RESUMO
Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to therapeutic resistance. We integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes and bulk multi-omic profiles across 11 adult IDH mutant or IDH wild-type gliomas to delineate sources of intratumoral heterogeneity. We showed that local DNA methylation disorder is associated with cell-cell DNA methylation differences, is elevated in more aggressive tumors, links with transcriptional disruption and is altered during the environmental stress response. Glioma cells under in vitro hypoxic and irradiation stress increased local DNA methylation disorder and shifted cell states. We identified a positive association between genetic and epigenetic instability that was supported in bulk longitudinally collected DNA methylation data. Increased DNA methylation disorder associated with accelerated disease progression and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work identified an epigenetically facilitated adaptive stress response process and highlights the importance of epigenetic heterogeneity in shaping therapeutic outcomes.
Assuntos
Neoplasias Encefálicas/genética , Plasticidade Celular/genética , Epigênese Genética , Glioma/genética , Análise de Célula Única , Estresse Fisiológico/genética , Evolução Clonal , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Genoma Humano , Humanos , Mutação/genética , Filogenia , Regiões Promotoras Genéticas/genética , Microambiente Tumoral/genéticaRESUMO
The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC's long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a "mixed" neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the "neuronal" genes are bound by both SOX2 and AP1.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Genoma , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição AP-1/genética , Animais , Sequência de Bases , Diferenciação Celular/genética , Regulação para Baixo/genética , Deleção de Genes , Lentivirus/metabolismo , Camundongos , Modelos Biológicos , Neuroglia/metabolismo , Neurônios/metabolismo , RNA-Seq , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
Extrachromosomal, circular DNA (ecDNA) is emerging as a prevalent yet less characterized oncogenic alteration in cancer genomes. We leverage ChIA-PET and ChIA-Drop chromatin interaction assays to characterize genome-wide ecDNA-mediated chromatin contacts that impact transcriptional programs in cancers. ecDNAs in glioblastoma patient-derived neurosphere and prostate cancer cell cultures are marked by widespread intra-ecDNA and genome-wide chromosomal interactions. ecDNA-chromatin contact foci are characterized by broad and high-level H3K27ac signals converging predominantly on chromosomal genes of increased expression levels. Prostate cancer cells harboring synthetic ecDNA circles composed of characterized enhancers result in the genome-wide activation of chromosomal gene transcription. Deciphering the chromosomal targets of ecDNAs at single-molecule resolution reveals an association with actively expressed oncogenes spatially clustered within ecDNA-directed interaction networks. Our results suggest that ecDNA can function as mobile transcriptional enhancers to promote tumor progression and manifest a potential synthetic aneuploidy mechanism of transcription control in cancer.
Assuntos
Cromossomos/genética , DNA de Neoplasias/genética , Glioblastoma/genética , Oncogenes/genética , Carcinogênese/genética , Cromatina/genética , HumanosRESUMO
Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA/genética , Glioma/genética , Mutação/genética , Animais , Cães , Exoma/genética , Humanos , Isocitrato Desidrogenase/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
HYPOTHESIS: We hypothesize that genomic variants including deletions, insertions, inversions, and tandem duplications beyond the changes in tumor suppressor NF2 gene affect gene expression of tumor-specific pathways in vestibular schwannomas (VS) patients with Neurofibromatosis type 2 (NF2), thus contributing to their clinical behavior. BACKGROUND: Genomic variation could reconfigure transcription in NF2 transformation process. Therefore, genome-wide high-resolution characterization of structural variants (SV) landscapes in NF2 tumors can expand our understanding of the genes regulating the clinical phenotypes in NF2-associated VS. METHODS: We performed whole-genome haplotype-specific structural variation analysis using synthetic linked reads generated through microfluidics-based barcoding of high molecular weight DNA followed by high-coverage Illumina paired-end whole-genome sequencing from 10 patients' tumors of different growth rates and their matching blood samples. RESULTS: NF2 tumor-specific deletions and large SVs were detected and can be classified based on their association with tumor growth rates. Through detailed annotation of these mutations, we uncover common alleles affected by these deletions and large SVs that can be associated with signaling pathways implicated in cell proliferation and tumorigenesis. CONCLUSION: The genomic variation landscape of NF2-related VS was investigated through whole-genome linked-read sequencing. Large SVs, in addition to deletions, were identified and may serve as modulators of clinical behavior.
