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1.
Cancer Cell ; 41(12): 2154-2165.e5, 2023 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-38039963

RESUMO

Circulating T cells from peripheral blood (PBL) can provide a rich and noninvasive source for antitumor T cells. By single-cell transcriptomic profiling of 36 neoantigen-specific T cell clones from 6 metastatic cancer patients, we report the transcriptional and cell surface signatures of antitumor PBL-derived CD8+ T cells (NeoTCRPBL). Comparison of tumor-infiltrating lymphocyte (TIL)- and PBL-neoantigen-specific T cells revealed that NeoTCRPBL T cells are low in frequency and display less-dysfunctional memory phenotypes relative to their TIL counterparts. Analysis of 100 antitumor TCR clonotypes indicates that most NeoTCRPBL populations target the same neoantigens as TILs. However, NeoTCRPBL TCR repertoire is only partially shared with TIL. Prediction and testing of NeoTCRPBL signature-derived TCRs from PBL of 6 prospective patients demonstrate high enrichment of clonotypes targeting tumor mutations, a viral oncogene, and patient-derived tumor. Thus, the NeoTCRPBL signature provides an alternative source for identifying antitumor T cells from PBL of cancer patients, enabling immune monitoring and immunotherapies.


Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Humanos , Estudos Prospectivos , Antígenos de Neoplasias , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Linfócitos do Interstício Tumoral , Receptores de Antígenos de Linfócitos T
2.
Science ; 375(6583): 877-884, 2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35113651

RESUMO

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8+ and CD4+ neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature-derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos do Interstício Tumoral/imunologia , Metástase Neoplásica , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Transcriptoma , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Redes Reguladoras de Genes , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , RNA-Seq , Análise de Célula Única
3.
J Clin Oncol ; 40(16): 1741-1754, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35104158

RESUMO

PURPOSE: Metastatic breast cancer (mBrCa) is most often an incurable disease with only modest responses to available immunotherapies. This study investigates the immunogenicity of somatic mutations in breast cancer and explores the therapeutic efficacy in a pilot trial of mutation-reactive tumor-infiltrating lymphocytes (TILs) in patients with metastatic disease. PATIENTS AND METHODS: Forty-two patients with mBrCa refractory to previous lines of treatment underwent surgical resection of a metastatic lesion(s), isolation of TIL cultures, identification of exomic nonsynonymous tumor mutations, and immunologic screening for neoantigen reactivity. Clinically eligible patients with appropriate reactivity were enrolled into one cohort of an ongoing phase II pilot trial of adoptive cell transfer of selected neoantigen-reactive TIL, with a short course of pembrolizumab (ClinicalTrials.gov identifier: NCT01174121). RESULTS: TILs were isolated and grown in culture from the resected lesions of all 42 patients with mBrCa, and a median number of 112 (range: 6-563) nonsynonymous mutations per patient were identified. Twenty-eight of 42 (67%) patients contained TIL that recognized at least one immunogenic somatic mutation (median: 3 neoantigens per patient, range: 1-11), and 13 patients demonstrated robust reactivity appropriate for adoptive transfer. Eight patients remained clinically eligible for treatment, and six patients were enrolled on a protocol of adoptive cell transfer of enriched neoantigen-specific TIL, in combination with pembrolizumab (≤ 4 doses). Objective tumor regression was noted in three patients, including one complete response (now ongoing over 5.5 years) and two partial responses (6 and 10 months). CONCLUSION: Most patients with breast cancer generated a natural immune response targeting the expressed products of their cancer mutations. Adoptive transfer of TIL is a highly personalized experimental option for patients with mBrCa shown to be capable of mediating objective responses in this pilot trial and deserves further study.


