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1.
Nucleic Acids Res ; 52(3): 1136-1155, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38038252

RESUMO

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.


Assuntos
Centrômero , Quebras de DNA de Cadeia Dupla , Chaperonas Moleculares , Proteínas Nucleares , Estruturas R-Loop , Proteína Nuclear Ligada ao X , Criança , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Centrômero/metabolismo , Cromatina , Proteínas Correpressoras/metabolismo , DNA , Histonas/genética , Histonas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo
2.
Prostate ; 84(2): 148-157, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37849074

RESUMO

BACKGROUND: Telomeres are terminal chromosomal elements that are essential for the maintenance of genomic integrity. The measurement of telomere content provides useful diagnostic and prognostic information, and fluorescent methods have been developed for this purpose. However, fluorescent-based tissue assays are cumbersome for investigators to undertake, both in research and clinical settings. METHODS: A robust chromogenic in situ hybridization (CISH) approach was developed to visualize and quantify telomere content at single cell resolution in human prostate tissues, both frozen and formalin-fixed, paraffin-embedded (FFPE). RESULTS: This new assay (telomere chromogenic in situ hybridization ["Telo-CISH"]) produces permanently stained slides that are viewable with a standard light microscope, thus avoiding the need for specialized equipment and storage. The assay is compatible with standard immunohistochemistry, thereby allowing simultaneous assessment of histomorphology, identification of specific cell types, and assessment of telomere status. In addition, Telo-CISH eliminates the problem of autofluorescent interference that frequently occurs with fluorescent-based methods. Using this new assay, we demonstrate successful application of Telo-CISH to help identify precancerous lesions in the prostate by the presence of markedly short telomeres specifically in the luminal epithelial cells. CONCLUSIONS: In summary, with fewer restrictions on the types of tissues that can be tested, and increased histologic information provided, the advantages presented by this novel chromogenic assay should extend the applicability of tissue-based telomere length assessment in research and clinical settings.


Assuntos
Lesões Pré-Cancerosas , Próstata , Masculino , Humanos , Hibridização in Situ Fluorescente/métodos , Hibridização In Situ , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/genética , Telômero
3.
Proc Natl Acad Sci U S A ; 118(32)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34341114

RESUMO

Prostate adenocarcinoma is the second most commonly diagnosed cancer in men worldwide, and the initiating factors are unknown. Oncogenic TMPRSS2:ERG (ERG+) gene fusions are facilitated by DNA breaks and occur in up to 50% of prostate cancers. Infection-driven inflammation is implicated in the formation of ERG+ fusions, and we hypothesized that these fusions initiate in early inflammation-associated prostate cancer precursor lesions, such as proliferative inflammatory atrophy (PIA), prior to cancer development. We investigated whether bacterial prostatitis is associated with ERG+ precancerous lesions in unique cases with active bacterial infections at the time of radical prostatectomy. We identified a high frequency of ERG+ non-neoplastic-appearing glands in these cases, including ERG+ PIA transitioning to early invasive cancer. These lesions were positive for ERG protein by immunohistochemistry and ERG messenger RNA by in situ hybridization. We additionally verified TMPRSS2:ERG genomic rearrangements in precursor lesions using tricolor fluorescence in situ hybridization. Identification of rearrangement patterns combined with whole-prostate mapping in three dimensions confirmed multiple (up to eight) distinct ERG+ precancerous lesions in infected cases. We further identified the pathogen-derived genotoxin colibactin as a potential source of DNA breaks in clinical cases as well as cultured prostate cells. Overall, we provide evidence that bacterial infections can initiate driver gene alterations in prostate cancer. In addition, our observations indicate that infection-induced ERG+ fusions are an early alteration in the carcinogenic process and that PIA may serve as a direct precursor to prostate cancer.


