RESUMO
BACKGROUND: Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer (NSCLC) is highly responsive to crizotinib. To determine whether ALK-positive NSCLC is also sensitive to pemetrexed, we retrospectively evaluated progression-free survival (PFS) of ALK-positive versus ALK-negative patients who had been treated with pemetrexed-based chemotherapy for advanced NSCLC. PATIENTS AND METHODS: We identified 121 patients with advanced, ALK-positive NSCLC in the USA, Australia, and Italy. For comparison, we evaluated 266 patients with advanced, ALK-negative, epidermal growth factor receptor (EGFR)-wild-type NSCLC, including 79 with KRAS mutations and 187 with wild-type KRAS (WT/WT/WT). We determined PFS on different pemetrexed regimens. RESULTS: Among 70 ALK-positive patients treated with a platinum/pemetrexed regimen, the median PFS (mPFS) was 7.3 months (95% confidence interval (CI) 5.5-9.5). The mPFS of 51 ALK-positive patients treated with single-agent pemetrexed or nonplatinum/pemetrexed combinations was 5.5 months (2.8-9.0). For ALK-negative patients, PFS on all pemetrexed-based regimens was similar to that of ALK-positive patients, except in the specific setting of first-line platinum/pemetrexed where the mPFS was only 4.2 and 5.4 months in KRAS and WT/WT/WT patients, respectively. However, among patients with a never/light-smoking history (0-10 pack-year smoking history) treated with first-line platinum/pemetrexed, there was no difference in PFS between ALK-positive and ALK-negative patients. CONCLUSIONS: PFS on pemetrexed or nonplatinum/pemetrexed combinations was similar in ALK-positive and ALK-negative patients. PFS on first-line platinum/pemetrexed may be prolonged in never/light-smoking patients regardless of ALK status.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Glutamatos/uso terapêutico , Guanina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Receptores Proteína Tirosina Quinases/metabolismo , Adulto , Idoso , Quinase do Linfoma Anaplásico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Feminino , Guanina/uso terapêutico , Humanos , Neoplasias Pulmonares/enzimologia , Masculino , Pessoa de Meia-Idade , Pemetrexede , Timidilato Sintase/metabolismo , Adulto JovemRESUMO
Myc and Mad family proteins regulate multiple biological processes through their capacity to influence gene expression directly. Here we show that the basic regions of Myc and Mad proteins are not functionally equivalent in oncogenesis, have separable E-box-binding activities and engage both common and distinct gene targets. Our data support the view that the opposing biological actions of Myc and Mxi1 extend beyond reciprocal regulation of common gene targets. Identification of differentially regulated gene targets provides a framework for understanding the mechanism through which the Myc superfamily governs the growth, proliferation and survival of normal and neoplastic cells.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Apoptose , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Sequência Conservada , Proteínas de Ligação a DNA/química , Sequências Hélice-Alça-Hélice , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Transfecção , Proteínas Supressoras de TumorRESUMO
BACKGROUND: Personalizing non-small-cell lung cancer (NSCLC) therapy toward oncogene addicted pathway inhibition is effective. Hence, the ability to determine a more comprehensive genotype for each case is becoming essential to optimal cancer care. METHODS: We developed a multiplexed PCR-based assay (SNaPshot) to simultaneously identify >50 mutations in several key NSCLC genes. SNaPshot and FISH for ALK translocations were integrated into routine practice as Clinical Laboratory Improvement Amendments-certified tests. Here, we present analyses of the first 589 patients referred for genotyping. RESULTS: Pathologic prescreening identified 552 (95%) tumors with sufficient tissue for SNaPshot; 51% had ≥1 mutation identified, most commonly in KRAS (24%), EGFR (13%), PIK3CA (4%) and translocations involving ALK (5%). Unanticipated mutations were observed at lower frequencies in IDH and ß-catenin. We observed several associations between genotypes and clinical characteristics, including increased PIK3CA mutations in squamous cell cancers. Genotyping distinguished multiple primary cancers from metastatic disease and steered 78 (22%) of the 353 patients with advanced disease toward a genotype-directed targeted therapy. CONCLUSIONS: Broad genotyping can be efficiently incorporated into an NSCLC clinic and has great utility in influencing treatment decisions and directing patients toward relevant clinical trials. As more targeted therapies are developed, such multiplexed molecular testing will become a standard part of practice.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Genótipo , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase Multiplex , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Ensaios Clínicos como Assunto , Testes Diagnósticos de Rotina , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Terapia de Alvo Molecular , Mutação , Adulto JovemRESUMO
