RESUMO
Previous studies showed that expression of the novel candidate tumor suppressor gene, DEC1 (Deleted in Esophageal Cancer 1), is reduced in esophageal carcinoma and suppresses cancer cell growth in vitro and tumor growth in vivo in nude mice. This study shows that DEC1 gene expression was downregulated in 100% of 16 esophageal squamous cell carcinoma (ESCC) cell lines and 52 and 45%, respectively, of esophageal tumor specimens from Hong Kong and a high-risk ESCC region of Henan, China. Using epitope tagging, the DEC1 protein was localized to both the cytoplasm and nucleus of the cell. In 3D Matrigel culture, no significant difference in colony numbers formed was observed for DEC1 stable transfectants, as compared to vector-alone transfectant controls. However, significantly smaller colony sizes were observed for the DEC1 transfectants. In in vitro cell migration, invasion and soft agar assays of DEC1 transfectants, only the soft agar assay showed statistically significant differences in colony numbers with the vector-alone controls, indicating that DEC1 may be involved in anchorage-independent cell growth. In addition, the global gene expression affected by DEC1 in tumor-suppressive stable transfectants was investigated using cDNA oligonucleotide microarray hybridization. Three candidate genes, TFPI-2, GDF15 and DUSP6, were identified through this approach; they are downregulated in tumor segregants of DEC1 stable transfectants, ESCC cell lines and esophageal tumors and have a potential role in tumor growth and progression. These studies show that DEC1 is involved in esophageal cancer development and help elucidate its functional role in tumor development.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Perfilação da Expressão Gênica , Proteínas Supressoras de Tumor/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Movimento Celular , Epitélio/metabolismo , Epitélio/patologia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Melanoma is the major cause of skin cancer deaths and melanoma incidence doubles every 10 to 20 years. However, little is known about melanoma pathway aberrations. Here we applied the robust Gene Identification Signature Paired End diTag (GIS-PET) approach to investigate the melanoma transcriptome and characterize the global pathway aberrations. METHODS: GIS-PET technology directly links 5' mRNA signatures with their corresponding 3' signatures to generate, and then concatenate, PETs for efficient sequencing. We annotated PETs to pathways of KEGG database and compared the murine B16F1 melanoma transcriptome with three non-melanoma murine transcriptomes (Melan-a2 melanocytes, E14 embryonic stem cells, and E17.5 embryo). Gene expression levels as represented by PET counts were compared across melanoma and melanocyte libraries to identify the most significantly altered pathways and investigate the expression levels of crucial cancer genes. RESULTS: Melanin biosynthesis genes were solely expressed in the cells of melanocytic origin, indicating the feasibility of using the PET approach for transcriptome comparison. The most significantly altered pathways were metabolic pathways, including upregulated pathways: purine metabolism, aminophosphonate metabolism, tyrosine metabolism, selenoamino acid metabolism, galactose utilization, nitrobenzene degradation, and bisphenol A degradation; and downregulated pathways: oxidative phosphorylation, ATPase synthesis, TCA cycle, pyruvate metabolism, and glutathione metabolism. The downregulated pathways concurrently indicated a slowdown of mitochondrial activities. Mitochondrial permeability was also significantly altered, as indicated by transcriptional activation of ATP/ADP, citrate/malate, Mg++, fatty acid and amino acid transporters, and transcriptional repression of zinc and metal ion transporters. Upregulation of cell cycle progression, MAPK, and PI3K/Akt pathways were more limited to certain region(s) of the pathway. Expression levels of c-Myc and Trp53 were also higher in melanoma. Moreover, transcriptional variants resulted from alternative transcription start sites or alternative polyadenylation sites were found in Ras and genes encoding adhesion or cytoskeleton proteins such as integrin, beta-catenin, alpha-catenin, and actin. CONCLUSION: The highly correlated results unmistakably point to a systematic downregulation of mitochondrial activities, which we hypothesize aims to downgrade the mitochondria-mediated apoptosis and the dependency of cancer cells on angiogenesis. Our results also demonstrate the advantage of using the PET approach in conjunction with KEGG database for systematic pathway analysis.