Assuntos
Neurofibromatose 2/genética , Neuroma Acústico/genética , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , MutaçãoRESUMO
Acquired genomic structural variants (SVs) are major hallmarks of cancer genomes, but they are challenging to reconstruct from short-read sequencing data. Here we exploited the long reads of the nanopore platform using our customized pipeline, Picky ( https://github.com/TheJacksonLaboratory/Picky ), to reveal SVs of diverse architecture in a breast cancer model. We identified the full spectrum of SVs with superior specificity and sensitivity relative to short-read analyses, and uncovered repetitive DNA as the major source of variation. Examination of genome-wide breakpoints at nucleotide resolution uncovered micro-insertions as the common structural features associated with SVs. Breakpoint density across the genome is associated with the propensity for interchromosomal connectivity and was found to be enriched in promoters and transcribed regions of the genome. Furthermore, we observed an over-representation of reciprocal translocations from chromosomal double-crossovers through phased SVs. We demonstrate that Picky analysis is an effective tool for comprehensive detection of SVs in cancer genomes from long-read data.
Assuntos
Regulação Neoplásica da Expressão Gênica , Variação Estrutural do Genoma , Nanoporos , Linhagem Celular Tumoral , Análise Mutacional de DNA/métodos , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , HumanosRESUMO
Panicum virgatum L. (switchgrass) is a polyploid, perennial grass species that is native to North America, and is being developed as a future biofuel feedstock crop. Switchgrass is present primarily in two ecotypes: a northern upland ecotype, composed of tetraploid and octoploid accessions, and a southern lowland ecotype, composed of primarily tetraploid accessions. We employed high-coverage exome capture sequencing (~2.4 Tb) to genotype 537 individuals from 45 upland and 21 lowland populations. From these data, we identified ~27 million single-nucleotide polymorphisms (SNPs), of which 1 590 653 high-confidence SNPs were used in downstream analyses of diversity within and between the populations. From the 66 populations, we identified five primary population groups within the upland and lowland ecotypes, a result that was further supported through genetic distance analysis. We identified conserved, ecotype-restricted, non-synonymous SNPs that are predicted to affect the protein function of CONSTANS (CO) and EARLY HEADING DATE 1 (EHD1), key genes involved in flowering, which may contribute to the phenotypic differences between the two ecotypes. We also identified, relative to the near-reference Kanlow population, 17 228 genes present in more copies than in the reference genome (up-CNVs), 112 630 genes present in fewer copies than in the reference genome (down-CNVs) and 14 430 presence/absence variants (PAVs), affecting a total of 9979 genes, including two upland-specific CNV clusters. In total, 45 719 genes were affected by an SNP, CNV, or PAV across the panel, providing a firm foundation to identify functional variation associated with phenotypic traits of interest for biofuel feedstock production.
Assuntos
Exoma/genética , Variação Genética , Panicum/genética , Análise de Sequência de DNA/métodos , Cromossomos de Plantas/genética , Variações do Número de Cópias de DNA , Ecossistema , Ecótipo , Genética Populacional , Genoma de Planta/genética , Genótipo , Geografia , Panicum/classificação , Panicum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Poliploidia , Especificidade da Espécie , Estados UnidosRESUMO
Anti-angiogenic therapy, inhibition of a co-operative process with vascular endothelial cells and pericytes could be an effective strategy to treat malignant tumours. Apart from vascular endothelial growth factor (VEGF), angiopoietin 2 (Ang2) is a promising target of anti-angiogenic therapy. Although inhibition of Ang2 has been shown to decrease tumour size in preclinical and phase I trials, its mechanisms of action remain largely unknown. To elucidate the mechanisms of Ang2 inhibition, we have focused on differentiation of the vessels as well as on growth of the vessels, especially in vivo. L1-10, a selective Ang2 inhibitor was used. The in vitro effects of Ang2 inhibition or addition of Ang2 using HUVECs were also examined. Growth and differentiation of tumour-associated vessels were investigated in xenografts derived from a colon cancer treated by L1-10. Effects of VEGF inhibition were also examined to discriminate Ang2-specific action on the tumour-associated vessels. In vitro studies showed that VEGF enhanced proliferation and tube formation of HUVECs, and caused a significant increase in Rac1 and CDC42 expression when cultured in the collagen matrix gel, whereas neither Ang2 nor L1-10 affected in vitro behaviour of HUVECs or levels of the proteins. In vivo, on the other hand, we found that Ang2 inhibition with treatment of L1-10 dosedependently decreased tumour growth. Furthermore, we found that L1-10 treatment extends the tumour-associated vessels whilst it suppressed a sound lumen formation. Histological analysis on xenografts suggests that Ang2 inhibition could have disturbed in vivo vascular differentiation. Our data provide a novel aspect that Ang2 may play an essential role in in vivo vascular differentiation, thus supporting a rationale for Ang2-targeted therapy against colon cancer.
