Publications by Year: 2013

MicroRNAs, a class of small, non-coding RNAs, are now widely known for their importance in many aspects of biology. These small regulatory RNAs have critical functions in diverse biological events, including development and disease. Recent findings show that microRNAs are essential for lifespan determination in the model organisms, Caenorhabditis elegans and Drosophila, suggesting that microRNAs are also involved in the complex process of ageing. Further, short RNA fragments derived from longer parental RNAs, such as transfer RNA cleavage fragments, have now emerged as a novel class of regulatory RNAs that inhibit translation in response to stress. In addition, the RNA editing pathway is likely to act in the double-stranded RNA-mediated silencing machinery to suppress unfavorable RNA interference activity in the ageing process. These multiple, redundant layers in gene regulatory networks may make it possible to both stably and flexibly regulate genetic pathways in ensuring robustness of developmental and ageing processes.

Once considered genetic "oddities", microRNAs (miRNAs) are now recognized as key epigenetic regulators of numerous biological processes, including some with a causal link to the pathogenesis, maintenance, and treatment of cancer. The crux of small RNA-based therapeutics lies in the antagonism of potent cellular targets; the main shortcoming of the field in general, lies in ineffective delivery. Inhibition of oncogenic miRNAs is a relatively nascent therapeutic concept, but as with predecessor RNA-based therapies, success hinges on delivery efficacy. This review will describes the canonical (e.g. pharmacokinetics and clearance, cellular uptake, endosome escape, etc.) and non-canonical (e.g. spatial localization and accessibility of miRNA, technical limitations of miRNA inhibition, off-target impacts, etc.) challenges to the delivery of antisense-based anti-miRNA therapeutics (i.e. antimiRs) for the treatment of cancer. Emphasis will be placed on how the current leading antimiR platforms-ranging from naked chemically modified oligonucleotides to nanoscale delivery vehicles-are affected by and overcome these barriers. The perplexity of antimiR delivery presents both engineering and biological hurdles that must be overcome in order to capitalize on the extensive pharmacological benefits of antagonizing tumor-associated miRNAs.

Radiotherapy is a form of cancer treatment that utilizes the ability of ionizing radiation to induce cell inactivation and cell death, generally via inflicting DNA double-strand breaks. However, different tumors and their normal surrounding tissues are not equally sensitive to radiation, posing a major challenge in the field: to seek out factors that influence radiosensitivity. In this review, we summarize the evidence for microRNA (miRNA) involvement in the radioresponse and discuss their potential as radiosensitizers. MicroRNAs are endogenous small, noncoding RNAs that regulate gene expression posttranscriptionally, influencing many processes including, as highlighted here, cellular sensitivity to radiation. Profiling studies demonstrate that miRNA expression levels change in response to radiation, while certain miRNAs, when overexpressed or knocked down, alter radiosensitivity. Finally, we discuss specific miRNA-target pairs that affect response to radiation and DNA damage as good potential targets for modulating radioresponsitivity.

Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of KrasLSL-G12D/+; p53LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra-G12D and p53R172 . While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

A select group of microRNAs identified in blood samples can differentiate between Alzheimer's disease, other neurological disorders and age-matched healthy controls with high accuracy.

MOTIVATION: MicroRNAs (miRNAs) play a crucial role in tumorigenesis and development through their effects on target genes. The characterization of miRNA-gene interactions will lead to a better understanding of cancer mechanisms. Many computational methods have been developed to infer miRNA targets with/without expression data. Because expression datasets are in general limited in size, most existing methods concatenate datasets from multiple studies to form one aggregated dataset to increase sample size and power. However, such simple aggregation analysis results in identifying miRNA-gene interactions that are mostly common across datasets, whereas specific interactions may be missed by these methods. Recent releases of The Cancer Genome Atlas data provide paired expression profiling of miRNAs and genes in multiple tumors with sufficiently large sample size. To study both common and cancer-specific interactions, it is desirable to develop a method that can jointly analyze multiple cancers to study miRNA-gene interactions without combining all the data into one single dataset.

RESULTS: We developed a novel statistical method to jointly analyze expression profiles from multiple cancers to identify miRNA-gene interactions that are both common across cancers and specific to certain cancers. The benefit of this joint analysis approach is demonstrated by both simulation studies and real data analysis of The Cancer Genome Atlas datasets. Compared with simple aggregate analysis or single sample analysis, our method can effectively use the shared information among different but related cancers to improve the identification of miRNA-gene interactions. Another useful property of our method is that it can estimate similarity among cancers through their shared miRNA-gene interactions.

AVAILABILITY AND IMPLEMENTATION: The program, MCMG, implemented in R is available at http://bioinformatics.med.yale.edu/group/.

For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters–not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals–e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death.

MicroRNAs (miRNAs) have emerged as key genetic regulators of a wide variety of biological processes, including growth, proliferation, and survival. Recent advances have led to the recognition that miRNAs can act as potent oncogenes and tumor suppressors, playing crucial roles in the initiation, maintenance, and progression of the oncogenic state in a variety of cancers. Determining how miRNA expression and function is altered in cancer is an important goal, and a necessary prerequisite to the development and adoption of miRNA-based therapeutics in the clinic. Highly promising clinical applications of miRNAs are the use of miRNA signatures as biomarkers for cancer (for example, for early detection or diagnosis), and therapeutic supplementation or inhibition of specific miRNAs to alter the cancer phenotype. In this review, we discuss the main methods used for miRNA profiling, and examine key miRNAs that are commonly altered in a variety of tumors. Current studies underscore the functional versatility and potency of miRNAs in various aspects of the cancer phenotype, pointing to their potential clinical applications. Consequently, we discuss the application of miRNAs as biomarkers, clinical agents, and therapeutic targets, highlighting both the enormous potential and major challenges in this field.

Targeted therapeutic approaches have seen tremendous advances in the last decade, for good reason. Specifically intervening with a disease-causing gene can revert the deleterious phenotype while eliminating the toxicity often associated with broad-spectrum agents. Unfortunately, because these selective agents hit one target in a single location, acquired resistance is often high. An arguably better treatment approach includes coupling multiple targeted agents or using an agent that hits an individual target in several independent locations and/or alters multiple relevant targets in the disease-causing pathway(s), precisely the approach taken by Nishimura and colleagues in their recent report aimed at identifying a better treatment option for ovarian cancer.