Non-coding RNAs and potential therapeutic targeting in cancer.

Recent advances have begun to clarify the physiological and pathological roles of non-coding RNAs (ncRNAs) in various diseases, including cancer. Among these, microRNAs (miRNAs) have been the most studied and have emerged as key players that are involved in the regulation of important growth regulatory pathways in cancer pathogenesis. The ability of a single ncRNA to modulate the expression of multiple downstream gene targets and associated pathways, have provided a rationale to pursue them for therapeutic drug development in cancer. In this context, early data from pre-clinical studies have demonstrated that synthetic miRNA-based therapeutic molecules, along with various protective coating approaches, has allowed for their efficient delivery and anti-tumor activity. In fact, some of the miRNA-based cancer therapeutic strategies have shown promising results even in early-phase human clinical trials. While the enthusiasm for ncRNA-based cancer therapeutics continue to evolve, the field is still in the midst of unraveling a more precise understanding of the molecular mechanisms and specific downstream therapeutic targets of other lesser studied ncRNAs such as the long-non-coding RNAs, transfer RNAs, circular RNAs, small nucleolar RNAs, and piwi-interacting RNAs. This review article provides the current state of knowledge and the evolving principles for ncRNA-based therapeutic approaches in cancer, and specifically highlights the importance of data to date and the approaches that are being developed to overcome the challenges associated with their delivery and mitigating the off-target effects in human cancers.

Circular RNA cir-ITCH Is a Potential Therapeutic Target for the Treatment of Castration-Resistant Prostate Cancer.

cir-ITCH, a well-known tumor-suppressive circular RNA, plays a critical role in different cancers. However, its expression and functional role in prostate cancer (PCa) are unclear. Herein, we explored the potential mechanism and tumor-inhibiting role of cir-ITCH in PCa. Using reverse transcriptase polymerase chain reaction assay, we analyzed the expression of cir-ITCH in PCa and paired adjacent nontumor tissue samples resected during surgical operation, as well as in two cell lines of human PCa (LNCaP and PC-3) and the immortalized normal prostate epithelial cell line (RWPE-1). Cell viability and migration of PCa cell lines were evaluated using CCK-8 and wound-healing assays. Expression of key proteins of the Wnt/ß-catenin and PI3K/AKT/mTOR pathways was detected using western blotting. We found that cir-ITCH expression was typically downregulated in the tissues and cell lines of PCa compared to that in the peritumoral tissue and in RWPE-1 cells, respectively. The results showed that cir-ITCH overexpression significantly inhibits the proliferation, migration, and invasion of human PCa cells and that reciprocal inhibition of expression occurred between cir-ITCH and miR-17. Proteins in the Wnt/ß-catenin and PI3K/AKT/mTOR pathways were downregulated by overexpression of cir-ITCH both in androgen receptor-positive LNCaP cells and androgen receptor-negative PC-3 cells. Taken together, these data demonstrated that cir-ITCH plays a tumor-suppressive role in human PCa cells, partly through the Wnt/ß-catenin and PI3K/AKT/mTOR pathways. Thus, cir-ITCH may serve as a novel therapeutic target for the treatment of PCa, especially castration-resistant prostate cancer.

Exosomes released upon mitochondrial ASncmtRNA knockdown reduce tumorigenic properties of malignant breast cancer cells.

During intercellular communication, cells release extracellular vesicles such as exosomes, which contain proteins, ncRNAs and mRNAs that can influence proliferation and/or trigger apoptosis in recipient cells, and have been proposed to play an essential role in promoting invasion of tumor cells and in the preparation of metastatic niches. Our group proposed the antisense non-coding mitochondrial RNA (ASncmtRNA) as a new target for cancer therapy. ASncmtRNA knockdown using an antisense oligonucleotide (ASO-1537S) causes massive death of tumor cells but not normal cells and strongly reduces metastasis in mice. In this work, we report that exosomes derived from ASO-1537S-treated MDA-MB-231 breast cancer cells (Exo-1537S) inhibits tumorigenesis of recipient cells, in contrast to exosomes derived from control-ASO-treated cells (Exo-C) which, in contrast, enhance these properties. Furthermore, an in vivo murine peritoneal carcinomatosis model showed that Exo-1537S injection reduced tumorigenicity compared to controls. Proteomic analysis revealed the presence of Lactadherin and VE-Cadherin in exosomes derived from untreated cells (Exo-WT) and Exo-C but not in Exo-1537S, and the latter displayed enrichment of proteasomal subunits. These results suggest a role for these proteins in modulation of tumorigenic properties of exosome-recipient cells. Our results shed light on the mechanisms through which ASncmtRNA knockdown affects the preparation of breast cancer metastatic niches in a peritoneal carcinomatosis model.

Editorial focus: understanding off-target effects as the key to successful RNAi therapy.

