Asunto(s)

Carcinoma de Células de Merkel/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/antagonistas & inhibidores , Neoplasias Cutáneas/metabolismo , Anciano , Anilidas/uso terapéutico , Biopsia , Carcinoma de Células de Merkel/tratamiento farmacológico , Carcinoma de Células de Merkel/patología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Resultado Fatal , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Piridinas/uso terapéutico , Transducción de Señal , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Tomografía Computarizada por Rayos X

RESUMEN

When using cell lines to study cancer, phenotypic similarity to the original tumor is paramount. Yet, little has been done to characterize how closely Merkel cell carcinoma (MCC) cell lines model native tumors. To determine their similarity to MCC tumor samples, we characterized MCC cell lines via gene expression microarrays. Using whole transcriptome gene expression signatures and a computational bioinformatic approach, we identified significant differences between variant cell lines (UISO, MCC13, and MCC26) and fresh frozen MCC tumors. Conversely, the classic WaGa and Mkl-1 cell lines more closely represented the global transcriptome of MCC tumors. When compared with publicly available cancer lines, WaGa and Mkl-1 cells were similar to other neuroendocrine tumors, but the variant cell lines were not. WaGa and Mkl-1 cells grown as xenografts in mice had histological and immunophenotypical features consistent with MCC, whereas UISO xenograft tumors were atypical for MCC. Spectral karyotyping and short tandem repeat analysis of the UISO cells matched the original cell line's description, ruling out contamination. Our results validate the use of transcriptome analysis to assess the cancer cell line representativeness and indicate that UISO, MCC13, and MCC26 cell lines are not representative of MCC tumors, whereas WaGa and Mkl-1 more closely model MCC.

Asunto(s)

Carcinoma de Células de Merkel/metabolismo , Regulación Neoplásica de la Expresión Génica , Animales , Carcinoma de Células de Merkel/genética , Línea Celular Tumoral , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Cariotipificación , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos NOD , Repeticiones de Microsatélite , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Transcriptoma

RESUMEN

Certain kindreds with low-penetrant (lp) retinoblastoma carry mutant alleles which retain partial tumor suppressor activity and we previously showed that these alleles exhibit defective, temperature-sensitive binding in yeast. To investigate the molecular basis for incomplete penetrance, we studied three recurrent lp alleles and observed approximately 50% of wildtype activity measured by (i) phosphorylation at key regulatory sites, S780, S795, S807/S811, (ii) transcriptional co-activation, and (iii) 'flat-cell' differentiation in mammalian cells in vivo. In addition, we studied a small-cell carcinoma that is homozygous for the R661W allele providing the first analysis of the effect of a naturally occurring lp allele in a human tumor. While we detected abundant expression of the R661W protein, we noted marked instability of both endogenous and recombinant R661W following treatment in vivo with the Hsp90 inhibitor, geldanamycin and stabilization of R661W following heat shock. In addition, we observed a discordant phenotype in the tumor cells with induction of p16 and loss of cyclin D1 consistent with a null RB status combined with homozygous expression of mutant ras which had not been reported previously for RB (-) small-cell cancer. These findings show that a recurrent missense lp allele retains greater functional activity in vivo than predicted from earlier in vitro assays, proposing a role for stabilizing chaperone-like activity in vivo. In addition, these data suggest that reversible protein instability and the requirement for a cooperating mutation may provide a stochastic explanation for the molecular basis of incomplete penetrance in kindreds carrying these alleles.

