Littérature scientifique sur le sujet « Cancer, p53, lncRNA, non-coding RNA »

Abstract Long non-coding RNAs (lncRNAs) are involved in controlling regulatory networks critical for gene expression, cellular growth, and development. Altered expression of lncRNAs are associated with tumor progression in multiple types of human cancers, but the mechanisms by which lncRNAs may control growth and proliferation in cancer cells remain unknown in most cases. Development of interventions targeting non-coding RNA regulation of cell growth would open new avenues for cancer treatment. To identify growth regulatory pathways controlled by non-coding RNAs, we used proteomics and genomics tools to study the effect of changes in lncRNA expression levels in human cancer cell lines. First, we identified five growth regulator lncRNAs based on data mining of CRISPR/Cas9 screens and high throughput sequencing studies from patient tumor and normal cells. These lncRNAs have been associated with progression and metastasis in breast, lung and colon cancers. We hypothesized that growth regulator lncRNAs play a role in cancer development by recruiting effector proteins to regulate gene expression. Next, we evaluated the cellular growth phenotype for each lncRNA in overexpression and knockdown strains using multiple established cancer cell lines. We analyzed cellular transcriptome changes after lncRNA perturbation using high-throughput RNA sequencing. Finally, RNA-protein interactions for each growth regulator lncRNA were identified using an RNA hybridization capture method paired with mass spectrometry that enables purification of direct and specific endogenous RNA-binding proteins. Using this combination of transcriptomics and proteomics data, we discovered that perturbation of growth regulator lncRNAs results in dysregulation of growth signaling pathways and increased expression of p53. We are currently identifying the sequence and structure determinants of RNA-protein complex formation to uncover the mechanisms of action of lncRNAs in controlling cancer cell growth. Citation Format: Tong Su, Bobby Kong, Calvin Huang, Jonathan Zhu, Colleen McHugh. Long non-coding RNA control of cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1533.

e23016 Background: Long non-coding RNAs (LncRNAs) may serve as a biomarker and potential therapeutic target of cancers. Chromosome 16q22.1 is frequently deleted in breast cancer and may contribute to breast carcinogenesis by inactivation of tumor suppressor genes. This study characterized a new LncRNA tumor suppressor in this region, named p53 activating non-coding RNA (PANCR). This LncRNA consists of 1.5kb in length. Methods: Quantitative real-time PCR was used for examine the PANCR expression in breast cancer tissues. RNA-pull down and RNA-Immunopreicitation were used to analyze PANCR targeted protein. Results: Our data showed that PANCR was downregulated in breast cancer cell lines and tissues. In the breast cancer cell lines, PANCR expression appeared reversely correlated with cell malignancy, and in breast cancer tissues, PANCR was downregulated over 2 times in 31(62.0%) of 50 cases compared to adjacent normal breast tissues. In breast cancer cells MCF7 and immortalized human mammary epithelial cells MCF10A, ectopic expression of PANCR induced marked apoptosis, suppressing cell proliferation in culture and tumor growth in xenografts, but in contrast, shRNA–mediated silencing of PANCR promoted cell growth and proliferation. Mechanistic approaches revealed that in both MCF7 and MCF10A cell, PANCR activated p53 and upregulated pro-apoptotic proteins bid and bim and cell cycle inhibitors p21waf/cip1 and p27Kip1. We further found that the PANCR binds to and activates p53 by dissociating the p53-MDM2 complex. We further characterized the functional domain of PANCR that interacts with p53. Conclusions: The LncRNA PANCR located in the deleted Chromosome 16q22.1 region is a novel intracellular p53 activator and tumor suppressor, which may be used as a target for cancer therapy through mimicking its binding domain and activation of p53.

