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1.
Cancer Cell ; 35(6): 932-947.e8, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31130341

RESUMO

We performed genomic and transcriptomic sequencing of 133 combined hepatocellular and intrahepatic cholangiocarcinoma (cHCC-ICC) cases, including separate, combined, and mixed subtypes. Integrative comparison of cHCC-ICC with hepatocellular carcinoma and intrahepatic cholangiocarcinoma revealed that combined and mixed type cHCC-ICCs are distinct subtypes with different clinical and molecular features. Integrating laser microdissection, cancer cell fraction analysis, and single nucleus sequencing, we revealed both mono- and multiclonal origins in the separate type cHCC-ICCs, whereas combined and mixed type cHCC-ICCs were all monoclonal origin. Notably, cHCC-ICCs showed significantly higher expression of Nestin, suggesting Nestin may serve as a biomarker for diagnosing cHCC-ICC. Our results provide important biological and clinical insights into cHCC-ICC.

2.
Clin Immunol ; 194: 80-86, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30006120

RESUMO

HLA-G protein expression could play a role in evasion of tumor immune surveillance. Accumulating evidence demonstrates that HLA-G is expressed in different types of malignancies, including colorectal cancer (CRC). The purpose of the current study was to further unravel whether HLA-G protein expression could play a role in immune evasion of CRC. Therefore, to firmly establish HLA-G protein expression, eight early passage human CRC cell lines and five human rectal cancer tissues were analyzed by western blot analysis. The results obtained by western blot analysis were compared with immunohistochemistry on tumor tissue sections of the same patient. Furthermore, multiple monoclonal antibodies (mAbs), 4H84, MEM-G/1 and 5A6G7, targeting HLA-G were used to unravel staining patterns. We showed that results obtained with immunohistochemistry did not correlate with protein expression detected by western blot analysis, using three different HLA-G targeting mAbs. Furthermore, with respect to the specificity of the mAbs employed, additional immune reactivity was detected using the mAbs MEM-G/1 and 5A6G7 in western blot analysis with K562 control cell lines overexpressing HLA-A2 or HLA-G, all tumor tissues and in two out of eight CRC cell lines. Based on the current study and our previously reported results, we conclude that claiming HLA-G plays a role in immune modulation of CRC seems premature, as results from anti-body based detection of HLA-G protein remain inconclusive. Until the time that detection of HLA-G is sensitive enough to detect all aspects of HLA-G expression in biological samples, rather than transfected cells or long time cultured cell lines, conclusions should be drawn with great care.

3.
Genome Res ; 28(5): 654-665, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29632087

RESUMO

Cisplatin reacts with DNA and thereby likely generates a characteristic pattern of somatic mutations, called a mutational signature. Despite widespread use of cisplatin in cancer treatment and its role in contributing to secondary malignancies, its mutational signature has not been delineated. We hypothesize that cisplatin's mutational signature can serve as a biomarker to identify cisplatin mutagenesis in suspected secondary malignancies. Knowledge of which tissues are at risk of developing cisplatin-induced secondary malignancies could lead to guidelines for noninvasive monitoring for secondary malignancies after cisplatin chemotherapy. We performed whole genome sequencing of 10 independent clones of cisplatin-exposed MCF-10A and HepG2 cells and delineated the patterns of single and dinucleotide mutations in terms of flanking sequence, transcription strand bias, and other characteristics. We used the mSigAct signature presence test and nonnegative matrix factorization to search for cisplatin mutagenesis in hepatocellular carcinomas and esophageal adenocarcinomas. All clones showed highly consistent patterns of single and dinucleotide substitutions. The proportion of dinucleotide substitutions was high: 8.1% of single nucleotide substitutions were part of dinucleotide substitutions, presumably due to cisplatin's propensity to form intra- and interstrand crosslinks between purine bases in DNA. We identified likely cisplatin exposure in nine hepatocellular carcinomas and three esophageal adenocarcinomas. All hepatocellular carcinomas for which clinical data were available and all esophageal cancers indeed had histories of cisplatin treatment. We experimentally delineated the single and dinucleotide mutational signature of cisplatin. This signature enabled us to detect previous cisplatin exposure in human hepatocellular carcinomas and esophageal adenocarcinomas with high confidence.


