18 research outputs found
Tert hypermethylated oncologic region (THOR) as a biomarker for breast cancer
Breast cancer (BC) is the most frequently diagnosed cancer and a leading cause of
death among women worldwide. Early BC is potentially curable, nevertheless, the
mortality rates still observed among BC patients, demonstrates the urgent need of
novel and more effective diagnostic and therapeutic options.
Limitless self-renewal is a hallmark of cancer governed by telomere maintenance. In
around 95% of BC cases, this process is achieved by telomerase reactivation through
upregulation of human Telomerase Reverse Transcriptase (hTERT). The
hypermethylation of a specific region within hTERT promoter, termed TERT
Hypermethylated Oncological Region (THOR) has been associated with increased
hTERT expression in cancer. However, its biological role and clinical potential in BC
has never been studied. Therefore, we aimed to investigate the role of THOR as a
biomarker, explore the functional impact of THOR in hTERT upregulation, and also
identify other potential DNA methylation-based markers in BC.
Firstly, we demonstrated that THOR is significantly hypermethylated in malignant
breast tissue when compared to benign tissue (40.23% vs. 12.81%), representing a
potential candidate biomarker for future application in BC screening and early
diagnosis. Importantly, as DNA methylation marks can be determined from blood
samples, assessing THOR methylation status may constitute a non-invasive assay to
help in BC management.
Next, using a reporter assay, we revealed that THOR acts as a repressive regulatory
element of hTERT, and that THOR hypermethylation might be relevant for hTERT
upregulation in BC. To further investigate its biological impact on hTERT transcription,
targeted THOR demethylation was performed using the CRISPR-dCas9 system.
Although, THOR demethylation was achieved, hTERT mRNA levels were not
significantly reduced. Surprisingly, cells previously demethylated on THOR region led
to a remarkable reduction in tumor development in vivo. Therefore, additional studies
are required to validate these observations and to unravel the causality between
THOR hypermethylation and hTERT upregulation in BC.
Finally, through a genome-wide methylation analysis, we identified three novel DNA
methylation markers, located on the PRAC2, TDRD10 and TMEM132C genes that
showed diagnostic and prognostic value in BC, as well as in other cancer types. This work evidences the importance of DNA methylation in breast tumorigenesis and, more importantly, their clinical value as promising diagnostic, prognostic and therapeutic targets in BC.O cancro da mama (CM) é o tumor maligno mais frequentemente diagnosticado, e
uma das principais causas de morte entre as mulheres, em todo o mundo. Em 2018,
cerca de 2,1 milhões de novos casos foram diagnosticados e 626.679 pessoas
morreram por CM, em todo o mundo. No geral, o CM é o segundo tipo de cancro mais
comum, a seguir ao cancro do pulmão, sendo considerada a quinta causa mais
comum de morte devido a cancro.
O CM consiste numa doença altamente heterogénea, que compreende diversos
subtipos histológicos e moleculares, os quais diferem em termos de resposta à
terapêutica e prognóstico da doença. A deteção precoce e os avanços no tratamento
conduziram a uma melhoria significativa na sobrevivência e qualidade de vida dos
pacientes, no entanto, o CM ainda está entre as principais causas de morte por
cancro. Atualmente, quando detetado numa fase inicial da doença, é potencialmente
curável mas as taxas de mortalidade ainda observadas entre os pacientes,
demonstram a necessidade urgente de novas e mais eficazes opções de diagnóstico
e terapêutica.
A capacidade de autorrenovação ilimitada das células cancerígenas é uma
característica fundamental do cancro, sendo alcançada pela manutenção dos
telómeros. Em cerca de 95% dos casos de CM, este processo depende da reativação
da enzima telomerase. A telomerase consiste num complexo ribonucleoproteico,
constituído por uma subunidade catalítica, a transcriptase reversa da telomerase
humana (hTERT) e uma subunidade de RNA, conhecida como componente de RNA
da telomerase humana (hTERC). Tem sido proposto que a atividade da telomerase é
determinada principalmente pela re-expressão da sua subunidade catalítica, a hTERT
e, visto que, quer a atividade da telomerase, como a expressão da hTERT, estão
aumentadas na maioria dos tumores malignos da mama, e ausentes na maioria dos
tecidos somáticos normais, a telomerase/hTERT, bem como os seus mecanismos
regulatórios, são considerados potenciais biomarcadores de cancro com implicações
relevantes na prática clínica.
