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1.
Cancer Res ; 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38832931

ABSTRACT

DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that decrease 5'-cytosine methylation. DNMTi are used clinically based on the hypothesis that cytosine demethylation will lead to re-expression of tumor suppressor genes. 5-Aza-4'-thio-2'-deoxycytidine (Aza TdCyd or ATC) is a recently described thiol substituted DNMTi that has been shown to have anti-tumor activity in solid tumor models. Here, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to transformation of transplanted wild-type bone marrow nucleated cells into lymphoid leukemia, and healthy mice treated with ATC also developed lymphoid leukemia. Whole exome sequencing revealed thousands of acquired mutations, almost all of which were C>G transversions in a specific 5'-NCG-3' context. These mutations involved dozens of genes involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Human cells treated in vitro with ATC showed thousands of acquired C>G transversions in a similar context. Deletion of Dck, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C>G transversions. Taken together, these findings demonstrate a highly penetrant mutagenic and leukemogenic phenotype associated with ATC.

2.
Genome Med ; 13(1): 93, 2021 05 25.
Article in English | MEDLINE | ID: mdl-34034815

ABSTRACT

BACKGROUND: Many carcinomas have recurrent chromosomal aneuploidies specific to the tissue of tumor origin. The reason for this specificity is not completely understood. METHODS: In this study, we looked at the frequency of chromosomal arm gains and losses in different cancer types from the The Cancer Genome Atlas (TCGA) and compared them to the mean gene expression of each chromosome arm in corresponding normal tissues of origin from the Genotype-Tissue Expression (GTEx) database, in addition to the distribution of tissue-specific oncogenes and tumor suppressors on different chromosome arms. RESULTS: This analysis revealed a complex picture of factors driving tumor karyotype evolution in which some recurrent chromosomal copy number reflect the chromosome arm-wide gene expression levels of the their normal tissue of tumor origin. CONCLUSIONS: We conclude that the cancer type-specific distribution of chromosomal arm gains and losses is potentially "hardwiring" gene expression levels characteristic of the normal tissue of tumor origin, in addition to broadly modulating the expression of tissue-specific tumor driver genes.


Subject(s)
Aneuploidy , Biomarkers, Tumor , Chromosome Mapping , Gene Expression Regulation, Neoplastic , Neoplasms/genetics , Algorithms , Cluster Analysis , Computational Biology/methods , DNA Methylation , Databases, Genetic , Gene Expression Profiling , Humans , Mutation , Oncogenes , Organ Specificity/genetics
3.
J Immunol ; 203(9): 2532-2544, 2019 11 01.
Article in English | MEDLINE | ID: mdl-31548328

ABSTRACT

Dual oxidase 2 (DUOX2) generates H2O2 that plays a critical role in both host defense and chronic inflammation. Previously, we demonstrated that the proinflammatory mediators IFN-γ and LPS enhance expression of DUOX2 and its maturation factor DUOXA2 through STAT1- and NF-κB‒mediated signaling in human pancreatic cancer cells. Using a panel of colon and pancreatic cancer cell lines, we now report the induction of DUOX2/DUOXA2 mRNA and protein expression by the TH2 cytokine IL-4. IL-4 activated STAT6 signaling that, when silenced, significantly decreased induction of DUOX2. Furthermore, the TH17 cytokine IL-17A combined synergistically with IL-4 to increase DUOX2 expression in both colon and pancreatic cancer cells mediated, at least in part, by signaling through NF-κB. The upregulation of DUOX2 was associated with a significant increase in the production of extracellular H2O2 and DNA damage-as indicated by the accumulation of 8-oxo-dG and γH2AX-which was suppressed by the NADPH oxidase inhibitor diphenylene iodonium and a DUOX2-specific small interfering RNA. The clinical relevance of these experiments is suggested by immunohistochemical, microarray, and quantitative RT-PCR studies of human colon and pancreatic tumors demonstrating significantly higher DUOX2, IL-4R, and IL-17RA expression in tumors than in adjacent normal tissues; in pancreatic adenocarcinoma, increased DUOX2 expression is adversely associated with overall patient survival. These data suggest a functional association between DUOX2-mediated H2O2 production and induced DNA damage in gastrointestinal malignancies.


