RESUMEN
BACKGROUND: Aberrant expressions of desmoglein 2 (Dsg2) and desmocollin 2(Dsc2), the two most widely distributed desmosomal cadherins, have been found to play various roles in cancer in a context-dependent manner. Their specific roles on breast cancer (BC) and the potential mechanisms remain unclear. METHODS: The expressions of Dsg2 and Dsc2 in human BC tissues and cell lines were assessed by using bioinformatics analysis, immunohistochemistry and western blotting assays. Wound-healing and Transwell assays were performed to evaluate the cells' migration and invasion abilities. Plate colony-forming and MTT assays were used to examine the cells' capacity of proliferation. Mechanically, Dsg2 and Dsc2 knockdown-induced malignant behaviors were elucidated using western blotting assay as well as three inhibitors including MK2206 for AKT, PD98059 for ERK, and XAV-939 for ß-catenin. RESULTS: We found reduced expressions of Dsg2 and Dsc2 in human BC tissues and cell lines compared to normal counterparts. Furthermore, shRNA-mediated downregulation of Dsg2 and Dsc2 could significantly enhance cell proliferation, migration and invasion in triple-negative MDA-MB-231 and luminal MCF-7 BC cells. Mechanistically, EGFR activity was decreased but downstream AKT and ERK pathways were both activated maybe through other activated protein tyrosine kinases in shDsg2 and shDsc2 MDA-MB-231 cells since protein tyrosine kinases are key drivers of triple-negative BC survival. Additionally, AKT inhibitor treatment displayed much stronger capacity to abolish shDsg2 and shDsc2 induced progression compared to ERK inhibition, which was due to feedback activation of AKT pathway induced by ERK inhibition. In contrast, all of EGFR, AKT and ERK activities were attenuated, whereas ß-catenin was accumulated in shDsg2 and shDsc2 MCF-7 cells. These results indicate that EGFR-targeted therapy is not a good choice for BC patients with low Dsg2 or Dsc2 expression. Comparatively, AKT inhibitors may be more helpful to triple-negative BC patients with low Dsg2 or Dsc2 expression, while therapies targeting ß-catenin can be considered for luminal BC patients with low Dsg2 or Dsc2 expression. CONCLUSION: Our finding demonstrate that single knockdown of Dsg2 or Dsc2 could promote proliferation, motility and invasion in triple-negative MDA-MB-231 and luminal MCF-7 cells. Nevertheless, the underlying mechanisms were cellular context-specific and distinct.
Asunto(s)
Movimiento Celular , Proliferación Celular , Desmocolinas , Desmogleína 2 , Neoplasias de la Mama Triple Negativas , Humanos , Desmocolinas/metabolismo , Desmocolinas/genética , Desmogleína 2/metabolismo , Desmogleína 2/genética , Femenino , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/genética , Línea Celular Tumoral , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Invasividad Neoplásica , Regulación Neoplásica de la Expresión Génica , beta Catenina/metabolismo , Transducción de SeñalRESUMEN
Epithelial-mesenchymal transition (EMT) is regulated by multiple signal transduction pathways. Twist-1 is one of the most important transcription factors in these pathways. In a previous study, we found that Bcl-2 enhanced the role of Twist-1 in EMT. Coexpression of Twist-1 and Bcl-2 may play an import role in vasculogenic mimicry (VM) through regulation of EMT. Moreover, regulators of EMT and VM are known to be important targets for microRNAs (miRNAs). To better understand how these critical pathways are induced by coexpression of Twist-1 and Bcl-2, we performed a comprehensive comparative bioinformatics analysis using microarrays on HCCs that overexpressed Twist-1 and Bcl-2. Eleven miRNAs associated with coexpression of Twist-1 and Bcl-2 were selected from the comprehensive analysis of miRNA microarray and ChIP-seq analysis. Changes in miRNAs were associated with significant differences in the expression of genes involved in signal transduction pathways related to processes including tumor invasion, metastasis, angiogenesis, and tumor cell shape. We confirmed the role of Twist-1 and Bcl-2 coexpression in HCC cells using wound healing assays, invasion assays, and 3D Matrigel assays. Furthermore, the role of miR-27a as a crucial regulator of EMT and VM was confirmed in HCC cells by RT-PCR and western blot analysis. These findings provide evidence that Bcl-2 enhances the role of Twist-1 in VM and EMT through miRNAs.
Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Hepáticas/genética , MicroARNs/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Proteína 1 Relacionada con Twist/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Humanos , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
Melanoma is the least common but most serious form of skin cancer. The leading cause of death in melanoma patients is widespread metastasis caused by increased cell motility and a rich blood supply for tumor cells. A unique form of microcirculation called vasculogenic mimicry, which efficiently supplies blood to tumor cells, has been reported recently. Apoptosis-related protein performs a nonapoptotic function to promote migration and invasion of tumor cells. This study focuses on the nonapoptotic role of caspase-3 in melanoma and its effects on the migration, invasion, and vasculogenic mimicry formation of melanoma cells. Human melanoma samples were used to detect active caspase-3 expression and determine its relationship with clinicopathologic parameters. In addition, a human melanoma A375 cell line was used to determine the role of caspase-3 in migration and invasion using z-DEVD-fmk, a selective caspase-3 inhibitor, to inhibit caspase-3 activity. The findings suggest that active caspase-3 is expressed in nonapoptotic melanoma cells and is related to metastasis and vasculogenic mimicry formation in patients with melanoma. Low doses of caspase-3 inhibitor reduced caspase-3 activity without affecting cell apoptosis. Inhibition of caspase-3 activity using low-dose z-DEVD-fmk decreased the migration, invasion, and vasculogenic mimicry formation of melanoma cells in vitro. Similarly, downregulation of caspase-3 by specific small interfering RNA also inhibited the migratory, invasive, and tube-forming potential of melanoma cells. The caspase-3-mediated promotion of melanoma cell motility may be because of the cleavage of matrix metalloproteinase-2.
Asunto(s)
Caspasa 3/metabolismo , Adhesión Celular , Movimiento Celular , Proliferación Celular , Melanoma/patología , Neovascularización Patológica/patología , Animales , Apoptosis , Western Blotting , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Neovascularización Patológica/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Células Tumorales CultivadasRESUMEN
Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Hypoxia plays a pivotal role in the formation of VM. Hypoxia-induced Bcl-2 overexpression is observed in many types of tumors including melanoma, in which it is associated with tumorigenicity and angiogenesis. VE-cadherin, the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation, is also overexpressed in melanoma. Despite these connections, whether hypoxia induces VM formation via VE-cadherin regulation by Bcl-2 is not confirmed. We used human melanoma cells to upregulate or knockdown the expression of Bcl-2 to investigate the possible molecular mechanism of VM formation under hypoxia. Bcl-2 overexpression increased VE-cadherin expression and VM formation under normoxia, whereas Bcl-2 siRNA significantly decreased VE-cadherin expression and VM formation under hypoxia. We then demonstrated that Bcl-2 regulated VE-cadherin transcription activity by Western blot, three-dimensional cultures, reporter gene assay, and clinical analysis. Therefore, Bcl-2-dependent VE-cadherin overexpression may be an important mechanism by which hypoxia induces VM.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Hipoxia de la Célula/fisiología , Melanoma/metabolismo , Imitación Molecular/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Vasos Sanguíneos , Western Blotting , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Melanoma/patologíaRESUMEN
The anti-apoptosis factor Bcl-2 is known to contribute to tumorigenesis and metastasis. Epithelial-mesenchymal transition (EMT) may also participate in tumor invasion and metastasis. This study investigated the relationship between coexpression profiles of Bcl-2 and EMT regulators in hepatocellular carcinoma (HCC) tumor samples and clinical outcome. The nuclear (Nu) and cytoplasmic (Cyt) expression of Bcl-2 and the EMT regulators Twist-1, Twist-2, and Snail were determined by immunohistochemical staining in tumor tissue isolated from 97 HCC patients. The clinical prognostic values of both individual protein expression and various expression combinations were investigated using univariate and multivariate survival analysis. Results showed that patients with nuclear expression of Bcl-2 had worse clinical outcomes than patients exhibiting cytoplasmic expression. Overall survival was significantly shorter in HCC patients individually expressing Bcl-2-Nu, Twist-1-Nu, Twist-1-Cyt, and Snail (all P<0.05). Patients coexpressing Bcl-2-Nu with Twist-1-Nu, Twist-1-Cyt, Twist-2, or Snail had even worse prognoses than those expressing no biomarker or any one biomarker alone (all P<0.05). Multivariate analysis showed that HCC patients coexpressing Bcl-2-Nu with Twist-1-Cyt had the worst prognosis. This study provides clinical evidence that nuclear expression of Bcl-2 combined with cytoplasmic expression of Twist-1 is a predictor of very poor prognosis in HCC. Coexpression profiles of Bcl-2 and EMT regulators might aid in the selection of the most efficacious therapy for patients with HCC.
