RESUMO
Lysyl hydroxylase 2 (LH2) regulates intermolecular cross-linking of collagen molecules. Accumulation of LH2-modified collagen, which is highly stable and resistant to collagenase cleavage, is one cause of fibrosis. We previously demonstrated that conventional LH2 knockout mice showed embryonic lethality. Here we established LH2 conditional knockout mice using a tamoxifen-inducible Cre system. Morphological analysis of LH2-deficient fibroblasts by microscopy showed a dramatic increase in the number of filopodia, the finger-like cell surface projections that enable cell movement. The tips and leading edges of these filopodia exhibited up-regulated expression of Myosin-X (Myo10), a regulator of filopodial integrity. Wound healing assays demonstrated that migration of LH2-deficient cells was significantly faster than that of control cells. Gene expression profiling data also supported this phenotype. Together these findings indicate that LH2 deficiency may prevent fibrosis through decreased accumulation of LH2-cross-linked collagen, and that fibroblasts with faster migration contribute to enhanced wound healing activity. In conclusion, our cellular models provide evidence that LH2 deficiency plays a critical role in cell migration mediated through filopodia formation. Understanding the precise role of this phenotype in LH2-deficient cells may be helpful to define the pathogenesis of fibrosis. As such, detailed analyses of fibrosis and wound healing using LH2-deficient mouse models are needed.
Assuntos
Fibroblastos/enzimologia , Miosinas/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pseudópodes/enzimologia , Animais , Movimento Celular , Colágeno/genética , Colágeno/metabolismo , Fibroblastos/citologia , Fibrose , Regulação da Expressão Gênica , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Miosinas/metabolismo , Fenótipo , Cultura Primária de Células , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/deficiência , Pseudópodes/ultraestrutura , Cicatrização/genéticaRESUMO
Radiotherapy is commonly used to treat oral squamous cell carcinoma (OSCC), and radioresistance is a critical factor resulting in poor outcomes. Several genes have been reported to be therapeutic targets for radioresistance; however, the involvement of chromatin accessibility in radioresistance has not been clarified in OSCC cells. Accordingly, in this study, we evaluated chromatin accessibility in radioresistant (HSC-3) and radiosensitive (KOSC-2) cells, identified from nine OSCC cell lines using clonogenic survival assays after irradiation. Chromatin accessibility in radioresistant OSCC cells was assessed using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Gene expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunoblot analysis. Viability was assessed by MTS assay. We found 1273 peaks (open chromatin regions by ATAC-seq) related to 8 Gy irradiation in HSC-3 but not KOSC-2 cells, among which 235 genes located around the chromatin open peaks were identified by ChIPpeakAnno analysis. Subsequently, 12 genes were selected as signal transduction-related genes by Gene Ontology analysis, and gene expression was confirmed by RT-qPCR. Among these genes, adenylate cyclase 2 (ADCY2) was significantly upregulated after treatment with irradiation in HSC-3 but not KOSC-2 cells. To further evaluate ADCY2 function in radioresistant cells, we performed ADCY2 knockdown by transfection of HSC-3 cells with small interfering RNA (siADCY2). Cell viability after irradiation was significantly decreased in siADCY2-transfected cells compared with that in control cells. These results suggested that ADCY2 expression was related to the open chromatin region in radioresistant OSCC cells and that ADCY2 may have therapeutic efficacy when used in combination with radiotherapy in patients with OSCC.
