RESUMO
BACKGROUND/AIM: Scirrhous-type gastric cancer (SGC), one of the most intractable cancer subtypes, is characterized by rapid cancer cell proliferation and infiltration accompanied by extensive stromal fibrosis. One of the reasons for its poor prognosis may be the lack of molecular target drugs for SGC, because of the unknown driver genes. Exploration of somatic mutations in the human samples of SGC using next-generation sequencing (NGS) has been hampered by abundant fibrous tissues in these samples. Therefore, this study aimed to determine a novel oncogene by RNA-sequencing using SGC cell lines, avoiding contamination with fibrosis. MATERIALS AND METHODS: In silico analysis of RNA-sequencing public data of the gastric cancer cell line, and RNA- sequencing using five of our unique SGC cell lines, OCUM1, OCUM2MLN, OCUM8, OCUM12, and OCUM14 were performed. RESULTS: We found three differentially expressed genes, ARHGAP4, NOS3, and OR51B5 that are significantly over-expressed in SGC cells. Immunohistochemical analysis indicated that the protein expression levels of these three genes were significantly higher in SGC than in other types of gastric cancer. The prognosis of patients with positive expression of these three genes was significantly poorer than those with negative expression. In particular, ARHGAP4 expression was an independent predictor of poor prognosis and recurrence. CONCLUSION: ARHGAP4, NOS3, and OR51B5 may be candidate driver genes for SGC. ARHGAP4 may be a promising molecular target for SGC.
Assuntos
Adenocarcinoma Esquirroso , Neoplasias Gástricas , Humanos , Adenocarcinoma Esquirroso/genética , Adenocarcinoma Esquirroso/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Fibrose , Proteínas Ativadoras de GTPase/genética , Óxido Nítrico Sintase Tipo III , Oncogenes , RNA , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Receptores Odorantes/metabolismoRESUMO
BACKGROUND/AIM: Cancer-associated fibroblasts (CAFs) may promote the malignancy of human scirrhous gastric cancer (SGC) cells. We conducted the present study to identify novel growth factors from CAFs. MATERIALS AND METHODS: OCUM-12 and 2 CAF cell lines were used. The proliferation of cancer cells was determined by the number of cancer cells or the MTT assay. The growth factor(s) were purified and characterized by the gel filtration chromatography and protein array. RESULTS: The molecular weight of the growth-stimulating factor was estimated to be approximately 66-669 kDa. Protein array of conditioned medium (CM) from CAFs indicated that dipeptidyl peptidase-4 (DPP-4) was one of the growth factors. The addition of CM increased the phosphorylation of C-X-C chemokine receptor 4 (CXCR4). The DPP-4 inhibitor significantly inhibited the growth-stimulating activity of CM. CONCLUSION: DPP-4 from CAFs might be one of the growth-stimulating factors for SGC through CXCR4.
Assuntos
Adenocarcinoma Esquirroso/genética , Dipeptidil Peptidase 4/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Receptores CXCR4/genética , Neoplasias Gástricas/genética , Adenocarcinoma Esquirroso/patologia , Fibroblastos Associados a Câncer/química , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/química , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Proteínas de Neoplasias/genética , Neoplasias Gástricas/patologiaRESUMO
OBJECTIVES: Scirrhous gastric cancer, which accounts for approximately 10% of all gastric cancers, often disseminates to the peritoneum, leading to intractable cases with poor prognosis. There is an urgent need for new treatment approaches for this difficult cancer. METHODS: We previously established an original cell line, HSC-60, from a scirrhous gastric cancer patient and isolated a peritoneal-metastatic cell line, 60As6, in nude mice following orthotopic inoculations. In the present study, we focused on the expression of long noncoding ribonucleic acid (RNA) (lncRNA) in the cell lines and investigated the mechanism on peritoneal dissemination. RESULTS: We demonstrated that an lncRNA, HOX transcript antisense RNA (HOTAIR), is expressed significantly more highly in 60As6 than HSC-60 cells. Then, using both HOTAIR knockdown and overexpression experiments, we showed that high-level expression of HOTAIR promotes epithelial-mesenchymal transition (EMT) in 60As6 cells. By luciferase assay, we found that HOTAIR directly targets and binds to miR-217, and that miR-217 directly binds to Zinc finger E-box-binding homeobox 1 (ZEB1). The knockdown of HOTAIR in 60As6 cells significantly reduced the invasion activity and peritoneal dissemination - and significantly prolonged the survival - in the orthotopic tumor mouse model. CONCLUSION: An EMT-associated pathway (the HOTAIR-miR-217-ZEB1 axis) appears to inhibit peritoneal dissemination and could lead to a novel therapeutic strategy against scirrhous gastric cancer in humans.