Assuntos
Infecções Bacterianas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/microbiologia , Serina Endopeptidases/genética , Atrofia , Infecções Bacterianas/complicações , Infecções Bacterianas/patologia , Quebras de DNA , Humanos , Masculino , Fusão Oncogênica , Peptídeos/genética , Policetídeos , Próstata/microbiologia , Próstata/patologia , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Prostatite/genética , Prostatite/microbiologia , Prostatite/patologia , Regulador Transcricional ERG/genética
4.
J Pathol ; 254(3): 279-288, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33870509

RESUMO

Epigenetic alterations are increasingly recognized as important contributors to the development and progression of pancreatic ductal adenocarcinoma. 5-hydroxymethylcytosine (5hmC) is an epigenetic DNA mark generated through the ten-eleven translocation (TET) enzyme-mediated pathway and is closely linked to gene activation. However, the timing of alterations in epigenetic regulation in the progression of pancreatic neoplasia is not well understood. In this study, we hypothesized that aberrant expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and subsequent global 5hmC alteration are linked to early tumorigenesis in the pancreas. Therefore, we evaluated alterations of 5hmC and TET1 levels using immunohistochemistry in pancreatic neoplasms (n = 380) and normal ducts (n = 118). The study cohort included representation of the full spectrum of precancerous lesions from low- and high-grade pancreatic intraepithelial neoplasia (n = 95), intraductal papillary mucinous neoplasms (all subtypes, n = 129), intraductal oncocytic papillary neoplasms (n = 12), and mucinous cystic neoplasms (n = 144). 5hmC and TET1 were significantly downregulated in all types of precancerous lesion and associated invasive pancreatic ductal adenocarcinomas compared with normal ductal epithelium (all p < 0.001), and expression of 5hmC positively correlated with expression of TET1. Importantly, downregulation of both 5hmC and TET1 was observed in most low-grade precancerous lesions. There were no clear associations between 5hmC levels and clinicopathological factors, thereby suggesting a common epigenetic abnormality across precancerous lesions. We conclude that downregulation of 5hmC and TET1 is an early event in pancreatic tumorigenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
5-Metilcitosina/análogos & derivados , Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Pancreáticas/metabolismo , 5-Metilcitosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/patologia , Carcinoma Ductal Pancreático/patologia , Regulação para Baixo , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxigenases de Função Mista/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas/metabolismo
5.
Proc Natl Acad Sci U S A ; 116(41): 20482-20488, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548407

RESUMO

A new evaluation of previously published data suggested to us that the accumulation of mutations might slow, rather than increase, as individuals age. To explain this unexpected finding, we hypothesized that normal stem cell division rates might decrease as we age. To test this hypothesis, we evaluated cell division rates in the epithelium of human colonic, duodenal, esophageal, and posterior ethmoid sinonasal tissues. In all 4 tissues, there was a significant decrease in cell division rates with age. In contrast, cell division rates did not decrease in the colon of aged mice, and only small decreases were observed in their small intestine or esophagus. These results have important implications for understanding the relationship between normal stem cells, aging, and cancer. Moreover, they provide a plausible explanation for the enigmatic age-dependent deceleration in cancer incidence in very old humans but not in mice.


Assuntos
Envelhecimento , Divisão Celular , Desaceleração , Mutação , Neoplasias/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Colo/citologia , Colo/metabolismo , Duodeno/citologia , Duodeno/metabolismo , Esôfago/citologia , Esôfago/metabolismo , Humanos , Incidência , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Seios Paranasais/citologia , Seios Paranasais/metabolismo , Adulto Jovem
6.
Mod Pathol ; 34(10): 1810-1819, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34103668

RESUMO

Subsets of high-grade gliomas, including glioblastoma (GBM), are known to utilize the alternative lengthening of telomeres (ALT) pathway for telomere length maintenance. However, the telomere maintenance profile of one subtype of GBM-giant cell GBM-has not been extensively studied. Here, we investigated the prevalence of ALT, as well as ATRX and SMARCAL1 protein loss, in a cohort of classic giant cell GBM and GBM with giant cell features. To determine the presence of ALT, a telomere-specific fluorescence in situ hybridization assay was performed on 15 cases of classic giant cell GBM, 28 additional GBMs found to have giant cell features, and 1 anaplastic astrocytoma with giant cell features. ATRX, SMARCAL1, and IDH1 protein status were assessed in a proportion of cases by immunohistochemistry and were compared to clinical-pathologic and molecular characteristics. In the overall cohort of 44 cases, 19 (43%) showed evidence of ALT. Intriguingly, of the ALT-positive cases, only 9 (47.4%) displayed loss of the ALT suppressor ATRX by immunohistochemistry. Since inactivating mutations in SMARCAL1 have been identified in ATRX wild-type ALT-positive gliomas, we developed an immunohistochemistry assay for SMARCAL1 protein expression using genetically validated controls. Of the 19 ALT-positive cases, 6 (31.5%) showed loss or mis-localization of SMARCAL1 by immunohistochemistry. Of these cases, four retained ATRX protein expression, while two cases also displayed ATRX loss. Additionally, we assessed five cases from which multiple temporal samples were available and ALT status was concordant between both tumor biopsies. In summary, we have identified a subset of giant cell GBM that utilize the ALT telomere maintenance mechanism. Importantly, in addition to ATRX loss, ALT-positive tumors harboring SMARCAL1 alterations are prevalent in giant cell GBM.


