Kara Batte

MicroRNA-126 inhibits invasion in non-small cell lung carcinoma cell lines

Crk is a member of a family of adaptor proteins that are involved in intracellular signal pathways altering cell adhesion, proliferation, and migration. Increased expression of Crk has been described in lung cancer and associated with increased tumor invasiveness. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-25 nt long) that are capable of targeting genes for either degradation of mRNA or inhibition of translation. Crk is a predicted putative target gene for miR-126. Over-expression of miR126 in a lung cancer cell line resulted in a decrease in Crk protein without any alteration in the associated mRNA. These lung cancer cells exhibit a decrease in adhesion, migration, and invasion. Decreased cancer cell invasion was also evident following targeted knockdown of Crk. MiR-126 alters lung cancer cell phenotype by inhibiting adhesion, migration, and invasion and the effects on invasion may be partially mediated through Crk regulation.

Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

Methodological challenges in utilizing miRNAs as circulating biomarkers.

MicroRNAs (miRNAs) have emerged as important regulators in the post‐transcriptional control of gene expression. The discovery of their presence not only in tissues but also in extratissular fluids, including blood, urine and cerebro‐spinal fluid, together with their changes in expression in various pathological conditions, has implicated these extracellular miRNAs as informative biomarkers of disease. However, exploiting miRNAs in this capacity requires methodological rigour. Here, we report several key procedural aspects of miRNA isolation from plasma and serum, as exemplified by research in cardiovascular and pulmonary diseases. We also highlight the advantages and disadvantages of various profiling methods to determine the expression levels of plasma‐ and serum‐derived miRNAs. Attention to such methodological details is critical, as circulating miRNAs become diagnostic tools for various human diseases.

Systems biology of interstitial lung diseases: integration of mRNA and microRNA expression changes

BackgroundThe molecular pathways involved in the interstitial lung diseases (ILDs) are poorly understood. Systems biology approaches, with global expression data sets, were used to identify perturbed gene networks, to gain some understanding of the underlying mechanisms, and to develop specific hypotheses relevant to these chronic lung diseases.MethodsLung tissue samples from patients with different types of ILD were obtained from the Lung Tissue Research Consortium and total cell RNA was isolated. Global mRNA and microRNA were profiled by hybridization and amplification-based methods. Differentially expressed genes were compiled and used to identify critical signaling pathways and potential biomarkers. Modules of genes were identified that formed a regulatory network, and studies were performed on cultured cells in vitro for comparison with the in vivo results.ResultsBy profiling mRNA and microRNA (miRNA) expression levels, we found subsets of differentially expressed genes that distinguished patients with ILDs from controls and that correlated with different disease stages and subtypes of ILDs. Network analysis, based on pathway databases, revealed several disease-associated gene modules, involving genes from the TGF-β, Wnt, focal adhesion, and smooth muscle actin pathways that are implicated in advancing fibrosis, a critical pathological process in ILDs. A more comprehensive approach was also adapted to construct a putative global gene regulatory network based on the perturbation of key regulatory elements, transcription factors and microRNAs. Our data underscores the importance of TGF-β signaling and the persistence of smooth muscle actin-containing fibroblasts in these diseases. We present evidence that, downstream of TGF-β signaling, microRNAs of the miR-23a cluster and the transcription factor Zeb1 could have roles in mediating an epithelial to mesenchymal transition (EMT) and the resultant persistence of mesenchymal cells in these diseases.ConclusionsWe present a comprehensive overview of the molecular networks perturbed in ILDs, discuss several potential key molecular regulatory circuits, and identify microRNA species that may play central roles in facilitating the progression of ILDs. These findings advance our understanding of these diseases at the molecular level, provide new molecular signatures in defining the specific characteristics of the diseases, suggest new hypotheses, and reveal new potential targets for therapeutic intervention.

Analyzing the circulating microRNAs in exosomes/extracellular vesicles from serum or plasma by qRT-PCR.