RESUMO
Advanced-stage oral squamous cell carcinoma (OSCC) patients are treated with combination therapies, such as surgery, radiation, chemotherapy, and immunotherapy. However, OSCC cells acquire resistance to these treatments, resulting in local recurrence and distant metastasis. The identification of genes involved in drug resistance is essential for improving the treatment of this disease. In this study, we applied chromatin immunoprecipitation sequencing (ChIP-Seq) to profile active enhancers. For that purpose, we used OSCC cell lines that had been exposed to cetuximab for a prolonged period. In total, 64 chromosomal loci were identified as active super-enhancers (SE) according to active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) ChIP-Seq. In addition, a total of 131 genes were located in SE regions, and 34 genes were upregulated in OSCC tissues by TCGA-OSCC analysis. Moreover, high expression of four genes (C9orf89; p = 0.035, CENPA; p = 0.020, PISD; p = 0.0051, and TRAF2; p = 0.0075) closely predicted a poorer prognosis for OSCC patients according to log-rank tests. Increased expression of the four genes (mRNA Z-score ≥ 0) frequently co-occurred in TCGA-OSCC analyses. The high and low expression groups of the four genes showed significant differences in prognosis, suggesting that there are clear differences in the pathways based on the underlying gene expression profiles. These data indicate that potential stratified therapeutic strategies could be used to overcome resistance to drugs (including cetuximab) and further improve responses in drug-sensitive patients.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Cetuximab , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Humanos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genéticaRESUMO
Recently, our studies revealed that some passenger strands of microRNAs (miRNAs) were closely involved in cancer pathogenesis. Analysis of miRNA expression signatures showed that the expression of miR-30e-3p (the passenger strand of pre-miR-30e) was significantly downregulated in cancer tissues. In this study, we focused on miR-30e-3p (the passenger strand of pre-miR-30e). We addressed target genes controlled by miR-30e-3p that were closely associated with the molecular pathogenesis of head and neck squamous cell carcinoma (HNSCC). Ectopic expression assays demonstrated that the expression of miR-30e-3p attenuated cancer cell malignant phenotypes (e.g., cell proliferation, migration, and invasive abilities). Our analysis of miR-30e-3p targets revealed that 11 genes (ADA, CPNE8, C14orf126, ERGIC2, HMGA2, PLS3, PSMD10, RALB, SERPINE1, SFXN1, and TMEM87B) were expressed at high levels in HNSCC patients. Moreover, they significantly predicted the short survival of HNSCC patients based on 5-year overall survival rates (p < 0.05) in The Cancer Genome Atlas (TCGA). Among these targets, SERPINE1 was found to be an independent prognostic factor for patient survival (multivariate Cox regression; hazard ratio = 1.6078, p < 0.05). Aberrant expression of SERPINE1 was observed in HNSCC clinical samples by immunohistochemical analysis. Functional assays by targeting SERPINE1 expression revealed that the malignant phenotypes (e.g., proliferation, migration, and invasion abilities) of HNSCC cells were suppressed by the silencing of SERPINE1 expression. Our miRNA-based approach will accelerate our understanding of the molecular pathogenesis of HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço , MicroRNAs , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genéticaRESUMO
We identified the microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC) tissues by RNA sequencing, in which 168 miRNAs were significantly upregulated, including both strands of the miR-31 duplex (miR-31-5p and miR-31-3p). The aims of this study were to identify networks of tumor suppressor genes regulated by miR-31-5p and miR-31-3p in HNSCC cells. Our functional assays showed that inhibition of miR-31-5p and miR-31-3p attenuated cancer cell malignant phenotypes (cell proliferation, migration, and invasion), suggesting that they had oncogenic potential in HNSCC cells. Our in silico analysis revealed 146 genes regulated by miR-31 in HNSCC cells. Among these targets, the low expression of seven genes (miR-31-5p targets: CACNB2 and IL34; miR-31-3p targets: CGNL1, CNTN3, GAS7, HOPX, and PBX1) was closely associated with poor prognosis in HNSCC. According to multivariate Cox regression analyses, the expression levels of five of those genes (CACNB2: p = 0.0189; IL34: p = 0.0425; CGNL1: p = 0.0014; CNTN3: p = 0.0304; and GAS7: p = 0.0412) were independent prognostic factors in patients with HNSCC. Our miRNA signature and miRNA-based approach will provide new insights into the molecular pathogenesis of HNSCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias de Cabeça e Pescoço/patologia , MicroRNAs/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Biomarcadores Tumorais/genética , Proliferação de Células , Perfilação da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