Assuntos
Neoplasias Encefálicas/metabolismo , DNA Helicases/metabolismo , Glioblastoma/metabolismo , Homeostase do Telômero/genética , Adolescente , Adulto , Idoso , Biópsia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Pré-Escolar , DNA Helicases/genética , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , Adulto Jovem
7.
Prostate ; 79(3): 320-330, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30488530

RESUMO

BACKGROUND: Prostate cancer is characterized by T-cell exclusion, which is consistent with their poor responses to immunotherapy. In addition, T-cells restricted to the adjacent stroma and benign areas are characterized by anergic and immunosuppressive phenotypes. In order for immunotherapies to produce robust anti-tumor responses in prostate cancer, this exclusion barrier and immunosuppressive microenvironment must first be overcome. We have previously identified mesenchymal stem cells (MSCs) in primary and metastatic human prostate cancer tissue. METHODS: An Opal Multiplex immunofluorescence assay based on CD73, CD90, and CD105 staining was used to identify triple-labeled MSCs in human prostate cancer tissue. T-cell suppression assays and flow cytometry were used to demonstrate the immunosuppressive potential of primary MSCs expanded from human bone marrow and prostate cancer tissue from independent donors. RESULTS: Endogenous MSCs were confirmed to be present at sites of human prostate cancer. These prostate cancer-infiltrating MSCs suppress T-cell proliferation in a dose-dependent manner similar to their bone marrow-derived counterparts. Also similar to bone marrow-derived MSCs, prostate cancer-infiltrating MSCs upregulate expression of PD-L1 and PD-L2 on their cell surface in the presence of IFNγ and TNFα. CONCLUSION: Prostate cancer-infiltrating MSCs suppress T-cell proliferation similar to canonical bone marrow-derived MSCs, which have well-documented immunosuppressive properties with numerous effects on both innate and adaptive immune system function. Thus, we hypothesize that selective depletion of MSCs infiltrating sites of prostate cancer should restore immunologic recognition and elimination of malignant cells via broad re-activation of cytotoxic pro-inflammatory pathways.


Assuntos
Células-Tronco Mesenquimais/imunologia , Neoplasias da Próstata/imunologia , Microambiente Tumoral/imunologia , Comunicação Celular/imunologia , Humanos , Tolerância Imunológica , Ativação Linfocitária , Masculino , Células-Tronco Mesenquimais/patologia , Células PC-3 , Neoplasias da Próstata/patologia , Linfócitos T/imunologia
8.
Clin Chem ; 65(1): 189-198, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30518666

RESUMO

BACKGROUND: Given translational research challenges, multidisciplinary team science is promoted to increase the likelihood of moving from discovery to health effect. We present a case study documenting the utility of multidisciplinary team science in prostate cancer tissue biomarker validation. METHODS: We used primary data generated by a team consisting of a pathologist, cancer biologists, a biostatistician, and epidemiologists. We examined their contributions by phase of biomarker evaluation to identify when, through the practice of team science, threats to internal validity were recognized and solved. Next, we quantified the extent of bias avoided in evaluating the association of Ki67 (immunohistochemistry), stromal cell telomere length (fluorescence in situ hybridization), and microRNA (miRNA) (miR-21, miR-141, miR-221; quantitative RT-PCR) with prostate cancer risk or recurrence in nested case-control studies. RESULTS: Threats to validity were tissue storage time (Ki67, miRNA) and laboratory equipment maintenance (telomeres). Solutions were all in the data analysis phase and involved using tissue storage-time specific cutpoints and/or batch-specific cutpoints. Bias in the regression coefficient for quantiles of each biomarker ranged from 24% to 423%, and the coefficient for the test for trend ranged from 15% to 910%. The interpretation of the associations changed as follows: Ki67, null to positive; stromal cell telomere length, null to positive; miR-21 and miR-141 remained null; miR-221, weak to moderate inverse. CONCLUSIONS: In this case study, we documented the inferential benefits of multidisciplinary team science when the team's collaboration and coordination led to the identification of threats to validity and the implementation of appropriate solutions.