BACKGROUND: AMP-activated protein kinase (AMPK, PRKA) has central roles in cellular metabolic sensing and energy balance homeostasis, and interacts with various pathways (e.g., TP53 (p53), FASN, MTOR and MAPK3/1 (ERK)). AMP-activated protein kinase activation is cytotoxic to cancer cells, supporting AMPK as a tumour suppressor and a potential therapeutic target. However, no study has examined its prognostic role in colorectal cancers. METHODS: Among 718 colon and rectal cancers, phosphorylated AMPK (p-AMPK) and p-MAPK3/1 expression was detected in 409 and 202 tumours, respectively, by immunohistochemistry. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical and tumoral features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. RESULTS: Phosphorylated AMPK expression was not associated with survival among all patients. Notably, prognostic effect of p-AMPK significantly differed by p-MAPK3/1 status (P(interaction)=0.0017). Phosphorylated AMPK expression was associated with superior colorectal cancer-specific survival (adjusted HR 0.42; 95% confidence interval (CI), 0.24-0.74) among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases (adjusted HR 1.22; 95% CI: 0.85-1.75). CONCLUSION: Phosphorylated AMPK expression in colorectal cancer is associated with superior prognosis among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases, suggesting a possible interaction between the AMPK and MAPK pathways influencing tumour behaviour.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/mortalidade , Metilação de DNA , Feminino , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Mutação , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent upon pRb.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Animais , Apoptose , Transporte Biológico , Núcleo Celular/metabolismo , Cristalinas , Ciclina D1/genética , Ciclina D2 , Ciclina E/genética , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p57 , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Feminino , Fase G1 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Fase SRESUMO
There are currently no effective targeted therapies for KRAS mutant cancers. Therapeutic strategies that combine MEK inhibitors with agents that target apoptotic pathways may be a promising therapeutic approach. We investigated combining MEK and MDM2 inhibitors as a potential treatment strategy for KRAS mutant non-small cell lung cancers (NSCLC) and colorectal carcinomas that harbor wild-type TP53. The combination of pimasertib (MEK inhibitor) and SAR405838 (MDM2 inhibitor) was synergistic and induced the expression of PUMA and BIM, led to apoptosis and growth inhibition in vitro, and tumor regression in vivo. Acquired resistance to the combination commonly resulted from the acquisition of TP53 mutations, conferring complete resistance to MDM2 inhibition. In contrast, resistant clones exhibited marked variability in sensitivity to MEK inhibition, which significantly impacted sensitivity to subsequent treatment with alternative MEK inhibitor-based combination therapies. These results highlight both the potential promise and limitations of combining MEK and MDM2 inhibitors for treatment of KRAS mutant NSCLC and colorectal cancers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , MAP Quinase Quinase Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Células A549 , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células , Neoplasias Colorretais/genética , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Indóis , Neoplasias Pulmonares/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , Mutação , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , RNA Interferente Pequeno , Compostos de Espiro , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Caveolae are small vesicular invaginations of the cell membrane. It is within this organelle that cells perform transcytosis, potocytosis and signal transduction. These "little caves" are composed of a mixture of lipids and proteins unlike those found in the plasma membrane proper. The chief structural proteins of caveolae are caveolins. To date, three caveolins (Cav-1, -2 and -3) with unique tissue distributions have been identified. Caveolins form a scaffold onto which many signalling molecules can assemble, to generate pre-assembled signalling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signalling molecules.