Small extracellular vesicles are released from both healthy and disease cells to facilitate cellular communication. They have a wide variety of names including exosomes, microvesicles and microparticles. Depending on their size, very small extracellular vesicles originating from the endocytic pathway have been called exosomes and in some cases nanovesicles. Collectively, extracellular vesicles are important mediators of a wide variety of functions including immune cell development and homeostasis. Encapsulated in the extracellular vesicles are proteins and nucleic acids including mRNA and microRNA molecules. MicroRNAs are small, non-coding RNA molecules implicated in the post-transcriptional control of gene expression that have emerged as important regulatory molecules and are involved in disease pathogenesis including cancer. In some diseases, not only does the quantity and the subpopulations of extracellular vesicles change in the peripheral blood but also microRNAs. Here, we described the analysis of peripheral blood extracellular vesicles by flow cytometry and the RNA extraction from extracellular vesicles isolated from the plasma or serum to profile microRNA expression.

HDAC8, A Potential Therapeutic Target for the Treatment of Malignant Peripheral Nerve Sheath Tumors (MPNST)

Introduction HDAC isoform-specific inhibitors may improve the therapeutic window while limiting toxicities. Developing inhibitors against class I isoforms poses difficulties as they share high homology among their catalytic sites; however, HDAC8 is structurally unique compared to other class I isoforms. HDAC8 inhibitors are novel compounds and have affinity for class I HDAC isoforms demonstrating anti-cancer effects; little is known about their activity in malignant peripheral nerve sheath tumors (MPNST). Recently, we demonstrated anti-MPNST efficacy of HDAC8i in human and murine-derived MPNST pre-clinical models; we now seek to consider the potential therapeutic inhibition of HDAC8 in MPNST. Methods Four Human MPNST cell lines, a murine-derived MPNST cell line, and two HDAC8 inhibitors (PCI-34051, PCI-48012; Pharmacyclics, Inc. Sunnyvale, CA) were studied. Proliferation was determined using MTS and clonogenic assays. Effects on cell cycle were determined via PI FACS analysis; effects on apoptosis were determined using Annexin V-PI FACS analysis and cleaved caspase 3 expression. In vivo growth effects of HDAC8i were evaluated using MPNST xenograft models. 2D gel electrophoresis and mass spectrometry were used to identify potential HDAC8 deacetylation substrates. Results HDAC8i induced cell growth inhibition and marked S-phase cell cycle arrest in human and murine-derived MPNST cells. Relative to control, HDAC8i induced apoptosis in both human and murine-derived MPNST cells. HDAC8i exhibited significant effects on MPNST xenograft growth (p=0.001) and tumor weight (p=0.02). Four potential HDAC8 substrate targets were identified using a proteomic approach: PARK7, HMGB1, PGAM1, PRDX6. Conclusions MPNST is an aggressive sarcoma that is notoriously therapy-resistant, hence the urgent need for improved anti-MPNST therapies. HDAC8 inhibition may be useful for MPNST by improving efficacy while limiting toxicities as compared to pan-HDACis.

Exosome-derived miR-25-3p and miR-92a-3p stimulate liposarcoma progression

Despite the development of combined modality treatments against liposarcoma in recent years, a significant proportion of patients respond only modestly to such approaches, possibly contributing to local or distant recurrence. Early detection of recurrent or metastatic disease could improve patient prognosis by triggering earlier clinical intervention. However, useful biomarkers for such purposes are lacking. Using both patient plasma samples and cell lines, we demonstrate here that miR-25-3p and miR-92a-3p are secreted by liposarcoma cells through extracellular vesicles and may be useful as potential biomarkers of disease. Both miR-25-3p and miR-92a-3p stimulated secretion of proinflammatory cytokine IL6 from tumor-associated macrophages in a TLR7/8-dependent manner, which in turn promoted liposarcoma cell proliferation, invasion, and metastasis via this interaction with the surrounding microenvironment. Our findings provide novel and previously unreported insight into liposarcoma progression, identifying communication between liposarcoma cells and their microenvironment as a process critically involved in liposarcoma progression. This study establishes the possibility that the pattern of circulating miRNAs may identify recurrence prior to radiological detectability while providing insight into disease outcome and as a possible approach to monitor treatment efficacy. Cancer Res; 77(14); 3846-56. ©2017 AACR.

