RESUMEN
Missense mutations in TP53 comprise >75% of all p53 alterations in cancer, resulting in highly stabilized mutant p53 proteins that not only lose their tumor-suppressor activity, but often acquire oncogenic gain-of-functions (GOFs). GOF manifests itself in accelerated tumor onset, increased metastasis, increased drug resistance and shortened survival in patients and mice. A known prerequisite for GOF is mutant p53 protein stabilization, which itself is linked to aberrant protein conformation. However, additional determinants for mutant p53 stabilization likely exist. Here we show that in initially heterozygous mouse tumors carrying the hotspot GOF allele R248Q (p53Q/+), another necessary prerequisite for mutant p53 stabilization and GOF in vivo is loss of the remaining wild-type p53 allele, termed loss-of-heterozygosity (LOH). Thus, in mouse tumors with high frequency of p53 LOH (osteosarcomas and fibrosarcomas), we find that mutant p53 protein is stabilized (16/17 cases, 94%) and tumor onset is significantly accelerated compared with p53+/- tumors (GOF). In contrast, in mouse tumors with low frequency of p53 LOH (MMTV-Neu breast carcinomas), mutant p53 protein is not stabilized (16/20 cases, 80%) and GOF is not observed. Of note, human genomic databases (TCGA, METABRIC etc.) show a high degree of p53 LOH in all examined tumor types that carry missense p53 mutations, including sarcomas and breast carcinomas (with and without HER2 amplification). These data - while cautioning that not all genetic mouse models faithfully represent the human situation - demonstrate for the first time that p53 LOH is a critical prerequisite for missense mutant p53 stabilization and GOF in vivo.
Asunto(s)
Neoplasias de la Mama/genética , Pérdida de Heterocigocidad/genética , Proteína p53 Supresora de Tumor/genética , Alelos , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Mutación Missense/genéticaRESUMEN
Mutations in the p53 tumor suppressor gene are the most prevalent genetic events in human Her2-positive breast cancer and are associated with poor prognosis and survival. Human clinical data and our in vitro and in vivo studies strongly suggest potent oncogenic cooperation between mutant p53 and Her2 (ErbB2). Yet, the translational significance of mutant p53 in Her2 positive breast cancer, especially with respect to Her2-targeted therapies, has not been evaluated. Our previous work identified novel oncogenic activity of mutant p53 whereby mutp53 amplifies ErbB2 signaling via the mutp53-HSF1-ErbB2 feed-forward loop. Here we report that pharmacological interception of this circuit by ErbB2 inhibitor lapatinib downregulates mutant p53 in vitro and in vivo. We found that ErbB2 inhibition by lapatinib inhibits transcription factor HSF1, and its target Hsp90, followed by mutant p53 degradation in MDM2 dependent manner. Thus, our data suggest that mutant p53 sensitizes cancer cells to lapatinib via two complementary mechanisms: mutant p53 mediated amplification of ErbB2 signaling, and simultaneous annihilation of both potent oncogenic drivers, ErbB2 and mutant p53. Hence, our study could provide valuable information for the optimization of therapeutic protocols to achieve superior clinical effects in the treatment of Her2 positive breast cancer.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinazolinas/administración & dosificación , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lapatinib , Ratones , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis , Quinazolinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The main tumor suppressor function of p53 as a "guardian of the genome" is to respond to cellular stress by transcriptional activation of apoptosis, growth arrest, or senescence in damaged cells. Not surprisingly, mutations in the p53 gene are the most frequent genetic alteration in human cancers. Importantly, mutant p53 (mutp53) proteins not only lose their wild-type tumor suppressor activity but also can actively promote tumor development. Two main mechanisms accounting for mutp53 proto-oncogenic activity are inhibition of the wild-type p53 in a dominant-negative fashion and gain of additional oncogenic activities known as gain-of-function (GOF). Here, we discuss a novel mechanism of mutp53 GOF, which relies on its oncogenic cooperation with the heat shock machinery. This coordinated adaptive mechanism renders cancer cells more resistant to proteotoxic stress and provides both, a strong survival advantage to cancer cells and a promising means for therapeutic intervention.