In humans, the coronin family is composed of seven proteins containing WD-repeat domains that regulate actin-based cellular processes. Some members of the coronin family are closely associated with cancer cell migration and invasion. The Cancer Genome Atlas (TCGA) analysis revealed that CORO1C, CORO2A, and CORO7 were significantly upregulated in oral squamous cell carcinoma (OSCC) tissues (p < 0.05). Moreover, the high expression of CORO2A was significantly predictive of the 5-year survival rate of patients with OSCC (p = 0.0203). Overexpression of CORO2A was detected in OSCC clinical specimens by immunostaining. siRNA-mediated knockdown of CORO2A suppressed cancer cell migration and invasion abilities. Furthermore, we investigated the involvement of microRNAs (miRNAs) in the molecular mechanism underlying CORO2A overexpression in OSCC cells. TCGA analysis confirmed that tumor-suppressive miR-125b-5p and miR-140-5p were significantly downregulated in OSCC tissues. Notably, these miRNAs bound directly to the 3'-UTR of CORO2A and controlled CORO2A expression in OSCC cells. In summary, we found that aberrant expression of CORO2A facilitates the malignant transformation of OSCC cells, and that downregulation of tumor-suppressive miRNAs is involved in CORO2A overexpression. Elucidation of the interaction between genes and miRNAs will help reveal the molecular pathogenesis of OSCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Movimento Celular , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Proteínas dos Microfilamentos/metabolismo , Neoplasias Bucais/patologia , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células , Humanos , Proteínas dos Microfilamentos/genética , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Invasividade Neoplásica , Prognóstico , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
We newly generated an RNA-sequencing-based microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC). Analysis of the signature revealed that both strands of some miRNAs, including miR-139-5p (the guide strand) and miR-139-3p (the passenger strand) of miR-139, were downregulated in HNSCC tissues. Analysis of The Cancer Genome Atlas confirmed the low expression levels of miR-139 in HNSCC. Ectopic expression of these miRNAs attenuated the characteristics of cancer cell aggressiveness (e.g., cell proliferation, migration, and invasion). Our in silico analyses revealed a total of 28 putative targets regulated by pre-miR-139 (miR-139-5p and miR-139-3p) in HNSCC cells. Of these, the GNA12 (guanine nucleotide-binding protein subunit alpha-12) and OLR1 (oxidized low-density lipoprotein receptor 1) expression levels were identified as independent factors that predicted patient survival according to multivariate Cox regression analyses (p = 0.0018 and p = 0.0104, respectively). Direct regulation of GNA12 and OLR1 by miR-139-3p in HNSCC cells was confirmed through luciferase reporter assays. Moreover, overexpression of GNA12 and OLR1 was detected in clinical specimens of HNSCC through immunostaining. The involvement of miR-139-3p (the passenger strand) in the oncogenesis of HNSCC is a new concept in cancer biology. Our miRNA-based strategy will increase knowledge on the molecular pathogenesis of HNSCC.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , MicroRNAs/genética , Oncogenes , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Perfilação da Expressão Gênica , Neoplasias de Cabeça e Pescoço/patologia , Humanos , MicroRNAs/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Cetuximab, an inhibitor of the epidermal growth factor receptor that is used widely to treat human cancers including oral squamous cell carcinoma (OSCC), has characteristic side effects of skin rash and hypomagnesemia. However, the mechanisms of and therapeutic agents for skin rashes and hypomagnesemia are still poorly understood. Our gene expression profiling analyses showed that cetuximab activates the p38 MAPK pathways in human skin cells (human keratinocyte cell line [HaCaT]) and inhibits c-Fos-related signals in human embryonic kidney cells (HEK293). We found that while the p38 inhibitor SB203580 inhibited the expression of p38 MAPK targets in HaCaT cells, flavagline reactivated c-Fos-related factors in HEK293 cells. It is noteworthy that, in addition to not interfering with the effect of cetuximab by both compounds, flavagline has additive effect for OSCC growth inhibition in vivo. Collectively, our results indicate that combination of cetuximab and these potential therapeutic agents for cetuximab-related toxicities could be a promising therapeutic strategy for patients with OSCC.