Assuntos
Angiopoietina-2/antagonistas & inibidores , Neoplasias do Colo/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Patológica/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Angiopoietina-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Tyrosine kinase B (TrkB) is associated with aggressive behavior and poor prognosis in various cancers. Here we examined the association between TrkB expression and distant metastases/prognosis in gastric carcinoma (GC). PATIENTS AND METHODS: We analyzed TrkB expression in 161 GC patients by immunohistochemistry and Western blot analysis. The correlation of TrkB mRNA and protein expression levels was examined in 10 patients by RT-PCR assay. RESULTS: TrkB expression was of level 1 in 97 (60.2%) and level 2 in 64 (39.8%) patients. Patients with level 2 expression had a significantly higher incidence of distant metastases (p < 0.0001), well-differentiated tumors (p < 0.005), deeper depth of invasion (p < 0.005) and poorer disease-free and overall survival (p < 0.0001 each) compared to patients with level 1. Multivariate analysis identified the level of TrkB expression as an independent prognostic factor for both disease-free and overall survival (p < 0.01 and p < 0.0001, respectively). Both lymph node metastasis (odds ratio = 10.7) and TrkB expression (odds ratio = 9.3) were independent predictors of distant metastases. CONCLUSION: A high level of TrkB expression was observed in well-differentiated GC subtypes and is a predictor for distant metastases and prognosis in GC.
Assuntos
Proteínas Tirosina Quinases/biossíntese , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Neoplasias Gástricas/secundário , Análise de SobrevidaRESUMO
Survivin has multiple functions including cytoprotection, inhibition of cell death, and cell-cycle regulation, especially at the mitotic process stage, all of which favor cancer survival. Many studies on clinical specimens have shown that survivin expression is invariably up-regulated in human cancers and is associated with resistance to chemotherapy or radiation therapy, and linked to poor prognosis, suggesting that cancer cells survive with survivin. It is also reported that survivin inhibition, alone or in combination with the other therapies, induces or enhances apoptosis and mitotic catastrophe in tumor cells. Moreover, certain antitumor agents can reduce survivin expression. These findings suggest that survivin may be a promising molecular target against human malignancies.
Assuntos
Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Proteínas Inibidoras de Apoptose , Survivina , Regulação para CimaRESUMO
PURPOSE: Connexin 26 (Cx26) is one of the gap junction-forming family members classically considered to be tumor suppressors. However, recent studies show association of elevated expression of Cx26 with poor prognosis in several human malignancies. Furthermore, Cx26 has been observed to be indispensable to spontaneous metastasis of melanoma cells. Here, we assessed Cx26 expression in primary colorectal cancer (CRC) and the metastatic lesions to elucidate its role in metastasis. EXPERIMENTAL DESIGN: Cx26 expression was assessed in 25 adenomas, 167 CRCs, and normal mucosa, together with the metastatic lesions. RESULTS: Normal mucosa and adenomatous tissue expressed Cx26 mainly in the plasma membrane, whereas cancer cells mostly contained Cx26 in the cytoplasm. The incidence of aberrant Cx26 expression varied widely in CRC (mean, 49.5 +/- 35.5%), and the expression levels were confirmed by Western blot and quantitative reverse transcription-PCR. Clinicopathologic survey revealed association of high expression with less differentiated histology and venous invasion (P = 0.0053 and P = 0.0084, respectively). Notably, high Cx26 expression was associated with shorter disease-free survival and shorter lung metastasis-free survival in 154 curatively resected CRC sets (P = 0.041 and P = 0.028, respectively). Survey of metastatic lesions revealed that lung metastasis, but not liver and lymph nodes metastases, expressed higher Cx26 than the CRC series or corresponding primary CRCs (P < 0.0001 and P = 0.0001, respectively). CONCLUSIONS: These findings suggest that aberrant expression of Cx26 plays an essential role in lung metastasis. Thus, Cx26 is a promising therapeutic target, particularly for CRC patients who develop lung metastasis.
