacetylase inhibitor (HDACi; vorinostat) responses were studied in murine and human lung cancer cell lines and genetically engineered mouse lung cancer models. Findings were compared with a window of opportunity trial in aerodigestive tract cancers. In human (HOP62, H522, and H23) and murine transgenic (ED-1, ED-2, LKR-13, and 393P, driven, respectively, by cyclin E, degradation-resistant cyclin E, KRAS, or KRAS/p53) lung cancer cell lines, vorinostat reduced growth, cyclin D1, and cyclin E levels, but induced p27, histone acetylation, and apoptosis. Other biomarkers also changed. Findings from transgenic murine lung cancer models were integrated with those from a window of opportunity trial that measured vorinostat pharmacodynamic responses in pre- versus posttreatment tumor biopsies. Vorinostat repressed cyclin D1 and cyclin E expression in murine transgenic lung cancers and significantly reduced lung cancers in syngeneic mice. Vorinostat also reduced cyclin D1 and cyclin E expression, but increased p27 levels in post- versus pretreatment human lung cancer biopsies. Notably, necrotic and inflammatory responses appeared in posttreatment biopsies. These depended on intratumoral HDACi levels. Therefore, HDACi treatments of murine genetically engineered lung cancer models exert similar responses (growth inhibition and changes in gene expression) as observed in lung cancer cell lines. Moreover, enhanced pharmacodynamic responses occurred in the window of opportunity trial, providing additional markers of response that can be evaluated in subsequent HDACi trials. Thus, combining murine and human HDACi trials is a strategy to translate preclinical HDACi treatment outcomes into the clinic. This study uncovered clinically tractable mechanisms to engage in future HDACi trials.
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Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias Pulmonares/genética , Anciano , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Apoptosis/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Inhibidores de Histona Desacetilasas/administración & dosificación , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ácidos Hidroxámicos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Estadificación de Neoplasias , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , VorinostatRESUMEN
New pharmacologic targets are needed for lung cancer. One candidate pathway to target is composed of the E1-like ubiquitin-activating enzyme (UBE1L) that associates with interferon-stimulated gene 15 (ISG15), which complexes with and destabilizes cyclin D1. Ubiquitin protease 43 (UBP43/USP18) removes ISG15 from conjugated proteins. This study reports that gain of UBP43 stabilized cyclin D1, but not other D-type cyclins or cyclin E. This depended on UBP43 enzymatic activity; an enzymatically inactive UBP43 did not affect cyclin D1 stability. As expected, small interfering RNAs that reduced UBP43 expression also decreased cyclin D1 levels and increased apoptosis in a panel of lung cancer cell lines. Forced cyclin D1 expression rescued UBP43 apoptotic effects, which highlighted the importance of cyclin D1 in conferring this. Short hairpin RNA-mediated reduction of UBP43 significantly increased apoptosis and reduced murine lung cancer growth in vitro and in vivo after transplantation of these cells into syngeneic mice. These cells also exhibited increased response to all-trans-retinoic acid, interferon, or cisplatin treatments. Notably, gain of UBP43 expression antagonized these effects. Normal-malignant human lung tissue arrays were examined independently for UBP43, cyclin D1, and cyclin E immunohistochemical expression. UBP43 was significantly (P < 0.01) increased in the malignant versus normal lung. A direct relationship was found between UBP43 and cyclin D1 (but not cyclin E) expression. Differential UBP43 expression was independently detected in a normal-malignant tissue array with diverse human cancers. Taken together, these findings uncovered UBP43 as a previously unrecognized antineoplastic target.
