Publications

The past decade has witnessed enormous progress, and has seen the noncoding RNAs (ncRNAs) turn from the so-called dark matter RNA to critical functional molecules, influencing most physiological processes in development and disease contexts. Many ncRNAs interact with each other and are part of networks that influence the cell transcriptome and proteome and consequently the outcome of biological processes. The regulatory circuits controlled by ncRNAs have become increasingly more relevant in cancer. Further understanding of these complex network interactions and how ncRNAs are regulated, is paving the way for the identification of better therapeutic strategies in cancer.

MicroRNAs (miRNA) are short, noncoding RNAs that have been implicated in many diseases, including cancers. Because miRNAs are dysregulated in disease, miRNAs show promise as highly stable biomarkers. Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable sample type to assay for biomolecules because it is a convenient storage method and is often used by pathologists for histological staining. However, extracting biomolecules from FFPE tissue is challenging because of the presence of cellular and extracellular proteins, formaldehyde cross-links, and paraffin. Moreover, most protocols to measure miRNA in FFPE tissue are time-consuming and laborious. Here, we report a simple protocol to directly measure miRNA from formalin-fixed cells, FFPE tissue sections after paraffin is removed, and FFPE tissue sections using encoded hydrogel microparticles fabricated using stop flow lithography. Measurements by these particles show agreement between formalin-fixed cells and fresh cells, and measurement of FFPE tissue with paraffin is 10% less than FFPE tissue when paraffin is removed before the assay. When normal and tumor FFPE tissue are compared using this microparticle assay, we observe differential miRNA signal for oncogenic miRNAs and tumor suppressing miRNAs. This approach reduces assay times, reduces the use of hazardous chemicals to remove paraffin, and provides a sensitive, quantitative, and multiplexed measurement of miRNA in FFPE tissue.

N6-methyladenosine (m6A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m6A homeostasis is linked to cancer1-5. m6A modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon5. METTL3 can promote translation but the mechanism and relevance of this process remain unknown1. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs-including bromodomain-containing protein 4-that is also m6A-modified in human primary lung tumours. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3-eIF3h as a potential therapeutic target for patients with cancer.

MicroRNAs (miRNA) are a class of small non-coding RNA that play a major role in various cellular processes by negatively regulating gene expression. In the past decade, miRNA dysregulation has been reported to be closely linked to inflammatory diseases. The immune response modulates cancer initiation and progression; miRNAs including let-7 family members have been shown to act as key regulators of the immune responses in various diseases and cancers. Notably, the let-7 miRNA has been reported to be closely associated with immunity, specifically with Toll-like receptors that mediate cytokine expression during pathogen infection and with the regulation of various other immune effectors. Areas covered: In this review, the authors describe the discovery of let-7 as the starting point of the RNA revolution and highlight let-7 as an efficient tool for cancer and immune therapy. Expert opinion: let-7 miRNA has emerged as a key player in cancer therapy and immune responses and it has potential role as a new immunotherapeutic target. However, while there are challenges regarding miRNA delivery, the exciting emergence of personalized medicine for cancer and immunotherapy could be beneficial for the development of let-7 therapeutics.

In just over two decades since the discovery of the first microRNA (miRNA), the field of miRNA biology has expanded considerably. Insights into the roles of miRNAs in development and disease, particularly in cancer, have made miRNAs attractive tools and targets for novel therapeutic approaches. Functional studies have confirmed that miRNA dysregulation is causal in many cases of cancer, with miRNAs acting as tumour suppressors or oncogenes (oncomiRs), and miRNA mimics and molecules targeted at miRNAs (antimiRs) have shown promise in preclinical development. Several miRNA-targeted therapeutics have reached clinical development, including a mimic of the tumour suppressor miRNA miR-34, which reached phase I clinical trials for treating cancer, and antimiRs targeted at miR-122, which reached phase II trials for treating hepatitis. In this article, we describe recent advances in our understanding of miRNAs in cancer and in other diseases and provide an overview of current miRNA therapeutics in the clinic. We also discuss the challenge of identifying the most efficacious therapeutic candidates and provide a perspective on achieving safe and targeted delivery of miRNA therapeutics.