Assuntos
Neoplasias Colorretais/patologia , Conexinas/genética , Neoplasias Pulmonares/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/genética , Neoplasias Colorretais/cirurgia , Conexina 26 , Primers do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Mucosa Intestinal/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Reação em Cadeia da PolimeraseRESUMO
Contrary to the previously purported role of gap junction (GJ) associated-protein connexin 26 (Cx26) as a tumor suppressor, increased expression of Cx26 has recently been demonstrated in several human malignancies. Surprisingly, this high expression is reportedly related to poor prognosis in squamous cell lung carcinoma and breast cancer. In this study, we examined levels of Cx26 in various human gastrointestinal (GI) carcinomas, with a focus on pancreatic carcinomas, using immunohistochemistry. Many GI carcinomas displayed abundant Cx26 expression, predominantly in the cytoplasm. Cx26 was detected in 5/8 gastric cancers (62.5%), 6/8 squamous cell carcinomas of the esophagus (75.0%), 7/8 pancreatic cancers (87.5%) and 7/8 colon cancer cases (87.5%). However, Cx26 expression was not present in hepatocellular carcinoma (HCC, 0/8). Extensive immunohistochemical examination was performed on pancreatic carcinomas, revealing strong expression of Cx26 protein in 30/43 cases (70%), weak expression in 6/43 (14%) and no expression in 7/43 (16%). The present study demonstrated up-regulated Cx26 expression in a considerable percentage of GI carcinomas, with the exception of HCC. Our findings suggest that Cx26 may be involved in some of the malignant processes of GI cancers, and especially in pancreatic carcinomas.
Assuntos
Carcinoma/metabolismo , Conexinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Conexina 26 , Neoplasias Gastrointestinais/metabolismo , Humanos , Imuno-Histoquímica , Regulação para CimaRESUMO
CDC25A is a cell cycle-activating phosphatase that promotes transition from the G1 to S phase. We previously reported that overexpression of CDC25A in human hepatocellular carcinoma (HCC) tissue samples was associated with poor prognosis. In this study, we attempted suppression of CDC25A in HCC cells to elucidate the therapeutic potential of this approach. Administration of CDC25A antisense (AS) oligonucleotide resulted in 25-50% inhibition of cell growth at 48 h, G0-G1 arrest, and significant inhibition of cancer cell invasion. To elucidate the underlying mechanism of the inhibitory effects of HCC cell invasion, we examined several invasion-associated molecules, and we found that membrane-type 3 (MT3)-matrix metalloproteinase (MMP) mRNA was greatly reduced following treatment with AS oligonucleotide to CDC25A or siRNA treatment. Notably, screening of a panel of gastrointestinal cancer cells indicated that MT3-MMP was generally expressed by HCC cells, whereas other cell types did not express this type of matrix metalloproteinase so frequently. We also found that CDC25A facilitated cellular differentiation by increasing albumin expression in the PLC cell line. These results suggest that CDC25A, by inhibiting HCC growth and invasion, may be a feasible therapeutic target for human HCC.
