RESUMO
Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation-related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment-induced chemokines C-X3-C motif chemokine ligand 1 (CX3CL1) and C-X-C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM-1) increased in HDLECs after acid treatment in a time-dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid-sensing receptors, HDLECs basally expressed G protein-coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM-1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti-VCAM-1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid-sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer.
Assuntos
Células Endoteliais , Metástase Linfática , Receptores Acoplados a Proteínas G , Animais , Humanos , Camundongos , Adesão Celular , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Concentração de Íons de Hidrogênio , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Microambiente Tumoral , Molécula 1 de Adesão de Célula Vascular/metabolismo , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Hepatocellular carcinoma (HCC) has a high rate of recurrence and poor prognosis, even after curative surgery. Multikinase inhibitors have been applied for HCC patients, but their effect has been restricted. This study aims to clarify the clinical impact of SUV420H1/KMT5B, one of the methyltransferases for histone H4 at lysine 20, and elucidate the novel mechanisms of HCC progression. We retrospectively investigated SUV420H1 expression using HCC clinical tissue samples employing immunohistochemical analysis (n = 350). We then performed loss-of-function analysis of SUV420H1 with cell cycle analysis, migration assay, invasion assay and RNA sequence for Gene Ontology (GO) pathway analysis in vitro, and animal experiments with xenograft mice in vivo. The SUV420H1-high-score group (n = 154) had significantly poorer prognosis for both 5-year overall and 2-year/5-year disease-free survival than the SUV420H1-low-score group (n = 196) (p < 0.001 and p < 0.05, respectively). The SUV420H1-high-score group had pathologically larger tumor size, more tumors, poorer differentiation, and more positive vascular invasion than the SUV420H1-low-score group. Multivariate analysis demonstrated that SUV420H1 high score was the poorest independent factor for overall survival. SUV420H1 knockdown could suppress cell cycle from G1 to S phase and cell invasion. GO pathway analysis showed that SUV420H1 contributed to cell proliferation, cell invasion, and/or metastasis. Overexpression of SUV420H1 clinically contributed to poor prognosis in HCC, and the inhibition of SUV420H1 could repress tumor progression and invasion both in vitro and in vivo; thus, further analyses of SUV420H1 are necessary for the discovery of future molecularly targeted drugs.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismo , Neoplasias Hepáticas/patologia , Metiltransferases/genética , Prognóstico , Estudos RetrospectivosRESUMO
Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS-RC-2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin-positive (ENG+ ) population exhibited growth dependency on endoglin in anchorage-independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor ß and bone morphogenetic protein-9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer-initiating cells, the OS5K-3 ENG+ population did not enrich other reported cancer-initiating cell markers or differentiate into the ENG- population. Mouse tumor inoculation models revealed that the tumor-forming capabilities of OS5K-3 ENG+ and ENG- cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG+ cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG+ cell-centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings.
Assuntos
Carcinoma de Células Renais/patologia , Endoglina/genética , Endoglina/metabolismo , Neoplasias Renais/patologia , Regulação para Cima , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Microambiente TumoralRESUMO
Tissue-clearing technology is an emerging imaging technique currently utilized not only in neuroscience research but also in cancer research. In our previous reports, tissue-clearing methods were used for the detection of metastatic tumors. Here, we showed that the cell cycles of primary and metastatic tumors were visualized by tissue-clearing methods using a reporter system. First, we established cancer cell lines stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) reporter with widely used cancer cell lines A549 and 4T1. Fluorescence patterns of the Fucci reporter were investigated in various tumor inoculation models in mice. Interestingly, fluorescence patterns of the Fucci reporter of tumor colonies were different between various organs, and even among colonies in the same organs. The effects of antitumor drugs were also evaluated using these Fucci reporter cells. Of the three antitumor drugs studied, 5-fluorouracil treatment on 4T1-Fucci cells resulted in characteristic fluorescent patterns by the induction of G2 /M arrest both in vitro and in vivo. Thus, the combination of a tissue-clearing method with the Fucci reporter is useful for analyzing the mechanisms of cancer metastasis and drug resistance.