Assuntos
Caveolinas , Membrana Celular/fisiologia , Proteínas de Membrana/fisiologia , Organelas/fisiologia , Transdução de Sinais , Caveolina 1 , Membrana Celular/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Organelas/ultraestruturaRESUMO
Tumor necrosis factor-alpha (TNFalpha) has been implicated as a contributing mediator of insulin resistance observed in pathophysiological conditions such as obesity, cancer-induced cachexia, and bacterial infections. Previous studies have demonstrated that TNFalpha confers insulin resistance by promoting phosphorylation of serine residues on insulin receptor substrate 1 (IRS-1), thereby diminishing subsequent insulin-induced tyrosine phosphorylation of IRS-1. However, little is known about which signaling molecules are involved in this process in adipocytes and about the temporal sequence of events that ultimately leads to TNFalpha-stimulated IRS-1 serine phosphorylation. In this study, we demonstrate that specific inhibitors of the MAP kinase kinase (MEK)1/2-p42/44 mitogen-activated protein (MAP) kinase pathway restore insulin signaling to normal levels despite the presence of TNFalpha. Additional experiments show that MEK1/2 activity is required for TNFalpha-induced IRS-1 serine phosphorylation, thereby suggesting a mechanism by which these inhibitors restore insulin signaling. We observe that TNFalpha requires 2.5-4 h to markedly reduce insulin-triggered tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes. Although TNFalpha activates p42/44 MAP kinase, maximal stimulation is observed within 10-30 min. To our surprise, p42/44 activity returns to basal levels well before IRS-1 serine phosphorylation and insulin resistance are observed. These activation kinetics suggest a mechanism of p42/44 action more complicated than a direct phosphorylation of IRS-1 triggered by the early spike of TNFalpha-induced p42/44 activity. Chronic TNFalpha treatment (>> 72 h) causes adipocyte dedifferentiation, as evidenced by the loss of triglycerides and down-regulation of adipocyte-specific markers. We observe that this longer term TNFalpha-mediated dedifferentiation effect utilizes alternative, p42/44 MAP kinase-independent intracellular pathways. This study suggests that TNFalpha-mediated insulin resistance, but not adipocyte dedifferentiation, is mediated by the MEK1/2-p42/44 MAP kinase pathway.
Assuntos
Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Resistência à Insulina , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Células 3T3 , Adipócitos/citologia , Animais , Anisomicina/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Flavonoides/farmacologia , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Cinética , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Transdução de SinaisRESUMO
Caveolins (Cav-1, -2, and -3) are a gene family of cytoplasmic membrane-anchored scaffolding proteins that: (i) help to sculpt caveolae membranes from the plasma membrane proper; and (ii) participate in the sequestration of inactive signaling molecules. In the adult, caveolin-1 and -2 are co-expressed and are most abundant in type I pneumocytes, endothelia, fibroblastic cells and adipocytes, while the expression of caveolin-3 is restricted to striated muscle cells. However, little is known regarding the genomic organization and developmental expression of the caveolin gene family. Here, using the mouse as a model system, we examine the chromosomal localization, the detailed intron-exon organization, and developmental expression pattern of the caveolin gene family. cDNAs encoding caveolin-1, -2, and -3 were used as probes to isolate murine genomic clones containing these genes. Fluorescence in situ hybridization (FISH) analysis using these genomic clones as probes reveals that all three caveolin genes are localized to murine chromosome 6. Specifically, caveolin-1 and -2 co-localize to chromosomal region 6-A2, while caveolin-3 is located within the chromosomal region 6-E1. Searches of the NCBI Human/Mouse Homology map indicate that murine region 6-A2 corresponds to human chromosome 7q31. As this region (6-A2/7q31) is the site of an as yet unidentified tumor suppressor gene(s), our mapping studies clearly define caveolin-1 and caveolin-2 as candidate genes that may be deleted at these loci. All three caveolin genes show similar intron-exon organization, with the last exon of each gene encoding the bulk of the known caveolin functional domains. The boundary position of the last exon is essentially identical in all three caveolin genes, suggesting that they may have arisen through gene duplication events. Developmentally, all three caveolins were expressed late during mouse embryogenesis as assessed by Northern and Western blot analysis. We examined the localization of the caveolin proteins in sections of day 16 mouse embryos using a well-characterized panel of antibody probes. Caveolin-1 and -2 were most abundantly expressed in the developing lung parenchyma, while caveolin-3 was most abundantly expressed in developing tissues that consist primarily of skeletal muscle cells. As the expression of all three caveolins in the adult is highest in terminally differentiated cell types, this is consistent with the idea that caveolins may be viewed as late markers of differentiation during embryogenesis.
