RESUMO
The study of genetic variation has been revolutionized by the advent of high-throughput technologies able to determine the complete genomic sequence of thousands of individuals. Understanding the functional relevance of variants is, however, still a difficult task, especially when focusing on non-coding variants. Most of the variants associated with disease by Genome-Wide Association Studies (GWAS) are indeed non-coding, and presumably exert their effects by altering gene regulation. Expression Quantitative Trait Loci (eQTL) studies represent an important step in understanding the functional relevance of regulatory variants. We propose a new strategy to detect and characterize eQTLs, based on the effect of variants on the Total Binding Affinity (TBA) profiles of regulatory regions. Using a large dataset of coupled genome and expression data, we show that TBA-based inference allows the identification of eQTLs not revealed by traditional methods and helps in their interpretation in terms of altered transcription factor binding.
Assuntos
Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Genômica , HumanosRESUMO
BACKGROUND: Post-transcriptional regulation is a complex mechanism that plays a central role in defining multiple cellular identities starting from a common genome. Modifications in the length of 3'UTRs have been found to play an important role in this context, since alternative 3' UTRs could lead to differences for example in regulation by microRNAs and cellular localization of the transcripts thus altering their fate. RESULTS: We propose a strategy to identify the genes undergoing regulation of 3' UTR length using RNA sequencing data obtained from standard libraries, thus widely applicable to data originally obtained to perform classical differential expression analyses. We decided to exploit previously annotated APA sites from public databases, in contrast with other approaches recently proposed in which the location of the APA site is inferred from the data together with the relative abundance of the isoforms. We demonstrate the reliability of our method by comparing it to the results of other microarray based or specific RNA-seq libraries methods and show that using APA sites databases results in higher sensitivity compared to de novo site prediction approach. CONCLUSIONS: We implemented the algorithm in a Bioconductor package to facilitate its broad usage in the scientific community. The ability of this approach to detect shortening from libraries with a number of reads comparable to that needed for differential expression analyses makes it useful for investigating if alternative polyadenylation is relevant in a certain biological process without requiring specific experimental assays.
Assuntos
Regiões 3' não Traduzidas/genética , Algoritmos , Encéfalo/metabolismo , Neoplasias da Mama/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Poliadenilação/genética , RNA Mensageiro/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Methylthioadenosine phosphorylase (MTAP) is an essential metabolic enzyme in the purine and methionine salvage pathway. In cancer, MTAP gene copy number loss (MTAP loss) confers a selective dependency on the related protein arginine methyltransferase 5. The impact of MTAP alterations in gastrointestinal (GI) cancers remains unknown although hypothetically druggable. Here, we aim to investigate the prevalence, clinicopathological features and prognosis of MTAP loss GI cancers. METHODS: Cases with MTAP alterations were retrieved from The Cancer Genome Atlas (TCGA) and a real-world cohort of GI cancers profiled by next-generation sequencing. If MTAP alterations other than loss were found, immunohistochemistry was performed. Finally, we set a case-control study to assess MTAP loss prognostic impact. RESULTS: Findings across the TCGA dataset (N=1363 patients) and our cohort (N=508) were consistent. Gene loss was the most common MTAP alteration (9.4%), mostly co-occurring with CDKN2A/B loss (97.7%). Biliopancreatic and gastro-oesophageal cancers had the highest prevalence of MTAP loss (20.5% and 12.7%, respectively), being mostly microsatellite stable (99.2%). In colorectal cancer, MTAP loss was rare (1.1%), while most MTAP alterations were mutations (5/7, 71.4%); among the latter, only MTAP-CDKN2B truncation led to protein loss, thus potentially actionable. MTAP loss did not confer worse prognosis. CONCLUSIONS: MTAP alterations are found in 5%-10% of GI cancers, most frequently biliopancreatic and gastro-oesophageal. MTAP loss is the most common alteration, identified almost exclusively in MSS, CDKN2A/B loss, upper-GI cancers. Other MTAP alterations were found in colorectal cancer, but unlikely to cause protein loss and drug susceptibility.
RESUMO
The bacterial genotoxin colibactin promotes colorectal cancer (CRC) tumorigenesis, but systematic assessment of its impact on DNA repair is lacking, and its effect on response to DNA-damaging chemotherapeutics is unknown. We find that CRC cell lines display differential response to colibactin on the basis of homologous recombination (HR) proficiency. Sensitivity to colibactin is induced by inhibition of ATM, which regulates DNA double-strand break repair, and blunted by HR reconstitution. Conversely, CRC cells chronically infected with colibactin develop a tolerant phenotype characterized by restored HR activity. Notably, sensitivity to colibactin correlates with response to irinotecan active metabolite SN38, in both cell lines and patient-derived organoids. Moreover, CRC cells that acquire colibactin tolerance develop cross-resistance to SN38, and a trend toward poorer response to irinotecan is observed in a retrospective cohort of CRCs harboring colibactin genomic island. Our results shed insight into colibactin activity and provide translational evidence on its chemoresistance-promoting role in CRC.