Assuntos
Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Humanos , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral , Mutação , Transplante Autólogo
5.
Sci Rep ; 8(1): 653, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29330521

RESUMO

Analysis of 501 melanoma exomes revealed RGS7, which encodes a GTPase-accelerating protein (GAP), to be a tumor-suppressor gene. RGS7 was mutated in 11% of melanomas and was found to harbor three recurrent mutations (p.R44C, p.E383K and p.R416Q). Structural modeling of the most common recurrent mutation of the three (p.R44C) predicted that it destabilizes the protein due to the loss of an H-bond and salt bridge network between the mutated position and the serine and aspartic acid residues at positions 58 as 61, respectively. We experimentally confirmed this prediction showing that the p.R44C mutant protein is indeed destabilized. We further show RGS7 p.R44C has weaker catalytic activity for its substrate Gαo, thus providing a dual mechanism for its loss of function. Both of these effects are expected to contribute to loss of function of RGS7 resulting in increased anchorage-independent growth, migration and invasion of melanoma cells. By mutating position 56 in the R44C mutant from valine to cysteine, thereby enabling the formation of a disulfide bridge between the two mutated positions, we slightly increased the catalytic activity and reinstated protein stability, leading to the rescue of RGS7's function as a tumor suppressor. Our findings identify RGS7 as a novel melanoma driver and point to the clinical relevance of using strategies to stabilize the protein and, thereby, restore its function.


Assuntos
Melanoma/genética , Mutação , Proteínas RGS/química , Proteínas RGS/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Dissulfetos/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligação de Hidrogênio , Melanoma/metabolismo , Modelos Moleculares , Invasividade Neoplásica , Conformação Proteica , Estabilidade Proteica , Proteínas RGS/genética
6.
PLoS One ; 12(5): e0176683, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28464039

RESUMO

PTEN is among the most commonly mutated tumor suppressor genes in human cancer. However, studying the role of PTEN in the pathogenesis of cancer has been limited, in part, by the paucity of human cell-based isogenic systems that faithfully model PTEN loss. In an effort to remedy this problem, gene editing was used to correct an endogenous mutant allele of PTEN in two human glioblastoma multiforme (GBM) cell lines- 42MGBA and T98G. PTEN correction resulted in reduced cellular proliferation that was Akt-dependent in 42MGBA cells and Akt-independent in T98G cells. This is the first report of human cancer cell lines in which mutant PTEN has been corrected by gene editing. The isogenic sets of gene edited cell lines reported here will likely prove useful for further study of PTEN mutations in the pathogenesis of cancer, and for the discovery and validation of novel therapeutics targeting the PTEN pathway.


Assuntos
Edição de Genes , Glioblastoma/genética , PTEN Fosfo-Hidrolase/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos , Glioblastoma/fisiopatologia , Células HEK293 , Humanos , Mutação , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo
7.
Biochim Biophys Acta ; 1866(1): 1-11, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27207471

RESUMO

Cohesin is a highly-conserved protein complex that plays important roles in sister chromatid cohesion, chromatin structure, gene expression, and DNA repair. In humans, cohesin is a ubiquitously expressed, multi-subunit protein complex composed of core subunits SMC1A, SMC3, RAD21, STAG1/2 and regulatory subunits WAPL, PDS5A/B, CDCA5, NIPBL, and MAU2. Recent studies have demonstrated that genes encoding cohesin subunits are somatically mutated in a wide range of human cancers. STAG2 is the most commonly mutated subunit, and in a recent analysis was identified as one of only 12 genes that are significantly mutated in four or more cancer types. In this review we summarize the findings reported to date and comment on potential functional implications of cohesin mutation in the pathogenesis of human cancer.


Assuntos
Antígenos Nucleares/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Complexos Multiproteicos/genética , Neoplasias/genética , Reparo do DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação/genética , Neoplasias/patologia , Troca de Cromátide Irmã/genética , Coesinas
8.
Nat Genet ; 47(12): 1408-10, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26502337

RESUMO

Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.


Assuntos
Biomarcadores Tumorais/genética , Exoma/genética , Melanoma/genética , Mutação/genética , Neoplasias Cutâneas/genética , Proteínas Ativadoras de ras GTPase/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Melanoma/mortalidade , Melanoma/patologia , Prognóstico , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Taxa de Sobrevida
9.
BMC Cancer ; 14: 506, 2014 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25012071