Assuntos
Adenocarcinoma de Pulmão/patologia , Neoplasias da Mama/patologia , Ciclo Celular , Medições Luminescentes/métodos , Neoplasias Pulmonares/patologia , Células A549 , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Fluoruracila/administração & dosagem , Genes Reporter , Vetores Genéticos/genética , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência/métodos , Transfecção , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Vermelha FluorescenteRESUMO
Although transforming growth factor beta (TGF-ß) is known to be involved in the pathogenesis and progression of many cancers, its role in renal cancer has not been fully investigated. In the present study, we examined the role of TGF-ß in clear cell renal carcinoma (ccRCC) progression in vitro and in vivo. First, expression levels of TGF-ß signaling pathway components were examined. Microarray and immunohistochemical analyses showed that the expression of c-Ski, a transcriptional corepressor of Smad-dependent TGF-ß and bone morphogenetic protein (BMP) signaling, was higher in ccRCC tissues than in normal renal tissues. Next, a functional analysis of c-Ski effects was carried out. Bioluminescence imaging of renal orthotopic tumor models demonstrated that overexpression of c-Ski in human ccRCC cells promoted in vivo tumor formation. Enhancement of tumor formation was also reproduced by the introduction of a dominant-negative mutant TGF-ß type II receptor into ccRCC cells. In contrast, introduction of the BMP signaling inhibitor Noggin failed to accelerate tumor formation, suggesting that the tumor-promoting effect of c-Ski depends on the inhibition of TGF-ß signaling rather than of BMP signaling. Finally, the molecular mechanism of the tumor-suppressive role of TGF-ß was assessed. Although TGF-ß signaling did not affect tumor angiogenesis, apoptosis of ccRCC cells was induced by TGF-ß. Taken together, these findings suggest that c-Ski suppresses TGF-ß signaling in ccRCC cells, which, in turn, attenuates the tumor-suppressive effect of TGF-ß.
Assuntos
Carcinoma de Células Renais/genética , Proteínas de Ligação a DNA/genética , Neoplasias Renais/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transplante HeterólogoRESUMO
Cancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell-like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer-initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer-initiating cells. Pancreatic cancer cells contained ALDH1(hi) cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-ß family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-ß and bone morphogenetic proteins. Treatment with TGF-ß attenuated ALDH1(hi) cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-ß regulated ALDH1A1 mRNA transcription through binding of Smad4 to its regulatory sequence. It appears that TGF-ß negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer-initiating cells.
Assuntos
Aldeído Desidrogenase/biossíntese , Regulação Neoplásica da Expressão Gênica/fisiologia , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Proteína Smad4/metabolismo , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Xenoenxertos , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , RNA Interferente Pequeno , Retinal Desidrogenase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta/metabolismoRESUMO
The epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Here, we demonstrate that transforming growth factor (TGF)-ß induced EMT and that long-term exposure to TGF-ß elicited the epithelial-myofibroblastic transition (EMyoT) by inactivating the MEK-Erk pathway. During the EMT process, TGF-ß induced isoform switching of fibroblast growth factor (FGF) receptors, causing the cells to become sensitive to FGF-2. Addition of FGF-2 to TGF-ß-treated cells perturbed EMyoT by reactivating the MEK-Erk pathway and subsequently enhanced EMT through the formation of MEK-Erk-dependent complexes of the transcription factor δEF1/ZEB1 with the transcriptional corepressor CtBP1. Consequently, normal epithelial cells that have undergone EMT as a result of combined TGF-ß and FGF-2 stimulation promoted the invasion of cancer cells. Thus, TGF-ß and FGF-2 may cooperate with each other and may regulate EMT of various kinds of cells in cancer microenvironment during cancer progression.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Actinas/genética , Actinas/metabolismo , Oxirredutases do Álcool/metabolismo , Processamento Alternativo/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Biológicos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/fisiologia , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