Assuntos
Biomarcadores Tumorais/metabolismo , Equipe de Assistência ao Paciente , Neoplasias da Próstata/metabolismo , Pesquisa Translacional Biomédica , Estudos de Casos e Controles , Humanos , Masculino , MicroRNAs/genética , Recidiva Local de Neoplasia , Prognóstico , Reprodutibilidade dos Testes , Fatores de Risco , Telômero
9.
Am J Pathol ; 188(6): 1478-1485, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29577933

RESUMO

Antibodies targeting the programmed cell death protein 1/programmed death-ligand 1 (PD-L1) interaction have shown clinical activity in multiple cancer types. PD-L1 protein expression is a clinically validated predictive biomarker of response for such therapies. Prior studies evaluating the expression of PD-L1 in primary prostate cancers have reported highly variable rates of PD-L1 positivity. In addition, limited data exist on PD-L1 expression in metastatic castrate-resistant prostate cancer (mCRPC). Here, we determined PD-L1 protein expression by immunohistochemistry using a validated PD-L1-specific antibody (SP263) in a large and representative cohort of primary prostate cancers and prostate cancer metastases. The study included 539 primary prostate cancers comprising 508 acinar adenocarcinomas, 24 prostatic duct adenocarcinomas, 7 small-cell carcinomas, and a total of 57 cases of mCRPC. PD-L1 positivity was low in primary acinar adenocarcinoma, with only 7.7% of cases showing detectable PD-L1 staining. Increased levels of PD-L1 expression were noted in 42.9% of small-cell carcinomas. In mCRPC, 31.6% of cases showed PD-L1-specific immunoreactivity. In conclusion, in this comprehensive evaluation of PD-L1 expression in prostate cancer, PD-L1 expression is rare in primary prostate cancers, but increased rates of PD-L1 positivity were observed in mCRPC. These results will be important for the future clinical development of programmed cell death protein 1/PD-L1-targeting therapies in prostate cancer.


Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Adenocarcinoma/cirurgia , Estudos de Coortes , Humanos , Masculino , Metástase Neoplásica , Valor Preditivo dos Testes , Neoplasias da Próstata/cirurgia
10.
J Pathol ; 244(1): 11-24, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28888037

RESUMO

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate-limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high-grade prostatic intraepithelial neoplasia or PIN) and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in eight cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma and that its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasia Prostática Intraepitelial/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/genética , RNA/genética , Telomerase/genética , Adulto , Idoso , Animais , Proliferação de Células , Genes Reporter , Humanos , Hibridização In Situ , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Próstata/patologia , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/patologia , Análise de Sequência de RNA , Telômero/genética
11.
Prostate ; 78(3): 233-238, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29164645

RESUMO

BACKGROUND: Current and recent smoking have been associated with a greater risk of prostate cancer recurrence and mortality, though the underlying mechanism is unknown. METHODS: To determine if telomere shortening, which has been associated with poor outcomes, may be a potential underlying mechanism, we prospectively evaluated the association between smoking status and telomere length in 567 participants in the Health Professionals Follow-up Study, who were surgically treated for prostate cancer. Using tissue microarrays (TMA), we measured telomere length in cancer and benign tissue, specifically stromal cells in the same TMA spot using a telomere-specific fluorescence in situ hybridization assay. Smoking status was collected via questionnaire 2-years before diagnosis. Adjusting for age, pathologic stage and grade, the median and standard deviation of the per-cell telomere signals were determined for each man for stromal cells and cancer cells by smoking categories. In sub-analyses, we restricted to men without major co-morbidities diagnosed before prostate cancer. RESULTS: Overall, there were no associations between smoking status and telomere length or variability in stromal cells or cancer cells. However, among men without comorbidities, current smokers and former smokers who quit <10 years ago had the most variable telomere length in stromal cells (29.3% more variable than never smokers; P-trend = 0.0005) and in cancer cells (27.7% more variable than never smokers; P-trend = 0.05). Among men without comorbidities, mean telomere length did not differ by smoking status in stromal cells or cancer cells. CONCLUSION: Telomere variability in prostate cells may be one mechanism through which smoking influences poor prostate cancer outcomes.