Assuntos
Cisplatino/envenenamento , Análise Mutacional de DNA/métodos , Mutação/efeitos dos fármacos , Sequenciamento Completo do Exoma/métodos , Adenocarcinoma/genética , Antineoplásicos/envenenamento , Carcinoma Hepatocelular/genética , Linhagem Celular , Neoplasias Esofágicas/genética , Genoma Humano/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Mutagênese/efeitos dos fármacos
5.
Endocr Relat Cancer ; 25(1): 83-97, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29066502

RESUMO

A near-homozygous genome (NHG) is especially seen in a subset of follicular thyroid cancer of the oncocytic type (FTC-OV). An NHG was also observed in the metabolically relatively quiescent cell lines XTC.UC1, a model for FTC-OV, and in FTC-133, -236 and -238, the latter three derived from one single patient with follicular thyroid cancer. FTC-236 subclones showed subtle whole-chromosome differences indicative of sustained reciprocal mitotic missegregations. Reactive oxygen species (ROS) scavenger experiments reduced the number of chromosomal missegregations in XTC.UC1 and FTC-236, while pCHK2 was downregulated in these cells. Treatment with antimycin A increased ROS indicated by enhanced MitoSOX Red and pCHK2 fluorescence in metaphase cells. In a selected set of oncocytic follicular thyroid tumors, increasing numbers of whole-chromosome losses were observed toward an aggressive phenotype, but with retention of chromosome 7. Together, ROS activates CHK2 and links to the stepwise loss of whole chromosomes during tumor progression in these lesions. We postulate that sequential loss of whole chromosomes is a dominant driver of the oncogenesis of a subset of follicular thyroid tumors.

6.
Sci Transl Med ; 9(412)2017 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-29046434

RESUMO

Many traditional pharmacopeias include Aristolochia and related plants, which contain nephrotoxins and mutagens in the form of aristolochic acids and similar compounds (collectively, AA). AA is implicated in multiple cancer types, sometimes with very high mutational burdens, especially in upper tract urothelial cancers (UTUCs). AA-associated kidney failure and UTUCs are prevalent in Taiwan, but AA's role in hepatocellular carcinomas (HCCs) there remains unexplored. Therefore, we sequenced the whole exomes of 98 HCCs from two hospitals in Taiwan and found that 78% showed the distinctive mutational signature of AA exposure, accounting for most of the nonsilent mutations in known cancer driver genes. We then searched for the AA signature in 1400 HCCs from diverse geographic regions. Consistent with exposure through known herbal medicines, 47% of Chinese HCCs showed the signature, albeit with lower mutation loads than in Taiwan. In addition, 29% of HCCs from Southeast Asia showed the signature. The AA signature was also detected in 13 and 2.7% of HCCs from Korea and Japan as well as in 4.8 and 1.7% of HCCs from North America and Europe, respectively, excluding one U.S. hospital where 22% of 87 "Asian" HCCs had the signature. Thus, AA exposure is geographically widespread. Asia, especially Taiwan, appears to be much more extensively affected, which is consistent with other evidence of patterns of AA exposure. We propose that additional measures aimed at primary prevention through avoidance of AA exposure and investigation of possible approaches to secondary prevention are warranted.


Assuntos
Ácidos Aristolóquicos/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Ásia , Geografia , Humanos , Imunoterapia , Neoplasias Hepáticas/genética , Mutagênese/genética , Mutação/genética , Taiwan
7.
PLoS One ; 12(9): e0184900, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28931069

RESUMO

Genetic and epigenetic alterations mark colorectal cancer (CRC). Global hypomethylation is observed in nearly all CRC, but a distinct subset of CRC show the CpG Island Methylator Phenotype (CIMP). These tumors show DNA hypermethylation of a specific subset of CpG islands, resulting in transcriptional downregulation of nearby genes. Recently we reported the establishment of novel CRC cell lines derived from primary and metastatic CRC tissues. In this study we describe the DNA methylation profiling of these low passage CRC cell lines. We generated global DNA methylation profiles with Infinium HumanMethylation450 BeadChips and analysed them in conjunction with matching gene expression profiles. Multidimensional scaling of the DNA methylation and gene expression datasets showed that BRAF mutated cell lines form a distinct group. In this group we investigated the 706 loci which we have previously identified to be hypermethylated in BRAF mutant CRC. We validated the significant findings in the The Cancer Genome Atlas colon adenocarcinoma dataset. Our analysis identified ELOVL5, FAM127B, MTERF1, ZNF606 to be subject to transcriptional downregulation through DNA hypermethylation in CRC. We further investigated ELOVL5 with qPCR and immunohistochemical staining, validating our results, but did not find a clear relation between ELOVL5 expression and tumor stage or relapse free survival. ELOVL5, FAM127B, MTERF1, ZNF606 are involved in important cellular processes such as apoptosis, lipogenesis and the downstream transcriptional effect of the MAPK-pathway. We have identified a DNA methylation profile regulating key cellular processes in CRC, resulting in a growth advantage to the tumor cells.


