Increased expression of microRNA-155-5p by alveolar type II cells contributes to development of lethal ARDS in H1N1 influenza A virus-infected mice.

Alveolar type II (ATII) cells are essential to lung function and a primary site of influenza A virus (IAV) replication. Effects of IAV infection on ATII cell microRNA (miR) expression have not been comprehensively investigated. Infection of C57BL/6 mice with 10,000 or 100 pfu/mouse of IAV A/WSN/33 (H1N1) significantly altered expression of 73 out of 1908 mature murine miRs in ATII cells at 2 days post-infection (d.p.i.) and 253 miRs at 6 d.p.i. miR-155-5p (miR-155) showed the greatest increase in expression within ATII cells at both timepoints and the magnitude of this increase correlated with inoculum size and pulmonary edema severity. Influenza-induced lung injury was attenuated in C57BL/6-congenic miR-155-knockout mice without affecting viral replication. Attenuation of lung injury was dependent on deletion of miR-155 from stromal cells and was recapitulated in ATII cell-specific miR-155-knockout mice. These data suggest that ATII cell miR-155 is a potential therapeutic target for IAV-induced ARDS.

Extracellular miRNAs as biomarkers in cancer.

Cancer is the leading cause of death worldwide. Despite significant progress in the field leading to identification of molecular signatures of individual tumors and the development of targeted therapies, early cancer diagnosis remains a clinical challenge. The emerging era of personalized medicine has intensified research towards biomarkers that can be obtained via noninvasive means. The recent discovery of extracellular vesicles (EVs), nano-vesicles secreted by the cell, in circulation has stimulated interest in their clinical utility as cancer biomarkers. EVs are secreted from all types of cells and their contents reflect the physiological and pathological state of the cell. Multiple clinical trials are underway investigating the clinical potential of EV content to serve as biomarkers and therapeutics. However, much work remains to translate EV content into clinical application.

Stromal Caveolin-1 Is Associated With Response and Survival in a Phase II Trial of nab-Paclitaxel With Carboplatin for Advanced NSCLC Patients.

UNLABELLED: In this phase II trial, carboplatin with nanoparticle albumin-bound (nab)-paclitaxel as first-line therapy for advanced non-small-cell lung cancer (NSCLC) was evaluated. Most patients had squamous cell histology. Tumor-associated stromal caveolin-1 (Cav-1) expression was correlated with improved response rate and survival in NSCLC patients who received nab-paclitaxel in this phase II trial. These results suggest Cav-1 might serve as a potential biomarker in this patient population. BACKGROUND: The combination of bevacizumab with platinum-based chemotherapy results in greater response rate (RR) and overall survival (OS) in advanced non-small-cell lung cancer (NSCLC). Bevacizumab is contraindicated in patients with squamous histology or hemoptysis. Nanoparticle albumin-bound (nab)-paclitaxel is a novel formulation of paclitaxel with greater dose tolerance and improved efficacy. We hypothesized that nab-paclitaxel and carboplatin would be superior to alternative doublets in advanced NSCLC patients ineligible for bevacizumab. PATIENTS AND METHODS: We conducted a single-arm phase II trial (NCT00729612) with carboplatin and nab-paclitaxel on day 1 of a 21-day cycle to evaluate RR (primary end point), safety, toxicity, and OS. Eligibility included: squamous histology, hemoptysis, or ongoing anticoagulation. Correlative studies included immunohistochemistry for secreted protein acid rich in cysteine (SPARC) and caveolin-1 (Cav-1). RESULTS: Sixty-three patients were enrolled. Most patients had squamous cell carcinoma (n = 48); other reasons for eligibility included hemoptysis (n = 11) and anticoagulation (n = 2). Toxicity Grade ≥ 3/4 included neuropathy, cytopenias, and fatigue. RR was 38% (24 partial response/0 complete response); 20 patients had stable disease (32%). Median progression-free survival was 5 months and median OS was 9.7 months. Immunohistochemistry for SPARC and Cav-1 was performed in 38 and 37 patients respectively. Although no association was found for SPARC expression in tumor or stroma with RR or OS, we found that higher Cav-1 levels in tumor-associated stroma was associated with improved RR and OS. CONCLUSION: Carboplatin and nab-paclitaxel every 21 days demonstrated promising efficacy with tolerable toxicity in NSCLC patients ineligible for bevacizumab therapy. Further analysis and validation of Cav-1 and SPARC expression in tumor and stromal compartments as prognostic and/or predictive biomarkers of NSCLC or nab-paclitaxel treatment is warranted.