Assuntos
Antineoplásicos Imunológicos/efeitos adversos , Carcinoma de Células Escamosas/tratamento farmacológico , Cetuximab/efeitos adversos , Inibidores do Crescimento/uso terapêutico , Imidazóis/uso terapêutico , Neoplasias Bucais/tratamento farmacológico , Piridinas/uso terapêutico , Animais , Carcinoma de Células Escamosas/complicações , Linhagem Celular Tumoral , Quimioterapia Combinada , Receptores ErbB/antagonistas & inibidores , Exantema/induzido quimicamente , Exantema/genética , Exantema/prevenção & controle , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Inibidores do Crescimento/efeitos adversos , Inibidores do Crescimento/antagonistas & inibidores , Células HEK293 , Humanos , Hipercalciúria/induzido quimicamente , Hipercalciúria/genética , Hipercalciúria/prevenção & controle , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Bucais/complicações , Neoplasias Bucais/genética , Nefrocalcinose/induzido quimicamente , Nefrocalcinose/genética , Nefrocalcinose/prevenção & controle , Erros Inatos do Transporte Tubular Renal/induzido quimicamente , Erros Inatos do Transporte Tubular Renal/genética , Erros Inatos do Transporte Tubular Renal/prevenção & controle , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tripartite motif family-like 2 (TRIML2), a member of the TRIM proteins family, is closely related to Alzheimer's disease, however, no studies of TRIML2 have been published in the cancer research literature. In the current study, we investigated the expression level of TRIML2 and its molecular mechanisms in human oral squamous cell carcinoma (OSCC); reverse transcriptase-quantitative polymerase chain reaction, immunoblot analysis, and immunohistochemistry showed that TRIML2 is up-regulated significantly in OSCCs in vitro and in vivo. TRIML2 knockdown OSCC cells showed decreased cellular proliferation by cell-cycle arrest at G1 phase that resulted from down-regulation of CDK4, CDK6, and cyclin D1 and up-regulation of p21Cip1 and p27Kip1. Surprisingly, resveratrol, a polyphenol, led to not only down-regulation of TRIML2 but also cell-cycle arrest at G1 phase similar to TRIML2 knockdown experiments. Taken together, we concluded that TRIML2 might play a significant role in tumoral growth and that resveratrol may be a new drug for treating OSCC by interfering with TRIML2 function.
Assuntos
Proteínas de Transporte/genética , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Proteínas de Transporte/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resveratrol/farmacologiaRESUMO
Unc-93 homolog B1 (UNC93B1), a transmembrane protein, is correlated with immune diseases, such as influenza, herpes simplex encephalitis, and the pathogenesis of systemic lupus erythematosus; however, the role of UNC93B1 in cancers including human oral squamous cell carcinomas (OSCCs) remains unknown. In the current study, we investigated the UNC93B1expression level in OSCCs using quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. Our data showed that UNC93B1 mRNA and protein expressions increased markedly (pâ¯<â¯0.05) in OSCCs compared with normal cells and tissues and that high expression of UNC93B1 in OSCCs was related closely to tumoral size. UNC93B1 knockdown (shUNC93B1) OSCC cells showed decreased cellular proliferation by cell-cycle arrest in the G1 phase with up-regulation of p21Cip1 and down-regulation of CDK4, CDK6, cyclin D1, and cyclin E. We also found that granulocyte macrophage colony-stimulating factor (GM-CSF) was down-regulated significantly (pâ¯<â¯0.05) in shUNC93B1 OSCC cells. Moreover, inactivation of GM-CSF using neutralization antibody led to cell-cycle arrest at the G1 phase similar to the phenotype of the shUNC93B1 cells. The current findings indicated that UNC93B1 might play a crucial role in OSCC by controlling the secretion level of GM-CSF involved in tumoral growth and could be a potential therapeutic target for OSCCs.
Assuntos
Carcinoma de Células Escamosas/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismoRESUMO
Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2-/-) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2-/- mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway.
