RESUMO
BACKGROUND: HER2 positive Breast Cancers (BC) have aggressive behavior and poor prognosis. Previously, we have identified miR-342-5p as an upstream regulator of HER2 signaling, as well as inhibitor of HER2 positive BC cell line growth. OBJECTIVE: Here, we aimed to further investigate the molecular mechanisms behind miR-342-5pinduced HER2 pathway deregulation. METHOD: Two HER2 amplified breast cancer cell lines were transiently transfected with miR-342-5p mimic or negative control, and gene expression was analyzed by Agilent microarrays. Three clinical datasets with BC patients were used to identify correlations between candidate genes and miR-342- 5p, and associations with survival. RESULTS: Pathway analyses of all deregulated genes revealed a significant suppression of the HER2 downstream pathways ERK/MAPK and SAPK/JNK, whereas the miR-342-5p predicted target genes were enriched for pathways associated with cell motility.Biological functions linked to mitochondrial stability were ranked among the top toxicological functions in both gene lists. Among the most deregulated genes, Cytochrome B5 Reductase 3 (CYB5R3) and Rap Guanine Nucleotide Exchange Factor 6 (RAPGEF6) significantly anticorrelated and correlated, respectively, with miR-342-5p in all three clinical BC datasets. Low CYB5R3 levels and high RAPGEF6 levels were significantly associated with survival, although this was not directly associated with HER2 expression. CONCLUSION: Our data suggest that miR-342-5p overexpression in HER2 positive BC cell lines elicits broad effects on HER2 downstream signaling, cell motility and mitochondrial stability. Together these effects may render cells less proliferative and more sensitive to cellular stress.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Citocromo-B(5) Redutase/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , MicroRNAs/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Citocromo-B(5) Redutase/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Receptor ErbB-2/metabolismoRESUMO
BACKGROUND: Despite the fact that metastases are the leading cause of colorectal cancer deaths, little is known about the underlying molecular changes in these advanced disease stages. Few have studied the overall gene expression levels in metastases from colorectal carcinomas, and so far, none has investigated the peritoneal carcinomatoses by use of DNA microarrays. Therefore, the aim of the present study is to investigate and compare the gene expression patterns of primary carcinomas (n = 18), liver metastases (n = 4), and carcinomatoses (n = 4), relative to normal samples from the large bowel. RESULTS: Transcriptome profiles of colorectal cancer metastases independent of tumor site, as well as separate profiles associated with primary carcinomas, liver metastases, or peritoneal carcinomatoses, were assessed by use of Bayesian statistics. Gains of chromosome arm 5p are common in peritoneal carcinomatoses and several candidate genes (including PTGER4, SKP2, and ZNF622) mapping to this region were overexpressed in the tumors. Expression signatures stratified on TP53 mutation status were identified across all tumors regardless of stage. Furthermore, the gene expression levels for the in vivo tumors were compared with an in vitro model consisting of cell lines representing all three tumor stages established from one patient. CONCLUSION: By statistical analysis of gene expression data from primary colorectal carcinomas, liver metastases, and carcinomatoses, we are able to identify genetic patterns associated with the different stages of tumorigenesis.
Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Peritoneais/metabolismo , Linhagem Celular Tumoral , Análise por Conglomerados , Perfilação da Expressão Gênica , Genes p53 , Predisposição Genética para Doença , Humanos , Neoplasias Hepáticas/secundário , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Peritoneais/secundário , Análise Serial de ProteínasRESUMO
BACKGROUND: Gene silencing through CpG island hypermethylation is a major mechanism in cancer development. In the present study, we aimed to identify and validate novel target genes inactivated through promoter hypermethylation in colorectal tumor development. METHODS: With the use of microarrays, the gene expression profiles of colon cancer cell lines before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine were identified and compared. The expression of the responding genes was compared with microarray expression data of primary colorectal carcinomas. Four of these down-regulated genes were subjected to methylation-specific PCR, bisulphite sequencing, and quantitative gene expression analysis using tumors (n=198), normal tissues (n=44), and cell lines (n=30). RESULTS: Twenty-one genes with a CpG island in their promoter responded to treatment in cell lines, and were simultaneously down-regulated in primary colorectal carcinomas. Among 20 colon cancer cell lines, hypermethylation was subsequently identified for three of four analyzed genes, ADAMTS1 (85%), CRABP1 (90%), and NR3C1 (35%). For the latter two genes, hypermethylation was significantly associated with absence or reduced gene expression. The methylation status of ADAMTS1, CRABP1, and NR3C1 was further investigated in 116 colorectal carcinomas and adenomas. Twenty-three of 63 (37%), 7/60 (12%), and 2/63 (3%) adenomas, as well as 37/52 (71%), 25/51 (49%), and 13/51 (25%) carcinomas were hypermethylated for the respective genes. These genes were unmethylated in tumors (n=82) from three other organs, prostate, testis, and kidney. Finally, analysis of normal colorectal mucosa demonstrated that the observed promoter hypermethylation was cancer-specific. CONCLUSION: By using a refined microarray screening approach we present three genes with cancer-specific hypermethylation in colorectal tumors, ADAMTS1, CRABP1, and NR3C1.
Assuntos
Proteínas ADAM/genética , Neoplasias Colorretais/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Receptores de Glucocorticoides/genética , Receptores do Ácido Retinoico/genética , Proteína ADAMTS1 , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilação de DNA , Humanos , Análise em Microsséries , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
In cancer biology, cell lines are often used instead of primary tumors because of their widespread availability and close reflection of the in vivo state. Cancer is a genetic disease, commonly caused by small- and large-scale DNA rearrangements. Therefore, it is essential to know the genomic profiles of tumor cell lines to enable their correct and efficient use as experimental tools. Here, we present a comprehensive study of the genomic profiles of 20 colon cancer cell lines combining conventional karyotyping (G-banding), comparative genomic hybridization (CGH), and multicolor fluorescence in situ hybridization (M-FISH). Major differences between the microsatellite instability (MSI) and chromosome instability (CIN) cell lines are shown; the CIN cell lines exhibited complex karyotypes involving many chromosomes (mean: 8.5 copy number changes), whereas the MSI cell lines showed considerably fewer aberrations (mean: 2.6). The 3 techniques complement each other to provide a detailed picture of the numerical and structural chromosomal changes that characterize cancer cells. Therefore, 7 of the cell lines (Colo320, EB, Fri, IS2, IS3, SW480, and V9P) are here completely karyotyped for the first time and, among these, 5 have not previously been cytogenetically described. By hierarchical cluster analysis, we show that the cell lines are representative models for primary carcinomas at the genome level. We also present the genomic profiles of an experimental model for tumor progression, including 3 cell lines (IS1, IS2, and IS3) established from a primary carcinoma, its corresponding liver- and peritoneal metastasis from the same patient. To address the question of clonality, we compared the genome of 3 common cell lines grown in 2 laboratories. Finally, we compared all our results with previously published CGH data and karyotypes of colorectal cell lines. In conclusion, the large variation in genetic complexity of the cell lines highlights the importance of a comprehensive reference of genomic profiles for investigators engaged in functional studies using these research tools.
Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Genoma Humano , Linhagem Celular Tumoral , Instabilidade Cromossômica , Aberrações Cromossômicas , Bandeamento Cromossômico , Células Clonais , Análise por Conglomerados , Neoplasias do Colo/patologia , Perfilação da Expressão Gênica , Variação Genética , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Repetições de Microssatélites , Metástase Neoplásica , Hibridização de Ácido Nucleico , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/secundário , PloidiasRESUMO
BACKGROUND: Until recently, chromosomal translocations and fusion genes have been an underappreciated class of mutations in solid tumors. Next-generation sequencing technologies provide an opportunity for systematic characterization of cancer cell transcriptomes, including the discovery of expressed fusion genes resulting from underlying genomic rearrangements. RESULTS: We applied paired-end RNA-seq to identify 24 novel and 3 previously known fusion genes in breast cancer cells. Supported by an improved bioinformatic approach, we had a 95% success rate of validating gene fusions initially detected by RNA-seq. Fusion partner genes were found to contribute promoters (5' UTR), coding sequences and 3' UTRs. Most fusion genes were associated with copy number transitions and were particularly common in high-level DNA amplifications. This suggests that fusion events may contribute to the selective advantage provided by DNA amplifications and deletions. Some of the fusion partner genes, such as GSDMB in the TATDN1-GSDMB fusion and IKZF3 in the VAPB-IKZF3 fusion, were only detected as a fusion transcript, indicating activation of a dormant gene by the fusion event. A number of fusion gene partners have either been previously observed in oncogenic gene fusions, mostly in leukemias, or otherwise reported to be oncogenic. RNA interference-mediated knock-down of the VAPB-IKZF3 fusion gene indicated that it may be necessary for cancer cell growth and survival. CONCLUSIONS: In summary, using RNA-sequencing and improved bioinformatic stratification, we have discovered a number of novel fusion genes in breast cancer, and identified VAPB-IKZF3 as a potential fusion gene with importance for the growth and survival of breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Fusão Oncogênica , Análise de Sequência de RNA/métodos , Linhagem Celular Tumoral , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Fator de Transcrição Ikaros/genética , Íntrons , Fenótipo , Reprodutibilidade dos Testes , Proteínas de Transporte Vesicular/genéticaRESUMO
INTRODUCTION: Few studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information. METHODS: We investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods. RESULTS: We identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process. CONCLUSION: This study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , MicroRNAs/análise , MicroRNAs/fisiologia , RNA Mensageiro/análise , Neoplasias da Mama/classificação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/classificação , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala/métodos , Humanos , Substâncias Macromoleculares/análise , Substâncias Macromoleculares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Análise em Microsséries , Modelos Biológicos , Mutação/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Integração de Sistemas , Proteína Supressora de Tumor p53/genética , Estudos de Validação como AssuntoRESUMO
Ductal carcinoma in situ (DCIS) is a non-invasive form of breast cancer where cells restricted to the ducts exhibit an atypical phenotype. Some DCIS lesions are believed to rapidly transit to invasive ductal carcinomas (IDCs), while others remain unchanged. Existing classification systems for DCIS fail to identify those lesions that transit to IDC. We studied gene expression patterns of 31 pure DCIS, 36 pure invasive cancers and 42 cases of mixed diagnosis (invasive cancer with an in situ component) using Agilent Whole Human Genome Oligo Microarrays 44k. Six normal breast tissue samples were also included as controls. qRT-PCR was used for validation. All DCIS and invasive samples could be classified into the "intrinsic" molecular subtypes defined for invasive breast cancer. Hierarchical clustering establishes that samples group by intrinsic subtype, and not by diagnosis. We observed heterogeneity in the transcriptomes among DCIS of high histological grade and identified a distinct subgroup containing seven of the 31 DCIS samples with gene expression characteristics more similar to advanced tumours. A set of genes independent of grade, ER-status and HER2-status was identified by logistic regression that univariately classified a sample as belonging to this distinct DCIS subgroup. qRT-PCR of single markers clearly separated this DCIS subgroup from the other DCIS, and contains samples from several histopathological and intrinsic molecular subtypes. The genes that differentiate between these two types of DCIS suggest several processes related to the re-organisation of the microenvironment. This raises interesting possibilities for identification of DCIS lesions both with and without invasive characteristics, which potentially could be used in clinical assessment of a woman's risk of progression, and lead to improved management that would avoid the current over- and under-treatment of patients.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Análise por Conglomerados , Hibridização Genômica Comparativa , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Pessoa de Meia-Idade , Família Multigênica , Invasividade Neoplásica , Receptor ErbB-2/genética , Receptores de Estrogênio/genéticaRESUMO