Assuntos
Acetiltransferases/genética , Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Recidiva Local de Neoplasia/genética , Adenocarcinoma/patologia , Idoso , Estudos de Coortes , Neoplasias Colorretais/patologia , Feminino , Seguimentos , Humanos , Masculino , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
8.
Cancer Discov ; 7(10): 1116-1135, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28667006

RESUMO

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analyzed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined 4 CCA clusters-fluke-positive CCAs (clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations; conversely, fluke-negative CCAs (clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3' untranslated region deletion as a mechanism of FGFR2 upregulation. Integration of noncoding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores-mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics, and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.Significance: Integrated whole-genome and epigenomic analysis of CCA on an international scale identifies new CCA driver genes, noncoding promoter mutations, and structural variants. CCA molecular landscapes differ radically by etiology, underscoring how distinct cancer subtypes in the same organ may arise through different extrinsic and intrinsic carcinogenic processes. Cancer Discov; 7(10); 1116-35. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.


Assuntos
Neoplasias dos Ductos Biliares/genética , Colangiocarcinoma/genética , Epigenômica/métodos , Estudo de Associação Genômica Ampla/métodos , Ilhas de CpG , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Receptor ErbB-2/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Proteína Supressora de Tumor p53/genética
9.
Br J Cancer ; 117(6): 1215-1223, 2017 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-28742792

RESUMO

BACKGROUND: A substantial fraction of familial colorectal cancer (CRC) and polyposis heritability remains unexplained. This study aimed to identify predisposing loci in patients with these disorders. METHODS: Homozygosity mapping was performed using 222 563 SNPs in 302 index patients with various colorectal neoplasms and 3367 controls. Linkage analysis, exome and whole-genome sequencing were performed in a family affected by microsatellite stable CRCs. Candidate variants were genotyped in 10 554 cases and 21 480 controls. Gene expression was assessed at the mRNA and protein level. RESULTS: Homozygosity mapping revealed a disease-associated region at 1q32.3 which was part of the linkage region 1q32.2-42.2 identified in the CRC family. This includes a region previously associated with risk of CRC. Sequencing identified the p.Asp1432Glu variant in the MIA3 gene (known as TANGO1 or TANGO) and 472 additional rare, shared variants within the linkage region. In both cases and controls the population frequency was 0.02% for this MIA3 variant. The MIA3 mutant allele showed predominant mRNA expression in normal, cancer and precancerous tissues. Furthermore, immunohistochemistry revealed increased expression of MIA3 in adenomatous tissues. CONCLUSIONS: Taken together, our two independent strategies associate genetic variations in chromosome 1q loci and predisposition to familial CRC and polyps, which warrants further investigation.


Assuntos
Polipose Adenomatosa do Colo/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Cromossomos Humanos Par 1/genética , Neoplasias Colorretais/genética , Predisposição Genética para Doença , Proteínas de Neoplasias/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Mapeamento Cromossômico , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Ligação Genética , Genótipo , Homozigoto , Humanos , Repetições de Microssatélites , Proteínas de Neoplasias/metabolismo , Polimorfismo de Nucleotídeo Único , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , RNA Mensageiro/metabolismo
10.
Genome Res ; 27(9): 1475-1486, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28739859

RESUMO

Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here, we present the first whole-genome data on the mutational signatures of AFB1 exposure from a total of >40,000 mutations in four experimental systems: two different human cell lines, in liver tumors in wild-type mice, and in mice that carried a hepatitis B surface antigen transgene-this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence.