Assuntos
Perda do Embrião/genética , Embrião de Mamíferos/patologia , Estresse do Retículo Endoplasmático , Cardiopatias Congênitas/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Animais , Apoptose , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Perda do Embrião/patologia , Embrião de Mamíferos/metabolismo , Coração/embriologia , Cardiopatias Congênitas/patologia , Camundongos , Camundongos KnockoutRESUMO
Multiple coagulation factor deficiency protein 2 (MCFD2), a binding partner of lectin mannose binding 1 (LMAN1), causes combined deficiencies of coagulation factors V and VIII. MCFD2 function in inherited hematologic disorders is well elucidated; however, little is known about its role in human tumorigenesis. The aim of the current study was to investigate the states of MCFD2 in oral squamous cell carcinoma (OSCC). The expression of MCFD2 was up-regulated significantly in all cell lines examined. Evaluation of the cellular functions associated with tumoral metastasis showed that MCFD2 knockdown (shMCFD2) cells exhibited significantly lower cellular invasiveness and migration and higher cellular adhesion compared with shControl cells. Of note, shMCFD2 cells also showed weak immunoreactivity of LMAN1 and a lower secretion level of galactoside-binding soluble 3 binding protein (LGALS3BP). In addition to in vitro validation, clinical data on 70 patients with OSCC indicated that state of MCFD2 expression level is associated with regional lymph node metastasis. Altogether, we have demonstrated that MCFD2 promotes cancer metastasis by regulating LMAN1 and LGALS3BP expression levels. Hence, MCFD2 may represent a promising candidate for a novel therapeutic target for patients with metastatic OSCCs.
Assuntos
Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Glicoproteínas/genética , Lectinas de Ligação a Manose/genética , Proteínas de Membrana/genética , Neoplasias Bucais/genética , Mutação de Sentido Incorreto/genética , Metástase Neoplásica/genética , Proteínas de Transporte Vesicular/genética , Cálcio/metabolismo , Carcinoma de Células Escamosas/genética , HumanosRESUMO
Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs.
Assuntos
Carcinogênese/genética , Carcinoma de Células Escamosas/patologia , Ciclo Celular/efeitos dos fármacos , Lipase Lipoproteica/metabolismo , Neoplasias Bucais/patologia , Animais , Carcinoma de Células Escamosas/enzimologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Lipase Lipoproteica/antagonistas & inibidores , Lipase Lipoproteica/genética , Camundongos , Camundongos Nus , Neoplasias Bucais/enzimologia , Orlistate/farmacologia , Cultura Primária de Células , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1) forms a complex with histone deacetylase (HDAC); however, the relevance of DNTTIP1 in cancer remains unknown. The aim of this study was to examine DNTTIP1 expression and its functional mechanisms in oral squamous cell carcinomas (OSCCs). DNTTIP1 expression was analyzed by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting analysis, and immunohistochemistry. The expression of DNTTIP1 was upregulated significantly in vitro and in vivo, and in patients with OSCC in whom DNTTIP1 was overexpressed and the expression level was correlated significantly (P < 0.05) with tumoral growth. DNTTIP1 knockdown (siDNTTIP1) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with high acetylation of p53 and upregulation of p21Cip1. Moreover, resveratrol, a HDAC inhibitor, controlled not only acetylated p53 status but also DNTTIP1 expression, leading to a similar phenotype of siDNTTIP1 cells. A marked (P < 0.05) reduction of tumoral growth in mouse xenograft models was observed with lower DNTTIP1 expression under the presence of this chemical reagent. Taken together, our results suggested that DNTTIP1-HDAC interaction promotes tumoral growth through deacetylation of p53 and that DNTTIP1 might be a critical therapeutic target in OSCCs.