Assuntos
Neoplasias Colorretais , Escherichia coli , Peptídeos , Policetídeos , Humanos , Irinotecano/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Estudos Retrospectivos , DNA/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologiaRESUMO
Multiple strategies are continuously being explored to expand the drug target repertoire in solid tumors. We devised a novel computational workflow for transcriptome-wide gene expression outlier analysis that allows the systematic identification of both overexpression and underexpression events in cancer cells. Here, it was applied to expression values obtained through RNA sequencing in 226 colorectal cancer (CRC) cell lines that were also characterized by whole-exome sequencing and microarray-based DNA methylation profiling. We found cell models displaying an abnormally high or low expression level for 3533 and 965 genes, respectively. Gene expression abnormalities that have been previously associated with clinically relevant features of CRC cell lines were confirmed. Moreover, by integrating multi-omics data, we identified both genetic and epigenetic alternations underlying outlier expression values. Importantly, our atlas of CRC gene expression outliers can guide the discovery of novel drug targets and biomarkers. As a proof of concept, we found that CRC cell lines lacking expression of the MTAP gene are sensitive to treatment with a PRMT5-MTA inhibitor (MRTX1719). Finally, other tumor types may also benefit from this approach.
Assuntos
Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Transcriptoma , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , Transcriptoma/genética , Perfilação da Expressão Gênica , Metilação de DNA/genéticaRESUMO
The emergence of drug resistance is the most substantial challenge to the effectiveness of anticancer therapies. Orthogonal approaches have revealed that a subset of cells, known as drug-tolerant 'persister' (DTP) cells, have a prominent role in drug resistance. Although long recognized in bacterial populations which have acquired resistance to antibiotics, the presence of DTPs in various cancer types has come to light only in the past two decades, yet several aspects of their biology remain enigmatic. Here, we delve into the biological characteristics of DTPs and explore potential strategies for tracking and targeting them. Recent findings suggest that DTPs exhibit remarkable plasticity, being capable of transitioning between different cellular states, resulting in distinct DTP phenotypes within a single tumour. However, defining the biological features of DTPs has been challenging, partly due to the complex interplay between clonal dynamics and tissue-specific factors influencing their phenotype. Moreover, the interactions between DTPs and the tumour microenvironment, including their potential to evade immune surveillance, remain to be discovered. Finally, the mechanisms underlying DTP-derived drug resistance and their correlation with clinical outcomes remain poorly understood. This Roadmap aims to provide a comprehensive overview of the field of DTPs, encompassing past achievements and current endeavours in elucidating their biology. We also discuss the prospect of future advancements in technologies in helping to unveil the features of DTPs and propose novel therapeutic strategies that could lead to their eradication.
RESUMO
Telomere maintenance is necessary to maintain cancer cell unlimited viability. However, the mechanisms maintaining telomere length in colorectal cancer (CRC) have not been extensively investigated. Telomere maintenance mechanisms (TMM) include the re-expression of telomerase or alternative lengthening of telomeres (ALT). ALT is genetically associated with somatic alterations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes. Cells displaying ALT present distinctive features including C-circles made of telomeric DNA, long and heterogenous telomeric tracts, and telomeric DNA co-localized with promyelocytic leukemia (PML) bodies forming so-called ALT-associated PML bodies (APBs). Here, we identified mutations in ATRX and/or DAXX genes in an extensive collection of CRC samples including 119 patient-derived organoids (PDOs) and 232 established CRC cell lines. C-circles measured in CRC PDOs and cell lines showed low levels overall. We also observed that CRC PDOs and cell lines did not display a significant accumulation of APBs or long telomeres with no appreciable differences between wild-type and mutated ATRX/DAXX samples. Overall, our extensive analyses indicate that CRC is not prone to engage ALT, even when carrying genetic lesions in ATRX and/or DAXX, and support the notion that ATRX/DAXX genomic footprints are not reliable predictors of ALT.
Assuntos
Neoplasias Colorretais , Deficiência Intelectual , Telomerase , Talassemia alfa , Humanos , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , Homeostase do Telômero/genética , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Telomerase/genética , Telomerase/metabolismo , Mutação/genética , Linhagem Celular , Telômero/genética , Telômero/metabolismo , Organoides/metabolismo , Neoplasias Colorretais/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMO
BACKGROUND: Colorectal cancer (CRC) remains largely incurable when diagnosed at the metastatic stage. Despite some advances in precision medicine for this disease in recent years, new molecular targets, as well as prognostic/predictive markers, are highly needed. Neuroligin 1 (NLGN1) is a transmembrane protein that interacts at the synapse with the tumor suppressor adenomatous polyposis Coli (APC), which is heavily involved in the pathogenesis of CRC and is a key player in the WNT/ß-catenin pathway. METHODS: After performing expression studies of NLGN1 on human CRC samples, in this paper we used in vitro and in vivo approaches to study CRC cells extravasation and metastasis formation capabilities. At the molecular level, the functional link between APC and NLGN1 in the cancer context was studied. RESULTS: Here we show that NLGN1 is expressed in human colorectal tumors, including clusters of aggressive migrating (budding) single tumor cells and vascular emboli. We found that NLGN1 promotes CRC cells crossing of an endothelial monolayer (i.e. Trans-Endothelial Migration or TEM) in vitro, as well as cell extravasation/lung invasion and differential organ metastatization in two mouse models. Mechanistically, NLGN1 promotes APC localization to the cell membrane and co-immunoprecipitates with some isoforms of this protein stimulates ß-catenin translocation to the nucleus, upregulates mesenchymal markers and WNT target genes and induces an "EMT phenotype" in CRC cell lines CONCLUSIONS: In conclusion, we have uncovered a novel modulator of CRC aggressiveness which impacts on a critical pathogenetic pathway of this disease, and may represent a novel therapeutic target, with the added benefit of carrying over substantial knowledge from the neurobiology field.
