Assuntos
Inibidores da Angiogênese/uso terapêutico , Paraganglioma/tratamento farmacológico , Pirimidinas/uso terapêutico , Neoplasias Retroperitoneais/tratamento farmacológico , Sulfonamidas/uso terapêutico , Biópsia , Humanos , Indazóis , Masculino , Pessoa de Meia-Idade , Paraganglioma/diagnóstico por imagem , Paraganglioma/patologia , Neoplasias Retroperitoneais/diagnóstico por imagem , Neoplasias Retroperitoneais/patologia , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
Aberrant expression of RNA-binding proteins has profound implications for cellular physiology and the pathogenesis of human diseases such as cancer. We previously identified the Fragile X-Related 1 gene (FXR1) as one amplified candidate driver gene at 3q26-29 in lung squamous cell carcinoma (SCC). FXR1 is an autosomal paralog of Fragile X mental retardation 1 and has not been directly linked to human cancers. Here we demonstrate that FXR1 is a key regulator of tumor progression and its overexpression is critical for nonsmall cell lung cancer (NSCLC) cell growth in vitro and in vivo. We identified the mechanisms by which FXR1 executes its regulatory function by forming a novel complex with two other oncogenes, protein kinase C, iota and epithelial cell transforming 2, located in the same amplicon via distinct binding mechanisms. FXR1 expression is a candidate biomarker predictive of poor survival in multiple solid tumors including NSCLCs. Because FXR1 is overexpressed and associated with poor clinical outcomes in multiple cancers, these results have implications for other solid malignancies.
Assuntos
Cromossomos Humanos Par 3/genética , Neoplasias Pulmonares/genética , Proteínas de Ligação a RNA/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Variações do Número de Cópias de DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Prognóstico , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Análise de Sobrevida , Resultado do TratamentoRESUMO
PURPOSE: Chromosome 3q26-29 is a critical region of genomic amplification in lung squamous cell carcinomas (SCC). Identification of candidate drivers in this region could help uncover new mechanisms in the pathogenesis and potentially new targets in SCC of the lung. EXPERIMENTAL DESIGN: We conducted a meta-analysis of seven independent datasets containing a total of 593 human primary SCC samples to identify consensus candidate drivers in 3q26-29 amplicon. Through integrating protein-protein interaction network information, we further filtered for candidates that may function together in a network. Computationally predicted candidates were validated using RNA interference (RNAi) knockdown and cell viability assays. Clinical relevance of the experimentally supported drivers was evaluated in an independent cohort of 52 lung SCC patients using survival analysis. RESULTS: The meta-analysis identified 20 consensus candidates, among which four (SENP2, DCUN1D1, DVL3, and UBXN7) are involved in a small protein-protein interaction network. Knocking down any of the four proteins led to cell growth inhibition of the 3q26-29-amplified SCC. Moreover, knocking down of SENP2 resulted in the most significant cell growth inhibition and downregulation of DCUN1D1 and DVL3. Importantly, a gene expression signature composed of SENP2, DCUN1D1, and DVL3 stratified patients into subgroups with different response to adjuvant chemotherapy. CONCLUSION: Together, our findings show that SENP2, DCUN1D1, and DVL3 are candidate driver genes in the 3q26-29 amplicon of SCC, providing novel insights into the molecular mechanisms of disease progression and may have significant implication in the management of SCC of the lung.