Subject(s)
Colonic Neoplasms/metabolism , DNA Damage , Dual Oxidases/genetics , Hydrogen Peroxide/metabolism , Interleukin-17/pharmacology , Interleukin-4/pharmacology , Pancreatic Neoplasms/metabolism , Cell Line, Tumor , Colonic Neoplasms/pathology , Humans , NF-kappa B/physiology , Oxidation-Reduction , Pancreatic Neoplasms/pathology , Receptors, Interleukin-4/physiology , STAT6 Transcription Factor/physiology , Signal Transduction , Up-Regulation
4.
Recent Results Cancer Res ; 200: 115-42, 2015.
Article in English | MEDLINE | ID: mdl-26376875

ABSTRACT

Chromosomal abnormalities are a defining feature of solid tumors. Such cytogenetic alterations are mainly classified into structural chromosomal aberrations and copy number alterations, giving rise to aneuploid karyotypes. The increasing detection of these genetic changes allowed the description of specific tumor entities and the associated patterns of gene expression. In fact, tumor-specific landscapes of gross genomic copy number changes, including aneuploidies of entire chromosome arms and chromosomes result in a global deregulation of the transcriptome of cancer cells. Furthermore, the molecular characterization of cytogenetic abnormalities has provided insights into the mechanisms of tumorigenesis and has, in a few instances, led to the clinical implementation of effective diagnostic and prognostic tools, as well as treatment strategies that target a specific genetic abnormality.


Subject(s)
Chromosome Aberrations , Neoplasms/genetics , Animals , Chromosome Deletion , Gene Amplification , Gene Dosage , Humans , Translocation, Genetic
5.
Carcinogenesis ; 34(8): 1929-39, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23619298

ABSTRACT

Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.


Subject(s)
Carcinogenesis/genetics , Epithelial Cells/physiology , Epithelial-Mesenchymal Transition/genetics , Gene Amplification , Oncogenes , Aneuploidy , Animals , Carcinogenesis/metabolism , Chromosomal Instability , Chromosome Aberrations , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Kidney/cytology , Male , Mice , Mice, Inbred C57BL , Spectral Karyotyping/methods , Transcription, Genetic , Transcriptome , Urinary Bladder/cytology
6.
Cancer Res ; 73(6): 2003-13, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23319804

ABSTRACT

Chromosomal copy number alterations (aneuploidy) define the genomic landscape of most cancer cells, but identification of the oncogenic drivers behind these imbalances remains an unfinished task. In this study, we conducted a systematic analysis of colorectal carcinomas that integrated genomic copy number changes and gene expression profiles. This analysis revealed 44 highly overexpressed genes mapping to localized amplicons on chromosome 13, gains of which occur often in colorectal cancers (CRC). RNA interference (RNAi)-mediated silencing identified eight candidates whose loss-of-function reduced cell viability 20% or more in CRC cell lines. The functional space of the genes NUPL1, LNX2, POLR1D, POMP, SLC7A1, DIS3, KLF5, and GPR180 was established by global expression profiling after RNAi exposure. One candidate, LNX2, not previously known as an oncogene, was involved in regulating NOTCH signaling. Silencing LNX2 reduced NOTCH levels but also downregulated the transcription factor TCF7L2 and markedly reduced WNT signaling. LNX2 overexpression and chromosome 13 amplification therefore constitutively activates the WNT pathway, offering evidence of an aberrant NOTCH-WNT axis in CRC.


Subject(s)
Carrier Proteins/physiology , Colorectal Neoplasms/metabolism , Receptors, Notch/physiology , Up-Regulation/physiology , Wnt Proteins/metabolism , beta Catenin/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Comparative Genomic Hybridization , Flow Cytometry , Humans , RNA Interference , Transcription, Genetic
7.
PLoS One ; 7(11): e49305, 2012.
Article in English | MEDLINE | ID: mdl-23166633

ABSTRACT

p53 is a tumor suppressor gene mutated in >50% of human cancers, while p53 deficiency in mice results in cancers and accelerated mortality. Thymic T cell lymphoma is the most common malignancy in p53-deficient mice, making it difficult to study the role of p53 in other malignancies. To overcome this limitation, we attempted to generate mice with a reversible p53 knockout (p53(rev/rev)) by inserting a floxed transcriptional stop into the first exon of p53, anticipating that this would allow tissue-specific Cre-mediated expression of p53. Contrary to expectations, functional p53 protein was expressed in the thymus and multiple other tissues of p53(rev/rev) mice in the absence of Cre, whereas B cells expressed p53 protein only in the presence of B cell-specific CD19-Cre. In the absence of Cre, 76% of p53(rev/rev) mice developed splenic marginal zone B cell lymphomas, indicating sensitivity of this B cell subset to transformation caused by p53 deficiency. 5'-RACE identified p53 mRNA transcribed from a novel start site utilized in thymocytes but not normal B cells or B cell lymphomas from p53(rev/rev) mice. The p53(rev/rev) mouse thus demonstrates an effect of p53 deficiency in development of splenic marginal zone lymphomas and provides a model for study of p53-deficient human B cell lymphomas.


