RESUMO
A fundamental question in the biology of sex differences has eluded direct study in humans: How does sex-chromosome dosage (SCD) shape genome function? To address this, we developed a systematic map of SCD effects on gene function by analyzing genome-wide expression data in humans with diverse sex-chromosome aneuploidies (XO, XXX, XXY, XYY, and XXYY). For sex chromosomes, we demonstrate a pattern of obligate dosage sensitivity among evolutionarily preserved X-Y homologs and update prevailing theoretical models for SCD compensation by detecting X-linked genes that increase expression with decreasing X- and/or Y-chromosome dosage. We further show that SCD-sensitive sex-chromosome genes regulate specific coexpression networks of SCD-sensitive autosomal genes with critical cellular functions and a demonstrable potential to mediate previously documented SCD effects on disease. These gene coexpression results converge with analysis of transcription factor binding site enrichment and measures of gene expression in murine knockout models to spotlight the dosage-sensitive X-linked transcription factor ZFX as a key mediator of SCD effects on wider genome expression. Our findings characterize the effects of SCD broadly across the genome, with potential implications for human phenotypic variation.
Assuntos
Aneuploidia , Cromossomos Humanos X , Cromossomos Humanos Y , Dosagem de Genes , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like , Modelos Genéticos , Animais , Cromossomos Humanos X/genética , Cromossomos Humanos X/metabolismo , Cromossomos Humanos Y/genética , Cromossomos Humanos Y/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos KnockoutRESUMO
Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age-22) and male (male:female ratio of 2:1) and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N-acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients.
Assuntos
Carcinoma Medular/patologia , Neoplasias Renais/patologia , Cultura Primária de Células/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Carcinoma Medular/tratamento farmacológico , Carcinoma Medular/genética , Autenticação de Linhagem Celular/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Camundongos , Camundongos Nus , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Células Tumorais CultivadasRESUMO
Genomic instability is a hallmark of human cancers, including the 5% caused by human papillomavirus (HPV). Here we report a striking association between HPV integration and adjacent host genomic structural variation in human cancer cell lines and primary tumors. Whole-genome sequencing revealed HPV integrants flanking and bridging extensive host genomic amplifications and rearrangements, including deletions, inversions, and chromosomal translocations. We present a model of "looping" by which HPV integrant-mediated DNA replication and recombination may result in viral-host DNA concatemers, frequently disrupting genes involved in oncogenesis and amplifying HPV oncogenes E6 and E7. Our high-resolution results shed new light on a catastrophic process, distinct from chromothripsis and other mutational processes, by which HPV directly promotes genomic instability.
Assuntos
Replicação do DNA/genética , Instabilidade Genômica , Papillomavirus Humano 16/genética , Neoplasias/genética , DNA Viral/genética , Feminino , Papillomavirus Humano 16/crescimento & desenvolvimento , Humanos , Masculino , Neoplasias/classificação , Neoplasias/patologia , Neoplasias/virologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Integração Viral/genéticaRESUMO
BACKGROUND: In 2003, this laboratory published an account of the human mast cell line LAD2 (Laboratory of Allergic Diseases 2) that expressed FcεRI, responded to recombinant human stem cell factor (rhSCF) and resembled CD34+-derived human mast cells. LAD2 cells have now been distributed worldwide. To study the impact of this transfer, we analyzed the number of investigators receiving LAD2 cells and resulting publications. METHODS: Records maintained in our laboratory, the Technology Transfer and Intellectual Property Office and Office of Technology Transfer, were reviewed for material transfer agreements (MTAs) and licensing agreements (LAs). Journals and impact factors were obtained from PubMed.gov by cross-referencing LAD2 and human mast cells from 2003 through November 2013. RESULTS: Over 300 MTAs and 40 LAs were approved. LAD2 cells were shipped to over 30 countries. More than 80 papers have been published in journals with impact factors from 1.31 to 13.21. Intended uses include the study of receptors, degranulation, and cell signaling. LAD2 cells continue to express described markers and have consistent FcεR1-mediated degranulation. CONCLUSIONS: Success of the LAD2 line reflects the demand for a human mast cell line in research, the uniqueness of this cell line, and that it continues to exhibit minimal variation from its original description. We hope that the awareness of the impact of this cell line on mast cell research will encourage others to develop and distribute other similar cell lines with additional characteristics so as to address the limitations of depending on the study of cultured human mast cells from tissues.