Assuntos
Adenocarcinoma Esquirroso/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Peritônio/patologia , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Adenocarcinoma Esquirroso/secundário , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Gástricas/secundárioRESUMO
Scirrhous-type gastric carcinoma (SGC), which is characterized by the rapid proliferation of cancer cells accompanied by extensive fibrosis, shows extremely poor survival. A reason for the poor prognosis of SGC is that the driver gene responsible for SGC has not been identified. To identify the characteristic driver gene of SGC, we examined the genomic landscape of six human SGC cell lines of OCUM-1, OCUM-2M, OCUM-8, OCUM-9, OCUM-12 and OCUM-14, using multiplex gene panel testing by next-generation sequencing. In this study, the non-synonymous mutations of serine threonine kinase 11/liver kinase B1 (STK11/LKB1) gene were detected in OCUM-12, OCUM-2M and OCUM-14 among the six SGC cell lines. Capillary sequencing analysis confirmed the non-sense or missense mutation of STK11/LKB1 in the three cell lines. Western blot analysis showed that LKB1 expression was decreased in OCUM-12 cells and OCUM-14 cells harboring STK11/LKB1 mutation. The mammalian target of rapamycin (mTOR) inhibitor significantly inhibited the proliferation of OCUM-12 and OCUM-14 cells. The correlations between STK11/LKB1 expression and clinicopathologic features of gastric cancer were examined using 708 primary gastric carcinomas by immunochemical study. The low STK11/LKB1 expression group was significantly associated with SGC, high invasion depth and frequent nodal involvement, in compared with the high STK11/LKB1 expression group. Collectively, our study demonstrated that STK11/LKB1 mutation might be responsible for the progression of SGC, and suggested that mTOR signaling by STK11/LKB1 mutation might be one of therapeutic targets for patients with SGC.
Assuntos
Adenocarcinoma Esquirroso/patologia , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Mutação , Proteínas Serina-Treonina Quinases/genética , Neoplasias Gástricas/patologia , Quinases Proteína-Quinases Ativadas por AMP , Adenocarcinoma Esquirroso/genética , Adenocarcinoma Esquirroso/metabolismo , Idoso , Apoptose , Proliferação de Células , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Scirrhous-type gastric cancer (SGC) is one of the most intractable cancer subtypes in humans, and its therapeutic targets have been rarely identified to date. Exploration of somatic mutations in the SGC genome with the next-generation sequencers has been hampered by markedly increased fibrous tissues. Thus, SGC cell lines may be useful resources for searching for novel oncogenes. Here we have conducted whole exome sequencing and RNA sequencing on 2 SGC cell lines, OCUM-8 and OCUM-9. Interestingly, most of the mutations thus identified have not been reported. In OCUM-8 cells, a novel CD44-IGF1R fusion gene is discovered, the protein product of which ligates the amino-terminus of CD44 to the transmembrane and tyrosine-kinase domains of IGF1R. Furthermore, both CD44 and IGF1R are markedly amplified in the OCUM-8 genome and abundantly expressed. CD44-IGF1R has a transforming ability, and the suppression of its kinase activity leads to rapid cell death of OCUM-8. To the best of our knowledge, this is the first report describing the transforming activity of IGF1R fusion genes. However, OCUM-9 seems to possess multiple oncogenic events in its genome. In particular, a novel BORCS5-ETV6 fusion gene is identified in the OCUM-9 genome. BORCS5-ETV6 possesses oncogenic activity, and suppression of its message partially inhibits cell growth. Prevalence of these novel fusion genes among SGC awaits further investigation, but we validate the significance of cell lines as appropriate reagents for detailed genomic analyses of SGC.