Subject(s)
Disease Models, Animal , Gene Expression Regulation/genetics , Lymphoma, B-Cell/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , B-Lymphocytes/metabolism , Blotting, Western , DNA Primers/genetics , Exons/genetics , Flow Cytometry , Genotype , Humans , Karyotyping , Lymphoma, B-Cell/genetics , Mice , Mice, Knockout , Reverse Transcriptase Polymerase Chain Reaction , Thymus Gland/metabolism , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics
8.
Clin Cancer Res ; 18(18): 4919-30, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22850566

ABSTRACT

PURPOSE: miRNAs play a prominent role in a variety of physiologic and pathologic biologic processes, including cancer. For rectal cancers, only limited data are available on miRNA expression profiles, whereas the underlying genomic and transcriptomic aberrations have been firmly established. We therefore, aimed to comprehensively map the miRNA expression patterns of this disease. EXPERIMENTAL DESIGN: Tumor biopsies and corresponding matched mucosa samples were prospectively collected from 57 patients with locally advanced rectal cancers. Total RNA was extracted, and tumor and mucosa miRNA expression profiles were subsequently established for all patients. The expression of selected miRNAs was validated using semi-quantitative real-time PCR. RESULTS: Forty-nine miRNAs were significantly differentially expressed (log(2)-fold difference >0.5 and P < 0.001) between rectal cancer and normal rectal mucosa. The predicted targets for these miRNAs were enriched for the following pathways: Wnt, TGF-beta, mTOR, insulin, mitogen-activated protein kinase, and ErbB signaling. Thirteen of these 49 miRNAs seem to be rectal cancer-specific, and have not been previously reported for colon cancers: miR-492, miR-542-5p, miR-584, miR-483-5p, miR-144, miR-2110, miR-652, miR-375, miR-147b, miR-148a, miR-190, miR-26a/b, and miR-338-3p. Of clinical impact, miR-135b expression correlated significantly with disease-free and cancer-specific survival in an independent multicenter cohort of 116 patients. CONCLUSION: This comprehensive analysis of the rectal cancer miRNAome uncovered novel miRNAs and pathways associated with rectal cancer. This information contributes to a detailed view of this disease. Moreover, the identification and validation of miR-135b may help to identify novel molecular targets and pathways for therapeutic exploitation.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Rectal Neoplasms/genetics , Cluster Analysis , Colonic Neoplasms/genetics , Gene Regulatory Networks , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Prognosis , Rectal Neoplasms/mortality , Rectal Neoplasms/pathology , Rectum/metabolism , Rectum/pathology , Reproducibility of Results
9.
Biochim Biophys Acta ; 1819(7): 784-93, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22426433

ABSTRACT

Chromosomal aneuploidies are a defining feature of carcinomas, i.e., tumors of epithelial origin. Such aneuploidies result in tumor specific genomic copy number alterations. The patterns of genomic imbalances are tumor specific, and to a certain extent specific for defined stages of tumor development. Genomic imbalances occur already in premalignant precursor lesions, i.e., before the transition to invasive disease, and their distribution is maintained in metastases, and in cell lines derived from primary tumors. These observations are consistent with the interpretation that tumor specific genomic imbalances are drivers of malignant transformation. Naturally, this precipitates the question of how such imbalances influence the expression of resident genes. A number of laboratories have systematically integrated copy number alterations with gene expression changes in primary tumors and metastases, cell lines, and experimental models of aneuploidy to address the question as to whether genomic imbalances deregulate the expression of one or few key genes, or rather affect the cancer transcriptome more globally. The majority of these studies showed that gene expression levels follow genomic copy number. Therefore, gross genomic copy number changes, including aneuploidies of entire chromosome arms and chromosomes, result in a massive deregulation of the transcriptome of cancer cells. This article is part of a Special Issue entitled: Chromatin in time and space.