Até à data, diversos mecanismos genéticos e epigenéticos demonstraram ser
responsáveis pela regulação da hTERT, no entanto, a complexidade por trás da sua
regulação no cancro não é ainda totalmente compreendida. Um dos mecanismos
associados à expressão da hTERT no cancro é a hipermetilação de uma região específica do seu promotor, denominada como Região Oncológica Hipermetilada da
hTERT (em inglês, THOR). Esta região demonstrou estar associada à progressão
tumoral e à sobrevivência dos pacientes em diversos tipos de cancro, tais como,
cancro da próstata, bexiga e pâncreas. No entanto, o seu papel biológico na ativação
da transcrição da hTERT, bem como o seu potencial clínico no CM não foi ainda
investigado.
Deste modo, o principal objetivo do presente estudo, foi investigar o papel do THOR
como biomarcador de doença em CM, bem como explorar o mecanismo pelo qual a
hipermetilação do THOR contribui para a regulação positiva da hTERT.
Adicionalmente, uma vez que vários estudos têm demonstrado que a metilação do
DNA desempenha um papel relevante na patogénese do CM, pretendeu-se também
identificar novos potenciais biomarcadores de diagnóstico e/ou prognóstico baseados
em alterações nos padrões de metilação do DNA.
Inicialmente, para avaliar o potencial do THOR como biomarcador clínico em CM, o
seu nível de metilação foi analisado na coorte de carcinoma invasivo da mama
disponível na plataforma de acesso público, The Cancer Genome Atlas (TCGA).
Posteriormente, confirmaram-se os resultados utilizando duas coortes compostas por
amostras de tecido de carcinoma invasivo de mama provenientes de mulheres
diagnosticadas com CM no Centro Hospitalar Universitário do Algarve (CHUAlgarve,
Faro, Portugal). Todas as coortes (TCGA e CHUAlgarve) revelaram que o THOR está
significativamente hipermetilado no tecido mamário maligno quando comparado com
o tecido benigno. A análise de metilação do THOR permitiu diferenciar cancro de
tecido normal a partir do estádio mais inicial da doença (AUC> 0,9574, p <0,0001),
evidenciando assim o seu potencial como biomarcador para deteção precoce do CM.
Para além disso, quando comparado com os biomarcadores séricos, CEA e CA 15-3,
o estado da metilação do THOR demonstrou ser mais representativo do estado atual
do tumor do que os biomarcadores acima mencionados e, portanto, poderia ser usado
no futuro como uma ferramenta valiosa para acompanhamento dos pacientes com
CM. É importante ressaltar que, apesar de no presente estudo a hipermetilação do
THOR ter sido determinada em amostras de tecido, esta pode constituir a base para
o desenvolvimento de um ensaio não invasivo, e assim, melhorar a prática clínica. Os
resultados obtidos revelam também que os pacientes com o THOR hipermetilado
apresentam níveis mais elevados de expressão da hTERT, sugerindo assim que a metilação do THOR atua como um mecanismo de regulação positiva da ativação
transcricional da hTERT.
Neste sentido, de forma a investigar o papel funcional da hipermetilação do THOR na
regulação génica da hTERT, foram realizados ensaios repórter de luciferase e a
desmetilação direcionada do THOR com recurso ao sistema CRISPR-dCas9 em
linhas celulares de CM. Os ensaios repórter revelaram que a região THOR atua como
um elemento regulador repressivo da hTERT e que a sua hipermetilação pode ser
relevante para a regulação positiva da hTERT no CM. Assim, para testar essa
hipótese, utilizou-se o sistema CRISPR-dCas9 fundido com a enzima TET1
desmetilase, para avaliar se a desmetilação direcionada do THOR poderia reverter a
regulação positiva de hTERT. Os resultados obtidos revelaram que a desmetilação
específica da região THOR, foi conseguida, tendo-se obtido uma redução nos níveis
de metilação de 15 a 70%, em determinadas CpGs. No entanto, apesar de se ter
observado uma redução significativa na metilação do THOR, os níveis de mRNA da
hTERT não foram significativamente reduzidos. Portanto, o presente estudo não nos
permite estabelecer um efeito de causalidade entre a desmetilação do THOR e a
inativação da transcrição da hTERT, sendo necessários estudos adicionais para
desvendar esta hipótese. Porém, surpreendentemente, as células previamente
desmetiladas na região THOR conduziram a uma notável redução no
desenvolvimento de tumors in vivo, sendo essencial no futuro validar e determinar a
razão biológica para estas observações.