RESUMEN
Intracellular macrophage migration inhibitory factor (MIF) often becomes stabilized in human cancer cells. MIF can promote tumor cell survival, and elevated MIF protein correlates with tumor aggressiveness and poor prognosis. However, the molecular mechanism facilitating MIF stabilization in tumors is not understood. We show that the tumor-activated HSP90 chaperone complex protects MIF from degradation. Pharmacological inhibition of HSP90 activity, or siRNA-mediated knockdown of HSP90 or HDAC6, destabilizes MIF in a variety of human cancer cells. The HSP90-associated E3 ubiquitin ligase CHIP mediates the ensuing proteasome-dependent MIF degradation. Cancer cells contain constitutive endogenous MIF-HSP90 complexes. siRNA-mediated MIF knockdown inhibits proliferation and triggers apoptosis of cultured human cancer cells, whereas HSP90 inhibitor-induced apoptosis is overridden by ectopic MIF expression. In the ErbB2 transgenic model of human HER2-positive breast cancer, genetic ablation of MIF delays tumor progression and prolongs overall survival of mice. Systemic treatment with the HSP90 inhibitor 17AAG reduces MIF expression and blocks growth of MIF-expressing, but not MIF-deficient, tumors. Together, these findings identify MIF as a novel HSP90 client and suggest that HSP90 inhibitors inhibit ErbB2-driven breast tumor growth at least in part by destabilizing MIF.
Asunto(s)
Apoptosis/fisiología , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Complejos Multiproteicos/metabolismo , Animales , Apoptosis/genética , Benzoquinonas/farmacología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cartilla de ADN/genética , Densitometría , Femenino , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Proteínas HSP90 de Choque Térmico/genética , Humanos , Procesamiento de Imagen Asistido por Computador , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Lactamas Macrocíclicas/farmacología , Ratones , Ratones Transgénicos , Estabilidad Proteica , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor ErbB-2 , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The tight control of wild-type p53 by mainly MDM2 in normal cells is permanently lost in tumors harboring mutant p53, which exhibit dramatic constitutive p53 hyperstabilization that far exceeds that of wild-type p53 tumors. Importantly, mutant p53 hyperstabilization is critical for oncogenic gain of function of mutant p53 in vivo. Current insight into the mechanism of this dysregulation is fragmentary and largely derived from ectopically constructed cell systems. Importantly, mutant p53 knock-in mice established that normal mutant p53 tissues have sufficient enzymatic reserves in MDM2 and other E3 ligases to maintain full control of mutant p53. We find that in human cancer cells, endogenous mutant p53, despite its ability to interact with MDM2, suffers from a profound lack of ubiquitination as the root of its degradation defect. In contrast to wild-type p53, the many mutant p53 proteins which are conformationally aberrant are engaged in complexes with the HSP90 chaperone machinery to prevent its aggregation. In contrast to wild-type p53 cancer cells, we show that in mutant p53 cancer cells, this HSP90 interaction blocks the endogenous MDM2 and CHIP (carboxy-terminus of Hsp70-interacting protein) E3 ligase activity. Interference with HSP90 either by RNA interference against HSF1, the transcriptional regulator of the HSP90 pathway, or by direct knockdown of Hsp90 protein or by pharmacologic inhibition of Hsp90 activity with 17AAG (17-allylamino-17-demethoxygeldanamycin) destroys the complex, liberates mutant p53, and reactivates endogenous MDM2 and CHIP to degrade mutant p53. Of note, 17AAG induces a stronger viability loss in mutant p53 than in wild-type p53 cancer cells. Our data support the rationale that suppression of mutant p53 levels in vivo in established cancers might achieve clinically significant effects.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Mutantes/metabolismo , Neoplasias/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Acetilación , Animales , Benzoquinonas , Línea Celular Tumoral , Proteínas HSP90 de Choque Térmico/genética , Humanos , Lactamas Macrocíclicas , Ratones , Ratones Noqueados , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteína p53 Supresora de Tumor/genética , UbiquitinaciónRESUMEN