Assuntos
Próstata/patologia , Neoplasias da Próstata/patologia , Fumar , Células Estromais/patologia , Homeostase do Telômero/fisiologia , Adulto , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Encurtamento do Telômero/fisiologia
12.
Cancer Causes Control ; 29(8): 759-767, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29980985

RESUMO

PURPOSE: Telomere length at birth sets the baseline for telomere shortening and may influence adult disease risk like cancer. Telomere length is heritable, but may also be a marker of exposures in utero, including those influencing racial differences in risk. We examined racial differences in telomere length in maternal and umbilical cord blood from male neonates, and maternal-neonate correlations to generate hypotheses. METHODS: Black and white pregnant women were recruited in 2006-2007 and followed to postpartum. Data came from questionnaires and medical records. Relative telomere length was measured by qPCR in leukocyte DNA. We estimated mean telomere length in mothers and neonates (n = 55 pairs) using linear regression and maternal-cord blood Spearman correlations, overall and by race. RESULTS: Black mothers had shorter age- and plate-adjusted telomere length (2.49, 95% CI 2.11-2.86) than whites (2.92, 95% CI 2.63-3.22; p = 0.1) and black neonates had shorter telomere length (2.58, 95% CI 2.16-3.01) than whites (3.13, 95% CI 2.79-3.47; p = 0.1), though not statistically significant. Differences were attenuated after further adjustment for maternal factors. Maternal-cord blood correlations were moderate (r = 0.53, p < 0.0001), and did not differ by race. CONCLUSION: Telomere length may differ by race at birth due to both inherited and racial differences in maternal factors. This study was for hypothesis generation and results should be followed up in larger studies.


Assuntos
População Negra , Sangue Fetal/citologia , Telômero , População Branca , População Negra/genética , População Negra/estatística & dados numéricos , Estudos de Coortes , Feminino , Humanos , Gravidez , Telômero/genética , Telômero/fisiologia , População Branca/genética , População Branca/estatística & dados numéricos
13.
J Biol Chem ; 291(18): 9690-9, 2016 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-26953344

RESUMO

ARID1A is a tumor suppressor gene that belongs to the switch/sucrose non-fermentable chromatin remodeling gene family. It is mutated in many types of human cancer with the highest frequency in endometrium-related ovarian and uterine neoplasms including ovarian clear cell, ovarian endometrioid, and uterine endometrioid carcinomas. We have previously reported that mutations in the promoter of human telomerase reverse transcriptase (TERT) rarely co-occur with the loss of ARID1A protein expression, suggesting a potential role of ARID1A in telomere biology. In this study, we demonstrate that ARID1A negatively regulates TERT transcriptional regulation and activity via binding to the regulatory element of TERT and promotes a repressive histone mode. Induction of ARID1A expression was associated with increased occupancy of SIN3A and H3K9me3, known transcription repressor and histone repressor marks, respectively. Thus, loss of ARID1A protein expression caused by inactivating mutations reactivates TERT transcriptional activity and confers a survival advantage of tumor cells by maintaining their telomeres.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Mutação , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Telomerase/biossíntese , Homeostase do Telômero , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Histonas/genética , Histonas/metabolismo , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3 , Telomerase/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética
14.
Prostate ; 77(4): 374-384, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27859428

RESUMO

BACKGROUND: There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. METHODS: A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. RESULTS: Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. CONCLUSIONS: The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic "drivers" for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374-384, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Células Epiteliais/metabolismo , Próstata/citologia , Próstata/metabolismo , Telomerase/biossíntese , Animais , Linhagem Celular Transformada , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Expressão Gênica , Humanos , Masculino , Camundongos , Telomerase/genética
15.
BMC Cancer ; 17(1): 32, 2017 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-28061773