BACKGROUND: A recent study reported that long non-coding RNA activated by TGF-ß (lncRNA-ATB) induced epithelial-mesenchymal transition (EMT) through the transforming growth factor-ß (TGF-ß)/miR-200s/ZEB axis in hepatocellular carcinoma. Herein, we focused on the clinical significance of lncRNA-ATB in gastric cancer (GC) patients. MATERIALS AND METHODS: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to examine expression of lncRNA-ATB, miR-200b, and miR-200c in GC tissues (n = 183). Patients were divided into high and low lncRNA-ATB expression groups using a cutoff of lncRNA-ATB/GAPDH ≥0.60 or <0.60 to determine the clinicopathological significance of lncRNA-ATB in GC. Moreover, we evaluated the expression of TGF-ß, lncRNA-ATB, miR-200s, and ZEB1 in GC cell lines by qRT-PCR. GC cell lines were treated by recombinant TGF-ß1 or TGF-ß receptor inhibitor to examine morphologic changes and genetic alterations, such as lncRNA-ATB, miR-200s, and ZEB1 levels, with respect to the EMT phenotype. RESULTS: The high lncRNA-ATB group experienced a lower overall survival rate compared with the low lncRNA-ATB group, and multivariate analysis indicated that lncRNA-ATB was an independent prognostic factor (hazard ratio 3.50; 95 % CI 1.73-7.44; p = 0.0004). miR-200c levels were lower and ZEB1 levels were higher in the high lncRNA-ATB group than in the low lncRNA-ATB group. Treatment with TGF-ß in GC cell lines resulted in morphological EMT changes, upregulation of lncRNA-ATB and ZEB1, and downregulation of miR-200c and CDH1. SB431542 reduced lncRNA-ATB expression. CONCLUSION: LncRNA-ATB plays an important role in EMT to promote invasion and metastasis through the TGF-ß/miR-200s/ZEB axis, resulting in a poor prognosis in GC. LncRNA-ATB is a novel biomarker of lncRNA, indicative of a poor prognosis in GC patients.
Assuntos
Adenocarcinoma Mucinoso/secundário , Adenocarcinoma/secundário , Carcinoma de Células em Anel de Sinete/secundário , Neoplasias Hepáticas/secundário , Neoplasias Peritoneais/secundário , RNA Longo não Codificante/genética , Neoplasias Gástricas/patologia , Fator de Crescimento Transformador beta/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/metabolismo , Idoso , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células em Anel de Sinete/genética , Carcinoma de Células em Anel de Sinete/metabolismo , Proliferação de Células , Feminino , Seguimentos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metástase Linfática , Masculino , MicroRNAs/genética , Invasividade Neoplásica , Estadiamento de Neoplasias , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Taxa de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Células Tumorais Cultivadas , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Transforming growth factor (TGF)-ß exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner. The anti-apoptotic function of TGF-ß is mediated by several downstream regulatory mechanisms, and has been implicated in the tumor-progressive phenotype of breast cancer cells. We conducted RNA sequencing of mouse mammary gland epithelial (NMuMG) cells and identified a long non-coding RNA, termed lncRNA-Smad7, which has anti-apoptotic functions, as a target of TGF-ß. lncRNA-Smad7 was located adjacent to the mouse Smad7 gene, and its expression was induced by TGF-ß in all of the mouse mammary gland epithelial cell lines and breast cancer cell lines that we evaluated. Suppression of lncRNA-Smad7 expression cancelled the anti-apoptotic function of TGF-ß. In contrast, forced expression of lncRNA-Smad7 rescued apoptosis induced by a TGF-ß type I receptor kinase inhibitor in the mouse breast cancer cell line JygMC(A). The anti-apoptotic effect of lncRNA-Smad7 appeared to occur independently of the transcriptional regulation by TGF-ß of anti-apoptotic DEC1 and pro-apoptotic Bim proteins. Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-ß-induced epithelial-mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-ß functions may be restricted to apoptosis. Our findings suggest a complex mechanism for regulating the anti-apoptotic and tumor-progressive aspects of TGF-ß signaling.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Linfotoxina-alfa/metabolismo , RNA Longo não Codificante/metabolismo , Proteína Smad7/metabolismo , Animais , Sequência de Bases , Northern Blotting , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad7/genéticaRESUMO
Loss or reduction in function of tumor suppressor genes contributes to tumorigenesis. Here, by allelic DNA copy number analysis using single-nucleotide polymorphism genotyping array and mass spectrometry, we report homozygous deletion in glioblastoma multiformes at chromosome 13q21, where DACH1 gene is located. We found decreased cell proliferation of a series of glioma cell lines by forced expression of DACH1. We then generated U87TR-Da glioma cells, where DACH1 expression could be activated by exposure of the cells to doxycycline. Both ex vivo cellular proliferation and in vivo growth of s.c. transplanted tumors in mice are reduced in U87TR-Da cells with DACH1 expression (U87-DACH1-high), compared with DACH1-nonexpressing U87TR-Da cells (U87-DACH1-low). U87-DACH1-low cells form spheroids with CD133 and Nestin expression in serum-free medium but U87-DACH1-high cells do not. Compared with spheroid-forming U87-DACH1-low cells, adherent U87-DACH1-high cells display lower tumorigenicity, indicating DACH1 decreases the number of tumor-initiating cells. Gene expression analysis and chromatin immunoprecipitation assay reveal that fibroblast growth factor 2 (FGF2/bFGF) is transcriptionally repressed by DACH1, especially in cells cultured in serum-free medium. Exogenous bFGF rescues spheroid-forming activity and tumorigenicity of the U87-DACH1-high cells, suggesting that loss of DACH1 increases the number of tumor-initiating cells through transcriptional activation of bFGF. These results illustrate that DACH1 is a distinctive tumor suppressor, which does not only suppress growth of tumor cells but also regulates bFGF-mediated tumor-initiating activity of glioma cells.