Assuntos
Caveolinas , Regulação da Expressão Gênica no Desenvolvimento , Genes Supressores de Tumor , Proteínas de Membrana/genética , Família Multigênica , Sequência de Aminoácidos , Animais , Caveolina 1 , Caveolina 2 , Caveolina 3 , Mapeamento Cromossômico , Éxons , Hibridização in Situ Fluorescente , Íntrons , Pulmão/embriologia , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The CA microsatellite repeat marker, D7S522, is located at the center of a approximately 1000 kb smallest common deleted region that is lost in many forms of human cancer. It has been proposed that a putative tumor suppressor gene lies in close proximity to D7S522, within this smallest common deleted region. However, the genes located in proximity to D7S522 have remained elusive. Recently, we identified five independent BAC clones (approximately 100-200 kb) containing D7S522 and the human genes encoding caveolins 1 and 2. Here, we present the detailed organization of the caveolin locus and its relationship to D7S522, as deduced using a shot-gun sequencing approach. We derived two adjacent contigs for a total coverage of approximately 250 kb. Analysis of these contigs reveals that D7S522 is located approximately 67 kb upstream of the caveolin-2 gene and that the caveolin-2 gene is located approximately 19 kb upstream of the caveolin-1 gene, providing for the first time a detailed genetic map of this region. Further sequence analysis reveals many interesting features of the caveolin genes; these include the intron-exon boundaries and several previously unrecognized CA repeats that lie within or in close proximity to the caveolin genes. The first and second exons of both caveolin genes are embedded within CpG islands. These results suggest that regulation of caveolin gene expression may be controlled, in part, by methylation of these CpG regions. In support of this notion, we show here that the CGs in the 5' promoter region of the caveolin-1 gene are functionally methylated in two human breast cancer cell lines (MCF7 and T-47D) that fail to express the caveolin-1 protein. In contrast, the same CGs in cultured normal human mammary epithelial cells (NHMECs) are non-methylated and these cells express high levels of the caveolin-1 protein. Comparison of the human locus with the same locus in the pufferfish Fugu rubripes reveals that the overall organization of the caveolin-1/-2 locus is conserved from pufferfish to man. In conclusion, our current studies provide a systematic basis for diagnostically evaluating the potential deletion, mutation, or methylation of the caveolin genes in a variety of human tumors.
Assuntos
Neoplasias da Mama/genética , Caveolinas , Ilhas de CpG/genética , Proteínas de Membrana/genética , Repetições de Microssatélites , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Caveolina 1 , Caveolina 2 , Cromossomos Humanos Par 7 , Metilação de DNA , Regulação para Baixo , Éxons , Peixes/genética , Biblioteca Gênica , Humanos , Íntrons , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The (CA)n microsatellite repeat marker D7S522 is located on human chromosome 7q31.1 and is frequently deleted in a variety of human cancers, including squamous cell carcinomas of the head and neck, prostate cancers, renal cell carcinomas, ovarian adenocarcinomas, colon carcinomas, and breast cancers. In addition, D7S522 spans FRA7G, a known common fragile site on human chromosome 7. Based on these studies, it has been proposed that an as yet unidentified tumor suppressor gene (or genes) is contained within or located in close proximity to this locus. However, the identity of the candidate tumor suppressor gene at the D7S522 locus remains unknown. Here, we show that the human genes encoding caveolins 1 and 2 are contained within the same human genomic BAC clones and co-localize to the q31.1-q31.2 region of human chromosome 7, as seen by FISH analysis. In addition, we determined the intron-exon boundaries of the human caveolin-1 and -2 genes. The human caveolin-1 gene contains three exons, while the human caveolin-2 gene contains two exons. Interestingly, the boundary of the last exon of the human caveolin-1 and caveolin-2 genes are analogous, suggesting that they arose through gene duplication at this locus. (CA)n microsatellite repeat marker analysis of these caveolin genomic clones indicates they contain the marker D7S522 (located at 7q31.1), but not other microsatellite repeat markers tested. The close proximity of caveolins 1 and 2 to the D7S522 locus was independently confirmed by using a panel of MIT/Whitehead human STS markers that are known to map in the neighborhood of the D7S522 locus. As it has been previously shown that caveolin 1 possesses transformation suppressor activity (Koleske, A.J., Baltimore, D. and M.P. Lisanti (1995) Proc. Natl. Acad. Sci. USA 92, 1381-1385; Engelman, J.A. et al. (1997) J. Biol. Chem. 272, 16374-16381), we propose that the caveolin-1 gene may represent the candidate tumor suppressor gene at the D7S522 locus on human chromosome 7q31.1.