Assuntos
Mastócitos , Fenótipo , Transferência de Tecnologia , Bibliometria , Biomarcadores/metabolismo , Linhagem Celular , Humanos , Cariotipagem , Mastócitos/imunologia , Mastócitos/metabolismo , Receptores de IgE/metabolismo , Estudos RetrospectivosRESUMO
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.
Assuntos
Carcinogênese/genética , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/genética , Amplificação de Genes , Oncogenes , Aneuploidia , Animais , Carcinogênese/metabolismo , Instabilidade Cromossômica , Aberrações Cromossômicas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Rim/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cariotipagem Espectral/métodos , Transcrição Gênica , Transcriptoma , Bexiga Urinária/citologiaRESUMO
Human carcinomas are defined by recurrent chromosomal aneuploidies, which result in a tissue-specific distribution of genomic imbalances. In order to develop models for these genome mutations and to determine their role in tumorigenesis, we generated 45 spontaneously transformed murine cell lines from normal epithelial cells derived from bladder, cervix, colon, kidney, lung, and mammary gland. Phenotypic changes, chromosomal aberrations, centrosome number, and telomerase activity were assayed in control uncultured cells and in three subsequent stages of transformation. Supernumerary centrosomes, binucleate cells, and tetraploidy were observed as early as 48 hr after explantation. In addition, telomerase activity increased throughout progression. Live-cell imaging revealed that failure of cytokinesis, not cell fusion, promoted genome duplication. Spectral karyotyping demonstrated that aneuploidy preceded immortalization, consisting predominantly of whole chromosome losses (4, 9, 12, 13, 16, and Y) and gains (1, 10, 15, and 19). After transformation, focal amplifications of the oncogenes Myc and Mdm2 were frequently detected. Fifty percent of the transformed lines resulted in tumors on injection into immunocompromised mice. The phenotypic and genomic alterations observed in spontaneously transformed murine epithelial cells recapitulated the aberration pattern observed during human carcinogenesis. The dominant aberration of these cell lines was the presence of specific chromosomal aneuploidies. We propose that our newly derived cancer models will be useful tools to dissect the sequential steps of genome mutations during malignant transformation, and also to identify cancer-specific genes, signaling pathways, and the role of chromosomal instability in this process.
Assuntos
Aneuploidia , Transformação Celular Neoplásica/genética , Instabilidade Cromossômica/genética , Células Epiteliais/patologia , Animais , Linhagem Celular Transformada , Células Epiteliais/metabolismo , Feminino , Genes myc , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Fenótipo , Proteínas Proto-Oncogênicas c-mdm2/genéticaRESUMO
BACKGROUND: MiT-Renal Cell Carcinoma (RCC) is characterized by genomic translocations involving microphthalmia-associated transcription factor (MiT) family members TFE3, TFEB, or MITF. MiT-RCC represents a specific subtype of sporadic RCC that is predominantly seen in young patients and can present with heterogeneous histological features making diagnosis challenging. Moreover, the disease biology of this aggressive cancer is poorly understood and there is no accepted standard of care therapy for patients with advanced disease. Tumor-derived cell lines have been established from human TFE3-RCC providing useful models for preclinical studies. METHODS: TFE3-RCC tumor derived cell lines and their tissues of origin were characterized by IHC and gene expression analyses. An unbiased high-throughput drug screen was performed to identify novel therapeutic agents for treatment of MiT-RCC. Potential therapeutic candidates were validated in in vitro and in vivo preclinical studies. Mechanistic assays were conducted to confirm the on-target effects of drugs. RESULTS: The results of a high-throughput small molecule drug screen utilizing three TFE3-RCC tumor-derived cell lines identified five classes of agents with potential pharmacological efficacy, including inhibitors of phosphoinositide-3-kinase (PI3K) and mechanistic target of rapamycin (mTOR), and several additional agents, including the transcription inhibitor Mithramycin A. Upregulation of the cell surface marker GPNMB, a specific MiT transcriptional target, was confirmed in TFE3-RCC and evaluated as a therapeutic target using the GPNMB-targeted antibody-drug conjugate CDX-011. In vitro and in vivo preclinical studies demonstrated efficacy of the