AbstractAimLong non-coding RNAs (lncRNAs) have been identified to regulate cancers by controlling the process of autophagy and by mediating the post-transcriptional and transcriptional regulation of autophagy-related genes. This study aimed to investigate the potential prognostic role of autophagy-associated lncRNAs in colorectal cancer (CRC) patients.MethodsLncRNA expression profiles and the corresponding clinical information of CRC patients were collected from The Cancer Genome Atlas (TCGA) database. Based on the TCGA dataset, autophagy-related lncRNAs were identified by Pearson correlation test. Univariate Cox regression analysis and the least absolute shrinkage and selection operator analysis (LASSO) Cox regression model were performed to construct the prognostic gene signature. Gene set enrichment analysis (GSEA) was used to further clarify the underlying molecular mechanisms.ResultsWe obtained 210 autophagy-related genes from the whole dataset and found 1187 lncRNAs that were correlated with the autophagy-related genes. Using Univariate and LASSO Cox regression analyses, eight lncRNAs were screened to establish an eight-lncRNA signature, based on which patients were divided into the low-risk and high-risk group. Patients’ overall survival was found to be significantly worse in the high-risk group compared to that in the low-risk group (log-rank p = 2.731E-06). ROC analysis showed that this signature had better prognostic accuracy than TNM stage, as indicated by the area under the curve. Furthermore, GSEA demonstrated that this signature was involved in many cancer-related pathways, including TGF-β, p53, mTOR and WNT signaling pathway.ConclusionsOur study constructed a novel signature from eight autophagy-related lncRNAs to predict the overall survival of CRC, which could assistant clinicians in making individualized treatment.

Abstract INTRODUCTION: The p53 tumor suppressor family is classically activated after DNA damage and plays a central role in cell fate decisions. Although, the p53 family activates many of the same genes in response to DNA damage, p73 plays distinct biological functions in development and metastasis. It is likely that p73 activates a unique transcriptional network which is critical for its anti-metastatic and anti-invasive action. Long non-coding RNAs (lncRNAs) are a class of mRNA-like transcripts longer than 200 nucleotides. They lack protein-coding ability and are believed to be involved in various kinds of biological processes. Increasing evidence suggests that lncRNA are frequently aberrantly expressed in cancers. Therefore, the roles of dysregulated functional lncRNA in human malignant tumors have attracted considerable scientific interest. The objective of our study is to find out novel long non-coding RNAs that can act as transcriptional targets of p73 and to delineate their role in p73-mediated anti-metastatic response. METHODS: For this purpose, we performed transcriptome sequencing in HCT116p73wt and HCT116p73KD cells and screened the data for modulation of expression of lncRNAs in differential manner. Quantitative Real Time PCR was further carried out to validate the data obtained after screening RNA seq Data. Promoter analysis was carried out for the identification of p73 binding sites in the selected upregulated or downregulated lncRNAs which was further confirmed by Luciferase reporter, ChIP and site directed mutagenesis assays. RESULTS: About six lncRNAs were observed to be significantly upregulated while four were down-regulated upon knockdown of p73. The promoters of selected lncRNAs were analysed in silico using TF Bind and JASPAR software for p73 binding sites and luciferase reporter assays suggested regulation of lncRNAs by p73. Chromatin immunoprecipitation showed promoter enrichment of the selected lncRNAs. Site directed mutagenesis further confirmed the exact binding sites of p73 onto the promoters of these novel long non coding RNAs. CONCLUSION: Together, our study provides insights into the differential regulation of long non-coding RNAs in p73 dependent manner which further will provide the mechanism of their action at the genome level. Citation Format: Chanchal Bareja, Apoorva Uboveja, Daman Saluja. Elucidating the differential regulation of novel long non coding RNAs and their mechanism of action in p73 dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1560.

Background/Aims: Long non-coding RNAs (lncRNAs) play an important role in tumorigenesis. However, the role of lncRNA expression in human Non-small cell lung cancer (NSCLC) biology, prognosis and molecular classification remains unknown. Methods: We established the IncRNA profile in NSCLC by re-annotation of microarrays from the Gene expression omnibus database. Quantitative real-time PCR was used to determine expression of LINC00342. Results: 6066 differentially expressed IncRNAs were identified and we found a novel IncRNA, LINC00342 was significantly up-regulated in NSCLC tissues compared with normal tissues. We confirmed the over-expression of LINC00342 in a cohort of NSCLC patients and found LINC00342 expression level was positively correlated with lymph node metastasis and TNM stages. Furthermore, in a large online database of 1942 NSCLC patients, high expression of LINC00342 indicated poor Overall survival (HR = 1.28, 95% CI: 1.13-1.45) and post progression survival (HR = 1.43, 95% CI: 1.09-1.88). Bioinformatics analyses showed that LINC00342 was co-expressed with different protein-coding genes in NSCLC and normal tissues. Additionally, gene set enrichment analyses found that PTEN and P53 pathways genes were enriched in the groups with higher LINC00342 expression level. By small interfering RNAs mediated silence of LINC00342, proliferation ability was significantly inhibited in lung cancer cell line. Conclusion: To summary, our findings indicate that a set of IncRNAs are differentially expressed in NSCLC and we characterized a novel IncRNA, LINC00342 which is significantly up-regulated in NSCLC and could be a prognostic biomarker.

