Immunopeptidomics-based identification of naturally presented non-canonical circRNA-derived peptides.

Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5' cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs. We increase the analytical stringency of our workflow by retaining peptides identified independently by two mass spectrometry search engines and/or by applying a group-specific FDR for canonical-derived and circRNA-derived peptides. A subset of circRNA-derived peptides specifically encoded by the region spanning the back-splice junction (BSJ) are validated with targeted MS, and with direct Sanger sequencing of the respective source transcripts. Our workflow identifies 54 unique BSJ-spanning circRNA-derived peptides in the immunopeptidome of melanoma and lung cancer samples. Our approach enlarges the catalog of source proteins that can be explored for immunotherapy.

Alveolar macrophages in early stage COPD show functional deviations with properties of impaired immune activation.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-ß1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation.

Adequacy of Severe Malaria Markers and Prognostic Scores in an Intensive Care Unit in Luanda, Angola: A Clinical Study.

Severe Plasmodium falciparum malaria remains the primary cause of mortality in several African countries, including Angola, where severe malaria patient admission into intensive care units (ICU) is mandatory. The present observational and prospective study enrolled 101 consecutive severe malaria patients admitted at the ICU of Américo Boavida University Hospital (Luanda, Angola). Malaria was confirmed by microscopy and RDT, and WHO criteria were used to define severe malaria. The Sequential Organ Failure Assessment (SOFA) score was used to monitor organ dysfunctions. Surviving and nonsurviving patients were compared using bivariate statistical methods. Two-step cluster analysis was used to find discriminant organ dysfunctions that may correlate better with the observed mortality (16.8%), which was much lower than the one generated by the SOFA score. The study population was young, and 87% of the patients were local native residents. There was no statistically significant correlation between the parasitemia and the outcome. Hematological and cerebral dysfunctions were prevalent but were not discriminant when cluster analyses were performed to detect homogeneous subgroups of patients. In conclusion, the SOFA score was readily applicable and efficient in monitoring daily organ dysfunction but was not effective enough in predicting the outcome of severe malaria patients.

Contributo da enfermagem de reabilitação na avaliação da qualidade de vida da pessoa idosa com deglutição comprometida / Contribution of rehabilitation nursing to the assessment of the quality of life of elderly people with impaired swallowing

Portugal atualmente apresenta uma população envelhecida, com mais idosos do que jovens, sendo que 21% de portugueses apresentam 65 ou mais anos. O envelhecimento humano, implica mudanças estruturais, funcionais e neuronais, com comprometimento de órgãos e funções, nomeadamente a deglutição, exigindo a adequação de padrões anteriormente adquiridos para não comprometer a qualidade de vida. Este relatório pretende descrever e avaliar a efetividade das intervenções de enfermagem de reabilitação na qualidade de vida da pessoa idosa com AVC e com disfagia, numa Unidade de Cuidados Prolongados. A população em estudo foi composta por trinta pessoas e os instrumentos de avaliação utilizados foram o Questionário SWAL-QOL e o Questionário SWAL-CARE, de modo a avaliar a satisfação da pessoa relativamente aos cuidados específicos realizados pelo enfermeiro de reabilitação. Após a implementação dos programas individualizados de Enfermagem de Reabilitação, aferimos que os resultados obtidos refletem uma melhoria significativa da qualidade de vida da população em estudo e da satisfação da pessoa face aos cuidados especializados.

Portugal currently has an aging population, with more elderly than young people, with 21% of Portuguese being 65 or older. Human aging implies structural, functional, and neuronal changes, with impairment of organs and functions, namely swallowing, requiring the adaptation of previously acquired standards so as not to compromise quality of life. This report intends to describe and evaluate the effectiveness of rehabilitation nursing interventions on the quality of life of the elderly with stroke and dysphagia, in an Extended Care Unit. The study population consisted of thirty people and the assessment instruments used were the SWAL-QOL Questionnaire and the SWAL-CARE Questionnaire, in order to assess the person's satisfaction with the specific care provided by the rehabilitation nurse. After the implementation of individualized Rehabilitation Nursing programs, we found that the results obtained reflect a significant improvement in the quality of life of the study population and in the person's satisfaction with specialized care.

Epigenetic silencing of TGFBI confers resistance to trastuzumab in human breast cancer.

BACKGROUND: Acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients. METHODS: We performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing. RESULTS: Epigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients. CONCLUSIONS: Our results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients.