RESUMO

BACKGROUND: The quantitative analysis of microRNA (miRNA) gene expression in archived formalin-fixed, paraffin embedded (FFPE) tissues has been instrumental to identifying their potential roles in cancer biology, diagnosis, and prognosis. However, it remains unclear whether miRNAs remain stable in FFPE tissues stored for long periods of time. METHODS: Here we report Taqman real-time RT-PCR quantification of miR-21, miR-141, miR-221, and RNU6B small nuclear RNA (snRNA) levels from 92 radical prostatectomy specimens stored for 12-20 years in FFPE blocks. The relative stability of each transcript over time was assessed using general linear models. The correlation between transcript quantities, sample age, and RNA integrity number (RIN) were determined utilizing Spearman rank correlation. RESULTS: All transcript levels linearly decreased with sample age, demonstrating a clear loss of miRNA stability and RNU6B snRNA stability over time. The most rapid rates of degradation were observed for RNU6B and miR-21, while miR-141 and miR-221 were more stable. RNA quality was not correlated with sample age or with miR-21, miR-221, or RNU6B snRNA levels. Conversely, miR-141 levels increased with RNA quality. CONCLUSIONS: MiRNA and snRNA levels gradually decreased over an eight year period in FFPE tissue blocks. Sample age was the most consistent feature associated with miRNA stability. The reference snRNA, RUN6B, was more rapidly degraded when compared to miR-141 and miR-221 miRNAs. Various miRNAs demonstrated differential rates of degradation. Quantitative miRNA studies from long-term archived FFPE tissues may therefore benefit from epidemiologic study design or statistical analysis methods that take into account differential storage-dependent transcript degradation.


Assuntos
MicroRNAs/análise , Inclusão em Parafina/métodos , Neoplasias da Próstata/metabolismo , RNA Nuclear Pequeno/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fixação de Tecidos/métodos , Formaldeído , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , RNA Nuclear Pequeno/genética , Fatores de Tempo
16.
Semin Cancer Biol ; 35 Suppl: S5-S24, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25869442

RESUMO

Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology.


Assuntos
Instabilidade Genômica/efeitos dos fármacos , Neoplasias/dietoterapia , Neoplasias/genética , Centrossomo/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Dieta , Instabilidade Genômica/genética , Humanos , Neoplasias/patologia , Prognóstico , Telomerase/antagonistas & inibidores , Telomerase/genética
17.
Proc Natl Acad Sci U S A ; 110(15): 6021-6, 2013 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-23530248

RESUMO

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.


Assuntos
Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Mutação , Telomerase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/metabolismo , Feminino , Predisposição Genética para Doença , Glioma/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Telômero/ultraestrutura , Adulto Jovem
18.
Prostate ; 75(11): 1160-6, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-25893825

RESUMO

BACKGROUND: Telomeres are repetitive nucleoproteins that help maintain chromosomal stability by inhibiting exonucleolytic degradation, prohibiting inappropriate homologous recombination, and preventing chromosomal fusions by suppressing double-strand break signals. We recently observed that men treated for clinically localized prostate cancer with shorter telomeres in their cancer-associated stromal cells, in combination with greater variation in cancer cell telomere lengths, were significantly more likely to progress to distant metastases, and die from their disease. Here, we hypothesized that shorter stromal cell telomere length would be associated with prostate cancer risk at time of biopsy. METHODS: Telomere-specific fluorescence in situ hybridization (FISH) analysis was performed in normal-appearing stromal, basal epithelial, and luminal epithelial cells in biopsies from men randomized to the placebo arm of the Prostate Cancer Prevention Trial. Prostate cancer cases (N = 32) were either detected on a biopsy performed for cause or at the end of the study per trial protocol, and controls (N = 50), defined as negative for cancer on an end-of-study biopsy performed per trial protocol (e.g., irrespective of indication), were sampled. Logistic regression was used to estimate the association between mean telomere length of the particular cell populations, cell-to-cell telomere length variability, and risk of prostate cancer. RESULTS: Men with short stromal cell telomere lengths (below median) had 2.66 (95% CI 1.04-3.06; P = 0.04) times the odds of prostate cancer compared with men who had longer lengths (at or above median). Conversely, we did not observe statistically significant associations for short telomere lengths in normal-appearing basal (OR = 2.15, 95% CI 0.86-5.39; P= 0 .10) or luminal (OR = 1.15, 95% CI 0.47-2.80; P = 0.77) cells. CONCLUSIONS: These findings suggest that telomere shortening in normal stromal cells is associated with prostate cancer risk. It is essential to extend and validate these findings, while also identifying the cellular milieu that comprises the subset of cells with short telomeres within the prostate tumor microenvironment.