MicroRNA as tools and therapeutics in lung cancer.

Lung cancer is the number one cause of cancer related deaths. The lack of specific and accurate tools for early diagnosis and minimal targeted therapeutics both contribute to poor outcomes. The recent discovery of microRNAs (miRNAs) revealed a novel mechanism for post-transcriptional regulation in cancer and has created new opportunities for the development of diagnostics, prognostics and targeted therapeutics. In lung cancer, miRNA expression profiles distinguish histological subtypes, predict chemotherapeutic response and are associated with prognosis, metastasis and survival. Furthermore, miRNAs circulate in body fluids and hence may serve as important biomarkers for early diagnosis or stratify patients for personalized therapeutic strategies. Here, we provide an overview of the miRNAs implicated in lung cancer, with an emphasis on their clinical utility.

MicroRNA regulation of tumorigenesis, cancer progression and interpatient heterogeneity: towards clinical use.

In the past two decades, microRNAs have emerged as crucial mediators of organ development and human disease. Here, we discuss their role as drivers or suppressors of the hallmarks of cancer during tumorigenesis and progression, in defining interpatient heterogeneity and the promise of therapeutic application.

MicroRNAs in respiratory disease. A clinician's overview.

Since their initial discovery in the early 1990s, microRNAs have now become the focus of a multitude of lines of investigation ranging from basic biology to translational applications in the clinic. Previously believed to be of no biological relevance, microRNAs regulate processes fundamental to human health and disease. In diseases of the lung, microRNAs have been implicated in developmental programming, as drivers of disease, potential therapeutic targets, and clinical biomarkers; however, several obstacles must be overcome for us to fully realize their potential therapeutic use. Here, we provide for the clinician an overview of microRNA biology in selected diseases of the lung with a focus on their potential clinical application.

A mathematical model for microRNA in lung cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Lack of early detection and limited options for targeted therapies are both contributing factors to the dismal statistics observed in lung cancer. Thus, advances in both of these areas are likely to lead to improved outcomes. MicroRNAs (miRs or miRNAs) represent a class of non-coding RNAs that have the capacity for gene regulation and may serve as both diagnostic and prognostic biomarkers in lung cancer. Abnormal expression patterns for several miRNAs have been identified in lung cancers. Specifically, let-7 and miR-9 are deregulated in both lung cancers and other solid malignancies. In this paper, we construct a mathematical model that integrates let-7 and miR-9 expression into a signaling pathway to generate an in silico model for the process of epithelial mesenchymal transition (EMT). Simulations of the model demonstrate that EGFR and Ras mutations in non-small cell lung cancers (NSCLC), which lead to the process of EMT, result in miR-9 upregulation and let-7 suppression, and this process is somewhat robust against random input into miR-9 and more strongly robust against random input into let-7. We elected to validate our model in vitro by testing the effects of EGFR inhibition on downstream MYC, miR-9 and let-7a expression. Interestingly, in an EGFR mutated lung cancer cell line, treatment with an EGFR inhibitor (Gefitinib) resulted in a concentration specific reduction in c-MYC and miR-9 expression while not changing let-7a expression. Our mathematical model explains the signaling link among EGFR, MYC, and miR-9, but not let-7. However, very little is presently known about factors that regulate let-7. It is quite possible that when such regulating factors become known and integrated into our model, they will further support our mathematical model.

