Non-Coding RNAs: Functional Aspects and Diagnostic Utility in Oncology.

Noncoding RNAs (ncRNAs) have been found to have roles in a large variety of biological processes. Recent studies indicate that ncRNAs are far more abundant and important than initially imagined, holding great promise for use in diagnostic, prognostic, and therapeutic applications. Within ncRNAs, microRNAs (miRNAs) are the most widely studied and characterized. They have been implicated in initiation and progression of a variety of human malignancies, including major pathologies such as cancers, arthritis, neurodegenerative disorders, and cardiovascular diseases. Their surprising stability in serum and other bodily fluids led to their rapid ascent as a novel class of biomarkers. For example, several properties of stable miRNAs, and perhaps other classes of ncRNAs, make them good candidate biomarkers for early cancer detection and for determining which preneoplastic lesions are likely to progress to cancer. Of particular interest is the identification of biomarker signatures, which may include traditional protein-based biomarkers, to improve risk assessment, detection, and prognosis. Here, we offer a comprehensive review of the ncRNA biomarker literature and discuss state-of-the-art technologies for their detection. Furthermore, we address the challenges present in miRNA detection and quantification, and outline future perspectives for development of next-generation biodetection assays employing multicolor alternating-laser excitation (ALEX) fluorescence spectroscopy.

Four-color alternating-laser excitation single-molecule fluorescence spectroscopy for next-generation biodetection assays.

BACKGROUND: Single-molecule detection (SMD) technologies are well suited for clinical diagnostic applications by offering the prospect of minimizing precious patient sample requirements while maximizing clinical information content. Not yet available, however, is a universal SMD-based platform technology that permits multiplexed detection of both nucleic acid and protein targets and that is suitable for automation and integration into the clinical laboratory work flow. METHODS: We have used a sensitive, specific, quantitative, and cost-effective homogeneous SMD method that has high single-well multiplexing potential and uses alternating-laser excitation (ALEX) fluorescence-aided molecule sorting extended to 4 colors (4c-ALEX). Recognition molecules are tagged with different-color fluorescence dyes, and coincident confocal detection of ≥2 colors constitutes a positive target-detection event. The virtual exclusion of the majority of sources of background noise eliminates washing steps. Sorting molecules with multidimensional probe stoichiometries (S) and single-molecule fluorescence resonance energy transfer efficiencies (E) allows differentiation of numerous targets simultaneously. RESULTS: We show detection, differentiation, and quantification-in a single well-of (a) 25 different fluorescently labeled DNAs; (b) 8 bacterial genetic markers, including 3 antibiotic drug-resistance determinants found in 11 septicemia-causing Staphylococcus and Enterococcus strains; and (c) 6 tumor markers present in blood. CONCLUSIONS: The results demonstrate assay utility for clinical molecular diagnostic applications by means of multiplexed detection of nucleic acids and proteins and suggest potential uses for early diagnosis of cancer and infectious and other diseases, as well as for personalized medicine. Future integration of additional technology components to minimize preanalytical sample manipulation while maximizing throughput should allow development of a user-friendly ("sample in, answer out") point-of-care platform for next-generation medical diagnostic tests that offer considerable savings in costs and patient sample.

C6orf176: a novel possible regulator of cAMP-mediated gene expression.

cAMP mediates diverse cellular signals including prostaglandin (PG) E(2)-mediated intraocular pressure (IOP)-lowering activity in human ocular ciliary smooth muscle cells (hCSM). We have identified gene regulatory networks and key genes upon activation of the cAMP pathway in hCSM, using novel agonists highly selective for PGE(2) receptor subtypes EP2 or EP4, which are G protein-coupled receptors well known to activate cAMP signaling. Here we describe a novel, EP2/EP4-induced, primate-specific gene of hitherto unknown function, also known as C6orf176 (chromosome 6 open reading frame 176) and recently reclassified as noncoding RNA in NCBI's database. Its expression, as determined by quantitative real-time RT-PCR (qRT-PCR), is dramatically upregulated (>2,000-fold) subsequent to transduction of EP2/EP4/Gs/cAMP signaling not only in hCSM, but also in HEK cells overexpressing the recombinant receptors. Moreover, activation of other IOP lowering, Gs-coupled prostanoid receptors, such as DP1 and IP, as well as a direct activator of adenylyl cyclase, forskolin, also substantially upregulated C6orf176 in hCSM, while FP and TP, which are Gq-coupled prostanoid receptor subtypes, did not. Novel transcript variants carrying open reading frames, derived from an at least 67 kb genomic locus on chromosome 6q27 with putative alternative transcription start sites, were identified. Transcriptional upregulation of transcript variants as well as of two genes expressed in antisense orientation that partially overlap the transcribed C6orf176 region was observed, to varying degrees, subsequent to induction of cAMP signaling using various agonists. Small interfering RNA-mediated C6orf176 gene silencing experiments showed modulation of several cAMP-responsive genes. These transcriptional activities identify C6orf176 as a potential biomarker and/or therapeutic target in context with diseases linked to deregulated cAMP signaling. Also, the cAMP-inducible C6orf176 gene locus could be useful as a model system for studying transcriptional regulation by chromatin and RNA polymerase II.