Background: Accumulating data suggest that long non-coding RNA (lncRNA) H19 and p53are closely related to the prognosis of lung cancer. This study aims to analyze the association and interaction betweenH19 and mutant p53 R175H in lung adenocarcinoma (LAC). Methods: Mutant-type (Mt) p53 R175H was assessed by using RT-PCR in LAC cells and 100 cases of LAC tissue samples for association with H19 expression. Western blot, RNA-pull down, immunoprecipitation-Western blot and animal experiments were used to evaluate the interaction between H19 and mtp53. Results: Mtp53 R175H and H19 were over-expressed in LAC tissues and cells, while H19 over-expression extended the p53 half-life and enhanced transcriptional activity. Combined with anti-p53, ShH19 can significantly inhibit tumor growth in vivo. Conclusions: H19 over-expression may induce the elevated expression of mtp53 and interact with mtp53, leading to LAC progression. In addition, the high expression of mtp53 R175H is associated with poor overall survival inpatients. The simultaneous inhibition of H19 and mtp53 may provide a novel strategy for the effective control of LAC clinically.

Background: Long intergenic non-coding RNA, regulator of reprogramming ( LINC-ROR) is a newly identified cytoplasmic long non-coding RNA (lncRNA), which has been found to be dysregulated in different cancers. The present work aimed to quantify LINC-ROR expression profile and assess the tumor proteins p53 and caspase 3 expressions in glioblastoma tissue specimens compared to non-cancer tissues, and to correlate these expression levels with the available clinicopathological and survival data. Methods: LINC-ROR relative expression in 57 glioblastoma cancer tissues and 10 non-cancer tissues was quantified by real-time polymerase chain reaction (qPCR). In addition, methylation-specific PCR of O-6-methylguanine-DNA methyltransferase ( MGMT) promoter and immunohistochemical expression of apoptosis related proteins: p53 and caspase 3 were performed. Results: The up-regulation of LINC-ROR was encountered in 89.5% of patients. The higher expression of LINC-ROR was associated with poor disease progression-free and overall survival as well as a younger age of patients ( P=0.036). p53 protein was expressed only in glioblastoma but not in non-cancer tissues while caspase 3 was weakly expressed in most non-cancer tissues and in varying degrees in glioblastoma (24% weak, 30% moderate, and 16% strong expression). The Kaplan–Meier survival plot illustrated poor survival in glioblastoma patients with over-expressed LINC-ROR ( P=0.010) and down-regulated p53 ( P=0.002). Multivariate analysis showed that glioblastoma patients were clustered into two distinct groups based on LINC-ROR expression profile, p53 staining levels and patients’ overall survival. Conclusions: LINC-ROR up-regulation may have a role in glioblastoma tumorigenesis and could be a potential prognostic marker for this fatal disease.

Long non-coding RNAs (lncRNAs) play important roles in gastric cancer (GC), but the mechanism is not fully clear. ERICH3-AS1 (ERICH3 antisense RNA1) is affiliated with the non-coding RNA class which has proven to be involved in the prognostic of GC, but the function of ERICH3-AS1 is still unclear. In this study, we aim to explore the potential function of ERICH3-AS1 in the development of GC and analyze the prognostic role of ERICH3-AS1 in GC. We found that the lncRNA ERICH3-AS1 was significantly up-regulated in GC tissues in the analysis of The Cancer Genome Atlas (TCGA) data; the Kaplan-Meier analysis showed that the higher the expression of ERICH3-AS1 was, the earlier the recurrence and the poorer the prognosis would be in patients. Cox univariate and multivariate analyses revealed that ERICH3-AS1 was a risk factor of disease-free survival (DFS) (p < 0.05) and overall survival (OS) (p < 0.05) of patients. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, it demonstrated that the ERBB pathways, the mitogen-activated protein kinase (MAPK) pathways, the MTOR pathways, p53 pathways and Wnt pathways were differentially enriched in ERICH3-AS1 high expression phenotype. Furthermore, the correlation analysis showed that ERICH3-AS1 had significant correlations with apoptosis-related proteins such as BCL2L10 and CASP14; cell cycle-associated proteins CDK14 and invasion and migration-associated proteins such as MMP20, MMP26 and MMP27. In summary, we identified that increased ERICH3-AS1 might be a potential biomarker for diagnosis and independent prognostic factor of GC. Moreover, ERICH3-AS1 might participate in the oncogenesis and development of tumors via cell cycle and apoptosis pathway mediated by ERBB, MAPK, MTOR, p53 and Wnt pathways.