Assuntos
Adenocarcinoma Esquirroso/genética , Oncogenes/genética , Neoplasias Gástricas/genética , Células 3T3 , Adenocarcinoma Esquirroso/patologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Proteínas de Membrana/genética , Camundongos , Mutação/genética , Receptor IGF Tipo 1/genética , Estômago/patologia , Neoplasias Gástricas/patologiaRESUMO
Scirrhous gastric cancer (SGC) exhibits aggressiveness of the rapid infiltrating tumor cells with abundant fibroblasts. Experimental studies using SGC cell lines have obtained useful information about this cancer. Our literature search divulged a total of 18 SGC cell lines; two cell lines were established from primary SGC and the other lines were established from a metastatic lesion of SGC. Fibroblast growth factor receptor 2 (FGFR2) and transforming growth factor-beta receptor (TßR) are linked to the rapid development of SGC. Cross-talk between the cancer cells and cancer-associated fibroblasts (CAFs) has been shown to contribute to the progression of SGC. Chemokine (C-X-C motif) receptor 1 (CXCR1) from SGC cells might be associated with the abundant CAFs in cancer microenvironments. The in vivo models established using SGC cell lines are expected to serve as a useful tool for the development of drugs such as FGFR2 inhibitors, TßR inhibitors, and CXCR1 inhibitors, which might be promising as SGC treatments. However, the number of available SGC cell lines is insufficient for the clarification of the entire biologic behavior of SGC. Since the mechanisms responsible for the characteristic aggressiveness of SGC are not fully elucidated, the establishment of new SGC cell lines could help clarify the biological behavior of SGC and contribute to its treatment.
Assuntos
Adenocarcinoma Esquirroso/genética , Adenocarcinoma Esquirroso/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Adenocarcinoma Esquirroso/terapia , Adulto , Idoso , Animais , Fibroblastos Associados a Câncer , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Interleucina-8A , Receptores de Fatores de Crescimento Transformadores beta , Microambiente Tumoral/genéticaRESUMO
OBJECTIVES: Scirrhous gastric cancers grow rapidly, and frequently invade the peritoneum. Such peritoneal dissemination properties markedly reduce patient survival. Thus, an effective means for inhibiting peritoneal dissemination is urgently required. METHODS: We previously established a cell line, HSC-58, from a scirrhous gastric cancer patient, and further successfully isolated a metastatic line, 58As9, in nude mice upon orthotopic inoculation. Using the lines, we examined the mechanism underlying peritoneal dissemination from the viewpoint of microRNA (miRNA) expression. RESULTS: miRNA array and qRT-PCR analysis showed that the expressions of epithelial-mesenchymal transition (EMT)-associated miRNAs such as miR-200c and miR-141 were significantly low in 58As9. Using 58As9 with stably overexpressing miR-200c, miR-141, or both, together with a luciferase reporter assay, we found that miR-200c targeted zinc finger E-box-binding homeobox 1 (ZEB1) and miR-141 targeted ZEB2. The overexpressed lines reversed the EMT status from mesenchymal to epithelial in 58As9, and significantly reduced the invasion activity and peritoneal dissemination for a significant prolongation of survival in the orthotopic tumor models in nude mice. CONCLUSIONS: EMT-associated miRNAs such as miR-200c and miR-141 and their target genes ZEB1/ZEB2 have good potential for antiperitoneal dissemination therapy in patients with scirrhous gastric cancers.