Assuntos
Caveolinas , Fragilidade Cromossômica , Cromossomos Humanos Par 7 , Deleção de Genes , Proteínas de Membrana/genética , Neoplasias/genética , Sequência de Aminoácidos , Sequência de Bases , Caveolina 1 , Caveolina 2 , Sítios Frágeis do Cromossomo , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA , Éxons , Feminino , Genes Supressores de Tumor , Marcadores Genéticos , Biblioteca Genômica , Humanos , Masculino , Proteínas de Membrana/química , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
The p42/44 mitogen-activated protein (MAP)-kinase cascade is a well-established signal transduction pathway that is initiated at the cell surface and terminates within the nucleus. More specifically, receptor tyrosine kinases can indirectly activate Raf, which in turn leads to activation of MEK and ERK and ultimately phosphorylation of Elk, a nuclear transcription factor. Recent reports have suggested that some members of p42/44 MAP kinase cascade can be sequestered within plasmalemmal caveolae in vivo. For example, morphological studies have directly shown that ERK-1/2 is concentrated in plasma membrane caveolae in vivo using immunoelectron microscopy. In addition, constitutive activation of the p42/44 MAP kinase cascade is sufficient to reversibly down-regulate caveolin-1 mRNA and protein expression. However, the functional relationship between the p42/44 MAP kinase cascade and caveolins remains unknown. Here, we examine the in vivo role of caveolins in regulating signaling along the MAP kinase cascade. We find that co-expression with caveolin 1 dramatically inhibits signaling from EGF-R, Raf, MEK-1 and ERK-2 to the nucleus. Using a variety of caveolin-1 deletion mutants, we mapped this in vivo inhibitory activity to caveolin-1 residues 32-95. Peptides derived from this region of caveolin 1 also inhibit the in vitro kinase activity of purified MEK-1 and ERK-2. Thus, we show here that caveolin-1 expression can inhibit signal transduction from the p42/44 MAP kinase cascade both in vitro and in vivo. Taken together with previous data, our results also suggest that a novel form of reciprocal negative regulation exists between p42/44 MAP kinase activation and caveolin-1 protein expression, i.e. up-regulation of caveolin-1 protein expression down-modulates p42/44 MAP kinase activity (this report) and up-regulation of p42/44 MAP kinase activity down-regulates caveolin-1 mRNA and protein expression.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caveolinas , Proteínas de Ligação a DNA , Proteínas de Membrana/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Transdução de Sinais/fisiologia , Fatores de Transcrição , Animais , Células CHO , Caveolina 1 , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Cricetinae , Receptores ErbB/biossíntese , Receptores ErbB/fisiologia , Cinética , MAP Quinase Quinase 1 , Proteínas de Membrana/química , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Coelhos , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas Elk-1 do Domínio etsRESUMO