PI3K/mTOR inhibitor NVP-BGT226, Mithramycin A, and CDX-011 as potential therapeutic options for treating advanced MiT-RCC as single agents or in combination. CONCLUSIONS: The results of the high-throughput drug screen and validation studies in TFE3-RCC tumor-derived cell lines have provided in vitro and in vivo preclinical data supporting the efficacy of the PI3K/mTOR inhibitor NVP-BGT226, the transcription inhibitor Mithramycin A, and GPNMB-targeted antibody-drug conjugate CDX-011 as potential therapeutic options for treating advanced MiT-RCC. The findings presented here should provide the basis for designing future clinical trials for patients with MiT-driven RCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Inibidores de MTOR , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Translocação Genética , Fosfatidilinositol 3-Quinase , Glicoproteínas de Membrana/genéticaRESUMO
Colorectal carcinomas (CRC) might be organized hierarchically and contain a subpopulation of tumorigenic, putative cancer stem cells that are CD133 positive. We studied the biological and genetic characteristics of such cells in CRC cell lines and primary tumors. Three CRC cell lines were sorted in CD133 positive and negative fractions. The respective genetic aberration profiles were studied using array comparative genomic hybridization (aCGH) and expression profiling. Tumorigenicity for each cellular population was tested by injection into nude mice. Additionally, we compared CD133+ and CD133- cells of 12 primary colorectal tumors using laser capture microdissection and aCGH. Three of five CRC cell lines displayed both CD133+ and CD133- cells, but tumorigenicity of these subfractions did not differ significantly and aCGH revealed essentially identical genomic imbalances. However, 96 genes were differentially expressed between the two populations. Array comparative genomic hybridization analysis after laser capture microdissection of CD133+ and CD133- areas in primary colorectal tumors revealed genetic differences in 7 of 12 cases. The use of cell lines for studying genomic alterations that define cancer stem cell characteristics, therefore, seems questionable. In contrast, CD133+ cells in primary cancer samples showed a unique genomic aberration profile. In conclusion, our data suggest that CD133 positivity defines a genetically distinct cellular compartment in primary CRC, which potentially includes tumor initiating cells.
Assuntos
Antígenos CD/biossíntese , Neoplasias Colorretais/metabolismo , Glicoproteínas/biossíntese , Antígeno AC133 , Animais , Biópsia/métodos , Células CACO-2 , Linhagem Celular Tumoral , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Citometria de Fluxo/métodos , Genoma , Genômica/métodos , Humanos , Lasers , Camundongos , Camundongos Nus , Microdissecção , Transplante de Neoplasias , Peptídeos , Transcrição GênicaRESUMO
BACKGROUND: In addition to clinical characteristics, DNA aneuploidy has been identified as a prognostic factor in epithelial malignancies in general and in endometrial cancers in particular. We mapped ploidy-associated chromosomal aberrations and identified corresponding gene and protein expression changes in endometrial cancers of different prognostic subgroups. METHODS: DNA image cytometry classified 25 endometrioid cancers to be either diploid (n = 16) or aneuploid (n = 9), and all uterine papillary serous cancers (UPSC) to be aneuploid (n = 8). All samples were subjected to comparative genomic hybridization and gene expression profiling. Identified genes were subjected to Ingenuity pathway analysis (IPA) and were correlated to protein expression changes. RESULTS: Comparative genomic hybridization revealed ploidy-associated specific, recurrent genomic imbalances. Gene expression analysis identified 54 genes between diploid and aneuploid endometrioid carcinomas, 39 genes between aneuploid endometrioid cancer and UPSC, and 76 genes between diploid endometrioid and aneuploid UPSC to be differentially expressed. Protein profiling identified AKR7A2 and ANXA2 to show translational alterations consistent with the transcriptional changes. The majority of differentially expressed genes and proteins belonged to identical molecular functions, foremost Cancer, Cell Death, and Cellular Assembly and Organization. CONCLUSIONS: We conclude that the grade of genomic instability rather than the histopathological subtype correlates with specific gene and protein expression changes. The identified genes and proteins might be useful as molecular targets for improved diagnostic and therapeutic intervention and merit prospective validation.