Strategies to induce p53 activation in tumors that retain wild-type p53 are promising for cancer therapy. Nutlin is a potent and selective pharmacological MDM2 inhibitor that competitively binds to its p53-binding pocket, thereby leading to non-genotoxic p53 stabilization and activation of growth arrest and apoptosis pathways. Nutlin-induced apoptosis is thought to occur via p53's transcriptional program. Here we report that the transcription-independent mitochondrial p53 program plays an important role in Nutlin-induced p53-mediated tumor cell death. Aside from nuclear stabilization, Nutlin causes cytoplasmic p53 accumulation and translocation to mitochondria. Monoubiquitinated p53, originating from a distinct cytoplasmic pool, is the preferred p53 species that translocates to mitochondria in response to stress. Nutlin does not interfere with MDM2's ability to monoubiquitinate p53, due to the fact that MDM2-p53 complexes are only partially disrupted and that Nutlin-stabilized MDM2 retains its E3 ubiquitin ligase activity. Nutlin-induced mitochondrial p53 translocation is rapid and associated with cytochrome C release that precedes induction of p53 target genes. Specific inhibition of mitochondrial p53 translocation by Pifithrin mu reduces the apoptotic Nutlin response by 2.5-fold, underlining the significance of p53's mitochondrial program in Nutlin-induced apoptosis. Surprisingly, blocking the transcriptional arm of p53, either via alpha-Amanitin or the p53-specific transcriptional inhibitor Pifithrin alpha, not only fails to inhibit, but greatly potentiates Nutlin-induced apoptosis. In sum, the direct mitochondrial program is a major mechanism in Nutlin-induced p53-mediated apoptosis. Moreover, at least in some tumors the transcriptional p53 activities in net balance not only are dispensable for the apoptotic Nutlin response, but appear to actively block its therapeutic effect.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Imidazoles/farmacología , Mitocondrias/genética , Piperazinas/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Benzotiazoles/farmacología , Línea Celular Tumoral , Humanos , Mitocondrias/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacología , Transcripción Genética , Translocación Genética , Proteína p53 Supresora de Tumor/genética , UbiquitinaciónRESUMEN
The protease HAUSP is a critical component of the p53-Mdm2 pathway and acts as a specific deubiquitinase for both p53 and Mdm2 and thus is important for p53 regulation. In knock-down and knock-out cellular systems it was observed that ablation of HAUSP induces profound stabilization of p53 due to enhanced degradation of Mdm2. Thus, inhibiting HAUSP by small compound interference has been proposed as a rational therapeutic strategy to activate p53 in p53 wild type tumors. However, HAUSP-mediated effects in the p53-Mdm2 axis are highly complex and non-linear and to date the role of HAUSP in tumor suppression in vivo remains unexplored. Here we investigate the effect of HAUSP up and downregulation on cell proliferation, apoptosis and tumor growth in vitro and in a xenograft model in vivo, using an inducible isogenic human colon carcinoma cell system. Importantly, in the absence of stress, both HAUSP up and downregulation inhibit cell proliferation in vitro and tumor growth in vivo due to constitutively elevated p53 levels. Moreover, tumors with HAUSP up and downregulation respond to radiotherapy with further growth inhibition. However, HAUSP downregulation causes resistance to Camptothecin- and irradiation-induced apoptosis, which correlates with suppressed mitochondrial translocation of p53. Our data suggest that changes in HAUSP modulate tumor growth and apoptotic sensitivity in vivo.