Assuntos
Adenocarcinoma Esquirroso/patologia , Transição Epitelial-Mesenquimal/genética , Invasividade Neoplásica/genética , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/patologia , Adenocarcinoma Esquirroso/genética , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , Neoplasias Peritoneais/genética , Neoplasias Gástricas/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/biossíntese , Homeobox 1 de Ligação a E-box em Dedo de Zinco/biossínteseRESUMO
Scirrhous type gastric cancer is characterized by diffuse infiltration of poorly differentiated adenocarcinoma cells and poor prognosis. Although association of poorly differentiated histology with reduction in E-cadherin expression, as well as association of microRNA (miR)-200c with E-cadherin through regulation of ZEB1/2, has been reported, participation of miR-200c in gastric carcinogenesis is not fully understood. We used 6 cell lines originating from gastric cancers, and investigated levels of miR-200c along with its target mRNAs ZEB1/2 and E-cadherin by qRT-PCR. ZEB1 and E-cadherin protein expression was also assessed via western blotting. Furthermore, we investigated the expression levels of miR200c by in situ hybridization, along with the expression of ZEB1 and E-cadherin by immunohistochemistry, in 97 gastric adenocarcinoma tissues. Inverse correlation between miR200c and ZEB1 levels were obtained by qRT-PCR in cell lines (P<0.05). Cell lines with low miR-200c and high ZEB1 exhibited low E-cadherin expression in both qRT-PCR and western blotting, and exhibited spindle-shaped morphology, in contrast to round cell morphology in those cell lines with high miR-200c levels. Inverse correlations were also obtained between miR-200c and ZEB1 as well as between ZEB1 and E-cadherin levels in tissue samples (P<0.001). Cancer tissues with low miR-200c, high ZEB1, and low E-cadherin expression were associated with poorly differentiated histology, in contrast to tubular form in cancers with high miR-200c expression levels (P<0.001). Our data revealed that downregulation of miR-200c primarily regulated cell morphology by downregulation of E-cadherin through upregulation of ZEB1, leading to poorly differentiated histology in gastric cancer.
Assuntos
Adenocarcinoma Esquirroso/patologia , Caderinas/metabolismo , MicroRNAs/genética , Neoplasias Gástricas/patologia , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Adenocarcinoma Esquirroso/genética , Adenocarcinoma Esquirroso/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD , Caderinas/genética , Diferenciação Celular , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Carga Tumoral , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
No disponible
Assuntos
Humanos , Feminino , Adulto , Flebite/sangue , Flebite/metabolismo , Parede Torácica/lesões , Parede Torácica/metabolismo , Adenocarcinoma Esquirroso/genética , Ultrassonografia/instrumentação , Ultrassonografia/métodos , Mamografia/métodos , Flebite/complicações , Flebite/diagnóstico , Parede Torácica/anormalidades , Parede Torácica/patologia , Adenocarcinoma Esquirroso/complicações , Ultrassonografia/normas , Ultrassonografia , Mamografia/instrumentaçãoRESUMO
Scirrhous gastric cancer, which has the worst prognosis among the various types of gastric cancer, is highly invasive and associated with abundant stromal fibroblasts. Although cancer-associated fibroblasts (CAFs) have been proposed to generate a tumor-supportive extracellular matrix that promotes the expansion of this type of cancer, the molecular mechanisms by which CAFs assist cancer cells are not yet fully understood. Here, we show for the first time that Asporin, a small leucine-rich proteoglycan (SLRP), is predominantly expressed in CAFs, and has essential roles in promoting co-invasion of CAFs and cancer cells. CAFs of scirrhous gastric cancer possess high potential for invasion, and invasion by CAFs frequently proceeded invasion by cancer cells, both in vitro and in vivo. Expression of Asporin was induced in fibroblasts by exposure to gastric cancer cells. Asporin secreted from CAFs activates Rac1 via an interaction with CD44 and promotes invasion by CAFs themselves. Moreover, Asporin promoted invasion by neighboring cancer cells, via paracrine effects mediated by activation of the CD44-Rac1 pathway. These results suggest that Asporin is a unique SLRP that promotes progression of scirrhous gastric cancer and is required for coordinated invasion by CAFs and cancer cells. Therefore, Asporin may represent a new therapeutic target molecule for the development of drugs aimed at manipulating the cancer microenvironment.