Assuntos
Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Fosfatases cdc25/antagonistas & inibidores , Albuminas/genética , Albuminas/metabolismo , Carcinoma Hepatocelular/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fluoresceína-5-Isotiocianato , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genética , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Oligonucleotídeos Antissenso/farmacologia , RNA Interferente Pequeno/farmacologia , TransfecçãoRESUMO
The platinum-based chemotherapeutic agent oxaliplatin displays a wide range of antitumor activities. However, the underlying molecular responses to oxaliplatin in esophageal cancer remain largely unknown. In the present study, we investigated the effect of oxaliplatin on two esophageal cancer cell lines, squamous cell carcinoma (TE3) and adenocarcinoma (TE7). Following cell-cycle arrest at G(2) phase after oxaliplatin treatment, TE3 cells died via apoptosis and TE7 cells died via mitotic catastrophe. Survivin was inhibited more in TE7 cells compared with TE3 cells, but inhibition of survivin using small interfering RNA induced mitotic catastrophe in both cell lines. Further investigations indicated that survivin promoter activity was also inhibited by oxaliplatin. Among mitotic catastrophe-associated proteins, 14-3-3 sigma was decreased in TE7 cells; no evident changes were observed for aurora kinases. Oxaliplatin-induced apoptosis in the TE3 cells was caspase dependent. However, downregulation of Bad, Bid, Puma, and Noxa, lack of cytochrome c release, and limited loss of mitochondrial membrane potential in early phase indicated possible initiation by pathways other than the mitochondrial pathway. Mechanistic studies showed that downregulation of survivin by oxaliplatin in TE7 cells was partially due to the proteasome-mediated protein degradation pathway and partially due to the downregulation of Sp1 transcription factor. Similar results were obtained for another gastric adenocarcinoma cell line, MKN45, in which survivin was previously shown to be inhibited by oxaliplatin. These data indicate that survivin may be a key target for oxaliplatin. The ability of oxaliplatin to induce different modes of cell death may contribute to its efficacy in esophageal cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Esofágicas/tratamento farmacológico , Compostos Organoplatínicos/farmacologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Mitose/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Oxaliplatina , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição Sp1/metabolismo , SurvivinaRESUMO
BACKGROUND AND OBJECTIVES: Adhesion molecules are implicated in the progression of colorectal cancer (CRC). Despite the evidence of association between their expression and patients' prognosis, the data have not been examined simultaneously in a same study; thus, the relative clinical value remained largely unknown. The aim of this study was to identify the adhesion factors that display the most significant prognostic value for CRC patients to guide clinical decision-making regarding appropriate treatment. PATIENTS AND METHODS: We examined by immunohistochemistry, the expression of E-cadherin and its associated catenins, alpha(alpha)-catenin and beta(beta)-catenin, DCC, and CD44 and its partner, MT1-MMP in a series of 140 CRC tissues at intermediate Stage II and Stage III to determine their prognostic significance. RESULTS: Clinicopathological survey indicated an inverse relationship between E-cadherin expression and tumor differentiation, and an association between CD44 expression and venous invasion. Univariate and multivariate analyses showed that loss of expression of E-cadherin and CD44 significantly correlated to poor survival, especially in Stage II. Combination studies indicated that loss of E-cadherin and loss of CD44 had the worst impact on patient prognosis, particularly in colon cancer. CONCLUSION: Immunohistochemical staining of E-cadherin and CD44 may help to identify a subgroup of high-risk patients with Stage II CRC, especially in colon cancer, who may need intensive follow-up and appropriate therapeutic strategy.