Assuntos
Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 3 , Amplificação de Genes , Genômica , Neoplasias Pulmonares/genética , Oncogenes , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/mortalidade , Resistencia a Medicamentos Antineoplásicos , Redes Reguladoras de Genes , Genômica/métodos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , TranscriptomaRESUMO
Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23-q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Metilação de DNA , Epigenômica , Regulação Neoplásica da Expressão Gênica , Kisspeptinas/genética , Melanoma/genética , Neoplasias Cutâneas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Ciclo Celular , Diferenciação Celular , Movimento Celular , Criança , Imunoprecipitação da Cromatina , Feminino , Inativação Gênica , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Kisspeptinas/metabolismo , Luciferases/metabolismo , Masculino , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/metabolismo , Adulto JovemRESUMO
Metastasis suppressors and other regulators of cell motility play an important role in tumor invasion and metastases. We previously identified that activation of the G protein coupled receptor 54 (GPR54) by the metastasis suppressor metastin inhibits cell migration in association with overexpression of Regulator of calcineurin 1 (RCAN1), an endogenous regulator of calcineurin. Calcineurin inhibitors also blocked cell migration in vitro and RCAN1 protein levels were reduced in nodal metastases in thyroid cancer. The purpose of the current study was to determine directly if RCAN1 functions as a motility suppressor in vitro. Several cancer cell lines derived from different cancer types with different motility rates were evaluated for RCAN1 expression levels. Using these systems we determined that reduction of endogenous RCAN1 using siRNA resulted in an increase in cancer cell motility while expression of exogenous RCAN1 reduced cell motility. In one cell line with a high migratory rate, the stability of exogenously expressed RCAN1 protein was reduced and was rescued by treatment with a proteasome inhibitor. Finally, overexpression of RCAN1 was associated with an increase in cell adhesion to collagen IV and reduced calcineurin activity. In summary, we have demonstrated that the expression of exogenous RCAN1 reduces migration and alters adhesion; and that the loss of endogenous RCAN1 leads to an increase in migration in the examined cancer cell lines. These results are consistent with a regulatory role for RCAN1 in cancer cell motility in vitro.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Musculares/fisiologia , Neoplasias/patologia , Inibidores de Calcineurina , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA , Doxiciclina/farmacologia , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genéticaRESUMO
BACKGROUND: Thiazolidinediones (TZDs) have been demonstrated to possess antitumor effects in breast cancer cells, although the mechanisms are not well established. We sought to better define TZDs' antitumor effects and molecular mechanisms to permit rational utilization of these agents. MATERIALS AND METHODS: We studied the effects of TZDs on DNA synthesis (BrdU enzyme-linked immunosorbent assay), gene expression (microarray, quantitative reverse transcription-polymerase chain reaction, and immunoblot), serine phosphorylation and localization of beta-catenin (nuclear/cytoplasmic fractionation and immunoblot), transactivation activity of beta-catenin/T cell factor 4 (TCF4) (luciferase assay with wild-type and mutant TCF4 responsive element), and beta-catenin/TCF4 complex (immunoprecipitation) in human breast cancer cells MDA-MB-231 and T47D. RESULTS: Troglitazone (TG) down-regulated DNA synthesis in MDA-MB-231 and T47D and decreased mRNA expression of the Wnt co-receptors frizzled-1 and low-density lipoprotein-related protein 6 (LRP6) as well as protein level of LRP6. TG also targets downstream Wnt signaling molecules in T47D cells. TG down-regulates p-beta-catenin (S33/S37/T41) and promotes translocation of beta-catenin into the nucleus. However, TG inhibits beta-catenin-mediated transactivation by down-regulating TCF4 protein levels, thereby inhibiting beta-catenin/TCF4 complex formation. Finally, we found that inhibition of Akt augments TG-mediated down-regulation of DNA synthesis and this result was accompanied with the decreased protein levels of Wnt signaling molecules: LRP6, beta-catenin, and TCF4. CONCLUSION: These results suggest that the Wnt/beta-catenin signaling pathway, which plays an important role in breast cancer pathogenesis, may be a target for TZD treatment. Moreover, a combination of TZDs and a specific Akt inhibitor may serve as a new approach to target Wnt/beta-catenin directly and via PI3K/Akt action on glycogen synthase-3beta.