Assuntos
Adenocarcinoma Esquirroso/genética , Proteínas da Matriz Extracelular/biossíntese , Invasividade Neoplásica/genética , Neoplasias Gástricas/genética , Adenocarcinoma Esquirroso/patologia , Animais , Técnicas de Cocultura , Matriz Extracelular/genética , Proteínas da Matriz Extracelular/genética , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Comunicação Parácrina/genética , Neoplasias Gástricas/patologia , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas rac1 de Ligação ao GTPRESUMO
Cancer-associated fibroblasts (CAFs) have recently been linked to the invasion and metastasis of gastric cancer. In addition, the microRNA (miR)-200 family plays a central role in the regulation of the epithelial-mesenchymal transition process during cancer metastasis, and aberrant DNA methylation is one of the key mechanisms underlying regulation of the miR-200 family. In this study, we clarified whether epigenetic changes of miR-200b by CAFs stimulate cancer invasion and peritoneal dissemination in gastric cancer. We evaluated the relationship between miR-200b and CAFs using a coculture model. In addition, we established a peritoneal metastasis mouse model and investigated the expression and methylation status of miR-200b. We also investigated the expression and methylation status of miR-200b and CAFs expression in primary gastric cancer samples. CAFs (CAF-37 and CAF-50) contributed to epigenetic changes of miR-200b, reduced miR-200b expression and promoted tumor invasion and migration in NUGC3 and OCUM-2M cells in coculture. In the model mice, epigenetic changes of miR-200b were observed in the inoculated high-frequency peritoneal dissemination cells. In the 173 gastric cancer samples, the low miR-200b expression group demonstrated a significantly poorer prognosis compared with the high miR-200b expression group and was associated with peritoneal metastasis. In addition, downregulation of miR-200b in cancer cells was significantly correlated with alpha-smooth muscle actin expression. Our data provide evidence that CAFs reduce miR-200b expression and promote tumor invasion through epigenetic changes of miR-200b in gastric cancer. Thus, CAFs might be a therapeutic target for inhibition of gastric cancer.
Assuntos
Adenocarcinoma Esquirroso/patologia , Epigênese Genética , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/patologia , Adenocarcinoma Esquirroso/genética , Adenocarcinoma Esquirroso/metabolismo , Animais , Apoptose , Western Blotting , Movimento Celular , Proliferação de Células , Ilhas de CpG , Metilação de DNA , Transição Epitelial-Mesenquimal , Feminino , Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Invasividade Neoplásica , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gastric cancer (GC) is one of the most common malignancies worldwide. In particular, scirrhous type GC is highly metastatic and is characterized clinically by rapid disease progression and poor prognosis. MicroRNAs (miRNAs) play crucial roles in cancer development and progression. We previously demonstrated by microarray analysis that microRNA-145 (miR-145) is one of the more highly expressed miRNAs in scirrhous type GC vs. non-scirrhous types of GC. In the present study, we investigated the role of miR-145 in scirrhous type GC. The expression levels of miR-145 assessed by quantitative RT-PCR were higher in scirrhous type GC tissue samples than in non-scirrhous type GC and corresponding normal tissues. GC patients with high miR-145 expression were at a more advanced tumor stage (P=0.0156) and had more scirrhous type histology (P=0.0054) than those with low miR-145 expression. Furthermore, miR-145 expression was significantly associated with poor prognosis in GC patients (P=0.0438). miR-145 expression was localized in stromal fibroblasts of scirrhous type GC but not in cancer cells. miR-145 was induced by treatment by transforming growth factor-ß, and it enhanced the expression of α-smooth muscle actin, a marker of myofibroblasts, in both normal gastric fibroblasts and cancer-associated fibroblasts. These data suggest that miR-145 may contribute to the progression of scirrhous type GC by regulating activation of peri-tumoral fibroblasts.