Assuntos
Neoplasias do Endométrio/genética , Perfilação da Expressão Gênica/métodos , Proteoma/genética , Transcriptoma , Aneuploidia , Neoplasias do Endométrio/classificação , Neoplasias do Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , HumanosRESUMO
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.
Assuntos
Neoplasias Colorretais/genética , Análise Citogenética/métodos , Linhagem Celular Tumoral , Aberrações Cromossômicas/estatística & dados numéricos , Bandeamento Cromossômico , Hibridização Genômica Comparativa/métodos , Reparo de Erro de Pareamento de DNA , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Translocação GenéticaRESUMO
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.
Assuntos
Aneuploidia , Biomarcadores Tumorais , Mapeamento Cromossômico , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Algoritmos , Análise por Conglomerados , Biologia Computacional/métodos , Metilação de DNA , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Mutação , Oncogenes , Especificidade de Órgãos/genéticaRESUMO
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.
Assuntos
Neoplasias Colorretais/genética , Variações do Número de Cópias de DNA , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Pontos de Quebra do Cromossomo , Cromossomos Humanos Par 8 , Neoplasias Colorretais/metabolismo , Hibridização Genômica Comparativa/métodos , Deleção de Genes , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Cariotipagem Espectral/métodos , Transcrição GênicaRESUMO
Barrett's esophagus (BE) is associated with reflux and is implicated the development of esophageal adenocarcinoma (EAC). Apoptosis induces cell death through mitochondrial outer membrane permeabilization (MOMP), which is considered an irreversible step in apoptosis. Activation of MOMP to levels that fail to reach the apoptotic threshold may paradoxically promote cancer-a phenomenon called "Minority MOMP." We asked whether reflux-induced esophageal carcinogenesis occurred via minority MOMP and whether compensatory resistance mechanisms prevented cell death during this process. We exposed preneoplastic, hTERT-immortalized Barrett's cell, CP-C and CP-A, to the oncogenic bile acid, deoxycholic acid (DCA), for 1 year. Induction of minority MOMP was tested via comet assay, CyQuant, annexin V, JC-1, cytochrome C subcellular localization, caspase 3 activation, and immunoblots. We used bcl-2 homology domain-3 (BH3) profiling to test the mitochondrial apoptotic threshold. One-year exposure of Barrett's cells to DCA induced a malignant phenotype noted by clone and tumor formation. DCA promoted minority MOMP noted by minimal release of cytochrome C and limited caspase 3 activation, which resulted in DNA damage without apoptosis. Upregulation of the antiapoptotic protein, Mcl-1, ROS generation, and NF-κB activation occurred in conjunction with minority MOMP. Inhibition of ROS blocked minority MOMP and Mcl-1 upregulation. Knockdown of Mcl-1 shifted minority MOMP to complete MOMP as noted by dynamic BH3 profiling and increased apoptosis. Minority MOMP contributes to DCA induced carcinogenesis in preneoplastic BE. Mcl-1 provided a balance within the mitochondria that induced resistance complete MOMP and cell death. Targeting Mcl-1 may be a therapeutic strategy in EAC.
