17ß-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17ß-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17ß-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17ß-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

Circulating Non-coding RNAs in Renal Cell Carcinoma-Pathogenesis and Potential Implications as Clinical Biomarkers.

Liquid biopsy-the determination of circulating cells, proteins, DNA or RNA from biofluids through a "less invasive" approach-has emerged as a novel approach in all cancer entities. Circulating non-(protein) coding RNAs including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and YRNAs can be passively released by tissue or cell damage or actively secreted as cell-free circulating RNAs, bound to lipoproteins or carried by exosomes. In renal cell carcinoma (RCC), a growing body of evidence suggests circulating non-coding RNAs (ncRNAs) such as miRNAs, lncRNAs, and YRNAs as promising and easily accessible blood-based biomarkers for the early diagnosis of RCC as well as for the prediction of prognosis and treatment response. In addition, circulating ncRNAs could also play a role in RCC pathogenesis and progression. This review gives an overview over the current study landscape of circulating ncRNAs and their involvement in RCC pathogenesis as well as their potential utility as future biomarkers in RCC diagnosis and treatment.

Non-Coding RNAs as Cancer Hallmarks in Chronic Lymphocytic Leukemia.

The discovery of non-coding RNAs (ncRNAs) and their role in tumor onset and progression has revolutionized the way scientists and clinicians study cancers. This discovery opened new layers of complexity in understanding the fine-tuned regulation of cellular processes leading to cancer. NcRNAs represent a heterogeneous group of transcripts, ranging from a few base pairs to several kilobases, that are able to regulate gene networks and intracellular pathways by interacting with DNA, transcripts or proteins. Deregulation of ncRNAs impinge on several cellular responses and can play a major role in each single hallmark of cancer. This review will focus on the most important short and long non-coding RNAs in chronic lymphocytic leukemia (CLL), highlighting their implications as potential biomarkers and therapeutic targets as they relate to the well-established hallmarks of cancer. The key molecular events in the onset of CLL will be contextualized, taking into account the role of the "dark matter" of the genome.

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

Targeting Epigenetic Dependencies in Solid Tumors: Evolutionary Landscape Beyond Germ Layers Origin.

Extensive efforts recently witnessed the complexity of cancer biology; however, molecular medicine still lacks the ability to elucidate hidden mechanisms for the maintenance of specific subclasses of rare tumors characterized by the silent onset and a poor prognosis (e.g., ovarian cancer, pancreatic cancer, and glioblastoma). Recent mutational fingerprints of human cancers highlighted genomic alteration occurring on epigenetic modulators. In this scenario, the epigenome dependency of cancer orchestrates a broad range of cellular processes critical for tumorigenesis and tumor progression, possibly mediating escaping mechanisms leading to drug resistance. Indeed, in this review, we discuss the pivotal role of chromatin remodeling in shaping the tumor architecture and modulating tumor fitness in a microenvironment-dependent context. We will also present recent advances in the epigenome targeting, posing a particular emphasis on how this knowledge could be translated into a feasible therapeutic approach to individualize clinical settings and improve patient outcomes.

miR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription-quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options.

Cancer-associated rs6983267 SNP and its accompanying long noncoding RNA CCAT2 induce myeloid malignancies via unique SNP-specific RNA mutations.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

Publisher Correction: Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

In the originally published version of this Article, the positions of the final two authors in the author list were inadvertently inverted during the production process. This error has now been corrected in both the PDF and HTML versions of the Article.

Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

The transcribed ultraconserved regions (T-UCRs) encode long non-coding RNAs implicated in human carcinogenesis. Their mechanisms of action and the factors regulating their expression in cancers are poorly understood. Here we show that high expression of uc.339 correlates with lower survival in 210 non-small cell lung cancer (NSCLC) patients. We provide evidence from cell lines and primary samples that TP53 directly regulates uc.339. We find that transcribed uc.339 is upregulated in archival NSCLC samples, functioning as a decoy RNA for miR-339-3p, -663b-3p, and -95-5p. As a result, Cyclin E2, a direct target of all these microRNAs is upregulated, promoting cancer growth and migration. Finally, we find that modulation of uc.339 affects microRNA expression. However, overexpression or downregulation of these microRNAs causes no significant variations in uc.339 levels, suggesting a type of interaction for uc.339 that we call "entrapping". Our results support a key role for uc.339 in lung cancer.

Understanding the Genomic Ultraconservations: T-UCRs and Cancer.