Assuntos
Proteínas do Olho/genética , Glioma/genética , Fatores de Transcrição/genética , Alelos , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromossomos Humanos Par 13/genética , Doxiciclina/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Deleção de Genes , Dosagem de Genes , Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor , Glioblastoma/genética , Glioblastoma/patologia , Glioma/patologia , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Polimorfismo de Nucleotídeo Único , Esferoides Celulares/patologia , Transplante HeterólogoRESUMO
Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Neoplasias Ósseas/metabolismo , Calcinose/metabolismo , Proteínas de Neoplasias/metabolismo , Osteoblastos/metabolismo , Neoplasias da Próstata/metabolismo , Transdução de Sinais , Microambiente Tumoral , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animais , Antígenos de Diferenciação/biossíntese , Proteína Morfogenética Óssea 4/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Calcinose/genética , Calcinose/patologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Proteínas de Neoplasias/genética , Osteoblastos/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteína Smad1/genética , Proteína Smad1/metabolismo , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Aldehyde dehydrogenase 1 (ALDH1) has been shown to serve as a marker for cancer-initiating cells (CICs), but little is known about the regulation of the CIC functions of ALDH1+ cancer cells. We isolated ALDH1+ cells from human diffuse-type gastric carcinoma cells and characterized these cells using an Aldefluor assay. ALDH1+ cells constituted 5-8% of the human diffuse-type gastric carcinoma cells, OCUM-2MLN and HSC-39; were more tumourigenic than ALDH1- cells; and were able to self-renew and generate heterogeneous cell populations. Using gene expression microarray analyses, we identified REG4 (regenerating islet-derived family, member 4) as one of the genes up-regulated in ALDH1+ cells, and thus as a novel marker for ALDH1+ tumour cells. Induced expression of REG4 enhanced the colony-forming ability of OCUM-2MLN cells, while knockdown of REG4 inhibited the tumourigenic potential of ALDH1+ cells. We further found that TGF-ß signalling reduces the expression of ALDH1 and REG4, and the size of the ALDH1+ cell population. In human diffuse-type gastric carcinoma tissues, the expression of ALDH1 and REG4 correlated with each other, as assessed by immunohistochemistry, and ALDH1 expression correlated inversely with Smad3 phosphorylation as a measure of TGF-ß signalling. These findings illustrate that, in diffuse-type gastric carcinoma, REG4 is up-regulated in ALDH1+ CICs, and that the increased tumourigenic ability of ALDH1+ cells depends on REG4. Moreover, TGF-ß down-regulates ALDH1 and REG4 expression, which correlates with a reduction in CIC population size and tumourigenicity. Targeting REG4 in ALDH1+ CICs may provide a novel strategy in the treatment of diffuse-type gastric carcinoma.