With the first RNA interference (RNAi) drug (ONPATTRO (patisiran)) on the market, we witness the RNAi therapy field reaching a critical turning point, when further improvements in drug candidate design and delivery pipelines should enable fast delivery of novel life changing treatments to patients. Nevertheless, ignoring parallel development of RNAi dedicated in vitro pharmacological profiling aiming to identify undesirable off-target activity may slow down or halt progress in the RNAi field. Since academic research is currently fueling the RNAi development pipeline with new therapeutic options, the objective of this article is to briefly summarize the basics of RNAi therapy, as well as to discuss how to translate basic research into better understanding of related drug candidate safety profiles early in the process.

Transcriptional landscape and clinical utility of enhancer RNAs for eRNA-targeted therapy in cancer.

Enhancer RNA (eRNA) is a type of noncoding RNA transcribed from the enhancer. Although critical roles of eRNA in gene transcription control have been increasingly realized, the systemic landscape and potential function of eRNAs in cancer remains largely unexplored. Here, we report the integration of multi-omics and pharmacogenomics data across large-scale patient samples and cancer cell lines. We observe a cancer-/lineage-specificity of eRNAs, which may be largely driven by tissue-specific TFs. eRNAs are involved in multiple cancer signaling pathways through putatively regulating their target genes, including clinically actionable genes and immune checkpoints. They may also affect drug response by within-pathway or cross-pathway means. We characterize the oncogenic potential and therapeutic liability of one eRNA, NET1e, supporting the clinical feasibility of eRNA-targeted therapy. We identify a panel of clinically relevant eRNAs and developed a user-friendly data portal. Our study reveals the transcriptional landscape and clinical utility of eRNAs in cancer.

Downregulated Long Noncoding RNA PART1 Inhibits Proliferation and Promotes Apoptosis in Bladder Cancer.

OBJECTIVE: In this study, we aimed to clarify the effects of long noncoding ribonucleic acid prostrate androgen-regulated transcript-1 on bladder cancer cell proliferation and apoptosis. METHODS: Microarrays were implemented to investigate the long noncoding ribonucleic acid expression profiles in bladder cancer tissue (N = 9) and in noncancer bladder tissue (N = 5). Relative prostrate androgen-regulated transcript-1 expression levels in tissue samples or cell lines were detected by real-time quantitative reverse transcription-polymerase chain reaction. Prostrate androgen-regulated transcript-1 expression was enhanced by the transfection of pcDNA3.1-prostrate androgen-regulated transcript-1 and downregulated by the infection with pcMV-sh prostrate androgen-regulated transcript-1. Additionally, cell proliferation and apoptosis were measured by the cell counting kit-8 assay and flow cytometry, respectively. Cell invasion was determined by a Transwell assay. RESULTS: Prostrate androgen-regulated transcript-1 expression was upregulated in bladder cancer tissues compared to adjacent nontumor tissues. Furthermore, prostrate androgen-regulated transcript-1 levels were successfully upregulated by pcDNA3.1-prostrate androgen-regulated transcript-1 and depleted by pCMV-sh prostrate androgen-regulated transcript-1 in bladder cancer cell lines (5637, T24). Enhanced prostrate androgen-regulated transcript-1 expression promoted cell proliferation and invasion and inhibited cell apoptosis. However, knockdown of prostrate androgen-regulated transcript-1 expression inhibited cell proliferation and invasion and induced cell apoptosis. CONCLUSION: In summary, these data suggest that the knockdown of prostrate androgen-regulated transcript-1 represents a tumor suppressor player in bladder cancer and contributes to the inhibition of tumor proliferation, the promotion of cell apoptosis, and the suppression of cell invasion. Prostrate androgen-regulated transcript-1 may function as a new prognostic biomarker and as a feasible therapeutic target for patients with bladder cancer.

Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase.

We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as 'negative' under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene's proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl2 concentrations (1.5-2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl2 concentrations (0.8-2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).

Targeted Degradation of a Hypoxia-Associated Non-coding RNA Enhances the Selectivity of a Small Molecule Interacting with RNA.

Small-molecule targeted recruitment of nucleases to RNA is a powerful method to affect RNA biology. Inforna, a sequence-based design approach to target RNA, enables the design of small molecules that bind to and cleave RNA in a selective and substoichiometric manner. Here, we investigate the ability of RNA-targeted degradation to improve the selectivity of small molecules targeting RNA. The microRNA-210 hairpin precursor (pre-miR-210) is overexpressed in hypoxic cancers. Previously, a small molecule (Targapremir-210 [TGP-210]) targeted this RNA in cells, but with a 5-fold window for DNA binding. Appendage of a nuclease recruitment module onto TGP-210 locally recruited ribonuclease L onto pre-miR-210, triggering its degradation. The chimera has enhanced selectivity compared with TGP-210 with nanomolar binding to the pre-miR-210, but no DNA binding, and is broadly selective for affecting RNA function in cells. Importantly, it cleaved pre-miR-210 substoichiometrically and induced apoptosis in breast cancer cells.