RESUMO

BACKGROUND: Grade IV glioblastomas exist in two forms, primary (de novo) glioblastomas (pGBM) that arise without precursor lesions, and the less common secondary glioblastomas (sGBM) which develop from earlier lower grade lesions. Genetic heterogeneity between pGBM and sGBM has been documented as have differences in the methylation of individual genes. A hypermethylator phenotype in grade IV GBMs is now well documented however there has been little comparison between global methylation profiles of pGBM and sGBM samples or of methylation profiles between paired early and late sGBM samples. METHODS: We performed genome-wide methylation profiling of 20 matched pairs of early and late gliomas using the Infinium HumanMethylation450 BeadChips to assess methylation at >485,000 cytosine positions within the human genome. RESULTS: Clustering of our data demonstrated a frequent hypermethylator phenotype that associated with IDH1 mutation in sGBM tumors. In 80% of cases, the hypermethylator status was retained in both the early and late tumor of the same patient, indicating limited alterations to genome-wide methylation during progression and that the CIMP phenotype is an early event. Analysis of hypermethylated loci identified 218 genes frequently methylated across grade II, III and IV tumors indicating a possible role in sGBM tumorigenesis. Comparison of our sGBM data with TCGA pGBM data indicate that IDH1 mutated GBM samples have very similar hypermethylator phenotypes, however the methylation profiles of the majority of samples with WT IDH1 that do not demonstrate a hypermethylator phenotype cluster separately from sGBM samples, indicating underlying differences in methylation profiles. We also identified 180 genes that were methylated only in sGBM. Further analysis of these genes may lead to a better understanding of the pathology of sGBM vs pGBM. CONCLUSION: This is the first study to have documented genome-wide methylation changes within paired early/late astrocytic gliomas on such a large CpG probe set, revealing a number of genes that maybe relevant to secondary gliomagenesis.


Assuntos
Metilação de DNA , Glioblastoma/genética , Glioblastoma/patologia , Ilhas de CpG , Progressão da Doença , Genoma Humano , Humanos , Isocitrato Desidrogenase/genética , Fenótipo
10.
J Invest Dermatol ; 134(9): 2390-2398, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24739903

RESUMO

The ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDARs)) are composed of large complexes of multi-protein subunits creating ion channels in the cell plasma membranes that allow for influx or efflux of mono- or divalent cations (e.g., Ca(2+)) important for synaptic transmissions, cellular migration, and survival. Recently, we discovered the high prevalence of somatic mutations within one of the ionotropic glutamate receptors, GRIN2A, in malignant melanoma. Functional characterization of a subset of GRIN2A mutants demonstrated a loss of NMDAR complex formation between GRIN1 and GRIN2A, increased anchorage-independent growth in soft agar, and increased migration. Somatic mutation of GRIN2A results in a dominant negative effect inhibiting the tumor-suppressive phenotype of wild-type (WT) GRIN2A in melanoma. Depletion of endogenous GRIN2A in melanoma cells expressing WT GRIN2A resulted in increased proliferation compared with control. In contrast, short-hairpin RNA depletion of GRIN2A in mutant cell lines slightly reduced proliferation. Our data show that somatic mutation of GRIN2A results in increased survival, and we demonstrate the functional importance of GRIN2A mutations in melanoma and the significance that ionotropic glutamate receptor signaling has in malignant melanoma.


Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Melanoma/genética , Receptores de N-Metil-D-Aspartato/genética , Neoplasias Cutâneas/genética , Proliferação de Células/genética , Genes Supressores de Tumor/fisiologia , Células HEK293 , Humanos , Melanoma/patologia , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Interferente Pequeno/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/patologia
11.
PLoS One ; 9(1): e85621, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24454902