Epigenetic regulation of miR-17~92 contributes to the pathogenesis of pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung fibrosis with a high mortality rate. In organ repair and remodeling, epigenetic events are important. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and can target epigenetic molecules important in DNA methylation. The miR-17~92 miRNA cluster is critical for lung development and lung epithelial cell homeostasis and is predicted to target fibrotic genes and DNA methyltransferase (DNMT)-1 expression. OBJECTIVES: We investigated the miR-17~92 cluster expression and its role in regulating DNA methylation events in IPF lung tissue. METHODS: Expression and DNA methylation patterns of miR-17~92 were determined in human IPF lung tissue and fibroblasts and fibrotic mouse lung tissue. The relationship between the miR-17~92 cluster and DNMT-1 expression was examined in vitro. Using a murine model of pulmonary fibrosis, we examined the therapeutic potential of the demethylating agent, 5'-aza-2'-deoxycytidine. MEASUREMENTS AND MAIN RESULTS: Compared with control samples, miR-17~92 expression was reduced in lung biopsies and lung fibroblasts from patients with IPF, whereas DNMT-1 expression and methylation of the miR-17~92 promoter was increased. Several miRNAs from the miR-17~92 cluster targeted DNMT-1 expression resulting in a negative feedback loop. Similarly, miR-17~92 expression was reduced in the lungs of bleomycin-treated mice. Treatment with 5'-aza-2'-deoxycytidine in a murine bleomycin-induced pulmonary fibrosis model reduced fibrotic gene and DNMT-1 expression, enhanced miR-17~92 cluster expression, and attenuated pulmonary fibrosis. CONCLUSIONS: This study provides insight into the pathobiology of IPF and identifies a novel epigenetic feedback loop between miR-17~92 and DNMT-1 in lung fibrosis.

Gene expression networks in COPD: microRNA and mRNA regulation.

BACKGROUND: The mechanisms underlying chronic obstructive pulmonary disease (COPD) remain unclear. MicroRNAs (miRNAs or miRs) are small non-coding RNA molecules that modulate the levels of specific genes and proteins. Identifying expression patterns of miRNAs in COPD may enhance our understanding of the mechanisms of disease. A study was undertaken to determine if miRNAs are differentially expressed in the lungs of smokers with and without COPD. miRNA and mRNA expression were compared to enrich for biological networks relevant to the pathogenesis of COPD. METHODS: Lung tissue from smokers with no evidence of obstructive lung disease (n=9) and smokers with COPD (n=26) was examined for miRNA and mRNA expression followed by validation. We then examined both miRNA and mRNA expression to enrich for relevant biological pathways. RESULTS: 70 miRNAs and 2667 mRNAs were differentially expressed between lung tissue from subjects with COPD and smokers without COPD. miRNA and mRNA expression profiles enriched for biological pathways that may be relevant to the pathogenesis of COPD including the transforming growth factor ß, Wnt and focal adhesion pathways. miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD compared with smokers without obstruction. miR-15b was increased in COPD samples compared with smokers without obstruction and localised to both areas of emphysema and fibrosis. miR-15b was differentially expressed within GOLD classes of COPD. Expression of SMAD7, which was validated as a target for miR-15b, was decreased in bronchial epithelial cells in COPD. CONCLUSIONS: miRNA and mRNA are differentially expressed in individuals with COPD compared with smokers without obstruction. Investigating these relationships may further our understanding of the mechanisms of disease.

Non-coding RNAs in cancer initiation and progression and as novel biomarkers.

Cancer represents a complex group of heterogeneous diseases. While many cancers share fundamental biological processes (hallmarks of cancer) necessary for their development and progression, cancers also distinguish themselves by their dependence on distinct oncogenic pathways. Over the last decade, targeted therapies have been introduced to the clinic with variable success. In truth, single targeted therapies may be successful in only a subset of malignancies but insufficient to address malignancies that often rely on multiple pathways, thus evading single targeted agents. Investigators have recently identified potentially functional components of the human genome that were previously thought to have no biological function. This discovery has added to the already established complexity of gene regulation in the pathogenesis of cancer. Non-coding RNAs represent key regulators of gene expression. Improved knowledge of their biogenesis and function may in turn lead to a better understanding of the heterogeneity of malignancies and eventually be leveraged as diagnostic, prognostic and therapeutic targets. MicroRNAs (miRNAs or miRs) for example, have the capacity for the regulation of multiple genes and thus redirection or reprogramming of biological pathways. However, several other members of the non-coding RNA family may be of equal biological relevance. In this review, we provide a perspective on emerging concepts in the clinical application of miRNA and other non-coding RNAs as biomarkers in cancer with an eye on the eventual integration of both miRNA and other non-coding RNA biology into our understanding of cancer pathogenesis and treatment.