Assuntos
Adenocarcinoma Esquirroso/genética , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Gástricas/genética , Adenocarcinoma Esquirroso/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Gástricas/patologiaRESUMO
Scirrhous type gastric cancer is highly aggressive and has a poorer prognosis than many other types of gastric carcinoma, due to its characteristic rapid cancer cell infiltration and proliferation, extensive stromal fibrosis, and frequent peritoneal dissemination. The aim of the present study was to identify novel prognostic markers or therapeutic targets for scirrhous type gastric cancer. We reviewed a list of genes with upregulated expression in scirrhous type gastric cancer and compared their expression with that in normal stomach from our previous Escherichia coli (E. coli) ampicillin secretion-trap (CAST) analysis. We focused on the ZDHHC14 gene, which encodes zinc finger, DHHC-type containing 14 protein. qRT-PCR analysis of ZDHHC14 in 41 gastric cancer cases revealed that compared to mRNA levels in normal non-neoplastic gastric mucosa, ZDHHC14 mRNA was overexpressed in 27% of gastric cancer tissue samples. The overexpression of ZDHHC14 was significantly associated with depth of tumor invasion, undifferentiated histology and scirrhous pattern. The invasiveness of ZDHHC14-knockdown HSC-44PE and 44As3 gastric cancer cells was decreased in comparison with that of the negative control siRNA-transfected cells, together with downregulation of MMP-17 mRNA. Integrins α5 and ß1 were also downregulated in ZDHHC14-knockdown 44As3 cells. Forced expression of ZDHHC14 activated gastric cancer cell migration and invasion in vitro. These results indicate that ZDHHC14 is involved in tumor progression in patients with scirrhous type gastric cancer.
Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Adenocarcinoma Esquirroso/patologia , Invasividade Neoplásica/genética , Neoplasias Gástricas/patologia , Adenocarcinoma Esquirroso/genética , Idoso , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Neoplasias Gástricas/genética , Regulação para CimaRESUMO
OBJECTIVE: Scirrhous-type gastric cancer (GC) is highly aggressive and has a poor prognosis due to rapid cancer cell infiltration accompanied by extensive stromal fibrosis. The aim of this study is to identify genes that encode transmembrane proteins frequently expressed in scirrhous-type GC. METHODS: We compared Escherichia coli ampicillin secretion trap (CAST) libraries from 2 human scirrhous-type GC tissues with a normal stomach CAST library. By sequencing 2,880 colonies from scirrhous CAST libraries, we identified a list of candidate genes. RESULTS: We focused on the TM9SF3 gene because it has the highest clone count, and immunohistochemical analysis demonstrated that 46 (50%) of 91 GC cases were positive for TM9SF3, which was observed frequently in scirrhous-type GC. TM9SF3 expression showed a significant correlation with the depth of invasion, tumor stage and undifferentiated GC. There was a strong correlation between TM9SF3 expression and poor patient outcome, which was validated in two separate cohorts by immunostaining and quantitative RT-PCR, respectively. Transient knockdown of the TM9SF3 gene by siRNA showed decreased tumor cell-invasive capacity. CONCLUSION: Our results indicate that TM9SF3 might be a potential diagnostic and therapeutic target for scirrhous-type GC.
Assuntos
Adenocarcinoma Esquirroso/fisiopatologia , Ampicilina , Escherichia coli , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Análise de Sequência de DNA/métodos , Neoplasias Gástricas/fisiopatologia , Adenocarcinoma Esquirroso/diagnóstico , Adenocarcinoma Esquirroso/genética , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/fisiologia , Proteínas de Transporte , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Masculino , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Prognóstico , Sinais Direcionadores de Proteínas , RNA Interferente Pequeno/farmacologia , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genéticaRESUMO
Scirrhous gastric cancer is associated with abundant stroma and frequently develops into peritoneal carcinomatosis with malignant ascites. Although malignant ascites is among the most deadly diseases worldwide, its molecular pathogenesis is poorly understood. We investigated the role of hepatocyte growth factor (HGF) in the production of peritoneal carcinomatosis with malignant ascites. We examined three scirrhous and three non-scirrhous human gastric cancer cell lines for the production of peritoneal carcinomatosis in vivo and responses to HGF in vitro. Furthermore, clinical scirrhous gastric cancer specimens were examined for HGF production. Among the six cell lines examined, only two scirrhous cell lines (NUGC4 and GCIY) produced peritoneal carcinomatosis with massive ascites after intraperitoneal injection in nude mice. Their proliferation was stimulated by exogenous HGF in vitro. On the other hand, a non-scirrhous cell line, MKN45, with MET amplification generated peritoneal tumors but not ascites. MET tyrosine kinase inhibitors, crizotinib and TAS-115, inhibited HGF-stimulated proliferation of NUGC4 and GCIY as well as constitutive proliferation of MKN45. Furthermore, crizotinib and TAS-115 prolonged the survival of mice bearing established tumors by NUGC4 or MKN45. In clinical specimens, HGF was markedly produced by stromal fibroblasts. Malignant ascitic fluids from patients with peritoneal carcinomatosis contained high levels of HGF. Our results strongly suggest that paracrine HGF-induced activation of MET-mediated signaling pathways plays an important role in the pathogenesis of peritoneal carcinomatosis in scirrhous gastric cancer. Thus, MET signaling pathway may be a potential therapeutic target for peritoneal carcinomatosis of gastric cancer, even without MET amplification.