Assuntos
Carcinoma de Células Escamosas/genética , Proteínas de Transporte/genética , Proliferação de Células/genética , Neoplasias Bucais/genética , Proteínas Nucleares/genética , Idoso , Animais , Antineoplásicos Fitogênicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Proteínas de Transporte/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Proteínas Nucleares/metabolismo , Interferência de RNA , Resveratrol/farmacologia , Fatores de Transcrição , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Tryptophan-aspartic acid (WD) repeat-containing protein 34 (WDR34), one of the WDR protein superfamilies with five WD40 domains, inhibits a transforming growth factor-beta (TGF-ß) activated kinase 1 (TAK1)-associated NF-κB activation pathway. Nevertheless, little is known about the roles of WDR34 in cancer. The current study sought to elucidate the clinical relevance of WDRsfb34 in oral squamous cell carcinoma (OSCC). We found WDR34 down-regulation in OSCCs compared with normal control tissues using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry. Models of overexpression of WDR34 (oeWDR34) showed depressed cellular growth through cell-cycle arrest at the G1 phase. To investigate the inhibitory function of WDR34, we challenged oeWDR34â¯cells with interleukin (IL)-1, a ligand for activation of the TAK1-NF-κB pathway and assessed the expression of a target gene of the pathway. oeWDR34 strongly inhibited IL-6 expression, which is closely related to tumoral growth, compared with control cells, suggesting that WDR34 would be a critical molecule for control of tumoral progression. In addition to the in vitro experiments, WDR34 negativity was correlated with tumoral growth of OSCCs. Our findings suggested that WDR34 inhibits OSCC progression and might be a potential tumor-suppressor molecule in OSCCs.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Transporte/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Apoptose/genética , Carcinoma de Células Escamosas/genética , Proteínas de Transporte/genética , Genes Supressores de Tumor , Humanos , Neoplasias Bucais/genética , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
Filamin-binding LIM protein 1 (FBLIM1) is related to regulation of inflammatory responses, such as chronic recurrent multifocal osteomyelitis; however, the relevance of FBLIM1 in oral squamous cell carcinoma (OSCC) is unknown. The aim of the current study was to elucidate the possible role of FBLIM1 in the carcinogenesis of OSCC. We analyzed FBLIM1 expression using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunoblot analysis, and immunohistochemistry. The expression levels of FBLIM1 were up-regulated significantly (P < 0.05) in OSCC-derived cell lines and primary OSCCs specimens compared with normal counterparts. FBLIM1 expression also was correlated with the primary tumoral size (P < 0.05) and vascular invasion (P < 0.05). We then assessed tumoral progression after treatment with FBLIM1 siRNA and clopidogrel, an antiplatelet agent. Similar to the FBLIM1 knockdown effect, clopidogrel-treated cells had attenuated functions of proliferation, migration, and invasiveness. Interestingly, clopidogrel treatment led to down-regulation of epidermal growth factor receptor (EGFR) and FBLIM1. These findings identify FBLIM1 as a putative therapeutic target by using clopidogrel for inhibiting over activation of EGFR signaling to prevent OSCC malignancy.
Assuntos
Carcinoma de Células Escamosas/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Clopidogrel/farmacologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Neoplasias Bucais/patologia , Transdução de Sinais/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Estadiamento de Neoplasias , Carga Tumoral , Regulação para CimaRESUMO
Signal-induced proliferation-associated protein 1 (SIPA1) is known to be a GTPase activating protein. Overexpressed SIPA1 is related to metastatic progression in breast and prostate cancers; however, the relevance of SIPA1 in oral squamous cell carcinoma (OSCC) is still unknown. The aim of this study was to examine SIPA1 expression and its functional mechanisms in OSCC. SIPA1 mRNA and protein expressions were analyzed by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. The expressions of SIPA1 were up-regulated significantly in vitro and in vivo. Moreover, SIPA1 expression was correlated with regional lymph node metastasis. We next assessed the cellular functions associated with tumoral metastasis using SIPA1 knockdown (shSIPA1) cells and analyzed the downstream molecules of SIPA1, i.e., bromodomain containing protein 4(BRD4), integrin beta1 (ITGB1), and matrix metalloproteinase 7 (MMP7). The shSIPA1 cells showed decreased invasiveness and migratory activities, however cellular adhesion ability was maintained at a high level. In addition, ITGB1 expression was greater in shSIPA1 cells, whereas MMP7 expression was lower than in control cells. This research is the first to establish that SIPA1 promotes cancer metastasis by regulating the ITGB1 and MMP7. Therefore, SIPA1 might be a novel therapeutic target for patients with lymph node metastasis of OSCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Movimento Celular , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Bucais/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metástase Linfática , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Proteínas Nucleares/genéticaRESUMO
