RESUMO
PURPOSE: Ectopic expression of squamous cell carcinoma-related oncogene (SCCRO or DCUN1D1) in NIH-3T3 cells induces invasion in vitro and produces highly invasive xenografts in nude mice with a propensity for regional lymphatical metastasis. The aim of this study was to identify the molecular mechanism underlying SCCRO-induced invasion and metastasis. EXPERIMENTAL DESIGN: The molecular mechanism of SCCRO-mediated effects on matrix metalloproteinase-2 (MMP2) levels and activity were assessed using a combination of cell biological and molecular methods, including real-time PCR, reporter assay, RNA interference, and chromatin immunoprecipitation assay. Tumor specimens from primary upper aerodigestive tract carcinomas (n = 89) were examined for levels of SCCRO, MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 mRNA by real-time PCR. RESULTS: Overexpression of SCCRO increases MMP2 levels and activity, which is required for SCCRO-induced invasion. Modified McKay assays reveal that SCCRO does not bind to the MMP2 promoter, suggesting that its transcriptional effects are indirect. Deletion or mutation of the activator protein-2 (AP2) and p53 binding element within the MMP2 promoter abrogates SCCRO-driven activation. Ectopic expression of SCCRO increases AP2 levels and promotes the binding of p53 to the MMP2 promoter. Consistent with these findings, SCCRO and MMP2 are coexpressed (P<0.0001; r(2)=0.58; 95% confidence interval, 0.46-0.69) in primary (upper aerodigestive tract) carcinomas (n=89), and this coexpression is associated with an increased prevalence of regional nodal metastasis (P=0.04; relative risk, 1.53). CONCLUSIONS: SCCRO-induced invasion involves activation of MMP2 transcription in an AP2- and p53-dependent manner. SCCRO is a potential marker for metastatic progression in affected cancers.
Assuntos
Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Matriz Extracelular/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Proteínas Oncogênicas/genética , Oncogenes , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Feminino , Genes p53 , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Proteínas , Proteínas Proto-Oncogênicas , Fator de Transcrição AP-2/fisiologiaRESUMO
PURPOSE: Amplification at chromosome 3q26.3 is a common and crucial event in head and neck squamous cell carcinoma (HNSCC), impacting significantly on tumor progression and clinical outcome. Two novel oncogenes, namely squamous cell carcinoma (SCC)-related oncogene (SCCRO) and PIK3CA (gene encoding phosphatidylinositol-3 kinase catalytic alpha-polypeptide), have been identified as targets of 3q26.3 amplification. This study aimed to delineate the role of SCCRO and PIK3CA in the pathogenesis of oral tongue SCC. EXPERIMENTAL DESIGN: The association between gene copy number for SCCRO and PIK3CA measured by fluorescence in situ hybridization and level of mRNA expression quantitated by real-time reverse transcription-PCR was assessed in a panel of human HNSCC cell lines. In addition, gene expression in 49 consecutive primary SCCs of the oral tongue was determined and correlated with clinicopathological characteristics and outcome. RESULTS: The mRNA level of SCCRO and PIK3CA was significantly correlated to the gene copy number in nine HNSCC cell lines. In addition, the expression level of SCCRO and PIK3CA was significantly greater in malignant tissues compared with those in histologically normal mucosae (2.17- and 2.46-fold, respectively; P < 0.001). Matched tumor normal control analysis revealed that 24.5 and 69.4% of patients expressed high levels of SCCRO and PIK3CA, respectively. Univariate analyses demonstrated that SCCRO overexpression correlated with nodal metastases (P = 0.05) and advanced stage (P = 0.02), whereas PIK3CA overexpression was associated with vascular invasion (P = 0.04). Only SCCRO overexpression was associated with disease-specific (P = 0.04) and overall survival (P = 0.02). Furthermore, SCCRO overexpression remained an independent predictor for cervical nodal metastasis on multivariate regression analysis (chi(2) likelihood ratio = 4.38; P = 0.04). CONCLUSIONS: Although both SCCRO and PIK3CA may play a role in the pathogenesis of oral tongue SCC through amplification at 3q26, SCCRO appears to be a significant predictor of regional metastasis and may be a marker for tumor aggressiveness and clinical outcome.