Transcribed ultraconserved regions (T-UCRs) are genomic regions conserved across large evolutionary distances, which encode for noncoding RNAs that serve as regulators of gene expression. Although T-UCRs have been linked to multiple aspects of mammalian gene regulation, the roles of their extreme evolutionary conservation remain largely unexplained. Growing body of literature is now focusing on T-UCRs as potential cancer biomarkers or as new drug targets. Here we present an overview of the discoveries so far published about the role of T-UCR in cancer and disease.

N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration.

BACKGROUND: Non-coding RNAs have been drawing increasing attention in recent years as functional data suggest that they play important roles in key cellular processes. N-BLR is a primate-specific long non-coding RNA that modulates the epithelial-to-mesenchymal transition, facilitates cell migration, and increases colorectal cancer invasion. RESULTS: We performed multivariate analyses of data from two independent cohorts of colorectal cancer patients and show that the abundance of N-BLR is associated with tumor stage, invasion potential, and overall patient survival. Through in vitro and in vivo experiments we found that N-BLR facilitates migration primarily via crosstalk with E-cadherin and ZEB1. We showed that this crosstalk is mediated by a pyknon, a short ~20 nucleotide-long DNA motif contained in the N-BLR transcript and is targeted by members of the miR-200 family. In light of these findings, we used a microarray to investigate the expression patterns of other pyknon-containing genomic loci. We found multiple such loci that are differentially transcribed between healthy and diseased tissues in colorectal cancer and chronic lymphocytic leukemia. Moreover, we identified several new loci whose expression correlates with the colorectal cancer patients' overall survival. CONCLUSIONS: The primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease. The presence of a functional pyknon within N-BLR and the related finding that many more pyknon-containing genomic loci in the human genome exhibit tissue-specific and disease-specific expression suggests the possibility of an alternative class of biomarkers and therapeutic targets that are primate-specific.

p27kip1 expression limits H-Ras-driven transformation and tumorigenesis by both canonical and non-canonical mechanisms.

The tumor suppressor protein p27Kip1 plays a pivotal role in the control of cell growth and metastasis formation.Several studies pointed to different roles for p27Kip1 in the control of Ras induced transformation, although no explanation has been provided to elucidate these differences. We recently demonstrated that p27kip1 regulates H-Ras activity via its interaction with stathmin.Here, using in vitro and in vivo models, we show that p27kip1 is an important regulator of Ras induced transformation. In H-RasV12 transformed cells, p27kip1 suppressed cell proliferation and tumor growth via two distinct mechanisms: 1) inhibition of CDK activity and 2) impairment of MT-destabilizing activity of stathmin. Conversely, in K-Ras4BV12 transformed cells, p27kip1 acted mainly in a CDK-dependent but stathmin-independent manner.Using human cancer-derived cell lines and primary breast and sarcoma samples, we confirmed in human models what we observed in mice.Overall, we highlight a pathway, conserved from mouse to human, important in the regulation of H-Ras oncogenic activity that could have therapeutic and diagnostic implication in patients that may benefit from anti-H-Ras therapies.

Targeting microRNAs as key modulators of tumor immune response.

The role of immune response is emerging as a key factor in the complex multistep process of cancer. Tumor microenvironment contains different types of immune cells, which contribute to regulate the fine balance between anti and protumor signals. In this context, mechanisms of crosstalk between cancer and immune cells remain to be extensively elucidated. Interestingly, microRNAs (miRNAs) have been demonstrated to function as crucial regulators of immune response in both physiological and pathological conditions. Specifically, different miRNAs have been reported to have a role in controlling the development and the functions of tumor-associated immune cells. This review aims to describe the most important miRNAs acting as critical modulators of immune response in the context of different solid tumors. In particular, we discuss recent studies that have demonstrated the existence of miRNA-mediated mechanisms regulating the recruitment and the activation status of specific tumor-associated immune cells in the tumor microenvironment. Moreover, various miRNAs have been found to target key cancer-related immune pathways, which concur to mediate the secretion of immunosuppressive or immunostimulating factors by cancer or immune cells. Modalities of miRNA exchange and miRNA-based delivery strategies are also discussed. Based on these findings, the modulation of individual or multiple miRNAs has the potential to enhance or inhibit specific immune subpopulations supporting antitumor immune responses, thus contributing to negatively affect tumorigenesis. New miRNA-based strategies can be developed for more effective immunotherapeutic interventions in cancer.

Circulating free xeno-microRNAs - The new kids on the block.