Epigenetic loss of the endoplasmic reticulum-associated degradation inhibitor SVIP induces cancer cell metabolic reprogramming.

The endoplasmic reticulum (ER) of cancer cells needs to adapt to the enhanced proteotoxic stress associated with the accumulation of unfolded, misfolded and transformation-associated proteins. One way by which tumors thrive in the context of ER stress is by promoting ER-Associated Degradation (ERAD), although the mechanisms are poorly understood. Here, we show that the Small p97/VCP Interacting Protein (SVIP), an endogenous inhibitor of ERAD, undergoes DNA hypermethylation-associated silencing in tumorigenesis to achieve this goal. SVIP exhibits tumor suppressor features and its recovery is associated with increased ER stress and growth inhibition. Proteomic and metabolomic analyses show that cancer cells with epigenetic loss of SVIP are depleted in mitochondrial enzymes and oxidative respiration activity. This phenotype is reverted upon SVIP restoration. The dependence of SVIP hypermethylated cancer cells on aerobic glycolysis and glucose was also associated with sensitivity to an inhibitor of the glucose transporter GLUT1. This could be relevant to the management of tumors carrying SVIP epigenetic loss, because these occur in high-risk patients who manifest poor clinical outcomes. Overall, our study provides insights into how epigenetics helps deal with ER stress and how SVIP epigenetic loss in cancer may be amenable to therapies that target glucose transporters.

Discovery of a Potent, Long-Acting, and CNS-Active Inhibitor (BIA 10-2474) of Fatty Acid Amide Hydrolase.

Fatty acid amide hydrolase (FAAH) can be targeted for the treatment of pain associated with various medical conditions. Herein we report the design and synthesis of a novel series of heterocyclic-N-carboxamide FAAH inhibitors that have a good alignment of potency, metabolic stability and selectivity for FAAH over monoacylglycerol lipase (MAGL) and carboxylesterases (CEs). Lead optimization efforts carried out with benzotriazolyl- and imidazolyl-N-carboxamide series led to the discovery of clinical candidate 8 l (3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide; BIA 10-2474) as a potent and long-acting inhibitor of FAAH. However, during a Phase I clinical trial with compound 8 l, unexpected and unpredictable serious neurological adverse events occurred, affecting five healthy volunteers, including the death of one subject.

Circular RNA CpG island hypermethylation-associated silencing in human cancer.

Noncoding RNAs (ncRNAs), such as microRNAs and long noncoding RNAs (lncRNAs), participate in cellular transformation. Work done in the last decade has also demonstrated that ncRNAs with growth-inhibitory functions can undergo promoter CpG island hypermethylation-associated silencing in tumorigenesis. Herein, we wondered whether circular RNAs (circRNAs), a type of RNA transcripts lacking 5'-3' ends and forming closed loops that are gaining relevance in cancer biology, are also a target of epigenetic inactivation in tumors. To tackle this issue, we have used cancer cells genetically deficient for the DNA methyltransferase enzymes in conjuction with circRNA expression microarrays. We have found that the loss of DNA methylation provokes a release of circRNA silencing. In particular, we have identified that promoter CpG island hypermethylation of the genes TUSC3 (tumor suppressor candidate 3), POMT1 (protein O-mannosyltransferase 1), ATRNL1 (attractin-like 1) and SAMD4A (sterile alpha motif domain containing 4A) is linked to the transcriptional downregulation of both linear mRNA and the hosted circRNA. Although some circRNAs regulate the linear transcript, we did not observe changes in TUSC3 mRNA levels upon TUSC3 circ104557 overexpression. Interestingly, we found circRNA-mediated regulation of target miRNAs and an in vivo growth inhibitory effect upon TUSC3 circ104557 transduction. Data mining for 5'-end CpG island methylation of TUSC3, ATRNL1, POMT1 and SAMD4A in cancer cell lines and primary tumors showed that the epigenetic defect was commonly observed among different tumor types in association with the diminished expression of the corresponding transcript. Our findings support a role for circRNA DNA methylation-associated loss in human cancer.

CpG Islands in Cancer: Heads, Tails, and Sides.