Assuntos
Adenocarcinoma Esquirroso/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Peritoneais/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma Esquirroso/tratamento farmacológico , Adenocarcinoma Esquirroso/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Crizotinibe , Fibroblastos/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias Peritoneais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/biossíntese , Pirazóis/farmacologia , Piridinas/farmacologia , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Células Estromais/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The molecular mechanisms underlying the peritoneal dissemination of gastric cancer remain unclear. Using in vivo metastatic models, this study attempted to clarify the role of hypoxia inducible factor (HIF)-1α in the development of peritoneal dissemination of gastric cancer. HIF-1α knockdown (KD) cells were established in the scirrhous gastric cancer cell line 58As9. Using KD and control (SC) cells, the presence of peritoneal dissemination was assessed in orthotopic implantation (o.i.) and intraperitoneal injection (i.p.) models. A series of in vitro analyses were also conducted. Finally, tumor angiogenesis was immunohistochemically analyzed. In the o.i. model, peritoneal dissemination was more frequently observed in the SC mice (93%) compared to the KD mice (13%) (P<0.001). In the i.p. model, peritoneal dissemination occurred at a high rate in both types of mice; however, a greater number of nodules was observed in the KD mice (P=0.017). The in vitro assays showed that HIF-1α exerts unfavorable effects on anoikis resistance and adhesion to extracellular matrix. Angiogenesis and vascular invasion were more aggressive in the SC gastric tumors. Vascular invasion was present in the intratumoral regions of the disseminated nodules in the SC o.i., but not the i.p., mice. HIF-1α was found to be crucial for the development of peritoneal dissemination in o.i. model, which mimics natural metastasis. In contrast, HIF-1α played an inhibitory role in suppressing peritoneal dissemination in the i.p. model. These results indicate that peritoneal dissemination in o.i. mice may not act through a seeding mechanism. An immunohistochemical analysis demonstrated HIF-1α-activated angiogenesis and vascular invasion in stomach tumors. Furthermore, the results showed that the disseminated nodules observed in SC o.i. mice were formed via extravasation of cancer cells. We provide a possible mechanism in which peritoneal dissemination of gastric cancer develops via a vascular network whereby HIF-1α activates tumor angiogenesis.
Assuntos
Adenocarcinoma Esquirroso/patologia , Apoptose , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Patológica , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/patologia , Adenocarcinoma Esquirroso/genética , Adenocarcinoma Esquirroso/metabolismo , Animais , Western Blotting , Adesão Celular , Proliferação de Células , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMO
BACKGROUND: A novel model system to study the cellular, molecular, and genetic characteristics of breast cancer stem cells is needed. Personalized prognostic models are indispensable for assessing the effect of so-called tailor-made adjuvant therapy. MATERIALS AND METHODS: Surgically-extirpated tissues were dispersed with Dispase and cultured in commonly used medium. The expression of tumor markers was detected with immunohistochemistry, and gene expression profiles of tissues and cells were analyzed using the Agilent Human Microarray. RESULTS: We established a primary culture, which exhibited mesenchymal morphology. The gene expression profile of the primary culture revealed that the cells underwent epithelial-mesenchymal transition and had cancer stem cell properties. CONCLUSION: A primary culture of breast cancer cells can be easily established and could be used for studying breast cancer stem cells and assessing treatment of a patient.