Assuntos
Perfilação da Expressão Gênica/métodos , Melaninas/biossíntese , Melanoma/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias Cutâneas/genética , Animais , Apoptose/genética , Vias Biossintéticas/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/genética , Melaninas/genética , Melanoma/patologia , Camundongos , RNA Mensageiro/genética , Elementos Reguladores de Transcrição/genética , Sensibilidade e Especificidade , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
Using oligonucleotide microarray analysis, THY1, mapping close to a previously defined 11q22-23 nasopharyngeal carcinoma (NPC) critical region was identified as showing consistent downregulated expression in the tumour segregants, as compared to their parental tumour-suppressing microcell hybrids (MCHs). Gene expression and protein analyses show that THY1 was not expressed in the NPC HONE1 recipient cells, tumour segregants, and other NPC cell lines; THY1 was exclusively expressed in the non-tumourigenic MCHs. The mechanism of THY1 gene inactivation in these cell lines was attributed to hypermethylation. Clinical study showed that in 65% of NPC specimens there was either downregulation or loss of THY1 gene expression. Using a tissue microarray and immunohistochemical staining, 44% of the NPC cases showed downregulated expression of THY1 and 9% lost THY1 expression. The frequency of THY1 downregulated expression in lymph node metastatic NPC was 63%, which was significantly higher than in the primary tumour (33%). After transfection of THY1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. These findings suggest that THY1 is a good candidate tumour suppressor gene in NPC, which is significantly associated with lymph node metastases.
Assuntos
Carcinoma/genética , Neoplasias Nasofaríngeas/genética , Antígenos Thy-1/genética , Carcinoma/patologia , Cromossomos Humanos Par 11 , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Humanos , Linfonodos/patologia , Metástase Linfática/genética , Análise em Microsséries , Neoplasias Nasofaríngeas/patologia , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antígenos Thy-1/metabolismo , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
Estrogen receptors ERalpha and ERbeta, members of the nuclear receptor superfamily, exert profound effects on the gene expression and biological response programs of their target cells. Herein, we explore the dynamic interplay between these two receptors in their selection of chromatin binding sites when present separately or together in MCF-7 breast cancer cells. Treatment of cells (containing ERalpha only, ERbeta only, or ERalpha and ERbeta) with estradiol or ER subtype-selective ligands was followed by chromatin immunoprecipitation analysis with a custom-designed tiling array for ER binding sites across the genome to examine the effects of ligand-occupied and unoccupied ERalpha and ERbeta on chromatin binding. There was substantial overlap in binding sites for these estradiol-liganded nuclear receptors when present alone, but many fewer sites were shared when both ERs were present. Each ER restricted the binding site occupancy of the other, with ERalpha generally being dominant. Binding sites of both receptors were highly enriched in estrogen response element motifs, but when both ERs were present, ERalpha displaced ERbeta, shifting it into new sites less enriched in estrogen response elements. Binding regions of the two ERs also showed differences in their enrichments for other transcription factor binding motifs. Studies with ER subtype-specific ligands revealed that it was the liganded subtype that principally determined the spectrum of chromatin binding. These findings highlight the dynamic interplay between the two ERs in their selection of chromatin binding sites, with competition, restriction, and site shifting having important implications for the regulation of gene expression by these two nuclear receptors.