Abstract Ferroptosis, with iron-dependent and ROS-dependent, is a novel type of cell death in a variety of diseases and some studies confirmed that ferroptosis-related lncRNAs are involved in the occurrence and development of several cancers. However, the ferroptosis-related lncRNA in the role of gliomas is unclear. Here, we constructed a prognostic scoring model of ferroptosis-related lncRNAs in gliomas. Data were downloaded from the Chinese glioma genome atlas (CGGA), the cancer genome atlas, and FerrDb database. In this study, we found 1051 lncRNAs associated with ferroptosis by Spearman's rank correlation analysis in CGGA653, and 547 lncRNAs were related to prognosis in gliomas. Subsequently, we identified 9 ferroptosis-related signatures (AC010729.2, AC062021.1, FAM225B, FAM66C, HOXA-AS2, LINC00662, LINC00665, MIR497HG, and TMEM72-AS1) by least absolute shrinkage and selection operator and Cox proportional hazards model. Next, all glioma patients were divided into high- and low-risk groups based on the median risk score based on these signatures, and the low-risk group had better prognosis significantly than the high-risk group by Kaplan-Meier curve. Moreover, the risk score can predict survival status with high sensitivity and specificity by receiver operating characteristic curve (area under the curve at 1, 3, 5 years: 0.791, 0.84, 0.856, respectively). In addition, some pathways (cell cycle, p53 signaling pathway, apoptosis, and oxidative phosphorylation) significantly enriched in KEGG enrichment pathway, and a nomogram was constructed by integrating some independent prognostic clinicopathological features to predict the overall survival in gliomas (C-index: 0.786). In summary, these 9 ferroptosis-related signatures have potential prognostic value and could be crucial factors for treating malignant gliomas.

Amongst the various gynecological malignancies affecting female health globally, ovarian cancer is one of the predominant and lethal among all. The identification and functional characterization of long non-coding RNAs (lncRNAs) are made possible with the advent of RNA-seq and the advancement of computational logarithm in understanding human disease biology. LncRNAs can interact with deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins and their combinations. Moreover, lncRNAs regulate orchestra of diverse functions including chromatin organization and transcriptional and post-transcriptional regulation. LncRNAs have conferred their critical role in key biological processes in human cancer including tumor initiation, proliferation, cell cycle, apoptosis, necroptosis, autophagy, and metastasis. The interwoven function of tumor-suppressor protein p53-linked lncRNAs in the ovarian cancer paradigm is of paramount importance. Several lncRNAs operate as p53 regulators or effectors and modulates a diverse array of functions either by participating in various signaling cascades or via interaction with different proteins. This review highlights the recent progress made in the identification of p53 associated lncRNAs while elucidating their molecular mechanisms behind the altered expression in ovarian cancer tumorigenesis. Moreover, the development of novel clinical and therapeutic strategies for targeting lncRNAs in human cancers harbors great promise.