Assuntos
Neoplasias da Mama/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Receptores Frizzled/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Tiazolidinedionas/uso terapêutico , Fator de Transcrição 4 , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
p21-Activated kinases (PAKs) are regulators of cell motility and proliferation. PAK activity is regulated in part by phosphoinositide-dependent kinase 1 (PDK1). We hypothesized that reduced PAK activity was involved in the effects of 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl} acetamide (OSU-03012), a previously characterized PDK1 inhibitor derived from celecoxib. In three human thyroid cancer cell lines, OSU-03012 inhibited cell proliferation with reduced AKT phosphorylation by PDK1. OSU-03012 unexpectedly inhibited PAK phosphorylation at lower concentrations than PDK1-dependent AKT phosphorylation in two of the three lines. In cell-free kinase assays, OSU-03012 was shown to inhibit PAK activity and compete with ATP binding. In addition, computer modeling predicted a docking site for OSU-03012 in the ATP binding motif of PAK1. Finally, overexpression of constitutively activated PAK1 partially rescued the ability of motile NPA thyroid cancer cells to migrate during OSU-03012 treatment, suggesting that inhibition of PAK may be involved in the cellular effects of OSU-03012 in these cells. In summary, OSU-03012 is a direct inhibitor of PAK, and inhibition of PAK, either directly or indirectly, may be involved in its biological effects in vitro.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Neoplasias da Glândula Tireoide/enzimologia , Quinases Ativadas por p21/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Celecoxib , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo , Quinases Ativadas por p21/metabolismoRESUMO
Papillary thyroid carcinomas (PTCs) that invade into local structures are associated with a poor prognosis, but the mechanisms for PTC invasion are incompletely defined, limiting the development of new therapies. To characterize biological processes involved in PTC invasion, we analyzed the gene expression profiles of microscopically dissected intratumoral samples from central and invasive regions of seven widely invasive PTCs and normal thyroid tissue by oligonucleotide microarray and performed confirmatory expression and functional studies. In comparison with the central regions of primary PTCs, the invasive fronts overexpressed TGF beta, NFkappaB and integrin pathway members, and regulators of small G proteins and CDC42. Moreover, reduced levels of mRNAs encoding proteins involved in cell-cell adhesion and communication were identified, consistent with epithelial-to-mesenchymal transition (EMT). To confirm that aggressive PTCs were characterized by EMT, 34 additional PTCs were examined for expression of vimentin, a hallmark of EMT. Overexpression of vimentin was associated with PTC invasion and nodal metastasis. Functional, in vitro studies demonstrated that vimentin was required both for the development and maintenance of a mesenchymal morphology and invasiveness in thyroid cancer cells. We conclude that EMT is common in PTC invasion and that vimentin regulates thyroid cancer EMT in vitro.
Assuntos
Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/patologia , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Mesoderma/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Núcleo Celular/metabolismo , Análise por Conglomerados , Humanos , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Vimentina/metabolismoRESUMO
OBJECTIVE: Tumor metastasis is a critical determinant of death from cancer. Metastin, a product of the KiSS-1 gene, is an endogenously expressed metastasis suppressor that is the ligand for G protein-coupled receptor 54 (GPR54), a Gq/11-coupled receptor. In the present study, our goal was to define the basis of GPR54 action using thyroid cancer cells as a model. DESIGN AND RESULTS: We used GPR54-null thyroid cancer cells to create a stable GPR54 overexpression model. Cell growth and cell migration of the GPR54-expressing lines were inhibited by recombinant metastin, and metastin stimulated the protein kinase C, ERK, and phosphatidylinositol-3-kinase pathways. To identify metastin-regulated genes, we performed microarray analyses using RNA isolated from GPR54 stable transfectants before and after 1 and 24 h of metastin stimulation. Consistent increases in expression of the gene encoding myocyte-enriched calcineurin interacting protein 1 (MCIP-1), an inhibitor of calcineurin, were identified and confirmed using real-time RT-PCR and Western blot. Functionally, metastin treatment of GPR54-expressing cells initially increased calcineurin activity, followed by a prolonged reduction in calcineurin activity for 24 and 48 h, consistent with the pattern of MCIP-1 expression. In addition, treatment with cyclosporin A, a calcineurin inhibitor, blocked cell migration. Lymph node metastasis in papillary thyroid cancers demonstrated loss of MCIP-1 expression in comparison with primary tumors. CONCLUSIONS: These data suggest a role for MCIP-1 and calcineurin inhibition in GPR54-mediated metastasis suppression in human cancers.