Assuntos
Aflatoxina B1/toxicidade , Carcinógenos/toxicidade , Análise Mutacional de DNA , Mutação/efeitos dos fármacos , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , China , Antígenos de Superfície da Hepatite B/genética , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Mutação/genética
11.
Int J Gynecol Cancer ; 27(8): 1571-1578, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28604461

RESUMO

OBJECTIVE: Serous adenocarcinomas that arise in patients with inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 are initially well treatable with platinum/paclitaxel. For recurrent disease in patients with BRCA1 or BRCA2 mutations, olaparib treatment is available. To study additional therapeutic regimens, a better understanding of the cellular and molecular mechanisms of the tumors in in vitro models is important. METHODS/MATERIALS: From a high-grade serous ovarian tumor of a BRCA1 mutation carrier, we established 3 distinct cell line subclones, OVCA-TR3.1, -2, and -3. Immunohistochemical characterization, flow cytometric analyses, chemosensitivity, and somatic mutation profiling were performed. RESULTS: The cell lines expressed AE1/AE3, Pax8, WT-1, OC125, estrogen receptor (ER), and p53, comparable to the primary tumor. Synergism could be shown in the combination treatment eremophila-1-(10)-11(13)-dien-12,8ß-olide (EPD), with cisplatin, whereas combination with olaparib did not show synergism. Eremophila-1-(10)-11(13)-dien-12,8ß-olide, a sesquiterpene lactone, is a novel chemotherapeutic agent. The inherited BRCA1 c.2989_2990dupAA mutation was confirmed in the cell lines. Loss of heterozygosity of BRCA1 was detected in each cell line, as well as a homozygous TP53 c.722C>A mutation. Flow cytometry showed that all cell lines had a distinct DNA index. CONCLUSIONS: Three new isogenic ovarian cancer cell lines were developed from a patient with a germ line BRCA1 mutation. Chemosensitivity profiling of the cell lines showed high tolerance for olaparib. Treatment with EPD proved synergistic with cisplatin. The effects of EPD will be further investigated for future clinical efficacy.


Assuntos
Proteína BRCA1/genética , Genes BRCA1 , Mutação em Linhagem Germinativa , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/genética , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Humanos , Imuno-Histoquímica , Lactonas/administração & dosagem , Lactonas/farmacologia , Ftalazinas/administração & dosagem , Ftalazinas/farmacologia , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Sesquiterpenos/administração & dosagem , Sesquiterpenos/farmacologia
12.
Int J Mol Sci ; 18(1)2016 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-28035987

RESUMO

Genome-wide DNA hypomethylation is associated with a worse prognosis in early-stage colorectal cancer. To measure genome-wide DNA methylation levels, long interspersed nucleotide element (LINE-1) repeats are used as a surrogate marker. Cohort studies on the clinical impact of genome-wide DNA methylation level in patients with only early-stage colon cancer, are currently lacking. This study aimed to investigate the prognostic value of LINE-1 methylation in a stage II colon cancer cohort (n = 164). Manual needle microdissection of tumor areas was performed on formalin-fixed paraffin-embedded tumor tissue sections followed by DNA extraction. Bisulfite converted DNA was used to assess tumor LINE-1 methylation level by qPCR. Patients with LINE-1 hypomethylated tumors had a significantly worse overall survival compared to patients with a higher level of LINE-1 tumor DNA methylation (HR 1.68, 95% CI 1.03-2.75; p = 0.04). This effect was more prominent in patients aged over 65 years (HR 2.00, 95% CI 1.13-3.52; p = 0.02), although the test for age interaction was not significant. No significant effect on recurrence-free survival was observed. Based on these results, tumor LINE-1 hypomethylation is associated with a worse overall survival in stage II colon cancer. Whether the origin of this causation is cancer-specific or age-related can be debated.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma/genética , Neoplasias do Colo/genética , Metilação de DNA , Elementos Nucleotídeos Longos e Dispersos , Idoso , Idoso de 80 Anos ou mais , Carcinoma/patologia , Neoplasias do Colo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida
13.
J Pathol ; 240(1): 72-83, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27265324