PART1, TITLE: A p53-inducible long non-coding RNA PICART1 mediating cancer cell proliferation and migration. Long non-coding RNAs (lncRNAs) function in the development and progression of cancer, but only a small portion of lncRNAs are characterized thus far. A novel lncRNA transcript with 2.53 kb in length was identified by a transcriptome sequencing analysis, named p53-inducible cancer-associated RNA transcript 1 (PICART1). This PICART1 is upregulated by p53 through a p53-binding site at -1808 to -1783bp. In breast and colorectal cancer cells and tissues, PICART1 expression was decreased. Ectopic expression of the PICART1 suppressed growth, proliferation, migration, and invasion of MCF7, MDA-MB-231 and HCT116 cells whereas silencing of PICART1 stimulated the cell growth and migration. In these cells, the expression of PICART1 lowered down the levels of p-AKT (Thr308 & Ser473) and p-GSK3β (Ser9), and accordingly, β-catenin, cyclin D1 and c-Myc expression were decreased, but p21cip1/Waf1 expression was increased. Together these data suggest that PICART1 is a novel p53-inducible tumor suppressor lncRNA, functioning through the AKT/GSK3β/β-catenin signaling cascade. PART2, TITLE: The novel long non-coding RNA PANCR is a tumor suppressor gene in breast cancer. Long non-coding RNAs (lncRNAs) function as oncogenes or tumor suppressors in development and progression of cancer. Chromosome 16q22.1 region is frequently deleted in breast cancer, which may contribute to breast carcinogenesis by inactivation of tumor suppressor genes. This study characterized a new lncRNA tumor suppressor, named p53 activating non-coding RNA (PANCR), located in this Chromosome 16q22.1 region. This PANCR lncRNA consists of 1.5kb in length. Our data showed that PANCR was downregulated in breast cancer tissues and cell lines. In the breast cancer cell lines, PANCR expression appeared reversely correlated with cell malignancy, and in breast cancer tissues, PANCR was downregulated over 2 times in 31 (62.0%) of 50 cases when compared to adjacent normal breast tissues. In breast cancer cells MCF7 cells, ectopic expression of PANCR suppressed cell proliferation in culture, but in contrast, shRNA–mediated silencing of PANCR promoted cell growth and proliferation.