Assuntos
Cromatina/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Ligação Competitiva , Neoplasias da Mama , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina/métodos , Estradiol/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Ligantes , Oxazóis/farmacologia , Fenóis , Pirazóis/farmacologia , RNA Interferente Pequeno , Elementos de Resposta , Fatores de Transcrição/metabolismoRESUMO
Aggressive forms of cancer are often defined by recurrent chromosomal alterations, yet in most cases, the causal or contributing genetic components remain poorly understood. Here, we utilized microarray informatics to identify candidate oncogenes potentially contributing to aggressive breast cancer behavior. We identified the Rab-coupling protein RCP (also known as RAB11FIP1), which is located at a chromosomal region frequently amplified in breast cancer (8p11-12) as a potential candidate. Overexpression of RCP in MCF10A normal human mammary epithelial cells resulted in acquisition of tumorigenic properties such as loss of contact inhibition, growth-factor independence, and anchorage-independent growth. Conversely, knockdown of RCP in human breast cancer cell lines inhibited colony formation, invasion, and migration in vitro and markedly reduced tumor formation and metastasis in mouse xenograft models. Overexpression of RCP enhanced ERK phosphorylation and increased Ras activation in vitro. As these results indicate that RCP is a multifunctional gene frequently amplified in breast cancer that encodes a protein with Ras-activating function, we suggest it has potential importance as a therapeutic target. Furthermore, these studies provide new insight into the emerging role of the Rab family of small G proteins and their interacting partners in carcinogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/genética , Genes ras , Proteínas de Membrana/genética , Oncogenes , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Neoplasias da Mama/etiologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Biologia Computacional , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Genes erbB-2 , Humanos , Imuno-Histoquímica , Proteínas de Membrana/análise , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Interferência de RNA , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
Functional studies to identify the potential role of a chromosome 3p14-21 gene, protein tyrosine phosphatase receptor type G (PTPRG), were performed. PTPRG was identified as a candidate tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) by differential gene profiling of tumorigenic and nontumorigenic NPC chromosome 3 microcell hybrids (MCH). Down-regulation of this gene was found in tumor segregants when compared with their corresponding tumor-suppressive MCHs, as well as in NPC cell lines and tumor biopsies. Promoter hypermethylation and loss of heterozygosity were found to be important mechanisms contributing to PTPRG silencing. PTPRG overexpression in NPC cell lines induces growth suppression and reduced anchorage-independent growth in vitro. This is the first study to use a tetracycline-responsive vector expression system to study PTPRG stable transfectants. Results indicate its ability to induce significant tumor growth suppression in nude mice under conditions activating transgene expression. These studies now provide functional evidence indicating critical interactions of PTPRG in the extracellular matrix milieu induce cell arrest and changes in cell cycle status. This is associated with inhibition of pRB phosphorylation through down-regulation of cyclin D1. These novel findings enhance our current understanding of how PTPRG may contribute to tumorigenesis.
Assuntos
Carcinoma/genética , Neoplasias Nasofaríngeas/genética , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/fisiologia , Animais , Células Cultivadas , Cromossomos Humanos Par 3 , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Genes cdc/fisiologia , Humanos , Perda de Heterozigosidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genéticaRESUMO
Previous studies transferring an intact chromosome 11 into HONE1 cells demonstrated the functional significance of chromosome regions, 11q13 and 11q22-23, in nasopharyngeal carcinoma (NPC) development. In our study the 11q22-23 region was comprehensively re-investigated by detailed microsatellite and single nucleotide polymorphism genotyping and by fluorescence in situ hybridization to map precisely the regions containing tumor suppressive activity. We observed 3 chromosomal intervals within 11q22-23 that were commonly lost in the tumor segregants derived from HONE1/chromosome 11 hybrids. One critical region of 0.36 Mb was mapped near the marker D11S2000 and a second 0.44 Mb region was located around the markers D11S1300 and D11S1391. In a third region high allelic loss was also observed at marker D11S4484, where a newly cloned tumor suppressor gene, TSLC1 (tumor suppressor in lung cancer 1), is located. The gene expression analysis showed absence or low expression levels of TSLC1 mRNA in 4 highly tumorigenic NPC cell lines. In addition, the methylation study results show that the TSLC1 promoter region was hypermethylated in all 4 NPC cell lines and re-expression of the gene occurs in HONE1 cells after 5-aza-2'-deoxycytidine treatment. Hence, the mode of silencing of this candidate TSG in NPC may be attributed to promoter hypermethylation. We have obtained functional evidence for multiple critical tumor suppressive regions in 11q22-23 by fine deletion mapping and for inactivation of TSLC1 being one of these candidate TSGs in NPC development.