Insights into the development of chemical probes for RNA.

Over the past decade, the RNA revolution has revealed thousands of non-coding RNAs that are essential for cellular regulation and are misregulated in disease. While the development of methods and tools to study these RNAs has been challenging, the power and promise of small molecule chemical probes is increasingly recognized. To harness existing knowledge, we compiled a list of 116 ligands with reported activity against RNA targets in biological systems (R-BIND). In this survey, we examine the RNA targets, design and discovery strategies, and chemical probe characterization techniques of these ligands. We discuss the applicability of current tools to identify and evaluate RNA-targeted chemical probes, suggest criteria to assess the quality of RNA chemical probes and targets, and propose areas where new tools are particularly needed. We anticipate that this knowledge will expedite the discovery of RNA-targeted ligands and the next phase of the RNA revolution.

Non-coding RNAs in cardiovascular diseases: diagnostic and therapeutic perspectives.

Recent research has demonstrated that the non-coding genome plays a key role in genetic programming and gene regulation during development as well as in health and cardiovascular disease. About 99% of the human genome do not encode proteins, but are transcriptionally active representing a broad spectrum of non-coding RNAs (ncRNAs) with important regulatory and structural functions. Non-coding RNAs have been identified as critical novel regulators of cardiovascular risk factors and cell functions and are thus important candidates to improve diagnostics and prognosis assessment. Beyond this, ncRNAs are rapidly emgerging as fundamentally novel therapeutics. On a first level, ncRNAs provide novel therapeutic targets some of which are entering assessment in clinical trials. On a second level, new therapeutic tools were developed from endogenous ncRNAs serving as blueprints. Particularly advanced is the development of RNA interference (RNAi) drugs which use recently discovered pathways of endogenous short interfering RNAs and are becoming versatile tools for efficient silencing of protein expression. Pioneering clinical studies include RNAi drugs targeting liver synthesis of PCSK9 resulting in highly significant lowering of LDL cholesterol or targeting liver transthyretin (TTR) synthesis for treatment of cardiac TTR amyloidosis. Further novel drugs mimicking actions of endogenous ncRNAs may arise from exploitation of molecular interactions not accessible to conventional pharmacology. We provide an update on recent developments and perspectives for diagnostic and therapeutic use of ncRNAs in cardiovascular diseases, including atherosclerosis/coronary disease, post-myocardial infarction remodelling, and heart failure.

In vivo knockdown of antisense non-coding mitochondrial RNAs by a lentiviral-encoded shRNA inhibits melanoma tumor growth and lung colonization.

The family of non-coding mitochondrial RNAs (ncmtRNA) is differentially expressed according to proliferative status. Normal proliferating cells express sense (SncmtRNA) and antisense ncmtRNAs (ASncmtRNAs), whereas tumor cells express SncmtRNA and downregulate ASncmtRNAs. Knockdown of ASncmtRNAs with oligonucleotides induces apoptotic cell death of tumor cells, leaving normal cells unaffected, suggesting a potential application for developing a novel cancer therapy. In this study, we knocked down the ASncmtRNAs in melanoma cell lines with a lentiviral-encoded shRNA approach. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. Moreover, the treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls. These results provide additional proof of concept to the knockdown of ncmtRNAs for cancer therapy and validate lentiviral-shRNA vectors for gene therapy.

Noncoding Rnas Emerging as Novel Biomarkers in Pancreatic Cancer.

Noncoding RNAs play important regulatory roles in diverse biological processes and their misregulation might lead to different diseases, including cancer. Previous studies have reported the evolving role of miRNAs as new potential biomarkers in cancer diagnosis, prognosis, as well as predictive biomarkers of chemotherapy response or therapeutic targets. In this review, we outline the involvement of noncoding RNA in pancreatic cancer, providing an overview of known miRNAs in its diagnosis, prognosis and chemoresistance. In addition, we discuss the influence of non-coding RNAs in the metastatic behavior of pancreatic cancer, as well as the role of diet in epigenetic regulation of non-coding RNAs in cancer, which can, in turn, lead the development of new prevention's techniques or novel targets for cancer therapy.

Small RNA Pathways That Protect the Somatic Genome.