Assuntos
Finasterida/administração & dosagem , Próstata/patologia , Neoplasias da Próstata , Células Estromais/patologia , Encurtamento do Telômero , Inibidores de 5-alfa Redutase/administração & dosagem , Biópsia , Instabilidade Cromossômica , Progressão da Doença , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologia , Fatores de Risco , Homeostase do Telômero
19.
BMC Cancer ; 15: 823, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26519084

RESUMO

BACKGROUND: Elevated mammographic density (MD) is a strong breast cancer risk factor but the mechanisms underlying the association are poorly understood. High MD and breast cancer risk may reflect cumulative exposures to factors that promote epithelial cell division. One marker of cellular replicative history is telomere length, but its association with MD is unknown. We investigated the relation of telomere length, a marker of cellular replicative history, with MD and biopsy diagnosis. METHODS: One hundred and ninety-five women, ages 40-65, were clinically referred for image-guided breast biopsies at an academic facility in Vermont. Relative peripheral blood leukocyte telomere length (LTL) was measured using quantitative polymerase chain reaction. MD volume was quantified in cranio-caudal views of the breast contralateral to the primary diagnosis in digital mammograms using a breast density phantom, while MD area (cm(2)) was measured using thresholding software. Associations between log-transformed LTL and continuous MD measurements (volume and area) were evaluated using linear regression models adjusted for age and body mass index. Analyses were stratified by biopsy diagnosis: proliferative (hyperplasia, in-situ or invasive carcinoma) or non-proliferative (benign or other non-proliferative benign diagnoses). RESULTS: Mean relative LTL in women with proliferative disease (n = 141) was 1.6 (SD = 0.9) vs. 1.2 (SD = 0.6) in those with non-proliferative diagnoses (n = 54) (P = 0.002). Mean percent MD volume did not differ by diagnosis (P = 0.69). LTL was not associated with MD in women with proliferative (P = 0.89) or non-proliferative (P = 0.48) diagnoses. However, LTL was associated with a significant increased risk of proliferative diagnosis (adjusted OR = 2.46, 95% CI: 1.47, 4.42). CONCLUSIONS: Our analysis of LTL did not find an association with MD. However, our findings suggest that LTL may be a marker of risk for proliferative pathology among women referred for biopsy based on breast imaging.


Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Leucócitos/metabolismo , Glândulas Mamárias Humanas/anormalidades , Telômero/genética , Adulto , Idoso , Densidade da Mama , Estudos Transversais , Feminino , Humanos , Biópsia Guiada por Imagem , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fatores de Risco
20.
J Neurooncol ; 121(3): 479-87, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25471051

RESUMO

Mutations in isocitrate dehydrogenase 1 (IDH1) have been found in the vast majority of low grade and progressive infiltrating gliomas and are characterized by the production of 2-hydroxyglutarate from α-ketoglutarate. Recent investigations of malignant gliomas have identified additional genetic and chromosomal abnormalities which cluster with IDH1 mutations into two distinct subgroups. The astrocytic subgroup was found to have frequent mutations in ATRX, TP53 and displays alternative lengthening of telomeres. The second subgroup with oligodendrocytic morphology has frequent mutations in CIC or FUBP1, and is linked to co-deletion of the 1p/19q arms. These mutations reflect the development of two distinct molecular pathways representing the majority of IDH1 mutant gliomas. Unfortunately, due to the scarcity of endogenously derived IDH1 mutant models, there is a lack of accurate models to study mechanism and develop new therapy. Here we report the generation of an endogenous IDH1 anaplastic astrocytoma in vivo model with concurrent mutations in TP53, CDKN2A and ATRX. The model has a similar phenotype and histopathology as the original patient tumor, expresses the IDH1 (R132H) mutant protein and exhibits an alternative lengthening of telomeres phenotype. The JHH-273 model is characteristic of anaplastic astrocytoma and represents a valuable tool for investigating the pathogenesis of this distinct molecular subset of gliomas and for preclinical testing of compounds targeting IDH1 mutations or alternative lengthening of telomeres.


Assuntos
Astrocitoma/genética , Astrocitoma/patologia , Isocitrato Desidrogenase/genética , Mutação , Telômero/patologia , Adulto , Animais , DNA Helicases/genética , Modelos Animais de Doenças , Genes p16 , Xenoenxertos , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Camundongos , Transplante de Neoplasias/métodos , Proteínas Nucleares/genética , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genética , Proteína Nuclear Ligada ao X
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