Assuntos
Caderinas/biossíntese , Moléculas de Adesão Celular/biossíntese , Neoplasias Colorretais/metabolismo , Receptores de Hialuronatos/biossíntese , Metaloproteinase 14 da Matriz/biossíntese , Idoso , Neoplasias Colorretais/patologia , Receptor DCC , Feminino , Humanos , Imuno-Histoquímica , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , Antígeno Nuclear de Célula em Proliferação/análise , Receptores de Superfície Celular/biossíntese , Análise de Sobrevida , Proteínas Supressoras de Tumor/biossínteseRESUMO
PURPOSE: Myofibroblasts, which are specifically differentiated fibroblasts, are thought to play a central role in the desmoplastic reaction, a dynamic stromal change closely associated with cancer development. Although fundamental studies suggest that myofibroblasts may either facilitate or inhibit cancer progression, cumulative evidence supports their role in promoting tumor progression. The aim of this study was to assess the value of myofibroblasts in the cancer stroma as an indicator of disease recurrence after colorectal cancer surgery. EXPERIMENTAL DESIGN: Using computer-assisted image analysis, we quantified myofibroblasts in the cancer-associated stroma of 192 colorectal cancers using alpha-smooth muscle actin as a marker. RESULTS: The cancer-associated stroma contained various numbers of myofibroblasts (0.35-19.0%; mean, 5.55 +/- 3.85%). Tumors with abundant myofibroblasts were associated with shorter disease-free survival rate (P = 0.001) for stage II and III colorectal cancer. Multivariate analysis indicated that alpha-smooth muscle actin was a significant prognostic factor comparable with lymph node metastasis and superior to other tumor and stromal components, including histology of the tumor invasive front, peritumoral lymphocytic infiltration, and Crohn's-like lymphoid reaction. Moreover, colorectal cancers with synchronous liver metastasis generally displayed an active desmoplastic reaction, which was retained in the metastatic lesion to a similar extent. CONCLUSIONS: The results suggest that the abundance of myofibroblasts in cancer-associated stroma may be a useful indicator of disease recurrence after curative colorectal cancer surgery.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Fibroblastos/metabolismo , Recidiva Local de Neoplasia/metabolismo , Actinas/metabolismo , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Células Estromais/metabolismoRESUMO
BACKGROUND: The enhanced antitumor effect of paclitaxel when used with oxaliplatin in gastric cancer is reported, however the underlying biological mechanism is unknown. METHODS: We tested the cytotoxic activity, apoptosis, and mitotic catastrophe of paclitaxel and oxaliplatin in MKN-28 and MKN-45 gastric cancer cell lines. The modulation of survivin expression was determined by Western blotting. RESULTS: WST-1 assay indicated that paclitaxel plus oxaliplatin showed better cytotoxicity than paclitaxel alone, even when low concentrations of oxaliplatin were used. Flow cytometry analysis revealed significantly greater increases in apoptotic cells after treatment with paclitaxel followed by low-dose oxaliplatin (1 microM) than after any single-reagent regimen in the MKN-45 cell line. In MKN-28, a difference existed only between combination treatment and oxaliplatin treatment. Morphologic examination showed that the cells undergoing mitotic catastrophe were highest in the combination groups in the both cell lines. Downregulation of survivin expression was found by Western blotting with treatment by paclitaxel, oxaliplatin, or their combination. CONCLUSION: Our findings suggest that the mechanism of enhanced cytotoxicity might be through enhanced mitotic catastrophe and apoptosis, which is possibly due to chemotherapy-induced downregulation of surviving. The combination of paclitaxel and low-dose oxaliplatin should be incorporated into the design of a clinical trial.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos Organoplatínicos/administração & dosagem , Paclitaxel/administração & dosagem , Neoplasias Gástricas/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas Inibidoras de Apoptose , Concentração Inibidora 50 , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Oxaliplatina , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , SurvivinaRESUMO
PURPOSE: Our aim was to determine the effects of cyclin D1 inhibition on tumor-associated neovascularization and endothelial cell growth. EXPERIMENTAL DESIGN: We have generated adenovirus system for antisense to cyclin D1 (AS CyD1) and evaluated in vitro and in vivo effects. Small interfering RNA against cyclin D1 was also used to analyze cyclin D1 inhibition-associated vascular endothelial growth factor (VEGF) regulation. RESULTS: The xenografts treated with adenoviral AS CyD1 showed less vessel density and displayed smaller tumor size in colon cancer cell lines HCT116 and DLD1. In vitro studies indicated that AS CyD1 decreased VEGF protein expression in DLD1 but not in HCT116. Cyclin D1 small interfering RNA caused a decrease in VEGF expression at protein and RNA levels in DLD1. A modest decrease was noted in the VEGF promoter activity, with inactivation of the STAT3 transcription factor through dephosphorylation. On the hand, the cyclin D1 inhibition plus STAT3 inhibitor markedly decreased VEGF expression in HCT116, although VEGF did not change by the STAT3 inhibitor alone. In cultures of human umbilical vein endothelial cells (HUVEC), VEGF augmented cyclin D1 expression and cell growth. AS CyD1 significantly inhibited HUVEC growth even in the presence of VEGF. AS CyD1 also significantly suppressed in vitro tube formation in VEGF-treated HUVEC and in vivo macroaneurysm formation in VEGF-treated Matrigel plug. CONCLUSIONS: Our results suggest that cyclin D1 may play a role in the maintenance of VEGF expression and that AS CyD1 could be potentially useful for targeting both cancer cells and their microenvironment of tumor vessels.