RESUMO

The genomes of a wide range of cancers, including colon, breast, and thyroid cancers, frequently show copy number gains of chromosome 7 and rarely show loss of heterozygosity. The molecular basis for this phenomenon is unknown. Strikingly, oncocytic follicular thyroid carcinomas can display an extreme genomic profile, with homozygosity of all chromosomes except for chromosome 7. The observation that homozygosity of chromosome 7 is never observed suggests that retention of heterozygosity is essential for cells. We hypothesized that cell survival genes are genetically imprinted on either of two copies of chromosome 7, which thwarts loss of heterozygosity at this chromosome in cancer cells. By employing a DNA methylation screen and gene expression analysis, we identified six imprinted genes that force retention of heterozygosity on chromosome 7. Subsequent knockdown of gene expression showed that CALCR, COPG2, GRB10, KLF14, MEST, and PEG10 were essential for cancer cell survival, resulting in reduced cell proliferation, G1 -phase arrest, and increased apoptosis. We propose that imprinted cell survival genes provide a genetic basis for retention of chromosome 7 heterozygosity in cancer cells. The monoallelically expressed cell survival genes identified in this study, and the cellular pathways that they are involved in, offer new therapeutic targets for the treatment of tumours showing retention of heterozygosity on chromosome 7. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Carcinoma Medular/genética , Sobrevivência Celular/genética , Cromossomos Humanos Par 7 , Regulação Neoplásica da Expressão Gênica , Impressão Genômica , Perda de Heterozigosidade , Neoplasias da Glândula Tireoide/genética , Proteína Semelhante a Receptor de Calcitonina/genética , Carcinoma Medular/patologia , Proliferação de Células/genética , Proteína Coatomer/genética , Metilação de DNA , Proteína Adaptadora GRB10/genética , Humanos , Proteínas/genética , Fatores de Transcrição Sp/genética , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia
14.
Hum Immunol ; 77(9): 764-72, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27245757

RESUMO

Expression of human leukocyte antigen-G (HLA-G) is a suggested mechanism used by tumor cells to escape from host immune recognition and destruction. Advances in the field have made it evident that HLA-G is expressed in different types of malignancies including colorectal cancer (CRC). We analyzed HLA-G expression in 21 low passage CRC cell lines. The level of DNA methylation of the HLA-G gene and the presence of mRNA encoding HLA-G was measured. Moreover, HLA-G protein expression was determined by flow cytometry and immunohistochemistry (IHC). IHC was performed with three different monoclonal antibodies (mAbs) (4H84, MEM-G/1 and MEM-G/2). In addition, HLA-G protein expression was measured in matching primary tumor tissues. RNA analysis using RT-PCR followed by sequencing in 6 samples indicated strong homology of the PCR product with HLA-G3 in 5 samples. In accordance, in none of the cell lines, HLA-G1 expression was detected by flow-cytometry. Furthermore, no association between HLA-G DNA methylation patterns and HLA-G mRNA expression was observed. In addition, different immunohistochemical staining profiles among various anti-HLA-G mAbs were observed. In conclusion, the results of this study show that the HLA-G3 isoform was expressed in some of the CRC cell lines irrespective of the level of DNA methylation of HLA-G.


Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Metilação de DNA , Antígenos HLA-G/genética , Regiões Promotoras Genéticas/genética , Adenocarcinoma/metabolismo , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Separação Celular , Neoplasias Colorretais/metabolismo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Antígenos HLA-G/imunologia , Antígenos HLA-G/metabolismo , Humanos , Imuno-Histoquímica , RNA Mensageiro/análise , Evasão Tumoral
15.
Oncotarget ; 7(12): 14499-509, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26894854

RESUMO

In vitro models are essential to understanding the molecular characteristics of colorectal cancer (CRC) and the testing of therapies for CRC. Many efforts to establish and characterize primary CRC cell lines have been published, most describing a small number of novel cell lines. However, there remains a lack of a large panel of uniformly established and characterized cell lines. To this end we established 20 novel CRC cell lines, of which six were derived from liver metastases. Genetic, genomic and transcriptomic profiling was performed in order to characterize these new cell lines. All data are made publically available upon publication.By combining mutation profiles with CNA and gene expression profiles, we generated an overall profile of the alterations in the major CRC-related signaling pathways. The combination of mutation profiles with genome, transcriptome and methylome data means that these low passage cell lines are among the best characterized of all CRC cell lines. This will allow researchers to select model cell lines appropriate to specific experiments, facilitating the optimal use of these cell lines as in vitro models for CRC. All cell lines are available for further research.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Neoplasias Hepáticas/genética , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Mutação , Células Tumorais Cultivadas
16.
Lancet Gastroenterol Hepatol ; 1(3): 207-216, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-28404093