Por último, pretendeu-se destacar a importância das alterações epigenéticas na
patogénese do CM e identificar novos biomarcadores de CM baseados na metilação
do DNA. Para tal, realizou-se uma análise genómica dos padrões de metilação do
DNA em CM e, avaliou-se o seu impacto na expressão génica. Estas análises foram
efetuadas com recurso aos dados de metilação do DNA e de expressão génica,
disponíveis nas bases de dados TCGA e METABRIC. De acordo com os resultados
obtidos, foram identificados sete novos genes associados ao CM. Destes, os genes,
PRAC2, TDRD10 e TMEM132C, apresentaram marcas de metilação do DNA com
valor diagnóstico e prognóstico. Estes três novos biomarcadores baseados na
metilação do DNA foram analisados em outras coortes de cancro disponíveis no
TCGA e demonstraram ter também potencial diagnóstico e prognóstico noutros tipos
de cancro. Em suma, o presente trabalho evidencia a importância dos padrões de metilação do
DNA na patogénese do cancro da mama e destaca o seu potencial valor clínico.
Particularmente, a hipermetilação do THOR é proposta como um alvo de diagnóstico
e de terapêutica promissor para o CM
Roadmap of DNA methylation in breast cancer identifies novel prognostic biomarkers
Background
Breast cancer is a highly heterogeneous disease resulting in diverse clinical behaviours and therapeutic responses. DNA methylation is a major epigenetic alteration that is commonly perturbed in cancers. The aim of this study is to characterize the relationship between DNA methylation and aberrant gene expression in breast cancer.
Methods
We analysed DNA methylation and gene expression profiles from breast cancer tissue and matched normal tissue in The Cancer Genome Atlas (TCGA). Genome-wide differential methylation analysis and methylation-gene expression correlation was performed. Gene expression changes were subsequently validated in the METABRIC dataset. The Oncoscore tool was used to identify genes that had previously been associated with cancer in the literature. A subset of genes that had not previously been studied in cancer was chosen for further analysis.
Results
We identified 368 CpGs that were differentially methylated between tumor and normal breast tissue (∆β > 0.4). Hypermethylated CpGs were overrepresented in tumor tissue and were found predominantly (56%) in upstream promoter regions. Conversely, hypomethylated CpG sites were found primarily in the gene body (66%). Expression analysis revealed that 209 of the differentially-methylated CpGs were located in 169 genes that were differently expressed between normal and breast tumor tissue. Methylation-expression correlations were predominantly negative (70%) for promoter CpG sites and positive (74%) for gene body CpG sites. Among these differentially-methylated and differentially-expressed genes, we identified 7 that had not previously been studied in any form of cancer. Three of these, TDRD10, PRAC2 and TMEM132C, contained CpG sites that showed diagnostic and prognostic value in breast cancer, particularly in estrogen-receptor (ER)-positive samples. A pan-cancer analysis confirmed differential expression of these genes together with diagnostic and prognostic value of their respective CpG sites in multiple cancer types.
Conclusion
We have identified 368 DNA methylation changes that characterize breast cancer tumor tissue, of which 209 are associated with genes that are differentially-expressed in the same samples. Novel DNA methylation markers were identified, of which cg12374721 (PRAC2), cg18081940 (TDRD10) and cg04475027 (TMEM132C) show promise as diagnostic and prognostic markers in breast cancer as well as other cancer types.info:eu-repo/semantics/publishedVersio
THOR is a targetable epigenetic biomarker with clinical implications in breast cancer
Breast cancer (BC) is the most frequently diagnosed cancer and a leading cause of death among
women worldwide. Early BC is potentially curable, but the mortality rates still observed among BC patients demon‑
strate the urgent need of novel and more efective diagnostic and therapeutic options. Limitless self-renewal is a
hallmark of cancer, governed by telomere maintenance. In around 95% of BC cases, this process is achieved by telom‑
erase reactivation through upregulation of the human telomerase reverse transcriptase (hTERT). The hypermethylation
of a specifc region within the hTERT promoter, termed TERT hypermethylated oncological region (THOR) has been
associated with increased hTERT expression in cancer. However, its biological role and clinical potential in BC have
never been studied to the best of our knowledge. Therefore, we aimed to investigate the role of THOR as a biomarker
and explore the functional impact of THOR methylation status in hTERT upregulation in BC.info:eu-repo/semantics/publishedVersio
Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer
Background
Limitless self-renewal is one of the hallmarks of cancer and is attained by telomere maintenance, essentially through telomerase (hTERT) activation. Transcriptional regulation of hTERT is believed to play a major role in telomerase activation in human cancers.