Assuntos
Apoptose , Esôfago de Barrett/patologia , Ácidos e Sais Biliares/farmacologia , Carcinogênese/patologia , Neoplasias Esofágicas/patologia , Mitocôndrias/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Esôfago de Barrett/tratamento farmacológico , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Citocromos c/metabolismo , Dano ao DNA , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Esôfago/efeitos dos fármacos , Esôfago/metabolismo , Esôfago/patologia , Fármacos Gastrointestinais/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transdução de SinaisRESUMO
PURPOSE: The standard treatment of patients with locally advanced rectal cancer consists of preoperative chemoradiotherapy (CRT) followed by surgery. However, the response of individual tumors to CRT is extremely diverse, presenting a clinical dilemma. This broad variability in treatment response is likely attributable to intratumor heterogeneity (ITH). EXPERIMENTAL DESIGN: We addressed the impact of ITH on response to CRT by establishing single-cell-derived cell lines (SCDCL) from a treatment-naïve rectal cancer biopsy after xenografting. RESULTS: Individual SCDCLs derived from the same tumor responded profoundly different to CRT in vitro. Clonal reconstruction of the tumor and derived cell lines based on whole-exome sequencing revealed nine separate clusters with distinct proportions in the SCDCLs. Missense mutations in SV2A and ZWINT were clonal in the resistant SCDCL, but not detected in the sensitive SCDCL. Single-cell genetic analysis by multiplex FISH revealed the expansion of a clone with a loss of PIK3CA in the resistant SCDCL. Gene expression profiling by tRNA-sequencing identified the activation of the Wnt, Akt, and Hedgehog signaling pathways in the resistant SCDCLs. Wnt pathway activation in the resistant SCDCLs was confirmed using a reporter assay. CONCLUSIONS: Our model system of patient-derived SCDCLs provides evidence for the critical role of ITH for treatment response in patients with rectal cancer and shows that distinct genetic aberration profiles are associated with treatment response. We identified specific pathways as the molecular basis of treatment response of individual clones, which could be targeted in resistant subclones of a heterogenous tumor.
Assuntos
Heterogeneidade Genética , Neoplasias Retais/etiologia , Neoplasias Retais/patologia , Análise de Célula Única , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Terapia Combinada , Hibridização Genômica Comparativa , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Neoplasias Retais/metabolismo , Neoplasias Retais/terapia , Transdução de Sinais , Resultado do Tratamento , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cisplatin, an anticancer drug, forms DNA interstrand cross-links (ICL) that interfere with replication, whereas TREX2 is a 3'-->5' exonuclease that removes 3' mismatched nucleotides and promotes cellular proliferation. Here, we show that TREX2 is depleted in human cells derived from cancer after exposure to cisplatin but not other genotoxins including another cross-linking agent, mitomycin C (MMC), indicating a potential role for TREX2 depletion in cisplatin-induced cytotoxicity. To better understand TREX2 cellular function, we deleted TREX2 in mouse embryonic stem (ES) cells by gene targeting and find these cells exhibit reduced proliferation and gross chromosomal rearrangements including Robertsonian translocations (RbT). Quite interestingly, ES cells exposed to cisplatin also exhibit RbTs. By contrast, RbTs are not observed for ES cells exposed to MMC, indicating that RbTs are not caused by ICLs but instead TREX2 depletion by either cisplatin exposure or mutation. Taken together, our results show that cisplatin depletes TREX2 and causes genomic instability that is similarly observed in TREX2-mutant cells. Thus, cisplatin has two potential cytotoxic activities: (a) the generation of ICLs and (b) the depletion of TREX2.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Dano ao DNA , Exodesoxirribonucleases/deficiência , Fosfoproteínas/deficiência , Translocação Genética/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Relação Dose-Resposta a Droga , Exodesoxirribonucleases/metabolismo , Células HeLa , Humanos , Mitomicina/farmacologia , Fosfoproteínas/metabolismoRESUMO
To characterize patterns of global transcriptional deregulation in primary colon carcinomas, we did gene expression profiling of 73 tumors [Unio Internationale Contra Cancrum stage II (n = 33) and stage III (n = 40)] using oligonucleotide microarrays. For 30 of the tumors, expression profiles were compared with those from matched normal mucosa samples. We identified a set of 1,950 genes with highly significant deregulation between tumors and mucosa samples (P < 1e-7). A significant proportion of these genes mapped to chromosome 20 (P = 0.01). Seventeen genes had a >5-fold average expression difference between normal colon mucosa and carcinomas, including up-regulation of MYC and of HMGA1, a putative oncogene. Furthermore, we identified 68 genes that were significantly