Asunto(s)
Carcinoma Hepatocelular/patología , Transición Epitelial-Mesenquimal , Neoplasias Hepáticas/patología , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Adulto , Anciano , Cadherinas/análisis , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/mortalidad , Femenino , Humanos , Neoplasias Hepáticas/química , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Proteínas Nucleares/análisis , Pronóstico , Modelos de Riesgos Proporcionales , Factores de Transcripción de la Familia Snail , Factores de Transcripción/análisis , Proteína 1 Relacionada con Twist/análisis , beta Catenina/análisisRESUMEN
Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The present study was designed to determine the role of VEGF in tumor growth and metastasis. The sequences for the VEGF gene were cloned into expression plasmids and then transfected into melanoma B16 cells. Overexpression of VEGF transfected with expression plasmids or given exogenous VEGF and epidermal growth factor (EGF) significantly enhanced tumor cell proliferation, migration, and invasion. Tumor growth and metastasis of melanoma B16 cells transfected with VEGF plasmid were significantly promoted compared with those of cells administered with exogenous VEGF or EGF. These results indicated that VEGF can be an effective antiangiogenic strategy for melanoma.
RESUMEN
UNLABELLED: The antiapoptotic protein Bcl-2 plays multiple roles in apoptosis, immunity, and autophagy. Its expression in tumors correlates with tumor grade and malignancy. The recapitulation of the normal developmental process of epithelial-mesenchymal transition (EMT) contributes to tumor cell plasticity. This process is also a characteristic of metastatic cells and vasculogenic mimicry. In the present study we report functional and structural interactions between Bcl-2 and the EMT-regulating transcription factor Twist1 and the relationship with metastasis and vascular mimicry. Bcl-2 and Twist1 are coexpressed under hypoxia conditions. The Bcl-2 can bind to Twist1 in vivo and in vitro. This interaction involves basic helix-loop-helix DNA binding domain within Twist1 and through two separate domains within Bcl-2 protein. Formation of the Bcl-2/Twist1 complex facilitates the nuclear transport of Twist1 and leads to transcriptional activation of wide ranges of genes that can increase the tumor cell plasticity, metastasis, and vasculogenic mimicry. Finally, nuclear expression of Bcl-2 and Twist1 is correlated with poor survival of these patients in a cohort of 97 cases of human hepatocellular carcinoma. CONCLUSION: The results describe a novel function of Bcl-2 in EMT induction, provide insight into tumor progression, and implicate the Bcl-2/Twist1 complex as a potential target for developing chemotherapeutics.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Neovascularización Patológica/fisiopatología , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/fisiopatología , Carcinoma Hepatocelular/secundario , Núcleo Celular/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Células Hep G2 , Humanos , Hipoxia/patología , Hipoxia/fisiopatología , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estructura Terciaria de Proteína/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal/fisiología , Proteína 1 Relacionada con Twist/química , Proteína 1 Relacionada con Twist/genéticaRESUMEN