Zrt-Irt-like protein 4 (ZIP4) is critical molecule for proper mammalian development and releasing zinc from vesicular compartments. Recent studies suggested that ZIP4 plays an important role of tumor progression in pancreatic, prostate, and hepatocellular cancers, however, little is known about the detail mechanism of ZIP4 in their cancers. In the present study, we examined the possibility of ZIP4 as a new molecular target for oral squamous cell carcinoma (OSCC). We evaluated ZIP4 expression in OSCC-derived cell lines and primary OSCC samples by quantitative RT-PCR, immunoblotting, and immunohistochemistry (IHC). We also analyzed the clinical correlation between ZIP4 status and clinical behaviors in patients with OSCC. In addition, ZIP4 knockdown cells (shZIP4 cells) and ZnCl2 treatment were used for functional experiments, including cellular proliferation assay, zinc uptake assay, and cell-cycle analysis. ZIP4 mRNA and protein were up-regulated significantly in OSCCs compared with normal counterparts in vitro and in vivo. IHC showed that ZIP4 expression in the primary OSCC was positively correlated with primary tumoral size. The shZIP4 cells showed decrease accumulation of intercellular zinc and decreased cellular growth by cell-cycle arrest at the G1 phase, resulting from up-regulation of cyclin-dependent kinase inhibitors and down-regulation of cyclins and cyclin-dependent kinases. Since cellular growth of OSCC cells after treatment with zinc was significantly greater than control cells, we speculated that intercellular ZnCl2 accumulation is an important factor for cellular growth. Consistent with our hypothesis, not only decreased zinc uptake by ZIP4 knockdown but also chelating agent, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), showed inhibitory effects of cellular proliferation. Therefore, our data provide evidence for an essential role of ZIP4 and intracellular zinc for tumoral growth in OSCC, suggesting that zinc uptake might be a potential therapeutic targeting event for OSCCs.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/metabolismo , Zinco/química , Carcinogênese , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Quelantes/química , Cloretos/química , Suplementos Nutricionais , Progressão da Doença , Relação Dose-Resposta a Droga , Etilenodiaminas/química , Humanos , Imuno-Histoquímica , RNA Interferente Pequeno/metabolismo , Compostos de Zinco/químicaRESUMO
TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma de Células Escamosas/genética , Proteínas de Ligação a DNA/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/genética , Proteínas Musculares/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/cirurgia , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Sinalização YAPRESUMO
Ubiquitin-conjugating enzyme E2S (UBE2S), a family of E2 protein in the ubiquitin-proteasome system, is highly expressed in several types of cancers; however, its roles in oral squamous cell carcinoma (OSCC) have not yet been well elucidated. The purpose of this study was to clarify the functional activities of UBE2S in OSCCs. We analyzed the expression levels of UBE2S in nine OSCC cell lines and primary OSCC tissues by quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry (IHC). The correlations between UBE2S expression and clinical classifications of OSCCs were analyzed using the IHC scoring system. We also used UBE2S knockdown OSCC cells for functional assays (proliferation assay, flow cytometry, and Western blotting). UBE2S was overexpressed in OSCCs in vitro and in vivo and was correlated significantly (P < 0.05) with the primary tumoral size. The cellular growth was decreased and the cell-cycle was arrested in the G2/M phase in the UBE2S knockdown (shUBE2S) cells. The expression level of P21, a target of the ubiquitin-proteasome system, was increased in the shUBE2S cells because of lower anaphase activity that promotes complex subunit 3 (APC3), an E3 ubiquitin ligase, compared with shMock cells. These findings might promote the understanding of the relationship between UBE2S overexpression and oral cancer proliferation, indicating that UBE2S would be a potential biomarker of and therapeutic target in OSCCs.