differentially expressed between lymph node-negative and lymph node-positive tumors (P < 0.001), the functional annotation of which revealed a preponderance of genes that play a role in cellular immune response and surveillance. The microarray-derived gene expression levels of 20 deregulated genes were validated using quantitative real-time reverse transcription-PCR in >40 tumor and normal mucosa samples with good concordance between the techniques. Finally, we established a relationship between specific genomic imbalances, which were mapped for 32 of the analyzed colon tumors by comparative genomic hybridization, and alterations of global transcriptional activity. Previously, we had conducted a similar analysis of primary rectal carcinomas. The systematic comparison of colon and rectal carcinomas revealed a significant overlap of genomic imbalances and transcriptional deregulation, including activation of the Wnt/beta-catenin signaling cascade, suggesting similar pathogenic pathways.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Aneuploidia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Linfonodos/patologia , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Colo/metabolismo , Colo/fisiologia , Neoplasias do Colo/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Família Multigênica , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Chromosomal aneuploidy is a defining feature of carcinomas and results in tumor-entity specific genomic imbalances. For instance, most sporadic colorectal carcinomas carry extra copies of chromosome 7, an aneuploidy that emerges already in premalignant adenomas, and is maintained throughout tumor progression and in derived cell lines. A comprehensive understanding on how chromosomal aneuploidy affects nuclear organization and gene expression, i.e., the nucleome, remains elusive. We now analyzed a cell line established from healthy colon mucosa with a normal karyotype (46,XY) and its isogenic derived cell line that acquired an extra copy of chromosome 7 as its sole anomaly (47,XY,+7). We studied structure/function relationships consequent to aneuploidization using genome-wide chromosome conformation capture (Hi-C), RNA sequencing and protein profiling. The gain of chromosome 7 resulted in an increase of transcript levels of resident genes as well as genome-wide gene and protein expression changes. The Hi-C analysis showed that the extra copy of chromosome 7 is reflected in more interchromosomal contacts between the triploid chromosomes. Chromatin organization changes are observed genome-wide, as determined by changes in A/B compartmentalization and topologically associating domain (TAD) boundaries. Most notably, chromosome 4 shows a profound loss of chromatin organization, and chromosome 14 contains a large A/B compartment switch region, concurrent with resident gene expression changes. No changes to the nuclear position of the additional chromosome 7 territory were observed when measuring distances of chromosome painting probes by interphase FISH. Genome and protein data showed enrichment in signaling pathways crucial for malignant transformation, such as the HGF/MET-axis. We conclude that a specific chromosomal aneuploidy has profound impact on nuclear structure and function, both locally and genome-wide. Our study provides a benchmark for the analysis of cancer nucleomes with complex karyotypes.
Assuntos
Aneuploidia , Núcleo Celular/genética , Expressão Gênica , Estudo de Associação Genômica Ampla , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Transformação Celular Neoplásica , Aberrações Cromossômicas , Mapeamento Cromossômico , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Humanos , Hibridização in Situ FluorescenteRESUMO
To identify genetic alterations underlying rectal carcinogenesis, we used global gene expression profiling of a series of 17 locally advanced rectal adenocarcinomas and 20 normal rectal mucosa biopsies on oligonucleotide arrays. A total of 351 genes were differentially expressed (P < 1.0e-7) between normal rectal mucosa and rectal carcinomas, 77 genes had a >5-fold difference, and 85 genes always had at least a 2-fold change in all of the matched samples. Twelve genes satisfied all three of these criteria. Altered expression of genes such as PTGS2 (COX-2), WNT1, TGFB1, VEGF, and MYC was confirmed, whereas our data for other genes, like PPARD and LEF1, were inconsistent with previous reports. In addition, we found deregulated expression of many genes whose involvement in rectal carcinogenesis has not been reported. By mapping the genomic imbalances in the tumors using comparative genomic hybridization, we could show that DNA copy number gains of recurrently aneuploid chromosome arms 7p, 8q, 13q, 18q, 20p, and 20q correlated significantly with their average chromosome arm expression profile. Taken together, our results show that both the high-level, significant transcriptional deregulation of specific genes and general modification of the average transcriptional activity of genes residing on aneuploid chromosomes coexist in rectal adenocarcinomas.