Assuntos
Transformação Celular Neoplásica/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Isoenzimas/fisiologia , Lectinas Tipo C/fisiologia , Células-Tronco Neoplásicas/fisiologia , Retinal Desidrogenase/fisiologia , Neoplasias Gástricas/fisiopatologia , Fator de Crescimento Transformador beta/farmacologia , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Família Aldeído Desidrogenase 1 , Animais , Biomarcadores Tumorais/fisiologia , Carcinoma de Células em Anel de Sinete/patologia , Carcinoma de Células em Anel de Sinete/fisiopatologia , Linhagem Celular Tumoral , Células Cultivadas , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Proteínas Associadas a Pancreatite , Transdução de Sinais/fisiologia , Neoplasias Gástricas/patologia , Fator de Crescimento Transformador beta/fisiologia , Transplante Heterólogo , Regulação para Cima/fisiologiaRESUMO
Bone morphogenetic proteins (BMPs) are multi-functional cytokines, which belong to the transforming growth factor-ß (TGF-ß) family. In some cancer tissues, aberrant expression of various BMP signal components has been detected. Here, we describe the divergent roles of BMPs during the progression of cancer. BMPs exhibit various effects on both cancer cells and on tumor microenvironments. BMPs inhibit the proliferation of cancer cells, with some exceptions. BMPs also induce the differentiation of certain cancer stem cells, and attenuate their aggressiveness. In parallel, BMPs play a critical role in the regulation of tumor angiogenesis and the metastasis of cancer cells. Some mouse xenograft models have revealed that cancer metastases are prevented by the inhibition of BMP signaling. Together, these findings imply that BMPs function as both suppressors and promoters of tumors in a context dependent manner. The bi-directional characteristics of BMPs in cancer are similar to those of TGF-ß, which was previously described as a molecular 'Jekyll and Hyde.'
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Citocinas/metabolismo , Neoplasias/patologia , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/imunologia , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Citocinas/imunologia , Progressão da Doença , Humanos , Metástase Neoplásica , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Neoplasias/metabolismo , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica , Microambiente TumoralRESUMO
Suppression of the antitumor cytokine interleukin-24 (IL-24) is critical for the survival of myxoid liposarcoma (MLS) cells. It has been previously demonstrated by the authors that an MLS-specific chimeric oncoprotein, translocated in liposarcoma-CCAAT/enhancer-binding protein homologous protein (TLS-CHOP), supresses IL24 mRNA expression via induction of proteoglycan 4 (PRG4) to sustain MLS cell proliferation. However, IL-24 has also been revealed to be suppressed by the ubiquitin-proteasome system in human ovarian and lung cancer cells. Therefore, the aim of the present study was to elucidate the mechanism of IL-24 suppression in MLS cells. The results revealed that the proteasome inhibitor, MG-132, induced cell death in MLS cells in vitro; this effect was reduced following IL-24 knockdown. This indicated that proteasomal degradation of IL-24 may be an important process for MLS cell survival. In addition, it was also previously revealed by the authors that knockdown of plasminogen activator inhibitor-1 (PAI-1), a TLS-CHOP downstream molecule, suppressed the growth of MLS cells, thus instigating the investigation of the effect of PAI-1 on IL-24 expression in MLS cells. Double knockdown of PAI-1 and IL-24 negated the growth-suppressive effect of PAI-1 single knockdown in MLS cells. Interestingly, PAI-1 single knockdown did not increase the mRNA expression of IL24, but it did increase the protein abundance of IL-24, indicating that PAI-1 suppressed IL-24 expression by promoting its proteasomal degradation. Moreover, treatment of MLS cells with a PAI-1 inhibitor, TM5275, induced IL-24 protein expression and apoptosis. Collectively, the results of the present as well as previous studies indicated that IL-24 expression may be suppressed at the transcriptional level by PRG4 and at the protein level by PAI-1 in MLS cells. Accordingly, PAI-1 may represent an effective therapeutic target for MLS treatment.
RESUMO
A relationship exists between defects in bone morphogenetic protein (BMP) signaling and formation of hamartoma and adenoma in the gastric epithelium; however, the role of BMP signaling in the progression of diffuse-type gastric carcinoma remains unknown. We investigated whether BMP functions as a tumor suppressor in human diffuse-type gastric carcinoma using three different human diffuse-type gastric carcinoma cell lines (OCUM-12, HSC-39, and OCUM-2MLN). Overexpression of the dominant-negative form of BMP-2/4-specific type I receptor (ALK-3) in OCUM-12 and HSC-39 cells accelerated their growth in vivo. BMP-4 induced cell cycle arrest in these cells via p21 induction through the SMAD pathway. Moreover, overexpression of the constitutively active form of ALK-3 in HSC-39 and OCUM-2MLN cells suppressed the proliferation of these cells in vitro and in vivo. Our findings suggest that BMP-2 and BMP-4 function as potent tumor suppressors in diffuse-type gastric carcinoma.