Many RNA species have been identified as important players in the development of chronic diseases, including cancer. Over the past decade, numerous studies have highlighted how regulatory RNAs such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) play crucial roles in the development of a disease state. It is clear that the aberrant expression of miRNAs promotes tumor initiation and progression, is linked with cardiac dysfunction, allows for the improper physiological response in maintaining glucose and insulin levels, and can prevent the appropriate integration of neuronal networks, resulting in neurodegenerative disorders. Because of this, there has been a major effort to therapeutically target these noncoding RNAs. In just the past 5 years, over 100 antisense oligonucleotide-based therapies have been tested in phase I clinical trials, a quarter of which have reached phase II/III. Most notable are fomivirsen and mipomersen, which have received FDA approval to treat cytomegalovirus retinitis and high blood cholesterol, respectively. The continued improvement of innovative RNA modifications and delivery entities, such as nanoparticles, will aid in the development of future RNA-based therapeutics for a broader range of chronic diseases. Here we summarize the latest promises and challenges of targeting noncoding RNAs in disease.

The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors - lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.

INTRODUCTION: Many RNA species have been identified as important players in the development of chronic diseases including cancer. Certain classes of regulatory RNAs such as microRNAs (miRNAs) have been investigated in such detail that bona fide tumor suppressive and oncogenic miRNAs have been identified. Because of this, there has been a major effort to therapeutically target these small RNAs. One in particular, a liposomal formulation of miR-34a (MRX34), has entered Phase I trials.

AREAS COVERED: This review aims to summarize miRNA biology, its regulation within normal versus disease states and how it can be targeted therapeutically, with a particular emphasis on miR-34a. Understanding the complexity of a single miRNA will aid in the development of future RNA-based therapeutics for a broader range of chronic diseases.

EXPERT OPINION: The potential of miRNAs to be developed into anti-cancer therapeutics has become an increasingly important area of research. miR-34a is a tumor suppressive miRNA across many tumor types through its ability to inhibit cellular proliferation, invasion and tumor sphere formation. miR-34a also shows promise within certain in vivo solid tumor models. Finally, as miR-34a moves into clinical trials it will be important to determine if it can further sensitize tumors to certain chemotherapeutic agents.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no clinically proven biologically targeted treatment options. The molecular heterogeneity of TNBC and lack of high frequency driver mutations other than TP53 have hindered the development of new and effective therapies that significantly improve patient outcomes. miRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are becoming promising therapeutic agents. We performed miRNA-profiling studies in different TNBC subtypes to identify miRNAs that significantly contribute to disease progression. We found that miR-34a was lost in TNBC, specifically within mesenchymal and mesenchymal stem cell-like subtypes, whereas expression of miR-34a targets was significantly enriched. Furthermore, restoration of miR-34a in cell lines representing these subtypes inhibited proliferation and invasion, activated senescence, and promoted sensitivity to dasatinib by targeting the proto-oncogene c-SRC. Notably, SRC depletion in TNBC cell lines phenocopied the effects of miR-34a reintroduction, whereas SRC overexpression rescued the antitumorigenic properties mediated by miR-34a. miR-34a levels also increased when cells were treated with c-SRC inhibitors, suggesting a negative feedback exists between miR-34a and c-SRC. Moreover, miR-34a administration significantly delayed tumor growth of subcutaneously and orthotopically implanted tumors in nude mice, and was accompanied by c-SRC downregulation. Finally, we found that miR-34a and SRC levels were inversely correlated in human tumor specimens. Together, our results demonstrate that miR-34a exerts potent antitumorigenic effects in vitro and in vivo and suggests that miR-34a replacement therapy, which is currently being tested in human clinical trials, represents a promising therapeutic strategy for TNBC.