Assuntos
Adenocarcinoma/genética , Aneuploidia , Neoplasias Retais/genética , Proteína Wnt2/fisiologia , beta Catenina/fisiologia , Adenocarcinoma/metabolismo , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Família Multigênica , Hibridização de Ácido Nucleico , Neoplasias Retais/metabolismo , Transdução de SinaisRESUMO
Atm-deficient mice die of malignant thymic lymphomas characterized by translocations within the Tcr alpha/delta locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA (dsDNA) breaks that occur during RAG-dependent V(D)J recombination. We recently demonstrated that development of thymic lymphoma in Atm(-/-) mice was not prevented by loss of RAG-2. Thymic lymphomas that developed in Rag2(-/-) Atm(-/-) mice contained multiple chromosomal abnormalities, but none of these involved the Tcr alpha/delta locus. These findings indicated that tumorigenesis in Atm(-/-) mice is mediated by chromosomal translocations secondary to aberrant responses to dsDNA breaks and that V(D)J recombination is an important, but not essential, event in susceptibility. In contrast to these findings, it was recently reported that Rag1(-/-) Atm(-/-) mice do not develop thymic lymphomas, a finding that was interpreted as demonstrating a requirement for RAG-dependent recombination in the susceptibility to tumors in Atm-deficient mice. To test the possibility that RAG-1 and RAG-2 differ in their roles in tumorigenesis, we studied Rag1(-/-) Atm(-/-) mice in parallel to our previous Rag2(-/-) Atm(-/-) study. We found that thymic lymphomas occur at high frequency in Rag1(-/-) Atm(-/-) mice and resemble those that occur in Rag2(-/-) Atm(-/-) mice. These results indicate that both RAG-1 and RAG-2 are necessary for tumorigenesis involving translocation in the Tcr alpha/delta locus but that Atm deficiency leads to tumors through a broader RAG-independent predisposition to translocation, related to a generalized defect in dsDNA break repair.
Assuntos
Proteínas de Ligação a DNA/genética , Rearranjo Gênico do Linfócito B/genética , Genes RAG-1/genética , Linfoma/genética , Proteínas Serina-Treonina Quinases/deficiência , Recombinação Genética/genética , Neoplasias do Timo/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização in Situ Fluorescente , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Análise de Sobrevida , Fatores de Tempo , Translocação Genética/genética , Proteínas Supressoras de TumorRESUMO
Adenoid cystic carcinomas (ACC) are rare salivary gland cancers with a high incidence of metastases. In order to study this tumor type, a reliable model system exhibiting the molecular features of this tumor is critical, but none exists, thereby inhibiting in-vitro studies and the analysis of metastatic behavior. To address this deficiency, we have coupled an efficient method to establish tumor cell cultures, conditional reprogramming (CR), with a rapid, reproducible and robust in-vivo zebrafish model. We have established cell cultures from two individual ACC PDX tumors that maintain the characteristic MYB translocation. Additional mutations found in one ACC culture also seen in the PDX tumor. Finally, the CR/zebrafish model mirrors the PDX mouse model and identifies regorafenib as a potential therapeutic drug to treat this cancer type that mimic the drug sensitivity profile in PDX model, further confirming the unique advantages of multiplex system.