The role of circulating free microRNAs (cfmiRNAs) as promising tools for cancer screening, prognosis and monitoring of anticancer therapies has been widely studied in the past decades. cfmiRNAs have all the characteristics of the perfect biomarkers owing high stability under storage and handling conditions and being detectable not only in plasma, but in almost all body fluids. Moreover, their levels in plasma are likely to resemble ones in the primary tumor. Recently, viral and plant miRNAs have been found in plasma of healthy individuals through deep sequencing technique, and subsequently the same ones were deregulated in patients. Growing body of literature is recently focusing on understanding the potential cross-kingdom regulation of human mRNAs by miRNAs most likely absorbed with food ingestion. In this article we will review the literature concerning the xenomiRs detected in plasma and their role in influencing cancer onset and progression. XenomiRs could potentially be used not only as early screening tool, but also for patients' prognosis.

The Potential of MicroRNAs as Prostate Cancer Biomarkers.

CONTEXT: Short noncoding RNAs known as microRNAs (miRNAs) control protein expression through the degradation of RNA or the inhibition of protein translation. The miRNAs influence a wide range of biologic processes and are often deregulated in cancer. This family of small RNAs constitutes potentially valuable markers for the diagnosis, prognosis, and therapeutic choices in prostate cancer (PCa) patients, as well as potential drugs (miRNA mimics) or drug targets (anti-miRNAs) in PCa management. OBJECTIVE: To review the currently available data on miRNAs as biomarkers in PCa and as possible tools for early detection and prognosis. EVIDENCE ACQUISITION: A systematic review was performed searching the PubMed database for articles in English using a combination of the following terms: microRNA, miRNA, cancer, prostate cancer, miRNA profiling, diagnosis, prognosis, therapy response, and predictive marker. EVIDENCE SYNTHESIS: We summarize the existing literature regarding the profiling of miRNA in PCa detection, prognosis, and response to therapy. The articles were reviewed with the main goal of finding a common recommendation that could be translated from bench to bedside in future clinical practice. CONCLUSIONS: The miRNAs are important regulators of biologic processes in PCa progression. A common expression profile characterizing each tumor subtype and stage has still not been identified for PCa, probably due to molecular heterogeneity as well as differences in study design and patient selection. Large-scale studies that should provide additional important information are still missing. Further studies, based on common clinical parameters and guidelines, are necessary to validate the translational potential of miRNAs in PCa clinical management. Such common signatures are promising in the field and emerge as potential biomarkers. PATIENT SUMMARY: The literature shows that microRNAs hold potential as novel biomarkers that could aid prostate cancer management, but additional studies with larger patient cohorts and common guidelines are necessary before clinical implementation.

p27kip1: An all-round tumor suppressor.

Our recent study has uncovered an additional mechanism by which the cell cycle inhibitor p27kip1 controls cell proliferation. Through its effect on the activity of the microtubule destabilizing protein Stathmin, p27kip1 modulates full H-Ras activation and, as a consequence, the MAPK signaling cascade. This regulatory mechanism influences the cell cycle in vitro and tissue and/or organ growth in mice, in vivo and, when unbalanced, may lead to uncontrolled proliferation and tumor onset.

p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability.

The cyclin-dependent kinase (CDK) inhibitor p27(kip1) is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27(kip1)-null mice is reverted by concomitant deletion of stathmin in p27(kip1)/stathmin double-KO mice, suggesting that a CDK-independent function of p27(kip1) contributes to the control of cell proliferation. Whether the regulation of MT stability by p27(kip1) impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27(kip1)-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27(kip1) and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27(kip1), by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27(kip1) and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.

Mutant AKT1-E17K is oncogenic in lung epithelial cells.

The hotspot E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6-2% of human lung cancers. In this manuscript, we sought to determine whether this AKT1 variant is a bona-fide activating mutation and plays a role in the development of lung cancer. Here we report that in immortalized human bronchial epithelial cells (BEAS-2B cells) mutant AKT1-E17K promotes anchorage-dependent and -independent proliferation, increases the ability to migrate, invade as well as to survive and duplicate in stressful conditions, leading to the emergency of cells endowed with the capability to form aggressive tumours at high efficiency. We provide also evidence that the molecular mechanism whereby AKT1-E17K is oncogenic in lung epithelial cells involves phosphorylation and consequent cytoplasmic delocalization of the cyclin-dependent kinase (cdk) inhibitor p27. In agreement with these results, cytoplasmic p27 is preferentially observed in primary NSCLCs with activated AKT and predicts poor survival.