Aneuploidy is among the most obvious differences between normal and cancer cells. However, mechanisms contributing to development and maintenance of aneuploid cell growth are diverse and incompletely understood. Functional genomics analyses have shown that aneuploidy in cancer cells is correlated with diffuse gene expression signatures and aneuploidy can arise by a variety of mechanisms, including cytokinesis failures, DNA endoreplication, and possibly through polyploid intermediate states. To identify molecular processes contributing to development of aneuploidy, we used a cell spot microarray technique to identify genes inducing polyploidy and/or allowing maintenance of polyploid cell growth in breast cancer cells. Of 5760 human genes screened, 177 were found to induce severe DNA content alterations on prolonged transient silencing. Association with response to DNA damage stimulus and DNA repair was found to be the most enriched cellular processes among the candidate genes. Functional validation analysis of these genes highlighted GINS2 as the highest ranking candidate inducing polyploidy, accumulation of endogenous DNA damage, and impairing cell proliferation on inhibition. The cell growth inhibition and induction of polyploidy by suppression of GINS2 was verified in a panel of breast cancer cell lines. Bioinformatic analysis of published gene expression and DNA copy number studies of clinical breast tumors suggested GINS2 to be associated with the aggressive characteristics of a subgroup of breast cancers in vivo. In addition, nuclear GINS2 protein levels distinguished actively proliferating cancer cells suggesting potential use of GINS2 staining as a biomarker of cell proliferation as well as a potential therapeutic target.
Assuntos
Proteínas Cromossômicas não Histona/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Poliploidia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Análise por Conglomerados , Feminino , Humanos , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Using array comparative genomic hybridization (aCGH), a large number of deleted genomic regions have been identified in human cancers. However, subsequent efforts to identify target genes selected for inactivation in these regions have often been challenging. METHODS: We integrated here genome-wide copy number data with gene expression data and non-sense mediated mRNA decay rates in breast cancer cell lines to prioritize gene candidates that are likely to be tumour suppressor genes inactivated by bi-allelic genetic events. The candidates were sequenced to identify potential mutations. RESULTS: This integrated genomic approach led to the identification of RIC8A at 11p15 as a putative candidate target gene for the genomic deletion in the ZR-75-1 breast cancer cell line. We identified a truncating mutation in this cell line, leading to loss of expression and rapid decay of the transcript. We screened 127 breast cancers for RIC8A mutations, but did not find any pathogenic mutations. No promoter hypermethylation in these tumours was detected either. However, analysis of gene expression data from breast tumours identified a small group of aggressive tumours that displayed low levels of RIC8A transcripts. qRT-PCR analysis of 38 breast tumours showed a strong association between low RIC8A expression and the presence of TP53 mutations (P = 0.006). CONCLUSION: We demonstrate a data integration strategy leading to the identification of RIC8A as a gene undergoing a classical double-hit genetic inactivation in a breast cancer cell line, as well as in vivo evidence of loss of RIC8A expression in a subgroup of aggressive TP53 mutant breast cancers.
RESUMO
A report on the Human Genome Organisation (HUGO) 11th Human Genome Meeting, Helsinki, Finland, 31 May-3 June 2006.
Assuntos
Epigênese Genética , Predisposição Genética para Doença , Genoma Humano , Metilação de DNA , Amplificação de Genes , Regulação da Expressão Gênica , Genes Neoplásicos , Variação Genética , HumanosRESUMO
To identify chromosomal aberrations that differentiate among the Dukes' stages of colorectal cancer (CRC) as well as those that are responsible for the progression into liver metastases, we performed a meta-analysis of data obtained from 31 comparative genomic hybridization (CGH) studies comprising a total of 859 CRCs. Individual copy number profiles for 373 primary tumors and 102 liver metastases were recorded and several statistical analyses, such as frequency, multivariate logistic regression, and trend tests, were performed. In addition, time of occurrence analysis was applied for the first time to copy number changes identified by CGH, and each genomic imbalance was thereby classified as an early or late event in colorectal tumorigenesis. By combining data from the different statistical tests, we present a novel genetic pathway for CRC progression that distinguishes the Dukes' stages and identifies early and late events in both primary carcinomas and liver metastases. Results from the combined analyses suggest that losses at 17p and 18 and gains of 8q, 13q, and 20 occur early in the establishment of primary CRCs, whereas loss of 4p is associated with the transition from Dukes' A to B-D. Deletion of 8p and gains of 7p and 17q are correlated with the transition from primary tumor to liver metastasis, whereas losses of 14q and gains of 1q, 11, 12p, and 19 are late events. We supplement these findings with a list of potential target genes for the specific alterations from a publicly available microarray expression dataset of CRC.