RESUMO

The recent publication of the TCGA Kidney Renal Clear Cell Carcinoma (KIRC) project has provided an immense wealth and breadth of data providing an invaluable tool for confirmation and expansion upon previous observations in a large data set containing multiple data types including DNA methylation, somatic mutation, and clinical information. In clear cell renal cell carcinoma (CCRCC) many genes have been demonstrated to be epigenetically inactivated by promoter hypermethylated and in a small number of cases to be associated with clinical outcome. This study created two cohorts based on the Illumina BeadChip array used to confirm the frequency of tumor-specific hypermethylation of these published hypermethylated genes, assess the impact of somatic mutation or chromosomal loss and provide the most comprehensive assessment to date of the association of this hypermethylation with patient survival. Hypermethylation of the Fibrillin 2 (FBN2) gene was the most consistent epigenetic biomarker for CCRCC across both cohorts in 40.2% or 52.5% of tumors respectively. Hypermethylation of the secreted frizzled-related protein 1 (SFRP1) gene and the basonuclin 1 (BNC1) gene were both statistically associated with poorer survival in both cohorts (SFRP1 - p = <0.0001 or 0.0010 and BNC1 - p = <0.0001 or 0.0380) and represented better independent markers of survival than tumor stage, grade or dimension in one cohort and tumor stage or dimension in the other cohort. Loss of the SFRP1 protein can potentially activate the WNT pathway and this analysis highlighted hypermethylation of several other WNT pathway regulating genes and demonstrated a poorer survival outcome for patients with somatic mutation of these genes. The success of demethylating drugs in hematological malignances and the current trials in solid tumors suggest that the identification of clinically relevant hypermethylated genes combined with therapeutic advances may improve the effectiveness and usefulness of such drugs in clear cell renal cell carcinoma.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Renais/genética , Proteínas de Membrana/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Estudos de Coortes , Ilhas de CpG , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Modelos de Riscos Proporcionais
12.
Annu Rev Genomics Hum Genet ; 14: 257-79, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23875803

RESUMO

Cutaneous malignant melanoma results from the interplay of genetic, host, and environmental factors. Genetic factors implicated in melanoma etiology include inherited high-, intermediate-, and low-risk susceptibility genes as well as numerous somatic mutations in melanoma tumors. CDKN2A is the major high-risk melanoma susceptibility gene identified to date. Recent identification of low-risk loci has been accomplished predominantly through genome-wide association studies. Whole-exome and whole-genome studies have identified numerous genes somatically altered in melanoma tumors and highlighted a higher mutation load in melanoma tumors compared with those in other cancers. This higher load is believed to be attributable to the preponderance of cytosine-to-thymine nucleotide substitutions as a result of UV radiation exposure. Technological advances, particularly next-generation sequencing, have increased the opportunities for germline and somatic gene discovery in melanoma and are opening up new avenues for understanding melanoma pathogenesis as well as leading to new opportunities for treatment.


Assuntos
Predisposição Genética para Doença , Melanoma/genética , Neoplasias Cutâneas/genética , Estudo de Associação Genômica Ampla , Humanos , Mutação
13.
Epigenetics ; 8(2): 149-56, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23291739

RESUMO

Glioblastoma (GBM) is the most common and malignant type of primary brain tumor in adults and prognosis of most GBM patients is poor. However, a small percentage of patients show a long term survival of 36 mo or longer after diagnosis. Epigenetic profiles can provide molecular markers for patient prognosis: recently, a G-CIMP positive phenotype associated with IDH1 mutations has been described for GBMs with good prognosis. In the present analysis we performed genome-wide DNA methylation profiling of short-term survivors (STS; overall survival < 1 y) and long-term survivors (LTS; overall survival > 3 y) by utilizing the HumanMethylation450K BeadChips to assess quantitative methylation at > 480,000 CpG sites. Cluster analysis has shown that a subset of LTS showed a G-CIMP positive phenotype that was tightly associated with IDH1 mutation status and was confirmed by analysis of the G-CIMP signature genes. Using high stringency criteria for differential hypermethylation between non-cancer brain and tumor samples, we identified 2,638 hypermethylated CpG loci (890 genes) in STS GBMs, 3,101 hypermethylated CpG loci (1,062 genes) in LTS (wild type IDH1) and 11,293 hypermethylated CpG loci in LTS (mutated for IDH1), reflecting the CIMP positive phenotype. The location of differentially hypermethylated CpG loci with respect to CpG content, neighborhood context and functional genomic distribution was similar in our sample set, with the majority of CpG loci residing in CpG islands and in gene promoters. Our preliminary study also identified a set of CpG loci differentially hypermethylated between STS and LTS cases, including members of the homeobox gene family (HOXD8, HOXD13 and HOXC4), the transcription factors NR2F2 and TFAP2A, and Dickkopf 2, a negative regulator of the wnt/ß-catenin signaling pathway.