Assuntos
Moléculas de Adesão Celular Neuronais , Neoplasias Colorretais , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
PURPOSE: Genomic instability is a hallmark of cancer and targeting DNA damage response (DDR) is emerging as a promising therapeutic strategy in different solid tumors. The effectiveness of targeting DDR in colorectal cancer has not been extensively explored. EXPERIMENTAL DESIGN: We challenged 112 cell models recapitulating the genomic landscape of metastatic colorectal cancer with ATM, ATR, CHK1, WEE1, and DNA-PK inhibitors, in parallel with chemotherapeutic agents. We focused then on ATR inhibitors (ATRi) and, to identify putative biomarkers of response and resistance, we analyzed at multiple levels colorectal cancer models highly sensitive or resistant to these drugs. RESULTS: We found that around 30% of colorectal cancers, including those carrying KRAS and BRAF mutations and unresponsive to targeted agents, are sensitive to at least one DDR inhibitor. By investigating potential biomarkers of response to ATRi, we found that ATRi-sensitive cells displayed reduced phospho-RPA32 foci at basal level, while ATRi-resistant cells showed increased RAD51 foci formation in response to replication stress. Lack of ATM and RAD51C expression was associated with ATRi sensitivity. Analysis of mutational signatures and HRDetect score identified a subgroup of ATRi-sensitive models. Organoids derived from patients with metastatic colorectal cancer recapitulated findings obtained in cell lines. CONCLUSIONS: In conclusion, a subset of colorectal cancers refractory to current therapies could benefit from inhibitors of DDR pathways and replication stress. A composite biomarker involving phospho-RPA32 and RAD51 foci, lack of ATM and RAD51C expression, as well as analysis of mutational signatures could be used to identify colorectal cancers likely to respond to ATRi.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Dano ao DNA , Replicação do DNA , Proteína Quinase Ativada por DNA/genética , Humanos , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The pro-tumourigenic role of epithelial TGFß signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFß signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFß signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFß signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFß signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC's with born to be bad traits.
Assuntos
Apoptose , Fator de Crescimento Transformador beta , Humanos , Apoptose/genéticaRESUMO
In the last decades, thousands of common genetic variants have been associated with human diseases by genome-wide association studies (GWAS). However, the functional interpretation of GWAS hits is usually nontrivial, especially because most of them lay outside the coding genome. These noncoding variants presumably exert their effect by altering gene expression levels; therefore, expression quantitative trait loci (eQTL) mapping analyses represent an important step in understanding their functional relevance and identifying the target genes. Here we describe an alternative strategy for the detection of eQTL that takes into account the combined effect of genetic variants within regulatory regions and leverages the idea that changes in gene expression often are the consequence of the alteration of transcription factor (TF) binding.
Assuntos
Sítios de Ligação , Mapeamento Cromossômico , Locos de Características Quantitativas , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Variação Genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Ligação ProteicaRESUMO
In the last decades, genome-wide association studies (GWAS) have uncovered tens of thousands of associations between common genetic variants and complex diseases. However, these statistical associations can rarely be interpreted functionally and mechanistically. As the majority of the disease-associated variants are located far from coding sequences, even the relevant gene is often unclear. A way to gain insight into the relevant mechanisms is to study the genetic determinants of intermediate molecular phenotypes, such as gene expression and transcript structure. We propose a computational strategy to discover genetic variants affecting the relative expression of alternative 3' untranslated region (UTR) isoforms, generated through alternative polyadenylation, a widespread posttranscriptional regulatory mechanism known to have relevant functional consequences. When applied to a large dataset in which whole genome and RNA sequencing data are available for 373 European individuals, 2,530 genes with alternative polyadenylation quantitative trait loci (apaQTL) were identified. We analyze and discuss possible mechanisms of action of these variants, and we show that they are significantly enriched in GWAS hits, in particular those concerning immune-related and neurological disorders. Our results point to an important role for genetically determined alternative polyadenylation in affecting predisposition to complex diseases, and suggest new ways to extract functional information from GWAS data.