Prostaglandin E2 receptor subtype EP2- and EP4-regulated gene expression profiling in human ciliary smooth muscle cells.

Prostanoids are an important class of intraocular pressure (IOP)-lowering antiglaucoma agents that act primarily via increased uveo-scleral aqueous humor outflow through the ciliary body. We have developed two novel PGE(2) analogs that are specific agonists for the PGE(2) receptor subtypes EP2 and EP4, respectively. To identify gene regulatory networks and key players that mediate the physiological effects observed in vivo, we performed genomewide expression studies using human ciliary smooth muscle cells. Quantitative real-time RT-PCR confirmed a largely overlapping gene expression profile subsequent to EP2 and EP4 agonist treatment, with 65 significantly regulated genes identified overall, 5 being specific for the EP2 agonist and 6 specific for the EP4 agonist. We found predicted functional cAMP-response elements in promoter regions of a large fraction of the predominantly upregulated genes, which suggests that the cAMP signaling pathway is the most important intracellular signaling pathway for these agonists in these cells. Several target genes were identified that, as part of complex regulatory networks, are implicated in tissue remodeling processes and osmoregulation (e.g., AREG, LOXL3, BMP2, AQP3) and thus may help elucidate the mechanism of action of these IOP-lowering drugs involving the uveo-scleral outflow path.

Retinoid-related molecule AGN193198 potently induces G2M arrest and apoptosis in bladder cancer cells.

The novel synthetic retinoid-related molecule 4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-oxo-propenyl]benzoic acid (AGN193198) neither binds effectively to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) nor transactivates in RAR- and RXR-mediated reporter assays. Even so, AGN193198 is potent in inducing apoptosis in human prostate and breast carcinoma cells (Keedwell et al., Cancer Res 2004;64:3302-12). Here, we extend these findings to show that AGN193198 potently and rapidly induces apoptosis in bladder carcinoma cell lines. One micromolar of AGN193198 completely abolished the growth of the transitional cell carcinoma lines UM-UC-3 and J82, and the squamous cell carcinoma line SCaBER; the transitional cell papilloma line RT-4 was slightly less sensitive to the growth inhibitory effect of AGN193198. Treated cells accumulated in the G2M phase of the cell cycle. This was accompanied by apoptosis, as revealed by staining cells for exposure of phosphatidylserine at their surface (binding of Annexin V) and FACS analysis of propidium iodide labeled cells. As reported for prostate cancer cells, AGN193198 provoked rapid activation of caspases-3 (by 6 hr), -8 (by 16 hr) and -9 (by 6 hr) in bladder cancer cells. These findings suggest that AGN193198 and related compounds, whose mechanism of action does not appear to involve RARs and RXRs, may be useful in the treatment of bladder cancer.

A retinoid-related molecule that does not bind to classical retinoid receptors potently induces apoptosis in human prostate cancer cells through rapid caspase activation.

Synthetic retinoid-related molecules, such as N-(4-hydroxyphenyl)retinamide (fenretinide) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induce apoptosis in a variety of malignant cells. The mechanism(s) of action of these compounds does not appear to involve retinoic acid receptors (RARs) and retinoid X receptors (RXRs), although some investigators disagree with this view. To clarify whether some retinoid-related molecules can induce apoptosis without involving RARs and/or RXRs, we used 4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-oxo-E-propenyl] benzoic acid (AGN193198) that neither binds effectively to RARs and RXRs nor transactivates in RAR- and RXR-mediated reporter assays. AGN193198 potently induced apoptosis in prostate, breast, and gastrointestinal carcinoma cells and in leukemia cells. AGN193198 also abolished growth (by 50% at 130-332 nM) and induced apoptosis in primary cultures established from prostatic carcinoma (13 patients) and gastrointestinal carcinoma (1 patient). Apoptosis was induced rapidly, as indicated by mitochondrial depolarization and DNA fragmentation. Molecular events provoked by AGN193198 included activation of caspase-3, -8, -9, and -10 (by 4-6 h) and the production of BID/p15 (by 6 h). These findings show that caspase-mediated induction of apoptosis by AGN193198 is RAR/RXR-independent and suggest that this compound may be useful in the treatment of prostate cancer.