Subject(s)
Aneuploidy , Neoplasms/metabolism , Transcriptome , Animals , Cell Line, Tumor , Chromosomes, Human , Gene Dosage , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/genetics
10.
Mol Cancer ; 11: 1, 2012 Jan 04.
Article in English | MEDLINE | ID: mdl-22216762

ABSTRACT

BACKGROUND: Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival. RESULTS: A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway. CONCLUSIONS: We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Calcium-Binding Proteins/genetics , Colorectal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Histones/genetics , RNA Interference , Transcription, Genetic , Transcriptome , Apoptosis/genetics , Apoptosis Regulatory Proteins/metabolism , Calcium-Binding Proteins/metabolism , Cell Line, Tumor , Cell Survival/genetics , Cluster Analysis , Colorectal Neoplasms/metabolism , Computational Biology/methods , DNA Replication , Gene Expression Profiling , Gene Silencing , Humans , Neurofilament Proteins/genetics
11.
Carcinogenesis ; 32(11): 1607-14, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21828058

ABSTRACT

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression by inhibiting translation or promoting degradation of specific target messenger RNAs (mRNAs). Alteration of the levels of a number of miRNAs is common in solid and hematological tumors. We have shown previously that miR-214 regulates Ezh2 in skeletal muscle and embryonic stem cells. The current study was aimed at examining the role of miR-214 in breast cancer where miR-214 levels are reduced but whether this phenomenon bears a functional relevance is unknown. MiR-214 expression was inversely correlated with Ezh2 mRNA and protein levels in breast cancer cell lines and at least one copy of the miR-214 alleles was found to be deleted in 24% (6/25) of primary breast tumors. Experimental increase of miR-214 in breast cancer cell lines correlated with reduction of Ezh2 protein levels, a known marker of invasion and aggressive breast cancer behavior. Supporting a direct targeting mechanism, miR-214 decreased luciferase activity from a construct containing the Ezh2 3' untranslated region. Expression of miR-214 specifically reduced cell proliferation of breast cancer cells and inhibited the invasive potential of a highly metastatic breast cancer cell line. These findings indicate that reduced miR-214 levels may contribute to breast tumorigenesis by allowing abnormally elevated Ezh2 accumulation and subsequent unchecked cell proliferation and invasion.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Movement , Cell Proliferation , DNA-Binding Proteins/metabolism , MicroRNAs/genetics , Transcription Factors/metabolism , Blotting, Western , Breast Neoplasms/enzymology , Cell Adhesion , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Enhancer of Zeste Homolog 2 Protein , Female , Humans , Immunoenzyme Techniques , Luciferases/metabolism , Neoplasm Invasiveness , Polycomb Repressive Complex 2 , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Tumor Cells, Cultured
12.
Cell Mol Life Sci ; 68(19): 3261-74, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21290163

ABSTRACT

DNA aneuploidy has been identified as a prognostic factor for epithelial malignancies. Further understanding of the translation of DNA aneuploidy into protein expression will help to define novel biomarkers to improve therapies and prognosis. DNA ploidy was assessed by image cytometry. Comparison of gel-electrophoresis-based protein expression patterns of three diploid and four aneuploid colorectal cancer cell lines detected 64 ploidy-associated proteins. Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in two overlapping high-ranked networks maintaining Cellular Assembly and Organization, Cell Cycle, and Cellular Growth and Proliferation. CAPZA1, TXNL1, and HDAC2 were significantly validated by Western blotting in cell lines and the latter two showed expression differences also in clinical samples using a tissue microarray of normal mucosa (n=19), diploid (n=31), and aneuploid (n=47) carcinomas. The results suggest that distinct protein expression patterns, affecting TXNL1 and HDAC2, distinguish aneuploid with poor prognosis from diploid colorectal cancers.