DNA methylation is a dynamic epigenetic mark that characterizes different cellular developmental stages, including tissue-specific profiles. This CpG dinucleotide modification cooperates in the regulation of the output of the cellular genetic content, in both healthy and pathological conditions. According to endogenous and exogenous stimuli, DNA methylation is involved in gene transcription, alternative splicing, imprinting, X-chromosome inactivation, and control of transposable elements. When these dinucleotides are organized in dense regions are called CpG islands (CGIs), being commonly known as transcriptional regulatory regions frequently associated with the promoter region of several genes. In cancer, promoter DNA hypermethylation events sustained the mechanistic hypothesis of epigenetic transcriptional silencing of an increasing number of tumor suppressor genes. CGI hypomethylation-mediated reactivation of oncogenes was also documented in several cancer types. In this chapter, we aim to summarize the functional consequences of the differential DNA methylation at CpG dinucleotides in cancer, focused in CGIs. Interestingly, cancer methylome is being recently explored, looking for biomarkers for diagnosis, prognosis, and predictors of drug response.

Non-coding RNAs, epigenetics, and cancer: tying it all together.

While only a small part of the human genome encodes for proteins, biological functions for the so-called junk genome are increasingly being recognized through high-throughput technologies and mechanistic experimental studies. Indeed, novel mechanisms of gene regulation are being discovered that require coordinated interaction between DNA, RNA, and proteins. Therefore, interdisciplinary efforts are still needed to decipher these complex transcriptional networks. In this review, we discuss how non-coding RNAs (ncRNAs) are epigenetically regulated in cancer and metastases and consequently how ncRNAs participate in the sculpting of the epigenetic profile of a cancer cell, thus modulating the expression of other RNA molecules. In the latter case, ncRNAs not only affect the DNA methylation status of certain genomic loci but also interact with histone-modifying complexes, changing the structure of the chromatin itself. We present several examples of epigenetic changes causing aberrant expression of ncRNAs in the context of tumor progression. Interestingly, there are also important epigenetic changes and transcriptional regulatory effects derived from their aberrant expression. As ncRNAs can also be used as biomarkers for diagnosis and prognosis or explored as potential targets, we present insights into the use of ncRNAs for targeted cancer therapy.

Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer.

BACKGROUND: One of the hallmarks of cancer is the disruption of gene expression patterns. Many molecular lesions contribute to this phenotype, and the importance of aberrant DNA methylation profiles is increasingly recognized. Much of the research effort in this area has examined proximal promoter regions and epigenetic alterations at other loci are not well characterized. RESULTS: Using whole genome bisulfite sequencing to examine uncharted regions of the epigenome, we identify a type of far-reaching DNA methylation alteration in cancer cells of the distal regulatory sequences described as super-enhancers. Human tumors undergo a shift in super-enhancer DNA methylation profiles that is associated with the transcriptional silencing or the overactivation of the corresponding target genes. Intriguingly, we observe locally active fractions of super-enhancers detectable through hypomethylated regions that suggest spatial variability within the large enhancer clusters. Functionally, the DNA methylomes obtained suggest that transcription factors contribute to this local activity of super-enhancers and that trans-acting factors modulate DNA methylation profiles with impact on transforming processes during carcinogenesis. CONCLUSIONS: We develop an extensive catalogue of human DNA methylomes at base resolution to better understand the regulatory functions of DNA methylation beyond those of proximal promoter gene regions. CpG methylation status in normal cells points to locally active regulatory sites at super-enhancers, which are targeted by specific aberrant DNA methylation events in cancer, with putative effects on the expression of downstream genes.

BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma.

Type II germ cell cancers (GCC) can be subdivided into seminomas and non-seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as seminomas with a non-seminoma component, raising the question if a CIS gives rise to seminomas and ECs at the same time or whether seminomas can be reprogrammed to ECs. In this study, we utilized the seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (GDF3, NODAL, DNMT3B, DPPA3, GAL, AK3L1) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state.

Epigenetic activation of a cryptic TBC1D16 transcript enhances melanoma progression by targeting EGFR.

Metastasis is responsible for most cancer-related deaths, and, among common tumor types, melanoma is one with great potential to metastasize. Here we study the contribution of epigenetic changes to the dissemination process by analyzing the changes that occur at the DNA methylation level between primary cancer cells and metastases. We found a hypomethylation event that reactivates a cryptic transcript of the Rab GTPase activating protein TBC1D16 (TBC1D16-47 kDa; referred to hereafter as TBC1D16-47KD) to be a characteristic feature of the metastatic cascade. This short isoform of TBC1D16 exacerbates melanoma growth and metastasis both in vitro and in vivo. By combining immunoprecipitation and mass spectrometry, we identified RAB5C as a new TBC1D16 target and showed that it regulates EGFR in melanoma cells. We also found that epigenetic reactivation of TBC1D16-47KD is associated with poor clinical outcome in melanoma, while conferring greater sensitivity to BRAF and MEK inhibitors.