Main body
The dominant interest in telomerase results from its role in cancer. The role of telomeres and telomere maintenance mechanisms is well established as a major driving force in generating chromosomal and genomic instability. Cancer cells have acquired the ability to overcome their fate of senescence via telomere length maintenance mechanisms, mainly by telomerase activation.
hTERT expression is up-regulated in tumors via multiple genetic and epigenetic mechanisms including hTERT amplifications, hTERT structural variants, hTERT promoter mutations and epigenetic modifications through hTERT promoter methylation. Genetic (hTERT promoter mutations) and epigenetic (hTERT promoter methylation and miRNAs) events were shown to have clinical implications in cancers that depend on hTERT activation. Knowing that telomeres are crucial for cellular self-renewal, the mechanisms responsible for telomere maintenance have a crucial role in cancer diseases and might be important oncological biomarkers. Thus, rather than quantifying TERT expression and its correlation with telomerase activation, the discovery and the assessment of the mechanisms responsible for TERT upregulation offers important information that may be used for diagnosis, prognosis, and treatment monitoring in oncology. Furthermore, a better understanding of these mechanisms may promote their translation into effective targeted cancer therapies.
Conclusion
Herein, we reviewed the underlying mechanisms of hTERT regulation, their role in oncogenesis, and the potential clinical applications in telomerase-dependent cancers.info:eu-repo/semantics/publishedVersio
Tert Hypermethylated Oncologic Region (THOR) como um biomarcador para cancro
O cancro é uma das principais causas de morte relacionada com doença, sendo responsável por cerca de 14 milhões de novos casos e 8,2 milhões de mortes em todo o mundo. Os tipos de cancro mais comuns são o cancro do pulmão, mama, colorretal e da próstata, sendo o cancro do pulmão, colorretal e da mama os mais mortais. Embora cada tipo de cancro apresente alterações únicas que são adquiridas durante a carcinogénese, biomarcadores universais de malignidade e métodos para estabelecer a progressão da doença em diferentes neoplasias não existem e continuam a ser um grande desafio em oncologia clínica. Uma característica do cancro é a manutenção dos telómeros, a qual é crucial para a autorrenovação de todos os tumores malignos. A ativação da telomerase ocorre através da expressão da transcriptase reversa humana (hTERT) e tem sido relatado que a sua expressão aumenta marcadamente na invasão tumoral. O mecanismo de regulação da hTERT não está completamente elucidado; no entanto, tem sido relatado que a hipermetilação de ilhas CpG apresenta um papel essencial na expressão da hTERT em células cancerígenas telomerase-positivas. O nosso grupo recentemente identificou uma região específica no promotor da hTERT (denominada THOR) que está hipermetilada e associada com a ativação da telomerase em tecido cancerígeno. THOR foi capaz de prever a progressão do tumor e evolução clínica do paciente em diversos tumores pediátricos e adultos. Objetivo do estudo - Pretendemos investigar se a metilação do THOR pode ser um biomarcador de doença maligna e de evolução clínica do paciente em diferentes cancros adultos
Epigenetic alterations in urothelial bladder cancer associated with disease outcomes
Objectives
Bladder cancer (BLCA) is a molecular heterogeneous disease with known genetic distinctive signatures. However, DNA methylation is highly prevalent across a wide range of tumors, suggesting its potential in oncogenesis. Here, we aimed to interrogate the role of nine epigenetic alterations as diagnostic and prognostic markers in BLCA.
Methods
DNA methylation, gene expression, and clinicopathological information were retrieved from The Cancer Genome Atlas data portal. Methylation values and gene expression were assessed to determine their association with normal and malignant tissue. Additionally, we studied the association between methylation values and clinicopathological variables. For the prognostic model, Kaplan–Meier Survival curves were generated. Lastly, univariate and multivariate analysis were performed to evaluate the simultaneous impact of methylation and clinicopathological variables on the risk of tumor progression and survival.
Results
Nine CpG sites' methylation
-values involved in our study demonstrated different methylation signatures between normal and malignant urothelium. Hypermethylated CpGs were overrepresented in tumor tissue (p < 0.0001). Opposingly, 4 CpG sites showed lower methylation values in tumor samples (p < 0.0001). Cg12743248high and cg17192862low are risk factors for progression-free survival, whereas cg12374721high (HR:3.003 (1.283–7.030)) also demonstrated to be the most valuable independent risk factor for disease progression and a risk factor for overall survival.