Subject(s)
Aneuploidy , Carcinoma/genetics , Colorectal Neoplasms/genetics , Diploidy , Histone Deacetylase 2/genetics , Thioredoxins/genetics , Blotting, Western , CapZ Actin Capping Protein/genetics , CapZ Actin Capping Protein/metabolism , CapZ Actin Capping Protein/physiology , Carcinoma/diagnosis , Carcinoma/pathology , Cell Line, Tumor , Cohort Studies , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/pathology , DNA, Neoplasm/chemistry , Genomic Instability , Histone Deacetylase 2/metabolism , Histone Deacetylase 2/physiology , Humans , Prognosis , Thioredoxins/metabolism , Thioredoxins/physiology
13.
Int J Cancer ; 128(5): 1069-79, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-20473941

ABSTRACT

Genes that are highly overexpressed in tumor cells can be required for tumor cell survival and have the potential to be selective therapeutic targets. In an attempt to identify such targets, we combined a functional genomics and a systems biology approach to assess the consequences of RNAi-mediated silencing of overexpressed genes that were selected from 140 gene expression profiles from colorectal cancers (CRCs) and matched normal mucosa. In order to identify credible models for in-depth functional analysis, we first confirmed the overexpression of these genes in 25 different CRC cell lines. We then identified five candidate genes that profoundly reduced the viability of CRC cell lines when silenced with either siRNAs or short-hairpin RNAs (shRNAs), i.e., HMGA1, TACSTD2, RRM2, RPS2 and NOL5A. These genes were further studied by systematic analysis of comprehensive gene expression profiles generated following siRNA-mediated silencing. Exploration of these RNAi-specific gene expression signatures allowed the identification of the functional space in which the five genes operate and showed enrichment for cancer-specific signaling pathways, some known to be involved in CRC. By comparing the expression of the RNAi signature genes with their respective expression levels in an independent set of primary rectal carcinomas, we could recapitulate these defined RNAi signatures, therefore, establishing the biological relevance of our observations. This strategy identified the signaling pathways that are affected by the prominent oncogenes HMGA1 and TACSTD2, established a yet unknown link between RRM2 and PLK1 and identified RPS2 and NOL5A as promising potential therapeutic targets in CRC.


Subject(s)
Colorectal Neoplasms/genetics , Genomics , Cell Line, Tumor , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , Gene Expression Profiling , Gene Silencing , Humans , Immunohistochemistry , RNA Interference
14.
Genes Chromosomes Cancer ; 49(3): 204-23, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19927377

ABSTRACT

In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer.


Subject(s)
Colorectal Neoplasms/genetics , Cytogenetic Analysis/methods , Cell Line, Tumor , Chromosome Aberrations/statistics & numerical data , Chromosome Banding , Comparative Genomic Hybridization/methods , DNA Mismatch Repair , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Translocation, Genetic
15.
BMC Genomics ; 10: 566, 2009 Nov 30.
Article in English | MEDLINE | ID: mdl-19948035

ABSTRACT

BACKGROUND: While attempting to reanalyze published data from Agilent 4 x 44 human expression chips, we found that some of the 60-mer olignucleotide features could not be interpreted as representing single human genes. For example, some of the oligonucleotides align with the transcripts of more than one gene. We decided to check the annotations for all autosomes and the X chromosome systematically using bioinformatics methods. RESULTS: Out of 42683 reporters, we found that 25505 (60%) passed all our tests and are considered "fully valid". 9964 (23%) reporters did not have a meaningful identifier, mapped to the wrong chromosome, or did not pass basic alignment tests preventing us from correlating the expression values of these reporters with a unique annotated human gene. The remaining 7214 (17%) reporters could be associated with either a unique gene or a unique intergenic location, but could not be mapped to a transcript in RefSeq. The 7214 reporters are further partitioned into three different levels of validity. CONCLUSION: Expression array studies should evaluate the annotations of reporters and remove those reporters that have suspect annotations. This evaluation can be done systematically and semi-automatically, but one must recognize that data sources are frequently updated leading to slightly changing validation results over time.


Subject(s)
Oligonucleotide Array Sequence Analysis/methods , Base Sequence , Computational Biology , Databases, Genetic , Humans , Internet , Molecular Sequence Data , RNA, Messenger/genetics , Reproducibility of Results , Software
16.
Environ Mol Mutagen ; 50(8): 672-96, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19768832