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Antineoplásicos/farmacología , Cisplatino/farmacología , Endopeptidasas/metabolismo , Neoplasias Pulmonares/enzimología , Terapia Molecular Dirigida , Sustitución de Aminoácidos , Animales , Línea Celular Tumoral , Ciclina D/genética , Ciclina D/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Citocinas/metabolismo , Endopeptidasas/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Interferones/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Mutagénesis Sitio-Dirigida , Trasplante de Neoplasias , Estabilidad Proteica , Interferencia de ARN , Análisis de Matrices Tisulares , Tretinoina/farmacología , Ubiquitina Tiolesterasa , Ubiquitinas/metabolismoRESUMEN
Hedgehog (HH) pathway Smoothened (Smo) inhibitors are active against Gorlin syndrome-associated basal cell carcinoma (BCC) and medulloblastoma where Patched (Ptch) mutations occur. We interrogated 705 epithelial cancer cell lines for growth response to the Smo inhibitor cyclopamine and for expressed HH pathway-regulated species in a linked genetic database. Ptch and Smo mutations that respectively conferred Smo inhibitor response or resistance were undetected. Previous studies revealed HH pathway activation in lung cancers. Therefore, findings were validated using lung cancer cell lines, transgenic and transplantable murine lung cancer models, and human normal-malignant lung tissue arrays in addition to testing other Smo inhibitors. Cyclopamine sensitivity most significantly correlated with high cyclin E (P=0.000009) and low insulin-like growth factor binding protein 6 (IGFBP6) (P=0.000004) levels. Gli family members were associated with response. Cyclopamine resistance occurred with high GILZ (P=0.002) expression. Newer Smo inhibitors exhibited a pattern of sensitivity similar to cyclopamine. Gain of cyclin E or loss of IGFBP6 in lung cancer cells significantly increased Smo inhibitor response. Cyclin E-driven transgenic lung cancers expressed a gene profile implicating HH pathway activation. Cyclopamine treatment significantly reduced proliferation of murine and human lung cancers. Smo inhibition reduced lung cancer formation in a syngeneic mouse model. In human normal-malignant lung tissue arrays cyclin E, IGFBP6, Gli1 and GILZ were each differentially expressed. Together, these findings indicate that Smo inhibitors should be considered in cancers beyond those with activating HH pathway mutations. This includes tumors that express genes indicating basal HH pathway activation.
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Antineoplásicos/farmacología , Carcinoma/genética , Mutación , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G/genética , Alcaloides de Veratrum/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina E/genética , Ciclina E/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog , Humanos , Ratones , Receptores Patched , Receptor Patched-1 , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Receptor SmoothenedRESUMEN
More effective treatments for acute promyelocytic leukemia (APL) are needed. APL cell treatment with all-trans-retinoic acid (RA) degrades the chimeric, dominant-negative-acting transcription factor promyelocytic leukemia gene (PML)/RARα, which is generated in APL by chromosomal translocation. The E1-like ubiquitin-activating enzyme (UBE1L) associates with interferon-stimulated gene ISG15 that binds and represses PML/RARα protein. Ubiquitin protease UBP43/USP18 removes ISG15 from conjugated proteins. In this study, we explored how RA regulates UBP43 expression and the effects of UBP43 on PML/RARα stability and APL growth, apoptosis, or differentiation. RA treatment induced UBE1L, ISG15, and UBP43 expression in RA-sensitive but not RA-resistant APL cells. Similar in vivo findings were obtained in a transgenic mouse model of transplantable APL, and in the RA response of leukemic cells harvested directly from APL patients. UBP43 knockdown repressed PML/RARα protein levels and inhibited RA-sensitive or RA-resistant cell growth by destabilizing the PML domain of PML/RARα. This inhibitory effect promoted apoptosis but did not affect the RA differentiation response in these APL cells. In contrast, elevation of UBP43 expression stabilized PML/RARα protein and inhibited apoptosis. Taken together, our findings define the ubiquitin protease UBP43 as a novel candidate drug target for APL treatment.
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Endopeptidasas/metabolismo , Leucemia Promielocítica Aguda/tratamiento farmacológico , Proteínas de Fusión Oncogénica/metabolismo , Tretinoina/farmacología , Animales , Apoptosis/efectos de los fármacos , Células COS , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Endopeptidasas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Ratones , Proteínas de Fusión Oncogénica/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Tumoral/efectos de los fármacos , Ubiquitina Tiolesterasa , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Spiroiminodihydantoin (Sp) is a hyperoxidized guanine base produced from oxidation of the mutagenic DNA lesion 7,8-dihydro-8-oxo-2'-deoxguanosine (8-oxoG) by a variety of species including peroxynitrite, singlet oxygen, and the high-valent metals Ir(IV) and Cr(V). In this study, the conformation and thermodynamic stability of a 15-mer DNA duplex containing an Sp lesion are examined using spectroscopic techniques and differential scanning calorimetry (DSC). The Sp lesion does not alter the global B-form conformation of the DNA duplex as determined by circular dichroism spectroscopy. Thermal denaturation experiments find that Sp significantly lowers the thermal stability of the duplex by approximately 20 degrees C. The enthalpies, entropies, and free energies of duplex formation for 15-mers containing guanine, 8-oxoG, and Sp were determined by performing DSC experiments as well as van't Hoff analysis of UV melting spectroscopic data. The thermodynamic stability of the Sp duplex is significantly reduced compared to that of both the 8-oxoG and parent G duplexes, with the thermodynamic destabilization being enthalpic in origin. The thermodynamic impact of the Sp lesion is compared to what is found for other types of DNA base damage and discussed in relation to how the presence of this lesion could affect cellular processes, in particular the recognition and repair of these adducts by the base excision repair enzymes.