Conclusions
We have identified that methylated cg12374721 shows promise as a diagnostic and independent prognostic marker in BLCA progression.F31D-D663-4EF2 | Anabela Mota Pintoinfo:eu-repo/semantics/publishedVersio
Epigenetic alterations in urothelial bladder cancer associated with disease outcomes
Objectives: Bladder cancer (BLCA) is a molecular heterogeneous disease with known genetic distinctive signatures. However, DNA methylation is highly prevalent across a wide range of tumors, suggesting its potential in oncogenesis. Here, we aimed to interrogate the role of nine epigenetic alterations as diagnostic and prognostic markers in BLCA.Methods: DNA methylation, gene expression, and clinicopathological information were retrieved from The Cancer Genome Atlas data portal. Methylation values and gene expression were assessed to determine their association with normal and malignant tissue. Additionally, we studied the association between methylation values and clinicopathological variables. For the prognostic model, Kaplan-Meier Survival curves were generated. Lastly, univariate and multivariate analysis were performed to evaluate the simultaneous impact of methylation and clinicopathological variables on the risk of tumor progression and survival.Results: Nine CpG sites' methylation beta-values involved in our study demonstrated different methylation signatures between normal and malignant urothelium. Hypermethylated CpGs were overrepresented in tumor tissue (p < 0.0001). Opposingly, 4 CpG sites showed lower methylation values in tumor samples (p < 0.0001). Cg12743248high and cg17192862low are risk factors for progression-free survival, whereas cg12374721high (HR:3.003 (1.283-7.030)) also demonstrated to be the most valuable independent risk factor for disease progression and a risk factor for overall survival.Conclusions: We have identified that methylated cg12374721 shows promise as a diagnostic and independent prognostic marker in BLCA progression.info:eu-repo/semantics/publishedVersio
Measuring healthy ageing: current and future tools
Human ageing is a complex, multifactorial process characterised by physiological damage, increased risk of age-related diseases and inevitable functional deterioration. As the population of the world grows older, placing significant strain on social and healthcare resources, there is a growing need to identify reliable and easy-to-employ markers of healthy ageing for early detection of ageing trajectories and disease risk. Such markers would allow for the targeted implementation of strategies or treatments that can lessen suffering, disability, and dependence in old age. In this review, we summarise the healthy ageing scores reported in the literature, with a focus on the past 5 years, and compare and contrast the variables employed. The use of approaches to determine biological age, molecular biomarkers, ageing trajectories, and multi-omics ageing scores are reviewed. We conclude that the ideal healthy ageing score is multisystemic and able to encompass all of the potential alterations associated with ageing. It should also be longitudinal and able to accurately predict ageing complications at an early stage in order to maximize the chances of successful early intervention.ALG-010145-FEDER-072586info:eu-repo/semantics/publishedVersio
Hot topics in epigenetic regulation of cancer self-renewal for pancreatic tumors: future trends
Self-renewal is critical in order for a cancer to proliferate, grow and eventually metastasize. Today, several molecular mechanisms are being studied, which may explain the emergence of this property in advanced cancer. Pancreatic cancer is both a deadly and highly recurrent tumor, relying heavily on accelerated proliferation. Understanding self-renewal is this tumor type is therefore likely to prove essential for reaching better disease control. Recent advances in our understanding of telomere maintenance pathways are beginning to yield those insights. Indeed, it is now known that telomerase reactivation in cancer is intimately related with expression of TERT gene. The multimodal regulation of this locus, along with transcriptional output itself, both serve as prognostic factors across a variety of tumor types
Hot topics in epigenetic regulation of cancer self-renewal for pancreatic tumors: future trends
Self-renewal is critical in order for a cancer to proliferate, grow and eventually metastasize. Today, several molecular mechanisms are being studied, which may explain the emergence of this property in advanced cancer. Pancreatic cancer is both a deadly and highly recurrent tumor, relying heavily on accelerated proliferation. Understanding self-renewal is this tumor type is therefore likely to prove essential for reaching better disease control. Recent advances in our understanding of telomere maintenance pathways are beginning to yield those insights. Indeed, it is now known that telomerase reactivation in cancer is intimately related with expression of TERT gene. The multimodal regulation of this locus, along with transcriptional output itself, both serve as prognostic factors across a variety of tumor types