Assuntos
Proteína Morfogenética Óssea 2/fisiologia , Proteína Morfogenética Óssea 4/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias Gástricas/etiologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Neoplasias Gástricas/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß (TGF-ß) family, are multifunctional cytokines. BMPs have a broad range of functions, and abnormalities in BMP signaling pathways are involved in cancer progression. BMPs activate the proliferation of certain cancer cells. Malignant phenotypes of cancer cells, such as increased motility, invasiveness, and stemness, are enhanced by BMPs. Simultaneously, BMPs act on various cellular components and regulate angiogenesis in the tumor microenvironment. Thus, BMPs function as pro-tumorigenic factors in various types of cancer. However, similar to TGF-ß, which shows both positive and negative effects on tumorigenesis, BMPs also act as tumor suppressors in other types of cancers. In this article, we review important findings published in the recent decade and summarize the pro-oncogenic functions of BMPs and their underlying mechanisms. The current status of BMP-targeted therapies for cancers is also discussed.
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We developed an in vivo serial passaging model for renal cancer with orthotopic renal subcapsular inoculation. We detail the procedures for renal subcapsular inoculation of cancer cells in mice, followed by in vivo and exvivo bioluminescence imaging, tumor-bearing kidney dissociation, and in vivo passaging. This protocol is capable of reproducing the coevolution between cancer cells and the primary tumor microenvironment. It enables us to unveil the systemic dynamics of metastasis and develop a therapeutic strategy for metastatic renal cancer. For complete details on the use and execution of this protocol, please refer to Nishida et al. (2020).
Assuntos
Neoplasias Renais , Transplantes , Animais , Diagnóstico por Imagem , Feminino , Humanos , Rim/patologia , Neoplasias Renais/patologia , Masculino , Camundongos , Transplantes/patologia , Microambiente TumoralRESUMO
DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5-aza-deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome-wide using methylation array in reference to RNA-sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor-suppressive role of UQCRH. Moreover, 5-aza-deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B-induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Apoptose/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Metilação de DNA/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Neoplasias Renais/patologiaRESUMO
The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks.
Assuntos
Análise de Dados , Vasos Linfáticos , Animais , Encéfalo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Vasos Linfáticos/diagnóstico por imagem , Camundongos , TecnologiaRESUMO
Pancreatic cancer is one of the cancers with the poorest prognosis, with a 5-year survival rate of approximately 5-10%. Thus, it is urgent to identify molecular targets for the treatment of pancreatic cancer. Using serial transplantations in a mouse pancreatic orthotopic inoculation model, we previously produced highly malignant pancreatic cancer sublines with increased tumor-forming abilities in vivo. Here, we used these sublines to screen molecular targets for the treatment of pancreatic cancer. Among the genes with increased expression levels in the sublines, we focused on those encoding cell surface receptors that may be involved in the interactions between cancer cells and the tumor microenvironment. Based on our previous RNA-sequence analysis, we found increased expression levels of neurotensin (NTS) receptor 1 (NTSR1) in highly malignant pancreatic cancer sublines. Furthermore, re-analysis of clinical databases revealed that the expression level of NTSR1 was increased in advanced pancreatic cancer and that high NTSR1 levels were correlated with a poor prognosis. Overexpression of NTSR1 in human pancreatic cancer cells Panc-1 and SUIT-2 accelerated their tumorigenic and metastatic abilities in vivo. In addition, RNA-sequence analysis showed that MAPK and NF-κB signaling pathways were activated upon NTS stimulation in highly malignant cancer sublines and also revealed many new target genes for NTS in pancreatic cancer cells. NTS stimulation increased the expression of MMP-9 and other pro-inflammatory cytokines and chemokines in pancreatic cancer cells. Moreover, the treatment with SR48692, a selective NTSR1 antagonist, suppressed the activation of the MAPK and NF-κB signaling pathways and induction of target genes in pancreatic cancer cells in vitro, while the administration of SR48692 attenuated the tumorigenicity of pancreatic cancer cells in vivo. These findings suggest that NTSR1 may be a prognostic marker and a molecular target for pancreatic cancer treatment.