Transposable elements (TEs) are DNA elements that can change their position within the genome, with the potential to create mutations and destabilize the genome. As such, special molecular systems have been adopted in animals to control TE activity in order to protect the genome. PIWI proteins, in collaboration with PIWI-interacting RNAs (piRNAs), are well known to play a critical role in silencing germline TEs. Although initially thought to be germline-specific, the role of PIWI-piRNA pathways in controlling TEs in somatic cells has recently begun to be explored in various organisms, together with the role of endogenous small interfering RNAs (endo-siRNAs). This review summarizes recent results suggesting that these small RNA pathways have been critically implicated in the silencing of somatic TEs underlying various physiological traits, with a special focus on the Drosophila model organism.

AS-IL6 promotes glioma cell invasion by inducing H3K27Ac enrichment at the IL6 promoter and activating IL6 transcription.

Interleukin-6 (IL-6) is widely expressed in a variety of malignant tumors; thus, targeting the IL-6/STAT3 pathway represents a promising therapeutic strategy for malignant cancers. In this study, we identified a noncoding RNA, AS-IL6, which is transcribed antisense to IL6 and induces IL6 expression in glioma cells. Knockdown of AS-IL6 attenuates LPS-induced IL6 transcription. Interestingly, AS-IL6 does not change IL6 mRNA stability, but induces the enrichment of histone H3 acetylated at lysine 27 (H3K27Ac) at the IL6 promoter. In addition, we found that depletion of AS-IL6 inhibits the invasive ability of glioblastoma cells, while treatment of cells with recombinant IL6 reverses this effect. Our results reveal a novel mechanism of IL6 regulation and demonstrate an oncogenic role for AS-IL6 in glioma cells.

Novel mechanisms and approaches to overcome multidrug resistance in the treatment of ovarian cancer.

Ovarian cancer remains the leading cause of gynecological cancer-related mortality despite the advances in surgical techniques and chemotherapy drugs over the past three decades. Multidrug resistance (MDR) to chemotherapy is the major cause of treatment failure. Previous research has focused mainly on strategies to reverse MDR by targeting the MDR1 gene encoded P-glycoprotein (Pgp) with small molecular compound inhibitors. However, prior Pgp inhibitors have shown very limited clinical success because these agents have relatively low potency and high toxicity. Therefore, identification of more specific and potent new inhibitors would be useful. In addition, emerging evidence suggests that cancer stem cells (CSCs), deregulated non-coding RNA (ncRNA), autophagy, and tumor heterogeneity also contribute significantly to drug sensitivity/resistance in ovarian cancer. This review summarizes these novel mechanisms of MDR and evaluates several new concepts to overcome MDR in the treatment of ovarian cancer. These new strategies include overcoming MDR with more potent and specific Pgp inhibitors, targeting CSCs and ncRNA, modulating autophagy signaling pathway, and targeting tumor heterogeneity.

Non-coding RNAs: Therapeutic Strategies and Delivery Systems.

The vast majority of the human genome is transcribed into RNA molecules that do not code for proteins, which could be small ones approximately 20 nucleotide in length, known as microRNAs, or transcripts longer than 200 bp, defined as long noncoding RNAs. The prevalent deregulation of microRNAs in human cancers prompted immediate interest on the therapeutic value of microRNAs as drugs and drug targets. Many features of microRNAs such as well-defined mechanisms, and straightforward oligonucleotide design further make them attractive candidates for therapeutic development. The intensive efforts of exploring microRNA therapeutics are reflected by the large body of preclinical studies using oligonucleotide-based mimicking and blocking, culminated by the recent entry of microRNA therapeutics in clinical trial for several human diseases including cancer. Meanwhile, microRNA therapeutics faces the challenge of effective and safe delivery of nucleic acid therapeutics into the target site. Various chemical modifications of nucleic acids and delivery systems have been developed to increase targeting specificity and efficacy, and reduce the associated side effects including activation of immune response. Recently, long noncoding RNAs become attractive targets for therapeutic intervention because of their association with complex and delicate phenotypes, and their unconventional pharmaceutical activities such as capacity of increasing output of proteins. Here I discuss the general therapeutic strategies targeting noncoding RNAs, review delivery systems developed to maximize noncoding RNA therapeutic efficacy, and offer perspectives on the future development of noncoding RNA targeting agents for colorectal cancer.

Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example.

Regulatory Roles of Non-Coding RNAs in Colorectal Cancer.

Non-coding RNAs (ncRNAs) have recently gained attention because of their involvement in different biological processes. An increasing number of studies have demonstrated that mutations or abnormal expression of ncRNAs are closely associated with various diseases including cancer. The present review is a comprehensive examination of the aberrant regulation of ncRNAs in colorectal cancer (CRC) and a summary of the current findings on ncRNAs, including long ncRNAs, microRNAs, small interfering RNAs, small nucleolar RNAs, small nuclear RNAs, Piwi-interacting RNAs, and circular RNAs. These ncRNAs might become novel biomarkers and targets as well as potential therapeutic tools for the treatment of CRC in the near future and this review may provide important clues for further research on CRC and for the selection of effective therapeutic targets.