Assuntos
Carcinoma de Células Escamosas/genética , Oncogenes/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Neoplasias da Língua/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Feminino , Dosagem de Genes , Humanos , Hibridização in Situ Fluorescente , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Subunidades Proteicas , Neoplasias da Língua/enzimologia , Neoplasias da Língua/patologiaAssuntos
Cristalino/metabolismo , Tiorredoxinas/genética , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Catarata/etiologia , Catarata/genética , Feminino , Regulação da Expressão Gênica , Humanos , Cristalino/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estresse Oxidativo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiorredoxina Dissulfeto Redutase/isolamento & purificação , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismoRESUMO
Chromosomal amplification at 3q is common to multiple human cancers, but has a specific predilection for squamous cell carcinomas (SCC) of mucosal origin. We identified and characterized a novel oncogene, SCC-related oncogene (SCCRO), which is amplified along the 3q26.3 region in human SCC. Amplification and overexpression of SCCRO in these tumors correlate with poor clinical outcome. The importance of SCCRO amplification in malignant transformation is established by the apoptotic response to short hairpin RNA against SCCRO, exclusively in cancer cell lines carrying SCCRO amplification. The oncogenic potential of SCCRO is underscored by its ability to transform fibroblasts (NIH-3T3 cells) in vitro and in vivo. We show that SCCRO regulates Gli1--a key regulator of the hedgehog (HH) pathway. Collectively, these data suggest that SCCRO is a novel component of the HH signaling pathway involved in the malignant transformation of squamous cell lineage.
Assuntos
Carcinoma de Células Escamosas/genética , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Animais , Apoptose/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Cromossomos Humanos Par 3/genética , Clonagem Molecular , Feminino , Proteínas Hedgehog/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células NIH 3T3/patologia , Células NIH 3T3/transplante , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Proteínas Oncogênicas/fisiologia , Proteínas , Proteínas Proto-Oncogênicas , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteína GLI1 em Dedos de ZincoRESUMO
BACKGROUND: Squamous cell carcinoma related oncogene expression (SCCRO) correlates with vascular endothelial growth factor-A expression. This data is validated in human lung tumors and provides a putative pathway for angiogenesis in a subset of squamous cell carcinomas. Squamous cell carcinoma related oncogene is a novel oncogene identified by positional cloning of a recurrent amplification at 3q26.3. It is over-expressed in 39.8% of lung, head and neck, cervical, and ovarian carcinomas. SCCRO imparts an aggressive phenotype to affected cancers, which may be related to increased angiogenesis due to SCCRO expression. Our previous work has demonstrated a link between SCCRO and vascular endothelial growth factor-A (VEGF-A) expression in vitro, suggesting a mechanism for SCCRO-induced angiogenesis. The present study aims to confirm and validate this link between SCCRO and VEGF-A expression in an ex vivo human tumor cohort. METHODS: Fresh tissue was collected at Memorial Sloan-Kettering Cancer Center from 34 patients undergoing primary resection of lung squamous cell carcinomas. RNA was extracted from this tissue, reverse-transcribed, and real-time polymerase chain reaction (RT-PCR) was carried out using a BioRad iQ iCycler with SYBR green fluorophore. Microvessel counting was performed on the tumor specimens using CD34 immunohistochemistry. RESULTS: The expression of both SCCRO and VEGF-A mRNA varies widely in both tumor and normal tissue. SCCRO and VEGF-A co-expression was significantly correlated (R(2) = 0.63; P < 0.032). Microvessel counts were not associated with expression of SCCRO or VEGF-A and failed to significantly predict survival. VEGF-A expression in this patient group is a predictor of overall survival (P < 0.032). CONCLUSIONS: VEGF-A expression correlates with SCCRO expression in these primary human lung squamous cell carcinomas and is a predictor of clinical behavior. This data supports the association of SCRRO and VEGF-A in the induction of angiogenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neovascularização Patológica/genética , Oncogenes/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Idoso , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Carcinoma de Células Escamosas/fisiopatologia , Estudos de Coortes , Humanos , Neoplasias Pulmonares/fisiopatologia , Fenótipo , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Análise de SobrevidaRESUMO
Interactions between the p53 and PI3K/AKT pathways play a significant role in the determination of cell death/survival. In benign cells these pathways are interrelated through the transcriptional regulation of PTEN by p53, which is required for p53-mediated apoptosis. PTEN exerts its effects by decreasing the phosphorylated AKT fraction, thereby diminishing prosurvival activities. However, the link between these pathways in cancer is not known. In this study, PIK3CA, encoding the p110alpha catalytic subunit of PI3K, is identified as an oncogene involved in upper aerodigestive tract (UADT) carcinomas. Simultaneous abnormalities in both pathways are rare in primary tumors, suggesting that amplification of PIK3CA and mutation of p53 are mutually exclusive events and either event is able to promote a malignant phenotype. Moreover, the negative effect of p53 induction on cell survival involves the transcriptional inhibition of PIK3CA that is independent of PTEN activity, as PTEN is not expressed in the primary tumors. Conversely, constitutive activation of PIK3CA results in resistance to p53-related apoptosis in PTEN deficient cells. Thus, p53 regulates cell survival by inhibiting the PI3K/AKT prosurvival signal independent of PTEN in epithelial tumors. This inhibition is required for p53-mediated apoptosis in malignant cells.