A comprehensive DNA methylation profile of epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) is a plastic process in which fully differentiated epithelial cells are converted into poorly differentiated, migratory and invasive mesenchymal cells, and it has been related to the metastasis potential of tumors. This is a reversible process and cells can also eventually undergo mesenchymal-to-epithelial transition. The existence of a dynamic EMT process suggests the involvement of epigenetic shifts in the phenotype. Herein, we obtained the DNA methylomes at single-base resolution of Madin-Darby canine kidney cells undergoing EMT and translated the identified differentially methylated regions to human breast cancer cells undergoing a gain of migratory and invasive capabilities associated with the EMT phenotype. We noticed dynamic and reversible changes of DNA methylation, both on promoter sequences and gene-bodies in association with transcription regulation of EMT-related genes. Most importantly, the identified DNA methylation markers of EMT were present in primary mammary tumors in association with the epithelial or the mesenchymal phenotype of the studied breast cancer samples.

Epigenetic loss of the PIWI/piRNA machinery in human testicular tumorigenesis.

Although most cancer research has focused in mRNA, non-coding RNAs are also an essential player in tumorigenesis. In addition to the well-recognized microRNAs, recent studies have also shown that epigenetic silencing by CpG island hypermethylation of other classes of non-coding RNAs, such as transcribed ultraconserved regions (T-UCRs) or small nucleolar RNAs (snoRNAs), also occur in human neoplasia. Herein we have studied the putative existence of epigenetic aberrations in the activity of PIWI proteins, an Argonaute family protein subclass, and the small regulatory PIWI-interacting RNAs (piRNAs) in testicular cancer, as the PIWI/piRNA pathway plays a critical role in male germline development. We have observed the existence of promoter CpG island hypermethylation-associated silencing of PIWIL1, PIWIL2, PIWIL4, and TDRD1 in primary seminoma and non-seminoma testicular tumors, in addition to testicular germ cell tumor cell lines. Most importantly, these epigenetic lesions occur in a context of piRNA downregulation and loss of DNA methylation of the LINE-1 repetitive sequences, one of the target genomic loci where the PIWI/piRNA machinery acts as a caretaker in non-transformed cells.

DNA methylation profiling in breast cancer discordant identical twins identifies DOK7 as novel epigenetic biomarker.

Using whole blood from 15 twin pairs discordant for breast cancer and high-resolution (450K) DNA methylation analysis, we identified 403 differentially methylated CpG sites including known and novel potential breast cancer genes. Confirming the results in an independent validation cohort of 21 twin pairs determined the docking protein DOK7 as a candidate for blood-based cancer diagnosis. DNA hypermethylation of the promoter region was also seen in primary breast cancer tissues and cancer cell lines. Hypermethylation of DOK7 occurs years before tumor diagnosis, suggesting a role as a powerful epigenetic blood-based biomarker as well as providing insights into breast cancer pathogenesis.

Epigenetic disruption of the PIWI pathway in human spermatogenic disorders.

Epigenetic changes are involved in a wide range of common human diseases. Although DNA methylation defects are known to be associated with male infertility in mice, their impact on human deficiency of sperm production has yet to be determined. We have assessed the global genomic DNA methylation profiles in human infertile male patients with spermatogenic disorders by using the Infinium Human Methylation27 BeadChip. Three populations were studied: conserved spermatogenesis, spermatogenic failure due to germ cell maturation defects, and Sertoli cell-only syndrome samples. A disease-associated DNA methylation profile, characterized by targeting members of the PIWI-associated RNA (piRNA) processing machinery, was obtained. Bisulfite genomic sequencing and pyrosequencing in a large cohort (n = 46) of samples validated the altered DNA methylation patterns observed in piRNA-processing genes. In particular, male infertility was associated with the promoter hypermethylation-associated silencing of PIWIL2 and TDRD1. The downstream effects mediated by the epigenetic inactivation of the PIWI pathway genes were a defective production of piRNAs and a hypomethylation of the LINE-1 repetitive sequence in the affected patients. Overall, our data suggest that DNA methylation, at least that affecting PIWIL2/TDRD1, has a role in the control of gene expression in spermatogenesis and its imbalance contributes to an unsuccessful germ cell development that might explain a group of male infertility disorders.