Asunto(s)
Apoptosis/genética , Carcinoma/metabolismo , Neoplasias del Colon/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Carcinoma/genética , Carcinoma/fisiopatología , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/genética , Neoplasias del Colon/fisiopatología , Modelos Animales de Enfermedad , Regulación hacia Abajo/fisiología , Resistencia a Antineoplásicos/genética , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica/genética , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Radioterapia , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Peptidasa Específica de Ubiquitina 7 , Ubiquitinación , Regulación hacia Arriba/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
There are several unorthodox features, which distinguish the non-redundant and unique novel matrix metalloproteinase-26 (MMP-26) (an enzyme that has recently evolved and does not exist in rodents but is present in humans) from other members of the MMP superfamily. This report describes our recent efforts to gain a better understanding of the mechanisms which restrict expression of MMP-26 to certain cell/tissue types. We examined transcriptional regulation of the human MMP-26 gene in normal and malignant cells. The AP-1 and Tcf-4 sites of the MMP-26 promoter appear most potent in regulating the expression of the MMP-26-luciferase chimera in HEK293 embryonic kidney and MCF7 breast carcinoma cells. Key regulators of the Wnt pathway (beta-catenin and lymphoid enhancer-binding factor/T-cell factor with which beta-catenin associates) enhanced the transcriptional activity of MMP-26 suggesting that the MMP-26 gene is a likely target of the Wnt pathway. Immunostaining, gene arrays and reverse-transcriptase polymerase chain reaction (RT-PCR) confirm the presence of MMP-26 in normal cells, including the apical epithelial conjunctiva cells of the human eye, as well as in malignant cells of epithelial origin. MMP-26 predominantly accumulates in its proenzyme form in the intracellular milieu of the transfected breast carcinoma MCF7 cells. This study brings us a step forward towards a better understanding of the unconventional role, regulation and functions of epithelial cell MMP-26 in physiological conditions and in neoplasms.
Asunto(s)
Carcinoma/enzimología , Proteínas del Citoesqueleto/metabolismo , Metaloproteinasas de la Matriz/genética , Transactivadores/metabolismo , Carcinoma/patología , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/enzimología , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Humanos , Inmunoquímica , Luciferasas/análisis , Factor de Unión 1 al Potenciador Linfoide , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Secretadas , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Esclerótica/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta Catenina , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in cell locomotion and tissue remodeling. Trafficking to the plasma membrane and internalization into the transient storage compartment both regulate the cell surface presentation of MT1-MMP. Our data indicate that mutant MT1-MMP lacking the cytoplasmic tail is recruited to the caveolae-enriched lipid raft membrane microdomains in breast carcinoma MCF7 cells. In contrast, the wild-type protease is not permanently associated with lipid rafts. Trafficking to lipid rafts correlated with poor internalization and the persistent presentation of MT1-MMP at the cell surface. The tail mutant efficiently functioned in inducing the activation of the latent proMMP-2 zymogen, matrix remodeling, and contraction of three-dimensional collagen lattices. Recruitment of the tail mutant to lipid raft antagonized, however, the cleavage of the plasma membrane-associated E-cadherin. These events limited the contribution of the tail mutant to cell locomotion and malignant growth. It is conceivable that the tail peptide sequence plays a crucial role in the translocations of MT1-MMP across the cell and contributes to coordinated cellular functions. It is tempting to hypothesize that the mechanisms involved in trafficking of MT1-MMP to caveolin-enriched lipid rafts may be targeted in a clinically advantageous manner.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Microdominios de Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Neoplasias/metabolismo , Sustitución de Aminoácidos , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/fisiología , Carcinoma/patología , Adhesión Celular , Agregación Celular , Línea Celular Tumoral , Movimiento Celular , Trasplante de Células , Colágeno/metabolismo , Activación Enzimática , Femenino , Glioma/genética , Glioma/metabolismo , Humanos , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Ratones , Ratones Desnudos , Neoplasias/genética , Trasplante HeterólogoRESUMEN
Matrix metalloproteinases (MMPs) play key roles in tissue remodelling under normal development and, especially, in diseases ranging from malignancies to stroke. We cloned and thoroughly characterized the novel human and mouse MMP gene encoding MMP-21. MMP-21 is the last uncharacterized MMP coded by the human genome. Human and mouse MMP-21 is the orthologue of Xenopus laevis X-MMP. The latent proenzyme of MMP-21 (569 amino acid residues) consists of the prodomain, the catalytic domain and the haemopexin-like domain, and is potentially capable of being activated in its secretory pathway to the extracellular milieu by furin-like proprotein convertases. Human MMP-21 is the probable target gene of the Wnt pathway. In addition, the expression of MMP-21 is controlled uniquely by Pax and Notch transcription factors known to be critical for organogenesis. MMP-21 is expressed transiently in mouse embryogenesis and increased in embryonic neuronal tissues. Our observations clearly indicate that there is an important specific function for MMP-21 in embryogenesis, especially in neuronal cells.