A instabilidade genómica é uma propriedade fundamental subjacente ao processo de carcinogénese, uma vez que confere às células cancerígenas a capacidade para a sua expansāo clonal. A aquisiçāo de um genótipo mutante permite nāo só a expansāo destas células, assim como, a manipulaçāo do ambiente em que estāo inseridas. O genoma possui, no entanto, sistemas de vigilância que detetam e corrigem defeitos na cadeia de DNA. O gene supressor tumoral TP53, mutado em cerca de metade de tumores humanos, é um fator de transcriçāo considerado o ‘guardiāo do genoma’ (Lane, 1992). A ativaçāo multifactorial do gene TP53, que atua via induçāo da transcriçāo de diferentes genes-alvo, classicamente culmina na paragem do ciclo celular, senescência e/ou apoptose das células cancerígenas. Até recentemente, era considerado que a extensa rede de genes-alvo regulada pelo gene p53 era composta apenas por genes codificadores de proteínas. No entanto, tem sido cada vez mais aceite, que os genes codificadores de proteínas regulados pelo p53 nāo sāo causa suficiente para explicar o seu amplo papel como supressor tumoral (Brady et al., 2011). O desenvolvimento de novas técnicas de sequenciação de última geração veio revelar nāo só que a maior parte do genoma de eucariotas é composto por DNA nāo codificante, mas também que estas regiões sāo transcritas numa nova classe de reguladores genómicos, os long non-coding RNAs (lncRNAs). O termo enhancer RNAs, uma classe de lncRNAs transcritos apartir de enhancers (em inglês) e reguladores da expressão génica, foi correlacionado com a expressāo positiva de genes-codificantes de proteínas localizados nas imediações da regiāo nāo-codificante (Kim et al., 2010). Sendo assim, formulamos a hipótese de que o p53 exerça parte da sua actividade reguladora via interaçāo com domínios produtores de enhancer-RNAs. Recorremos à técnica de genome-wide chromatin-binding profiles para testar a nossa hipótese e descobrimos regiões-genómicas ligadas a p53, mas que se localizam a grandes distâncias em relaçāo a qualquer um dos genes-alvo de p53. A análise destas regiões permitiu identificar a presença de elementos conservados de ativaçāo de p53 em domínios epigeneticamente caraterizados como enhancers. Nestas regiões, que designámos de p53-bound enhancer regions (p53BERs), constatámos que sāo produtoras de eRNAs, em resposta à activaçāo de p53. O nosso estudo demonstrou também que os p53BERs aumentam a transcriçāo regulada pelo p53 através de interacções intra-cromossómicas com múltiplos genes-vizinhos. Adicionalmente, mostrámos nāo só que uma das funções dos eRNAs consiste no aumento da transcriçāo mas também na relevância do papel dos eRNAs na resposta regulada pelo p53 (Melo et al., 2013). No seguimento dos nossos resultados, mapeámos todos os enhancers regulados pelo p53 (p53RERs, em inglês) e descobrimos que, enquanto a maioria dos enhancers induzidos por p53 possuem domínios de ligaçāo ao p53 (p53BERs, em inglês), na sua maioria estes domínios estão ausentes (p53FERs, em inglês) o que sugere um modelo de activaçāo diferente. De facto, observámos que para um subconjunto de p53RERs é necessária a presença de um lncRNA activado pelo stress, que denominamos por LED (lncRNA activator of enhancer domains). Demonstrámos que o LED é um factor-alvo de p53 e é importante para a deposiçāo do marcador epigenético de acetilação da histona 3 na lisina 9 (H3K9ac, em inglês) (um marcador de enhancer) no subconjunto de p53RERs. A presença de LED é indispensável para a correcta paragem do ciclo celular, induzida pelo p53. Finalmente, detetamos a presença de LED em diversas linhas celulares cancerígenas e tumores humanos. O trabalho desenvolvido nesta Tese demonstra que os lncRNAs representam elementos funcionais do genoma não-codificante e que desempenham um papel crucial na biologia do cancro, podendo vir a ser utilizados em futuras abordagens terapêuticas.
Underlying the multiple steps characteristic of cancer progression is genomic instability, as it provides the requirements necessary for the selective clonal advantage of cancer cells. The acquisition of a mutant genotype allows the outgrowth of these cells and manipulation of their local environment. However, the genome possesses efficient surveillance systems to detect and resolve defects in the DNA. The tumor suppressor gene TP53, which was found to be mutated in about half of all human cancers, represents a known transcription factor often referred to as the “guardian of the genome” (Lane, 1992). The multifactorial activation of p53 results in a complex transcriptional response that classically culminates in: cell cycle arrest, senescence and/or a programmed cell death (apoptosis), achieved through the regulation of different target genes. Until recently, the p53 transcriptional network mainly revolved around its protein-coding target genes. However, it has been increasingly recognized that the thus-far known protein-coding p53 target genes cannot fully explain its tumor suppression activity (Brady et al., 2011). The advent of next-generation sequencing techniques unraveled the existence of a large portion of non-coding elements in the genome. These elements can be transcribed into a new class of genomic regulators, the long non-coding RNAs (lncRNAs). The expression of enhancer RNAs (eRNAs), a class of long ncRNAs transcribed from enhancer elements that regulate gene expression, was show to positively correlate with nearby protein-coding genes (Kim et al., 2010), which led us to hypothesize that part of the p53-regulatory network could be mediated through its interaction with enhancer-RNA producing domains. For that, we used genome-wide chromatin-binding profiles and discovered p53-bound genome regions located far from any known p53 target gene. These regions essentially revealed, the presence of conserved p53 response elements and presented hallmarks of enhancer domains. We named these new elements as p53-bound enhancer regions (p53BERs), and observed the production of eRNAs in response to p53 induction. Our study further demonstrates that p53BERs enhance p53 transcriptional response accomplished through intra-chromosomal interactions with multiple neighboring target genes. Additionally, we provide evidence concerning the functionality of eRNAs in the enhancement of transcription and demonstrate the importance of these new RNA molecules required for a proper p53 response (Melo et al., 2013a). We then set out to globally map all p53-regulated enhancers (p53RERs) and found that while many p53-induced enhancers contained p53-binding sites (p53BERs), most did not (p53-free enhancer regions, p53FERs), suggesting a different mode of activation. Indeed, we were able to observe that for a subset of p53RERs the presence of a stress-activated lncRNA, termed LED (lncRNA activator of enhancer domains), is required. We show that LED is a direct p53-target that is important for the deposition of histone 3 lysine 9 acetylation (H3K9ac) (a transcriptional active enhancer mark) at a subset of p53RERs. Furthermore, LED’s presence is indispensable for a proper p53-induded cell-cycle arrest. Finally, we uncover the promoter-associated hypermehtylation of LED in several cancer cell lines and human tumors. The findings in this thesis provide evidence that lncRNAs represent functional elements embedded in the non-coding portion of our genome. We show that these newly emerging RNA molecules are important players in cancer biology, and can be a future therapeutic approach in the fight against cancer.