Assuntos
Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/mortalidade , Metilação de DNA , Glioblastoma/genética , Glioblastoma/mortalidade , Ilhas de CpG , Humanos , Isocitrato Desidrogenase/genética , Mutação , Prognóstico , Sobreviventes
14.
Cancer Res ; 71(8): 2988-99, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21363912

RESUMO

Epigenetic profiling of tumor DNAs may reveal important new theranostic targets to improve prognosis and treatment of advanced cancer patients. In this study, we performed a genome-wide profile of DNA methylation patterns in sporadic breast tumors by using the HumanMethylation27 BeadChips to assess relationships between DNA methylation changes and patient tumor characteristics. The arrays identified 264 hypermethylated loci/genes present in genomic CpG islands. Hierarchical clustering based on methylation levels divided the specimens into three distinct groups, within which certain clinical features also clustered. Statistically significant differences were determined between overall methylation levels of these clusters and estrogen receptor and progesterone receptor (ER/PR) status (P = 0.001), tumor relapse (P = 0.035), and lymph node metastasis (P = 0.042). We identified several individual methylated genes associated with clinical features, including six genes (RECK, SFRP2, UAP1L1, ACADL, ITR, and UGT3A1) that showed statistical significance between methylation and relapse-free survival. Notably, the RECK gene in this group has been associated in other cancers with poorest prognosis. Among the leading relapse-associated genes and the genes associated with ER/PR status, we sequenced an independent set of paired normal/tumor breast DNA samples to confirm tumor specificity of methylation. Further, we carried out quantitative real-time reverse transcriptase PCR to confirm reduced expression in methylated tumors. Our findings suggest the utility for the DNA methylation patterns in these genes as clinically useful surrogate markers in breast cancer, as well as new molecular pathways for further investigation as therapeutic targets.


Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ilhas de CpG , Feminino , Estudo de Associação Genômica Ampla , Humanos , Estadiamento de Neoplasias , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo
15.
Epigenetics ; 6(3): 326-32, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21173572

RESUMO

The WW-domain containing protein KIBRA has recently been identified as a new member of the Salvador/Warts/Hippo (SWH) pathway in Drosophila and is shown to act as a tumor suppressor gene in Drosophila. This pathway is conserved in humans and members of the pathway have been shown to act as tumor suppressor genes in mammalian systems. We determined the methylation status of the 5' CpG island associated with the KIBRA gene in human cancers. In a large panel of cancer cell lines representing common epithelial cancers KIBRA was unmethylated. But in pediatric acute lymphocytic leukemia (ALL) cell lines KIBRA showed frequent hypermethylation and silencing of gene expression, which could be reversed by treatment with 5-aza-2'-deoxycytidine. In ALL patient samples KIBRA was methylated in 70% B-ALL but was methylated in < 20% T-ALL leukemia (p = 0.0019). In B-ALL KIBRA methylation was associated with ETV6/RUNX1 [t(12;21) (p13;q22)] chromosomal translocation (p = 0.0082) phenotype, suggesting that KIBRA may play an important role in t(12;21) leukemogenesis. In ALL paired samples at diagnosis and remission KIBRA methylation was seen in diagnostic but not in any of the remission samples accompanied by loss of KIBRA expression in disease state compared to patients in remission. Hence KIBRA methylation occurs frequently in B-cell acute lymphocytic leukemia but not in epithelial cancers and is linked to specific genetic event in B-ALL.


Assuntos
Epigênese Genética , Genes Supressores de Tumor , Leucemia de Células B/genética , Proteínas/genética , Doença Aguda , Linhagem Celular Tumoral , Metilação de DNA , Proteínas de Drosophila/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfoproteínas , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais
16.
Mol Cancer ; 9: 51, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20205715

RESUMO

BACKGROUND: There are several high throughput approaches to identify methylated genes in cancer. We utilized one such recently developed approach, MIRA (methylated-CpG island recovery assay) combined with CpG island arrays to identify novel genes that are epigenetically inactivated in breast cancer. RESULTS: Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers. CONCLUSION: The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy.


Assuntos
Neoplasias da Mama/genética , Metilação de DNA/genética , Epitélio/patologia , Genes Neoplásicos/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Epitélio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida
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