Caveolae are vesicular organelles with a characteristic uniform diameter in the range of 50-100 nm. Although recombinant expression of caveolin-1 is sufficient to drive caveolae formation, it remains unknown what controls the uniform diameter of these organelles. One hypothesis is that specific caveolin-caveolin interactions regulate the size of caveolae, as caveolin-1 undergoes two stages of self-oligomerization. To test this hypothesis directly, we have created two caveolin-1 deletion mutants that lack regions of caveolin-1 that are involved in directing the self-assembly of caveolin-1 oligomers. More specifically, Cav-1 delta61-100 lacks a region of the N-terminal domain that directs the formation of high molecular mass caveolin-1 homo-oligomers, while Cav-1 deltaC lacks a complete C-terminal domain that is required to allow caveolin homo-oligomers to interact with each other, forming a caveolin network. It is important to note that these two mutants retain an intact transmembrane domain. Our current results show that although Cav-1 delta61-100 and Cav-1 deltaC are competent to drive vesicle formation, these vesicles vary widely in their size and shape with diameters up to 500-1000 nm. In addition, caveolin-induced vesicle formation appears to be isoform-specific. Recombinant expression of caveolin-2 under the same conditions failed to drive the formation of vesicles, while caveolin-3 expression yielded caveolae-sized vesicles. These results are consistent with the previous observation that in transformed NIH 3T3 cells that lack caveolin-1 expression, but continue to express caveolin-2, no morphologically distinguishable caveolae are observed. In addition, as caveolin-2 alone exists mainly as a monomer or homo-dimer, while caveolins 1 and 3 exist as high molecular mass homo-oligomers, our results are consistent with the idea that the formation of high molecular mass oligomers of caveolin are required to regulate the formation of uniform caveolae-sized vesicles. In direct support of this notion, regulated induction of caveolin-1 expression in transformed NIH 3T3 cells was sufficient to recruit caveolin-2 to caveolae membranes. The ability of caveolin-1 to recruit caveolin-2 most likely occurs through a direct interaction between caveolins 1 and 2, as caveolins 1 and 2 are normally co-expressed and interact with each other to form high molecular mass hetero-oligomers containing both caveolins 1 and 2.
Assuntos
Caveolinas , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Proteínas de Membrana/genética , Células 3T3 , Animais , Caveolina 1 , Caveolina 2 , Análise Mutacional de DNA , Dimerização , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de SequênciaRESUMO
Hyperactivation of phosphatidylinositol-3 kinase (PI3K) can occur as a result of somatic mutations in PIK3CA, the gene encoding the p110α subunit of PI3K. The HER2 oncogene is amplified in 25% of all breast cancers and some of these tumors also harbor PIK3CA mutations. We examined mechanisms by which mutant PI3K can enhance transformation and confer resistance to HER2-directed therapies. We introduced the PI3K mutations E545K and H1047R in MCF10A human mammary epithelial cells that also overexpress HER2. Both mutants conferred a gain of function to MCF10A/HER2 cells. Expression of H1047R PI3K, but not E545K PI3K, markedly upregulated the HER3/HER4 ligand heregulin (HRG). HRG siRNA inhibited growth of H1047R but not E545K-expressing cells and synergized with the HER2 inhibitors trastuzumab and lapatinib. The PI3K inhibitor BEZ235 markedly inhibited HRG and pAKT levels and, in combination with lapatinib, completely inhibited growth of cells expressing H1047R PI3K. These observations suggest that PI3K mutants enhance HER2-mediated transformation by amplifying the ligand-induced signaling output of the ErbB network. This also counteracts the full effect of therapeutic inhibitors of HER2. These data also suggest that mammary tumors that contain both HER2 gene amplification and PIK3CA mutations should be treated with a combination of HER2 and PI3K inhibitors.