Myc deregulation critically contributes to many cancer etiologies. Recent work suggests that Myc and its direct interactors can confer a distinct epigenetic state. Our goal is to better understand the Myc-conferred epigenetic status of cells. We have previously identified the long non-coding RNA (lncRNA), H19, as a target of Myc regulation and shown it to be important for transformation in lung and breast cells. These results prompted further analysis to identify similarly important Myc-regulated lncRNAs. Myc-regulated lncRNAs associated with the cell cycle and transformation have been identified by microarray analysis. A small number of candidate lncRNAs that were differentially expressed in both the cell cycle and transformation have been validated. Given the increasing importance of lncRNAs and epigenetics to cancer biology, the discovery of Myc-induced, growth associated lncRNAs could provide insight into the mechanisms behind Myc-related epigenetic signatures in both normal and disease states.

Bladder cancer (BC) is the sixth most common cause of cancer; BC risk increases with age and is more common among men than women. Upon diagnosis, the 5-year relative survival rate for patients is approximately 77%. The treatment options available for bladder cancer include chemotherapy, radiation therapy, immunotherapy, targeted therapy, and surgery. Despite the advances in therapeutically novel approaches, BC remains an important problem of public health. Long non-coding RNA (lncRNA) is defined as non-protein-coding RNA molecule longer than 200 nucleotides. Recent findings have highlighted that lncRNA contributes to the regulation of multiple signaling pathways in bladder cancer, suggesting that lncRNA exerts its roles during the biological processes of tumorigenesis, tumor proliferation, differentiation, apoptosis, invasion, migration, and stemness. In our laboratory, we described a family of mitochondrial long non-coding RNAs containing stem-loop structures, named sense and antisense. These transcripts are found outside the organelle, in the cytosol and nucleus in normal and tumor cells, and are differentially expressed according to proliferative status of cells. The antisense transcript seems to be a novel target for BC treatment based in modified antisense oligonucleotides. In this chapter, the novel biology and role of these RNAs as therapeutical targets will be discussed.

Introduction: Long RNAs are non-coding RNAs with more than 200 nucleotides in length, with essential regulatory roles in several biological processes, including in breast cancer (BC). The human genome contains 481 ultraconserved regions, which are genomic stretches of over 200 base pairs conserved among humans, rats, and mice. Most of these regions are transcriptionally active (T-UCRs), and several are differentially expressed in tumors. Some T-UCRs have been functionally characterized, but few have been associated with BC. Objectives: In this study, we aimed to expand the knowledge of T-UCRs in BC and characterize the lnc-uc.147, a long RNA transcribed from an ultraconserved region. Methods: We evaluated the expression level of 481 T-UCRs and their association with clinical parameters from TCGA data. For confirmation, 102 Brazilian BC samples were analyzed by RT-qPCR. Cytosolic and nuclear cell fractions and RT-qPCRs were done to determine the cell compartment of the transcript. Northern blotting and RACE were performed to determine the sequence and precise size of lnc-uc.147. Using two luminal cell lines (CAMA and BT474), a siRNA-based approach was applied to investigate the effects of lnc-uc.147 knockdown in cell viability, colony formation, and apoptosis level. To understand the interactions of lnc-uc.147 and proteins, we performed a pull-down assay. Results: Using TCGA (The Cancer Genome Atlas) data, we found 302 T-UCRs related to clinical features in BC: 43% were associated with molecular subtypes, 36% with estrogen-receptor positivity, 17% with HER2 expression, 12% with stage, and 10% with overall survival. We found that uc.147 is highly expressed in luminal A and B patients, which was also confirmed in Brazilian samples. For luminal A, a subtype usually associated with better prognosis, high uc.147 expression was associated with a poor prognosis and suggested as an independent prognostic factor. The lncRNA from uc.147 (lnc-uc.147) is in the nucleus. Northern blotting results show that uc.147 is a 2,8 kb monoexonic transcript. The silencing of uc.147 increases apoptosis, arrests the cell cycle and reduces cell viability and colony formation in luminal BC cell lines. Additionally, we identified 19 proteins that interact with uc.147 through mass spectrometry. These proteins are mainly involved in cytoskeletal and centrosome organization as well as in epithelial-mesenchymal transition. Conclusions: We show herein evidence that neoplastic BC cells exhibit a unique expression profile of T-UCRs. This study characterized the lnc-uc.147, a transcript that has never been described before. Indeed, lnc-uc.147 has an oncogenic effect in the luminal BC cell line and can interact with proteins. Furthermore, uc.147 has the potential as a BC prognostic marker in luminal patients.