Assuntos
Adenocarcinoma Esquirroso/patologia , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Mama/patologia , Transição Epitelial-Mesenquimal , Modelos Biológicos , Células-Tronco Neoplásicas/patologia , Adenocarcinoma Esquirroso/genética , Adenocarcinoma Esquirroso/metabolismo , Biomarcadores Tumorais/metabolismo , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The prognosis of patients with advanced diffuse-type gastric cancer (GC), especially scirrhous gastric cancer (SGC) remains extremely poor. Peritoneal carcinomatosis is a frequent form of metastasis of SGC. With survival rates of patients with peritoneal metastasis at 3 and 5 years being only 9.8% and 0%, respectively, development of a new treatment is urgently crucial. For such development, the establishment of a therapeutic mouse model is required. Among the 11 GC cell lines we examined, HSC-60 showed the most well-preserved expression profiles of the Hedgehog and epithelial-mesenchymal transition pathways found in primary SGCs. After six cycles of harvest of ascitic tumor cells and their orthotopic inoculation in scid mice, a highly metastatic subclone of HSC-60, 60As6 was obtained, by means of which we successfully developed peritoneal metastasis model mice. The mice treated with small interfering (si) RNA targeting NEDD1, which encodes a gamma-tubulin ring complex-binding protein, by the atelocollagen-mediated delivery system showed a significantly prolonged survival. Our mouse model could thus be useful for the development of a new therapeutic modality. Intraperitoneal administration of siRNAs of targeted genes such as NEDD1 could provide a new opportunity in the treatment of the peritoneal metastasis of SGC.
Assuntos
Adenocarcinoma Esquirroso/genética , Adenocarcinoma Esquirroso/terapia , Proteínas Associadas aos Microtúbulos/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/terapia , Adenocarcinoma Esquirroso/patologia , Adenocarcinoma Esquirroso/secundário , Animais , Linhagem Celular Tumoral , Colágeno/administração & dosagem , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos/métodos , Transição Epitelial-Mesenquimal/genética , Feminino , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos SCID , Metástase Neoplásica , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Elk-1 do Domínio ets/genéticaRESUMO
Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.
Assuntos
Adenocarcinoma Esquirroso/metabolismo , Adenocarcinoma Esquirroso/patologia , Proteínas de Ciclo Celular/metabolismo , Transição Epitelial-Mesenquimal , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Adenocarcinoma Esquirroso/genética , Animais , Caderinas/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Metástase Neoplásica , Peritônio/metabolismo , Peritônio/patologia , Regiões Promotoras Genéticas/genética , Neoplasias Gástricas/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Regulação para Cima , Vimentina/metabolismoRESUMO
The scirrhous subtype of gastric cancer is a highly infiltrative tumor with a poor outcome. To identify a transforming gene in this intractable disorder, we constructed a retroviral complementary DNA (cDNA) expression library from a cell line (OCUM-1) of scirrhous gastric cancer. A focus formation assay with the library and mouse 3T3 fibroblasts led to the discovery of a transforming cDNA, encoding for MAP2K1 with a glutamine-to-proline substitution at amino acid position 56. Interestingly, treatment with a MAP2K1-specific inhibitor clearly induced cell death of OCUM-1 but not of other two cells lines of scirrhous gastric cancer that do not carry MAP2K1 mutations, revealing the essential role of MAP2K1(Q56P) in the transformation mechanism of OCUM-1 cells. By using a next-generation sequencer, we further conducted deep sequencing of the MAP2K1 cDNA among 171 human cancer specimens or cell lines, resulting in the identification of one known (D67N) and four novel (R47Q, R49L, I204T and P306H) mutations within MAP2K1. The latter four changes were further shown to confer transforming potential to MAP2K1. In our experiments, a total of six (3.5%) activating mutations in MAP2K1 were thus identified among 172 of specimens or cell lines for human epithelial tumors. Given the addiction of cancer cells to the elevated MAP2K1 activity for proliferation, human cancers with such MAP2K1 mutations are suitable targets for the treatment with MAP2K1 inhibitors.