Assuntos
Ciclina D1/antagonistas & inibidores , Células Endoteliais/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica/metabolismo , RNA Interferente Pequeno/farmacologia , Relação Estrutura-Atividade , Transplante Heterólogo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The oncogenic beta-catenin/T-cell factor (TCF) signal is a common trigger inducing expressions of various cancer-related genes and is activated in various types of human malignancy. The aim of this study was to create an effective double-stranded DNA decoy that would interfere with endogenous TCF hyperactivity in tumor cells. We first established the TCF-activated model using nontumor human embryonic kidney 293 (HEK293) cells by introducing a beta-catenin cDNA. Based on a consensus TCF-binding sequence in the cyclin D1 and c-myc promoters, several double-stranded oligodeoxynucleotides were designed and tested for their ability to inhibit TCF activity in the HEK293 model. Among them, the 18-mer oligodeoxynucleotide stably formed double-stranded DNA and efficiently inhibited TCF activity. FITC-labeled oligodeoxynucleotide was efficiently incorporated into the nucleus at 6 hours and remained within cells for up to 72 to 96 hours. When compared with scrambled oligodeoxynucleotide, we found that the 18-mer TCF decoy significantly inhibited TCF activity and promoter activities of the downstream target genes, such as cyclin D1, c-myc, and matrix metalloproteinase 7 in HCT116 colon cancer cells. Reverse transcription-PCR assays indicated that mRNA expression of these genes decreased with treatment of the TCF decoy. Proliferation assay showed that the TCF decoy significantly inhibited growth of HCT116 tumor cells, but not of nontumor HEK293 cells. Our data provide evidence that the TCF decoy reduced both TCF activity and transcriptional activation of downstream target genes. Thus, this TCF decoy is potentially an efficient and nontoxic molecular targeting therapy for controlling malignant properties of cancer cells.
Assuntos
Fatores de Transcrição TCF/fisiologia , beta Catenina/fisiologia , Pareamento Incorreto de Bases , Sequência de Bases , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias do Colo , Primers do DNA , Humanos , Rim , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição TCF/antagonistas & inibidores , Fatores de Transcrição TCF/química , Fatores de Transcrição TCF/genética , beta Catenina/antagonistas & inibidores , beta Catenina/genéticaRESUMO
Angiogenesis is a compensatory mechanism that enables malignant tumors to survive in an oxygen-deficient environment. To test our hypothesis that hypoxia stimulates the production of angiopoietin-2 (Ang-2) in colorectal cancer (CRC), we investigated the expression of Ang-2 in three cultured CRC cell lines, and in specimens from 11 CRC metastatic liver tumors. Hypoxia-induced Ang-2 mRNA expression was clearly evident in HCT116 cells that did not express Ang-2 under normoxic conditions. Ang-2 mRNA was detected only after 48 h in hypoxic serum-deprived cultures in a LoVo cell line, and under both normoxic and hypoxic conditions without any noticeable difference in the HT29 cells. There was a stepwise increase in Ang-2 expression from the periphery to the central part of the liver metastatic foci, whereas an inverse result was noted in tumor blood vessels, with a gradual decrease in CD31-positive ECs from the edge to the central region of the metastatic lesion. An expression pattern similar to Ang-2 was found in glucose transporter 1 (Glut-1), a known hypoxia-induced factor. These findings suggest that hypoxia plays an important role in inducing the expression of Ang-2 in CRC.