The epithelial-mesenchymal transition regulator Twist1 has been implicated in tumor invasion, metastasis, and vasculogenic mimicry formation of human hepatocellular carcinoma. However, the relationship between Twist1 expression and endothelium-dependent angiogenesis is not clear. In this study, to investigate the role of Twist1 in hepatocellular carcinoma angiogenesis, we measured the microvessel density by CD31 immunohistochemistry stain and explored the microvessel density as an angiogenesis indicator. The microvessel density in paraffin sections from 97 patients was correlated with Twist1 expression up-regulation. Nuclear relocation was also identified based on immunohistochemistry stain, presenting a significant clinical pattern in hepatocellular carcinoma metastasis and prognosis. Twist1 expression, which is both located in the cytoplasm and relocated into the nucleus, was associated with matrix metalloproteinase 9 up-regulation; matrix metalloproteinase 2 did not appear to present these effects in hepatocellular carcinoma. An assessment of microvessel density could provide an estimate of the degree of angiogenic activity in tissues, as well as its association with Twist1 up-regulated expression. To the best of our knowledge, not only is Twist1 related to metastasis by tumor cells, but vasculogenic mimicry is also significantly related to microvessel density; this process is also associated with matrix metalloproteinase 9 up-regulated expression. This work provides a better understanding of the role of Twist1 in hepatocellular carcinoma angiogenesis and metastasis, suggesting that our findings could represent tumor cell epithelial-mesenchymal transition and endothelium-dependent angiogenesis, as can be seen in hepatocellular carcinoma.
Asunto(s)
Carcinoma Hepatocelular/irrigación sanguínea , Neoplasias Hepáticas/irrigación sanguínea , Neovascularización Patológica/metabolismo , Proteínas Nucleares/fisiología , Proteína 1 Relacionada con Twist/fisiología , Adulto , Biomarcadores , Carcinoma Hepatocelular/mortalidad , Núcleo Celular/metabolismo , Núcleo Celular/patología , China/epidemiología , Citoplasma/metabolismo , Citoplasma/patología , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Hepáticas/mortalidad , Masculino , Metaloproteinasa 8 de la Matriz/metabolismo , Microvasos/metabolismo , Microvasos/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Neovascularización Patológica/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Pronóstico , Tasa de Supervivencia , Regulación hacia ArribaRESUMEN
E-cadherin loss is a key biological mechanism in tumour invasion. As a main regulator of epithelial-mesenchymal transition (EMT) mechanism-mediated invasion and metastasis, Twist1 plays an important role through its regulation of E-cadherin expression. However, whether or not Twist2 has the same function in tumour metastasis remains unclear. The purpose of this study is to investigate the expressions and different roles of Twist1 and Twist2 in human hepatocellular carcinoma (HCC). The expressions of Twist1 and Twist2 in HCC tissue were evaluated by immunohistochemical staining. The role of Twist1 and Twist2 in invasiveness was also evaluated in vitro by using HCC cell lines. Twist1 nuclear overexpression is found to be correlated with HCC metastasis, and its expression is negatively correlated with E-cadherin expression in human tissue. Twist2, a Twist1 homology protein, only expresses in the cytoplasm and shows no significant correlation with HCC metastasis. By ectopic transfection of Twist1 and Twist2 into the HCC cells, HepG2 and PLC, Twist1 is able to down-regulate E-cadherin expression and promote matrix metalloproteinase (MMP) activation, specifically in MMP2 and MMP9. In functional assays, Twist1 is found to promote invasion in HepG2 and PLC cells, but the invasion ability of the groups is not affected Twist2. Our findings indicate that Twist1 induces HCC invasion via increased activity in MMPs, leading to poor clinical prognoses. The results of this study also demonstrate a novel cogitation in Twist2, which has no effect on HCC invasion and metastasis. Twist1 may contribute to HCC invasion and metastasis and may be used as a novel therapeutic target for the inhibition of HCC metastasis.