Electrophilic reactivity of tetrabromorhodamine 123 is bromine induced: convergent interpretation through complementary molecular descriptors.

Nucleophilic addition of water and of methanol to 3,6-diamino-2,4,5,7-tetrabromo-9-[2-(methoxycarbonyl) phenyl]-9H-xanthen-9-ylium, 4BrR123, yields respectively 2-(3,6-diamino-2,4,5,7-tetrabromo-9-hydroxy-9H-xanthen-9-yl)xanthyl benzoate, HO4BrR123 and 2-(3,6-diamino-2,4,5,7-tetrabromo-9-methoxy-9H-xanthen-9-yl)xanthyl benzoate, MeO4BrR123. The novel experimental results are addressed theoretically. The linear free energy relationship, LFER, second-order perturbation theory analysis of the natural bond orbital, NBO, and quantum theory of atoms in molecules, QTAIM, lead to the same conclusion: the electron-withdrawing effect of bonded Br atoms in 4BrR123 extremely enhances the molecular electrophilicity, as compared to 3,6-diamino-9-[2-(methoxycarbonyl) phenyl]-9H-xanthen-9-ylium, R123. The reactivity of these diaminoxanthylium cations is discussed in the context of local and global softness in extended conjugated systems.

Distinct DNA methylomes of newborns and centenarians.

Human aging cannot be fully understood in terms of the constrained genetic setting. Epigenetic drift is an alternative means of explaining age-associated alterations. To address this issue, we performed whole-genome bisulfite sequencing (WGBS) of newborn and centenarian genomes. The centenarian DNA had a lower DNA methylation content and a reduced correlation in the methylation status of neighboring cytosine--phosphate--guanine (CpGs) throughout the genome in comparison with the more homogeneously methylated newborn DNA. The more hypomethylated CpGs observed in the centenarian DNA compared with the neonate covered all genomic compartments, such as promoters, exonic, intronic, and intergenic regions. For regulatory regions, the most hypomethylated sequences in the centenarian DNA were present mainly at CpG-poor promoters and in tissue-specific genes, whereas a greater level of DNA methylation was observed in CpG island promoters. We extended the study to a larger cohort of newborn and nonagenarian samples using a 450,000 CpG-site DNA methylation microarray that reinforced the observation of more hypomethylated DNA sequences in the advanced age group. WGBS and 450,000 analyses of middle-age individuals demonstrated DNA methylomes in the crossroad between the newborn and the nonagenarian/centenarian groups. Our study constitutes a unique DNA methylation analysis of the extreme points of human life at a single-nucleotide resolution level.

CpG island hypermethylation-associated silencing of small nucleolar RNAs in human cancer.

Much effort in cancer research has focused on the tiny part of our genome that codes for mRNA. However, it has recently been recognized that microRNAs also contribute decisively to tumorigenesis. Studies have also shown that epigenetic silencing by CpG island hypermethylation of microRNAs with tumor suppressor activities is a common feature of human cancer. The importance of other classes of non-coding RNAs, such as long intergenic ncRNAs (lincRNAs) and transcribed ultraconserved regions (T-UCRs) as altered elements in neoplasia, is also gaining recognition. Thus, we wondered whether there were other ncRNAs undergoing CpG island hypermethylation-associated inactivation in cancer cells. We focused on the small nucleolar RNAs (snoRNAs), a subset of ncRNA with a wide variety of cellular functions, such as chemical modification of RNA, pre-RNA processing and control of alternative splicing. By data mining snoRNA databases and the scientific literature, we selected 49 snoRNAs that had a CpG island within ≤ 2 Kb or that were processed from a host gene with a 5'-CpG island. Bisulfite genomic sequencing of multiple clones in normal colon mucosa and the colorectal cancer cell line HCT-116 showed that 46 snoRNAs were equally methylated in the two samples: completely unmethylated (n = 26) or fully methylated (n = 20). Most interestingly, the host gene-associated 5'-CpG islands of the snoRNAs SNORD123, U70C and ACA59B were hypermethylated in the cancer cells but not in the corresponding normal tissue. CpG island hypermethylation was associated with the transcriptional silencing of the respective snoRNAs. Results of a DNA methylation microarray platform in a comprehensive collection of normal tissues, cancer cell lines and primary malignancies demonstrated that the observed hypermethylation of snoRNAs was a common feature of various tumor types, particularly in leukemias. Overall, our findings indicate the existence of a new subclass of ncRNAs, snoRNAs, that are targeted by epigenetic inactivation in human cancer.