ABSTRACT

Analysis of centrosome number and structure has become one means of assessing the potential for aberrant chromosome segregation and aneuploidy in tumor cells. Centrosome amplification directly causes multipolar catastrophic mitoses in mouse embryonic fibroblasts (MEFs) deficient for the tumor suppressor genes Brca1 or Trp53. We observed supernumerary centrosomes in cell lines established from aneuploid, but not from diploid, colorectal carcinomas; however, multipolar mitoses were never observed. This discrepancy prompted us to thoroughly characterize the centrosome abnormalities in these and other cancer cell lines with respect to both structure and function. The most striking result was that supernumerary centrosomes in aneuploid colorectal cancer cell lines were unable to nucleate microtubules, despite the presence of gamma-tubulin, pericentrin, PLK1, and AURKA. Analysis by scanning electron microscopy revealed that these supernumerary structures are devoid of centrioles, a result significantly different from observations in aneuploid pancreatic cancer cell lines and in Trp53 or Brca1 deficient MEFs. Thus, multipolar mitoses are dependent upon the ability of extra gamma-tubulin containing structures to nucleate microtubules, and this correlated with the presence of centrioles. The assessment of centrosome function with respect to chromosome segregation must therefore take into consideration the presence of centrioles and the capacity to nucleate microtubules. The patterns and mechanisms of chromosomal aberrations in hematologic malignancies and solid tumors are fundamentally different. The former is characterized by specific chromosome translocations, whose consequence is the activation of oncogenes. Most carcinomas, however, reveal variations in the nuclear DNA content. The observed genomic imbalances and gross variations in chromosome number can result from unequal chromosome segregation during mitotic cell division. It is therefore fundamental to elucidate mechanisms involved in distribution of the genome to daughter cells. Prior to cell division, the centrosome organizes microtubules and the mitotic spindle. Deciphering the consequences of alterations in centrosome number, structure, and function is an important step towards understanding how a diploid genome is maintained. Although extra centrosomes have now been observed in carcinomas and were correlated with aneuploidy, a careful functional investigation of these structures and their role in generating chromosome imbalances may lead to the identification of distinct mechanistic pathways of genomic instability. Understanding these pathways will also be important in determining whether they are potential molecular targets of therapeutic intervention.


Subject(s)
Centrosome , Colorectal Neoplasms/pathology , Animals , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/ultrastructure , Gene Expression Profiling , Humans , Immunohistochemistry , Mice , Microscopy, Electron/methods
17.
Genes Chromosomes Cancer ; 48(11): 1002-17, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19691111

ABSTRACT

To evaluate the mechanisms and consequences of chromosomal aberrations in colorectal cancer (CRC), we used a combination of spectral karyotyping, array comparative genomic hybridization (aCGH), and array-based global gene expression profiling on 31 primary carcinomas and 15 established cell lines. Importantly, aCGH showed that the genomic profiles of primary tumors are recapitulated in the cell lines. We revealed a preponderance of chromosome breakpoints at sites of copy number variants (CNVs) in the CRC cell lines, a novel mechanism of DNA breakage in cancer. The integration of gene expression and aCGH led to the identification of 157 genes localized within high-level copy number changes whose transcriptional deregulation was significantly affected across all of the samples, thereby suggesting that these genes play a functional role in CRC. Genomic amplification at 8q24 was the most recurrent event and led to the overexpression of MYC and FAM84B. Copy number dependent gene expression resulted in deregulation of known cancer genes such as APC, FGFR2, and ERBB2. The identification of only 36 genes whose localization near a breakpoint could account for their observed deregulated expression demonstrates that the major mechanism for transcriptional deregulation in CRC is genomic copy number changes resulting from chromosomal aberrations.


Subject(s)
Colorectal Neoplasms/genetics , DNA Copy Number Variations , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , Chromosome Breakpoints , Chromosomes, Human, Pair 8 , Colorectal Neoplasms/metabolism , Comparative Genomic Hybridization/methods , Gene Deletion , Gene Expression Profiling/methods , Gene Regulatory Networks , Humans , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Oligonucleotide Array Sequence Analysis/methods , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Virus/genetics , Receptors, Virus/metabolism , Spectral Karyotyping/methods , Transcription, Genetic
18.
Cancer Genet Cytogenet ; 193(1): 19-28, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19602460

ABSTRACT

Standard treatment of rectal cancer patients comprises preoperative chemoradiotherapy followed by radical surgery. However, clinicians are faced with the problem that response rates vary from one individual to another. Predictive biomarkers would therefore be helpful. To identify genomic imbalances that might assist in stratifying tumors into responsive or nonresponsive categories, we used metaphase comparative genomic hybridization to prospectively analyze pretherapeutic biopsies from 42 patients with locally advanced rectal cancers. These patients were subsequently treated with 5-fluorouracil-based preoperative chemoradiotherapy. Based on downsizing of the T-category, 21 rectal cancers were later classified as responsive, while the other 21 were nonresponsive. Comparing these two groups, we could show that gains of chromosomal regions 7q32 approximately q36 and 7q11 approximately q31, as well as amplifications of 20q11 approximately q13, were significantly associated with responsiveness to preoperative chemoradiotherapy (P<0.05). However, the probability of detecting these copy number changes by chance is high (P=0.21). Our primary results suggest that pretherapeutic evaluation of chromosomal copy number changes may be of value for response prediction of rectal cancers to preoperative chemoradiotherapy. This will require validation in a larger cohort of patients.