Mocetinostat combined with gemcitabine for the treatment of leiomyosarcoma: Preclinical correlates

Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastatic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of mocetinostat alone and in combination with gemcitabine in LMS. Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included the class I HDAC inhibitor, mocetinostat, and nucleoside analog, gemcitabine. MTS and clonogenic assays were used to evaluate the effect of mocetinostat on LMS cell growth. Cleaved caspase 3/7 analysis was used to determine the effects of mocetinostat on apoptosis. Compusyn software was used to determine in vitro synergy studies for the combination of mocetinostat plus gemcitabine. A LMS xenograft model in SCID mice was used to test the impact of mocetinostat alone, gemcitabine alone and the combination of mocetinostat plus gemcitabine. Mocetinostat abrogated LMS cell growth and clonogenic potential, and enhanced apoptosis in LMS cell lines. The combination of mocetinostat plus gemcitabine exhibited a synergistic effect in LMS cells in vitro. Similarly, mocetinostat combined with gemcitabine resulted in superior anti-LMS effects in vivo. Mocetinostat reduced the expression of gemcitabine-resistance markers RRM1, RRM2, and increased the expression of gemcitabine-sensitivity marker, hENT1, in LMS cells. LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of mocetinostat combined with gemcitabine for the treatment of LMS.

Abstract 966: Circulating exosomal miRNAs as potential biomarker in liposarcoma

Liposarcoma (LPS) is the most common soft tissue sarcoma histological subtype. Despite the development of combined modality treatments in recent years, a significant proportion of patients respond poorly to chemotherapy, leading to local recurrence or distant metastasis. Thus, early detection of recurrent or metastatic disease or early decision making could improve patient prognosis. However, there are no useful biomarkers for these purposes. Thus, the discovery of novel biomarkers to detect tumors, predict their drug sensitivity, and monitor their progression is one of the most important challenges that must be overcome. miRNAs are short (approximately 22 nucleotides in length) non-coding RNAs (ncRNAs) that regulate gene expression by binding to specific mRNA targets and promoting their degradation and/or translational inhibition; since their discovery in body fluids (secreted in cell-borne membrane vesicles or associated with macromolecular complexes), circulating blood-borne microRNAs are being investigated as potent minimally invasive biomarkers of several diseases including tumors. In this study, our goal is to identify potential biomarkers in the peripheral blood of LPS patients that could be useful for LPS diagnostic, prognostic and therapeutic purposes. To determine the miRNA expression profiles of LPS in peripheral blood (PB) and in peripheral blood exosomes (PBX), a series of 16 human LPS patient specimens and 8 healthy controls was analyzed on a miRNA microarray platform capable of detecting 800 human miRNAs (nCounter Human microRNA expression Assay Nanostring v3). Validation of the array was performed by qRT-PCR on a new set of samples (10 human LPS and 9 healthy controls). To identify differentially expressed miRNAs, we compared miRNAs expression profiles from PBX of 15 patients to 8 healthy samples. A total of 26 miRNAs were significantly upregulated and 3 miRNAs were significantly down regulated (p Our study has identified a specific miRNA signature in circulating exosomes that may have a novel role in the diagnosis, prognosis and or treatment of LPS patients in a clinical setting. Citation Format: Lucia Casadei, Chad Creighton, Kara Batte, Dina Chelouche-Lev, Carlo M. Croce, Raphael E. Pollock. Circulating exosomal miRNAs as potential biomarker in liposarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 966.