Asunto(s)

Benzoquinonas/farmacología , Carcinoma de Células Pequeñas/genética , Genes de Retinoblastoma , Genes ras , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/genética , Proteína de Retinoblastoma/metabolismo , Alelos , Antibióticos Antineoplásicos/uso terapéutico , Secuencia de Bases , Benzoquinonas/uso terapéutico , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/metabolismo , Dominio Catalítico , Células Cultivadas , Ciclina D , Quinasa 4 Dependiente de la Ciclina/metabolismo , Ciclinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Lactamas Macrocíclicas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Proteínas Mutantes/efectos de los fármacos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Penetrancia , Fosforilación , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Proteína de Retinoblastoma/efectos de los fármacos

RESUMEN

Malignant salivary gland tumors can arise from a t(11;19) translocation that fuses 42 residues from Mect1/Torc1, a cyclic AMP (cAMP)/cAMP-responsive element binding protein (CREB)-dependent transcriptional coactivator, with 982 residues from Maml2, a NOTCH receptor coactivator. To determine if the Mect1-Maml2 fusion oncogene mediates tumorigenicity by disrupting cAMP/CREB signaling, we have generated in-frame deletions within the CREB-binding domain of Mect1/Torc1 for testing transformation activity and have also developed a doxycycline-regulated Mect1-Maml2 mammalian expression vector for global gene expression profiling. We observed that small deletions within the CREB-binding domain completely abolished transforming activity in RK3E epithelial cells. Further, we have shown that the ectopic induction of Mect1-Maml2 in HeLa cells strongly activated the expression of a group of known cAMP/CREB-regulated genes. In addition, we detected candidate cAMP-responsive element sites within 100 nucleotides of the transcriptional start sites of other genes activated by Mect1-Maml2 expression. In contrast, we did not observe alterations of known Notch-regulated target genes in these expression array profile experiments. We validated the results by reverse transcription-PCR in transfected HeLa, RK3E, and H2009 lung tumor cells and in mucoepidermoid cancer cells that endogenously express the fusion oncopeptide. Whereas overexpression of components of the cAMP pathway has been associated with a subset of human carcinomas, these data provide a direct genetic link between deregulation of cAMP/CREB pathways and epithelial tumorigenesis and suggest future therapeutic strategies for this group of salivary gland tumors.

Asunto(s)

AMP Cíclico/genética , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción/genética , Carcinoma Mucoepidermoide/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Doxiciclina/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Estructura Terciaria de Proteína , Neoplasias de las Glándulas Salivales/genética , Transfección

RESUMEN

Truncation of Notch1 has been shown to cause a subtype of acute leukemia, and activation of Notch4 has been associated with mammary and salivary gland carcinomas of mice. Here we identify a new mechanism for disrupting Notch signaling in human tumorigenesis, characterized by altered function of a new ortholog of the Drosophila melanogaster Notch co-activator molecule Mastermind. We cloned the t(11;19) translocation that underlies the most common type of human malignant salivary gland tumor. This rearrangement fuses exon 1 from a novel gene of unknown function at 19p13, termed mucoepidermoid carcinoma translocated 1 (MECT1), with exons 2-5 of a novel member of the Mastermind-like gene family (MAML2) at 11q21 (ref. 3). Similar to D. melanogaster Mastermind and MAML1 (refs. 4,5), full-length MAML2 functioned as a CSL (CBF-1, suppressor of hairless and Lag-1)-dependent transcriptional co-activator for ligand-stimulated Notch. In contrast, MECT1-MAML2 activated transcription of the Notch target gene HES1 independently of both Notch ligand and CSL binding sites. MECT1-MAML2 induced foci formation in RK3E epithelial cells, confirming a biological effect for the fusion product. These data suggest a new mechanism to disrupt the function of a Notch co-activator in a common type of malignant salivary gland tumor.

Asunto(s)

Fusión Artificial Génica , Carcinoma Mucoepidermoide/genética , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 19/genética , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Translocación Genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Carcinoma Mucoepidermoide/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Regulación de la Expresión Génica , Reordenamiento Génico , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hibridación Fluorescente in Situ , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-2 , Cariotipificación , Ligandos , Luciferasas/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/metabolismo , Regiones Promotoras Genéticas , Receptores Notch , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ribonucleasa Pancreática/metabolismo , Neoplasias de las Glándulas Salivales/genética , Neoplasias de las Glándulas Salivales/metabolismo , Transducción de Señal , Transactivadores , Factor de Transcripción HES-1 , Factores de Transcripción , Transcripción Genética , Activación Transcripcional , Transfección , Células Tumorales Cultivadas