Assuntos
Genes erbB-2 , Mutação , Neuregulina-1/biossíntese , Fosfatidilinositol 3-Quinases/fisiologia , Receptor ErbB-3/metabolismo , Divisão Celular , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Ligantes , Neuregulina-1/genética , Fosfatidilinositol 3-Quinases/genética , Interferência de RNA , Receptor ErbB-3/genéticaRESUMO
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib are effective therapies against mutant non-small cell lung cancers (NSCLCs). Treatment is limited by the development of resistance in part explained by the gain of a secondary EGFR mutation, T790M, at the gatekeeper residue. Irreversible EGFR inhibitors, including PF00299804, are effective in vitro and in vivo against EGFR mutant tumors that contain EGFR T790M and are currently under clinical development. In this study, we generate models of resistance to PF00299804, using cell lines with EGFR T790M and show that the PF00299804-resistant models develop focal amplification of EGFR that preferentially involves the T790M-containing allele. These PF00299804-resistant cell lines remain dependent on EGFR for growth as downregulation of EGFR by shRNA compromises their viability. We show that resistance to PF00299804 arises, at least in part, through selection of a pre-existing EGFR T790M-amplified clone both in vitro and using a xenograft model in vivo. Our findings show that EGFR T790M is a common resistance mechanism to both reversible, and when amplified, the irreversible EGFR kinase inhibitors further emphasizing the need to develop more potent therapies against EGFR T790M. These findings can be used to guide studies of patient tumor specimens from ongoing clinical trials of irreversible EGFR kinase inhibitors.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Amplificação de Genes , Inibidores de Proteínas Quinases/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Gefitinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Fosforilação , Quinazolinas/farmacologia , Quinazolinonas/farmacologiaRESUMO
SB203580 and SB202190, pyridinyl imidazoles that selectively inhibit p38 mitogen-activated protein (MAP) kinase, are widely utilized to assess the physiological roles of p38. Here, we demonstrate that treatment of 3T3-L1 fibroblasts with these p38 MAP kinase inhibitors prevents their differentiation into adipocytes as judged by an absence of lipid accumulation, a lack of expression of adipocyte-specific genes, and a fibroblastic morphological appearance. In 3T3-L1 fibroblasts and developing adipocytes, p38 is active. p38 activity decreases dramatically during later stages of differentiation. In accordance with the time course of p38 activity, p38 inhibitor treatment during only the early stages of differentiation is sufficient to block adipogenesis. In addition, we constructed a 3T3-L1 cell line harboring an inducible dominant negative p38 mutant. The induction of this dominant negative mutant of p38 prevents adipocyte differentiation. Thus, it is likely that the antiadipogenic activity of SB203580 and SB202190 is indeed due to inhibition of p38 MAP kinase. This study points out that CCAAT/enhancer-binding protein beta (C/EBPbeta), a transcription factor critical for the initial stages of 3T3-L1 adipogenesis, bears a consensus site for p38 phosphorylation and serves as a substrate for p38 MAP kinase in vitro. Although the induction of C/EBPbeta is not significantly altered in the presence of the p38 inhibitor, the amount of in vivo phosphorylated C/EBPbeta is reduced by SB203580. The transcriptional induction of PPARgamma, a gene whose expression is induced by C/EBPbeta, and a factor critically involved in terminal differentiation of adipocytes, is impaired in the presence of p38 inhibitors. Thus, transcription factors such as C/EBPbeta that promote adipocyte differentiation may be p38 targets during adipogenesis. Collectively, the data in this study suggest that p38 MAP kinase activity is important for proper 3T3-L1 differentiation.
Assuntos
Adipócitos/citologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno , Piridinas/farmacologia , Células 3T3 , Adipócitos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Indução Enzimática , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are down-regulated in NIH 3T3 cells in response to transformation by activated oncogenes, such as H-Ras(G12V) and v-Abl. The mechanisms governing this down-regulation event remain unknown. Here, we show that caveolin-1 gene expression is directly regulated by activation of the Ras-p42/44 MAP kinase cascade. Down regulation of caveolin-1 protein expression by Ras is independent of (i) the type of activating mutation (G12V versus Q61L) and (ii) the form of activated Ras transfected (H-Ras versus K-Ras versus N-Ras). Treatment of Ras or Raf-transformed NIH 3T3 cells with a well characterized MEK inhibitor (PD 98059) restores caveolin-1 protein expression. In contrast, treatment of v-Src and v-Abl transformed NIH 3T3 cells with PD 98059 does not restore caveolin-1 expression. Thus, there must be at least two pathways for down-regulating caveolin-1 expression: one that is p42/44 MAP kinase-dependent and another that is p42/44 MAP kinase-independent. We focused our efforts on the p42/44 MAP kinase-dependent pathway. The activity of a panel of caveolin-1 promoter constructs was evaluated using transient expression in H-Ras(G12V) transformed NIH 3T3 cells. We show that caveolin-1 promoter activity is up-regulated approximately 5-fold by inhibition of the p42/44 MAP kinase cascade. Using electrophoretic mobility shift assays we provide evidence that the caveolin-1 promoter (from -156 to -561) is differentially bound by transcription factors in normal and H-Ras(G12V)-transformed cells. We also show that activation of protein kinase A (PKA) signaling is sufficient to down-regulate caveolin-1 protein expression and promoter activity. Thus, we have identified two signaling pathways (Ras-p42/44 MAP kinase and PKA) that transcriptionally down-regulate caveolin-1 gene expression.