Assuntos
Aberrações Cromossômicas , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Dosagem de Genes , Análise por Conglomerados , Humanos , Neoplasias Hepáticas/secundário , Masculino , Análise Multivariada , Estadiamento de Neoplasias , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
TP53 mutations play an important role in the development of several cancers and are present in 20-40% of all breast carcinomas, contributing to increased genomic instability. In order to address the relationship of mutated TP53 to genomic complexity, the present study analysed 61 breast carcinomas for TP53 mutations and compared mutation status with the pattern of genomic imbalances as assessed by comparative genomic hybridization (CGH). Twenty per cent of the present series of breast carcinomas harboured TP53 mutations. An increasing number of abnormalities, as identified by CGH (higher genomic complexity), correlated significantly with mutant TP53. Among the chromosome arms most commonly altered (in more than 20% of the tumours), loss of 8p and gain of 8q were associated with TP53 mutations, whereas loss of 16q was associated with wild-type TP53. By performing supervised hierarchical clustering analysis of the CGH data, a cluster of chromosome imbalances was observed that showed differences between wild-type and mutant TP53 cases. Among these, loss of chromosome arm 5q revealed the strongest correlation with altered TP53. To investigate further the most commonly deleted region of 5q, gene expression patterns from two publicly available microarray data sets of breast carcinomas were evaluated statistically. The expression data sets identified potential target genes, including genes involved in ubiquitination and the known TP53 target CSPG2. The genomic complexity of breast carcinomas as assessed by CGH is associated with TP53 mutation status; breast cancers with TP53 mutations display more complex genomes than do those with wild-type TP53. The pattern of genomic imbalances associated with mutant TP53 is non-random, with loss of chromosome arm 5q being particularly closely associated with TP53 mutations.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Genes p53/genética , Mutação , Aberrações Cromossômicas , Cromossomos Humanos Par 5 , Análise por Conglomerados , Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , Regulação para Baixo , Feminino , Humanos , Hibridização de Ácido NucleicoRESUMO
About 20% of breast carcinomas show no clonal chromosome abnormalities when analyzed after short-term culturing. An interesting question is whether this subset of breast carcinomas really is karyotypically normal or if selection for normal cells occurred in vitro. To address this issue, 26 breast carcinomas that had shown no cytogenetic changes by chromosome banding analysis were examined by comparative genomic hybridization (CGH), a technique that does not require culturing or tumor metaphase cells. All but one case showed copy number changes by CGH (median, four). A comparison of these findings with those of a karyotypically abnormal series analyzed using the same CGH protocol found that the cytogenetically "normal" cases were typically genetically less complex (median, four and eight, respectively; P = 0.0058). Although largely the same alterations were found in both series, some differences with respect to the frequencies of specific imbalances were seen. Gains of 3p and 6q and losses of 10q, 14q, and 17p more often were found in the cytogenetically abnormal series than in the normal tumors. We conclude that in most instances cells found to be normal by chromosome banding analysis after short-term culture do not belong to the tumor parenchyma. Furthermore, when we compared the distribution of the number of imbalances detected by CGH in the total data set according to the mitotic index in vivo (scored from 1 to 3), the median values were three, seven, and 18, respectively (P < 0.001). These data indicate not only that karyotypically normal breast carcinomas may represent a genetically simpler subgroup that grows poorly in vitro but also that this subset of tumors already has a slow growth rate in vivo.