Asunto(s)
Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Dominio Catalítico , ADN Complementario/química , ADN Complementario/genética , Ensayo de Cambio de Movilidad Electroforética , Desarrollo Embrionario y Fetal/genética , Escherichia coli/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Reguladores/genética , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Secretadas , Ratones , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
A quantitative assessment of rate-limiting steps in metastasis has always been challenging because of the difficulty of detecting small tumor cell populations. We have developed a highly sensitive assay for monitoring the metastatic dissemination of human tumor cells in the chick embryo and used this assay to investigate the relative efficacy of sequential stages in the metastatic cascade for two malignant human tumor cells lines, HEp3 and HT1080. This assay is based on the real-time PCR amplification of human alu sequences and exhibits a high sensitivity (25 cells/lung) with a large linear range (50-100,000 cell/lung). The assay is optimized for a high number of replicate in vivo assays (50-100 animals/assay) and can be applied in both experimental and spontaneous metastasis models. Using quantitative alu PCR, we determined that HEp3 spontaneously metastasizes very efficiently and rapidly, generating secondary growth in the lung exceeding 1-2 x 10(4) cells/lung in 7 days. In contrast, spontaneous HT1080 metastasis is 50-100-fold less efficient, resulting in only 200-400 cells/lung in 7 days. By taking advantage of the sensitivity and specificity of the real-time alu PCR assay we were also able to quantitatively assess multiple steps in metastasis including intravasation, arrest of tumor cells in secondary organs of the embryo, and the initial growth and expansion of the arrested tumor cells. A comparative analysis of HEp3 and HT1080 metastasis demonstrates that the relatively low-to-moderate metastatic rate of HT1080 is caused by two distinct deficiencies, an 8-10-fold lower rate of intravasation and a delayed onset of HT1080 growth expansion in the secondary organ. Thus, a very facile metastasis model system coupled with the sensitive, real-time PCR-based assay allows for the identification and quantification of rate-limiting steps in the metastatic cascade for select human tumor cell lines.
Asunto(s)
Metástasis de la Neoplasia/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Animales , Embrión de Pollo , Fibrosarcoma/patología , Fibrosarcoma/secundario , Células HeLa , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundarioRESUMEN
A novel matrix metalloproteinase-26 (MMP-26) is known to be specifically expressed in epithelial carcinomas. To facilitate studies of MMP-26 transcriptional regulation, we have cloned and characterized a 1 kb 5'-flanking region of the human MMP-26 gene. Altogether, our findings indicate that the MMP-26 promoter has distinctive structural and functional features among MMP genes. An unusual polyadenylation site proximal to the transcription-factor-binding sites protects transcription of the MMP-26 gene from the upstream promoters and represents a part of the stringent transcriptional regulation of the gene. The MMP-26 gene has a consensus TATA-box and one transcriptional start site located 60 and 35 nucleotides upstream of the translational start site, respectively. The MMP-26 promoter was able to drive luciferase expression in human A549 lung carcinoma, HT1080 fibrosarcoma and HEK293 embryonic kidney cells. The basal transcription efficiency of the MMP-26 promoter is relatively low, thereby explaining the minute expression of the gene in most cells and tissues. When compared with other MMP genes, the MMP-26 promoter contains binding sites for a few transcription factors. Sequential deletion and mutation analysis, and electrophoretic mobility-shift assay have identified the T-cell factor-4 (Tcf-4) motif and the activator protein-1 site as the major regulatory elements of the MMP-26 promoter. Since previous studies have established that the Tcf-4 transcription factor is subjected exclusively to regulation through the beta-catenin/E(epithelial)-cadherin pathway, this implies the specific expression of MMP-26 in cancer cells of epithelial origin.