Assuntos
Caveolinas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Células 3T3 , Animais , Caveolina 1 , Transformação Celular Viral , Ativação Enzimática , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Fatores de Transcrição/metabolismoRESUMO
Caveolin-3 is the principal structural protein of caveolae membrane domains in striated muscle cells. Caveolin-3 mRNA and protein expression are dramatically induced during the differentiation of C2C12 skeletal myoblasts, coincident with myoblast fusion. In these myotubes, caveolin-3 localizes to the sarcolemma (muscle cell plasma membrane), where it associates with the dystrophin-glycoprotein complex. However, it remains unknown what role caveolin-3 plays in myoblast differentiation and myotube formation. Here, we employ an antisense approach to derive stable C2C12 myoblasts that fail to express the caveolin-3 protein. We show that C2C12 cells harboring caveolin-3 antisense undergo differentiation and express normal amounts of four muscle-specific marker proteins. However, C2C12 cells harboring caveolin-3 antisense fail to undergo myoblast fusion and, therefore, do not form myotubes. Interestingly, treatment with specific p38 mitogen-activated protein kinase inhibitors blocks both myotube formation and caveolin-3 expression, but does not affect the expression of other muscle-specific proteins. In addition, we find that three human rhabdomyosarcoma cell lines do not express caveolin-3 and fail to undergo myoblast fusion. Taken together, these results support the idea that caveolin-3 expression is required for myoblast fusion and myotube formation, and suggest that p38 is an upstream regulator of caveolin-3 expression.
Assuntos
Caveolinas , Diferenciação Celular , Regulação para Baixo , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Esquelético/metabolismo , Caveolina 3 , Fusão Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Oligonucleotídeos Antissenso/farmacologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Caveolae are plasma membrane-attached vesicular organelles. Caveolin-1, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Both caveolae and caveolin are most abundantly expressed in terminally differentiated cells: adipocytes, endothelial cells, and muscle cells. Conversely, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes such as v-abl and H-ras (G12V); caveolae are absent from these cell lines. However, its remains unknown whether down-regulation of caveolin-1 protein and caveolae organelles contributes to their transformed phenotype. Here, we have expressed caveolin-1 in oncogenically transformed cells under the control of an inducible-expression system. Regulated induction of caveolin-1 expression was monitored by Western blot analysis and immunofluorescence microscopy. Our results indicate that caveolin-1 protein is expressed well using this system and correctly localizes to the plasma membrane. Induction of caveolin-1 expression in v-Abl-transformed and H-Ras (G12V)-transformed NIH 3T3 cells abrogated the anchorage-independent growth of these cells in soft agar and resulted in the de novo formation of caveolae as seen by transmission electron microscopy. Consistent with its antagonism of Ras-mediated cell transformation, caveolin-1 expression dramatically inhibited both Ras/MAPK-mediated and basal transcriptional activation of a mitogen-sensitive promoter. Using an established system to detect apoptotic cell death, it appears that the effects of caveolin-1 may, in part, be attributed to its ability to initiate apoptosis in rapidly dividing cells. In addition, we find that caveolin-1 expression levels are reversibly down-regulated by two distinct oncogenic stimuli. Taken together, our results indicate that down-regulation of caveolin-1 expression and caveolae organelles may be critical to maintaining the transformed phenotype in certain cell populations.