RESUMO

BACKGROUND: Precision cancer medicine depends on defining distinct tumour subgroups using biomarkers that may occur at very modest frequencies. One such subgroup comprises patients with exceptionally mutated (ultramutated) cancers caused by mutations that impair DNA polymerase epsilon (POLE) proofreading. METHODS: We examined the association of POLE proofreading domain mutation with clinicopathological variables and immune response in colorectal cancers from clinical trials (VICTOR, QUASAR2, and PETACC-3) and colorectal cancer cohorts (Leiden University Medical Centre 1 and 2, Oslo 1 and 2, Bern, AMC-AJCC-II, and Epicolon-1). We subsequently investigated its association with prognosis in stage II/III colorectal cancer by Cox regression of pooled individual patient data from more than 4500 cases from these studies. FINDINGS: Pathogenic somatic POLE mutations were detected in 66 (1·0%) of 6517 colorectal cancers, and were mutually exclusive with mismatch repair deficiency (MMR-D) in the 6277 cases for whom both markers were determined (none of 66 vs 833 [13·4%] of 6211; p<0·0001). Compared with cases with wild-type POLE, cases with POLE mutations were younger at diagnosis (median 54·5 years vs 67·2 years; p<0·0001), were more frequently male (50 [75·8%] of 66 vs 3577 [55·5%] of 6445; p=0·0010), more frequently had right-sided tumour location (44 [68·8%] of 64 vs 2463 [39·8%] of 6193; p<0·0001), and were diagnosed at an earlier disease stage (p=0·006, χ2 test for trend). Compared with mismatch repair proficient (MMR-P) POLE wild-type tumours, POLE-mutant colorectal cancers displayed increased CD8+ lymphocyte infiltration and expression of cytotoxic T-cell markers and effector cytokines, similar in extent to that observed in immunogenic MMR-D cancers. Both POLE mutation and MMR-D were associated with significantly reduced risk of recurrence compared with MMR-P colorectal cancers in multivariable analysis (HR 0·34 [95% CI 0·11-0·76]; p=0·0060 and 0·72 [0·60-0·87]; p=0·00035), although the difference between the groups was not significant. INTERPRETATION: POLE proofreading domain mutations identify a subset of immunogenic colorectal cancers with excellent prognosis. This association underscores the importance of rare biomarkers in precision cancer medicine, but also raises important questions about how to identify and implement them in practice. FUNDING: Cancer Research UK, Academy of Medical Sciences, Health Foundation, EU, ERC, NIHR, Wellcome Trust, Dutch Cancer Society, Dutch Digestive Foundation.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , DNA Polimerase II/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/mortalidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida
17.
Sci Rep ; 5: 16286, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26553438

RESUMO

Whilst common genetic variation in many non-coding genomic regulatory regions are known to impart risk of colorectal cancer (CRC), much of the heritability of CRC remains unexplained. To examine the role of recurrent coding sequence variation in CRC aetiology, we genotyped 12,638 CRCs cases and 29,045 controls from six European populations. Single-variant analysis identified a coding variant (rs3184504) in SH2B3 (12q24) associated with CRC risk (OR = 1.08, P = 3.9 × 10(-7)), and novel damaging coding variants in 3 genes previously tagged by GWAS efforts; rs16888728 (8q24) in UTP23 (OR = 1.15, P = 1.4 × 10(-7)); rs6580742 and rs12303082 (12q13) in FAM186A (OR = 1.11, P = 1.2 × 10(-7) and OR = 1.09, P = 7.4 × 10(-8)); rs1129406 (12q13) in ATF1 (OR = 1.11, P = 8.3 × 10(-9)), all reaching exome-wide significance levels. Gene based tests identified associations between CRC and PCDHGA genes (P < 2.90 × 10(-6)). We found an excess of rare, damaging variants in base-excision (P = 2.4 × 10(-4)) and DNA mismatch repair genes (P = 6.1 × 10(-4)) consistent with a recessive mode of inheritance. This study comprehensively explores the contribution of coding sequence variation to CRC risk, identifying associations with coding variation in 4 genes and PCDHG gene cluster and several candidate recessive alleles. However, these findings suggest that recurrent, low-frequency coding variants account for a minority of the unexplained heritability of CRC.