Asunto(s)
Metaloproteinasas de la Matriz/genética , Neoplasias Glandulares y Epiteliales/enzimología , Neoplasias Glandulares y Epiteliales/genética , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Quimera/genética , Mapeo Cromosómico , Clonación Molecular , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Genes Reguladores , Humanos , Luciferasas/genética , Metaloproteinasas de la Matriz Secretadas , Datos de Secuencia Molecular , Factores de Transcripción TCF , Proteína 2 Similar al Factor de Transcripción 7 , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
ProMMP-26 has the unique Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp cysteine-switch motif that discriminates this protease from all other matrix metalloproteinases (MMPs) known so far. The conserved, free cysteine residue of the conventional PRCXXPD sequence interacts with the zinc ion of the catalytic domain and provides the fourth coordination site for the catalytic zinc, thereby preventing latent proMMPs from becoming active. MMPs become functionally active when proteolytic cleavage releases the prodomain and the PRCXXPD sequence and exposes the zinc atom. Here, we report that the Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp motif is not functional in proMMP-26 and consequently is not involved in the activation mechanisms. Organomercurial treatment failed to activate proMMP-26. The autolytic Lys-Lys-Gln(59) downward arrow Gln(60)-Phe-His cleavage upstream of the Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp motif induced the proteolytic activity of recombinant proMMP-26 whereas any further cleavage inactivated the enzyme. The His(81) --> Arg(81) mutation restored the conventional cysteine-switch sequence in the prodomain but failed to induce the cysteine-switch activation mechanism. These data and computer modeling studies allowed us to hypothesize that the presence of His(81) significantly modified the fold of proMMP-26, abolished the functionality of the cysteine-switch motif, and stimulated an alternative intramolecular activation pathway of the proenzyme.
Asunto(s)
Secuencias de Aminoácidos , Cisteína/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Simulación por Computador , Cisteína/química , Activación Enzimática , Escherichia coli/genética , Humanos , Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz Secretadas , Datos de Secuencia Molecular , Mutagénesis , Células Tumorales CultivadasRESUMEN
The genes encoding aspartate kinase (ask), homoserine dehydrogenase (hom), homoserine kinase (thrB) and threonine synthase (thrC) from the obligate methylotroph Methylobacillus flagellatus were cloned. In maxicells hom and thrC directed synthesis of 51 and 48 kDa polypeptides, respectively. The hom, thrB and thrC genes and adjacent DNA areas were sequenced. Of the threonine biosynthesis genes, only hom and thrC were tightly linked in the order hom-thrC. The gene for thymidylate synthase (thyA) followed thrC and the gene for aspartate aminotransferase (aspC) preceded hom. All four genes (aspC-hom-thrC-thyA) were transcribed in the same direction. mRNA analysis indicated that hom-thrC are apparently transcribed in one 7.5 kb transcript in M. flagellatus. Promoter analysis showed the presence of a functional promoter between aspC and hom. No functional promoter was found to be associated with the DNA stretch between hom and thrC. The thrB gene encoded an unusual type of homoserine kinase and was not linked to other threonine biosynthesis genes.