Subject(s)
Gene Dosage , Rectal Neoplasms/genetics , Adult , Aged , Chemotherapy, Adjuvant , Chromosomes, Human, Pair 7/genetics , Comparative Genomic Hybridization , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Radiotherapy, Adjuvant , Rectal Neoplasms/drug therapy , Rectal Neoplasms/radiotherapy , Rectal Neoplasms/surgery
19.
Cell Cycle ; 8(15): 2408-12, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19556863

ABSTRACT

Translocations involving the T cell receptor alpha/delta (TCRalpha/delta) chain locus, which bring oncogenes in the proximity of the TCRalpha enhancer, are one of the hallmark features of human T cell malignancies from ataxia telangiectasia (AT) and non-AT patients. These lesions are frequently generated by the fusion of DNA breaks at the TCRalpha/delta locus to a disperse region centromeric of the immunoglobulin heavy chain (IgH) locus. Aberrant VDJ joining accounts for TCRalpha/delta associated DNA cleavage, but the molecular mechanism that leads to generation of the "oncogene partner" DNA break is unclear. Here we show that in ATM deficient primary mouse T cells, IgH/TCRalpha/delta fusions arise at a remarkably similar frequency as in human AT lymphocytes. Recombinase-activating gene (RAG) is responsible for both TCRalpha/delta as well as IgH associated breaks on chromosome 12 (Chr12), which are subject to varying degrees of chromosomal degradation. We suggest a new model for how oncogenic translocations can arise from two non-concerted physiological DSBs.


Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Immunoglobulin Heavy Chains/metabolism , Protein Serine-Threonine Kinases/metabolism , T-Lymphocytes/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Recombination, Genetic/physiology , Translocation, Genetic/physiology , Tumor Suppressor Proteins/genetics
20.
Cancer Genet Cytogenet ; 190(2): 57-65, 2009 Apr 15.
Article in English | MEDLINE | ID: mdl-19380020

ABSTRACT

Preoperative chemoradiotherapy is recommended for locally advanced rectal cancer (UICC stage II/III). We recently demonstrated that responsive and nonresponsive tumors showed differential expression levels of 54 genes. In this follow-up study, we investigated the relationship between this gene set and disease-free (DFS) and overall survival (OS). Pretherapeutic biopsies from 30 participants in the CAO/ARO/AIO-94 trial of the German Rectal Cancer Study Group were analyzed using gene expression microarrays. Statistical analysis was performed to identify differentially expressed genes between recurrent and nonrecurrent tumors and to correlate these changes with disease recurrence and outcome. After a median follow-up of 59 months, seven of eight patients with recurrent disease was a nonresponder, and one responsive tumor recurred. Response to chemoradiotherapy was significantly correlated with an improved DFS (log rank P=0.028), whereas OS did not differ significantly (P=0.11). Applying a class comparison analysis, we identified 20 genes that were differentially expressed between recurrent and nonrecurrent tumors (P<0.001). Analyzing the first two principal components of the 54 genes previously identified to predict response, we observed that this response signature correlated with an increased risk of cancer recurrence. These data suggest that the genetic basis of local response also affects the genetic basis of tumor recurrence. Genes that are indicative of nonresponse to preoperative chemoradiotherapy might also be linked to an increased risk of tumor recurrence.


Subject(s)
Gene Expression Profiling , Neoplasm Recurrence, Local/therapy , Rectal Neoplasms/therapy , Aged , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Chemotherapy, Adjuvant , Combined Modality Therapy , Disease-Free Survival , Follow-Up Studies , Humans , Middle Aged , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/surgery , Preoperative Care , Radiotherapy, Adjuvant , Rectal Neoplasms/genetics , Rectal Neoplasms/surgery
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