Assuntos
Neoplasias Colorretais/genética , Variação Genética , Fator 1 Ativador da Transcrição/genética , Alelos , Caderinas/genética , Estudos de Casos e Controles , Neoplasias Colorretais/patologia , Grupo com Ancestrais do Continente Europeu/genética , Frequência do Gene , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Desequilíbrio de Ligação , Razão de Chances , Polimorfismo de Nucleotídeo Único , Proteínas/genética
18.
J Exp Clin Cancer Res ; 34: 38, 2015 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-25907439

RESUMO

BACKGROUND: Ovarian cancer remains still the leading cause of death of gynecological malignancy, in spite of first-line chemotherapy with cisplatin and paclitaxel. Although initial response is favorably, relapses are common and prognosis for women with advanced disease stays poor. Therefore efficacious approaches are needed. METHODS: Previously, an anti-cancer agent, EPD exhibited potent cytotoxic effects towards ovarian cancer and not towards normal cells. Cell viability and cell cycle analysis studies were performed with EPD, in combination with cisplatin and/or paclitaxel, using the ovarian carcinoma cell lines: SK-OV-3, OVCAR-3, JC, JC-pl and normal fibroblasts. Cell viability was measured using Presto Blue and cell cycle analysis using a flow cytometer. Apoptosis was measured in JC and JC-pl , using the caspase 3 assay kit. RESULTS: In JC-pl, SK-OV-3 and JC, synergistic interactions between either EPD and cisplatin or EPD and paclitaxel were observed. For the first time the effects of EPD on the cell cycle of ovarian cancer cells and normal cells was studied. EPD and combinations of EPD with cisplatin and/ or paclitaxel showed cell cycle arrest in the G2/M phase. The combination of EPD and cisplatin showed a significant synergistic effect in cell line JC-pl, while EPD with paclitaxel showed synergistic interaction in JC. Additionally, synergistic drug combinations showed increased apoptosis. CONCLUSIONS: Our results showed a synergistic effect of EPD and cisplatin in an ovarian drug resistant cell line as well as a synergistic effect of EPD and paclitaxel in two other ovarian cell lines. These results might enhance clinical efficacy, compared to the existing regimen of paclitaxel and cisplatin.


Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Lactonas/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Sesquiterpenos/farmacologia , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Cisplatino/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia
19.
Anticancer Res ; 34(4): 1673-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24692696

RESUMO

BACKGROUND/AIM: The potential of indolizine derivatives as anticancer agents has been shown through recent studies. Herein, we present our experimental results, showing that pyrido[2,3-b]indolizine derivatives are effective against colorectal cancer (CRC) cell lines. MATERIALS AND METHODS: Several pyrido[2,3-b]indolizine derivatives were synthesized and their anticancer potential was evaluated against three CRC cell lines and two normal fibroblast cultures. RESULTS: Our experiments identified 4-(3,4)-dihydroxyphenyl)-2-phenylpyrido[2,3-b]indolizine-10-carbonitrile (4f) as being active against all CRC cell lines at concentrations non-cytotoxic against fibroblast cultures. Additionally, cell-cycle analysis indicated that pyrido[2,3-b]indolizines can affect cell-cycle progression, with treated cells accumulating in the S- and G2/M-phase. CONCLUSION: The hydroxyl groups in both the 3- and 4- positions of the aromatic substituent on C4 of the indolizine nucleus are crucial for activity against CRC cell lines. Further manipulation of the number and position of hydroxyl substituents on the aromatic rings may lead to improved anticancer activity of this class of compounds.


Assuntos
Antineoplásicos/farmacologia , Indolizinas/farmacologia , Antineoplásicos/química , Antineoplásicos/toxicidade , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias Colorretais , Relação Dose-Resposta a Droga , Células HCT116 , Células HT29 , Humanos , Indolizinas/química , Indolizinas/toxicidade , Concentração Inibidora 50 , Fenóis/farmacologia
20.
PLoS One ; 8(11): e79898, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244575

RESUMO

Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAF (p.V600E) mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAF (p.V600E) mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAF (p.V600E) with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10(-9)). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAF (p.V600E) and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1 D27 , which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAF (p.V600E) tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAF (p.V600E) stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAF (p.V600E) colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.


Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas B-raf/genética , Idoso , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Metilação de DNA , Análise Mutacional de DNA , Éxons , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteína Meis1 , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Valina/genética , Valina/metabolismo
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