MicroRNAs as therapeutic targets in cancer.

Cancer remains a worldwide epidemic. An improved understanding of the underlying molecular mechanisms and development of effective targeted therapies are still required for many deadly cancers. The discovery of microRNAs (miRNAs or miRs) nearly 20 years ago introduced a new layer of complexity to gene regulation, but it also afforded us the opportunity to further our understanding of the molecular pathogenesis of cancers. Dysregulation of miRNAs is fundamental to the pathogenesis of many cancers based on their involvement in basic cellular functions. In addition, these previously underrecognized, noncoding RNAs have the capacity to target tens to hundreds of genes simultaneously. Thus, they are attractive candidates as prognostic biomarkers and therapeutic targets in cancer. However, several challenges remain in translating our current understanding of miRNAs to clinical therapies. Herein, we provide a review of the current knowledge of miRNAs in both solid and hematological malignancies with a focus on their potential application as therapeutic targets in cancer.

MicroRNA in chronic lymphocytic leukemia: transitioning from laboratory-based investigation to clinical application.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia among adults in the Western world, with an incidence of approximately 1 out of 100,000 patients per year. CLL is characterized by the clonal expansion of immature CD5(+) B cells. Although cytotoxic agents remain the mainstay of therapy, the disease of up to 20% of patients is not controlled with standard therapies. Therefore, there remains a need for novel therapeutic strategies. MicroRNAs (miRNAs or miRs), first identified nearly two decades ago, are noncoding RNAs that have the capacity for simultaneous regulation of tens to hundreds of genes. An association between CLL-associated chromosomal abnormalities and miRNA deregulation is beginning to emerge. miRNAs may play a biological role in the pathogenesis of CLL: specific miRNAs (miR-15a and miR-16-1) are located at a chromosomal region (13q14.3) that is often absent in patients with CLL. These same miRNAs are relevant to cellular phenotype and in vivo development of disease. This finding has led to a rapidly expanding series of investigations linking miRNAs to CLL. As a result, miRNAs are currently under investigation as diagnostic and prognostic biomarkers as well as potential therapeutic targets in CLL.

MicroRNA dysregulation in cancer: opportunities for the development of microRNA-based drugs.

MicroRNAs (miRNAs) are 19- to 24-nucleotide, non-coding RNAs that have been implicated in both solid and hematological malignancies. Given the ability of miRNAs to target multiple genes and key biological processes, these molecules have received intensive research interest both as biomarkers and as therapeutic agents. The prospect of leveraging miRNAs to complement current therapeutic strategies in cancer is appealing, but is also complex. Simultaneously targeting multiple genes has both advantages and disadvantages for miRNA therapeutic delivery, and achieving target organ specificity while limiting off-target effects remains a major challenge in the translation of novel discoveries in research to the successful therapeutic delivery of miRNA in the clinic. This feature review discusses the current status of therapeutic approaches for miRNA in cancer, as well as potential challenges and future strategies.

MicroRNAs in cancer: personalizing diagnosis and therapy.

MicroRNAs (miRNAs) are 19-24nt noncoding RNAs that have been implicated in the pathogenesis of both solid and hematological malignancies. Frequently located in fragile chromosomal regions, miRNAs are essential to key biological functions, such as cellular differentiation, apoptosis, and growth. miRNAs may serve as either tumor suppressors or oncogenes. As a result, they have the potential to serve as both biomarkers and therapeutic agents in cancer. Based on our presentation at the recent Towards Personalized Cancer Medicine conference held in Barcelona, Spain, May 19-21, 2010, we provide an overview of the current knowledge of miRNA deregulation in solid and hematological malignancies and their application as biomarkers of disease.

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods.

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.