Tuesday, March 11, 2008

microRNA IN ONCOLOGY

8:00 Chairpersons' Remarks
Søren Møller, Ph.D., Vice President, Research and Development, Exiqon

8:10 Toward a Non-coding RNA Revolution in the Cancer Society
Riccardo Spizzo, M.D, Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center

8:40 miRNA in the Progression of Human Breast Oncogenesis
Alan Dombkowski, Assistant Professor, IEHS, Wayne State University
Breast cancer is believed to develop in a multistep process, with premalignant lesions preceding invasive carcinoma. While recent work has demonstrated the role of microRNAs in cancer, there is little known about the role of microRNAs in preneoplastic events that precede malignant transformation. In this work we characterize miRNA expression changes associated with breast oncogenesis using a multistep cell line model, representing benign to premalignant to malignant transformation, and with corresponding mouse xenografts. Global miRNA microarrays and bioinformatics analysis are used to identify miRNAs that exhibit changes in expression correlating with the progression of the disease, including premalignant events that may provide diagnostic and therapeutic targets.

9:10 The Novel microRNAs Cluster in a Genomically Unstable Region of Human chr8q24
Natalia Volfovsky, Ph.D., Bioinformatics Scientist, Advanced Biomedical Computing Center (ABCC), SAIC-Frederick/NCI-Frederick
Recent reports indicate that miRNAs may be associated with fragile sites and cancer-associated genomic regions. The PVT1 locus of 8q24 immediately downstream of c-MYC was originally identified as a "variant" breakpoint in association with chromosomal translocations found in 20% of Burkitt’s lymphomas. However the role of the region in the regulation of c-MYC is still unclear. The earlier discovery of transcriptional activity in the PVT1 region raised the possibility of a PVT1 product, but an inability to detect a protein associated with PVT1 derailed this hypothesis and left PVT1 as one of many non-coding RNAs in the transcriptome. Computational analysis of the entire PVT1 region (400kb), reveals a new cluster of the potential microRNA structures.13 candidates were identified based on criteria of species conservation and stable hairpin structure of which seven have been confirmed experimentally. One of them, miR-15PVT1, is found within a fusion transcript of PVT1 that is present in high copy number in MYC/PVT1-amlified tumors. Bioinformatics has further identified a number of potential target genes for miR-15PVT1 including members of the JAK-STAT, WNT and MAPK signaling pathways. Expression analysis of the miRNA precursors shows higher levels for virtually all transcripts in late-stage B cells compare to immature B cells suggesting possible roles in lymphoid development an/or lymphoma. Consistent with co-amplification of MYC/PVT1 in breast and colon cancer, high levels of expression of at least one precursor, miR-15PVT1, was seen in representative epithelial cancer cell lines indicating a potential broader role for these miRNAs in tumorigenesis.

9:40 The Role of let-7 families During Development and Cancer Progression
Aurora Esquela Kerscher, Ph.D., Assistant Professor, Department of Microbiology & Molecular Cell Biology, Eastern Virginia Medical School
Cancer is marked by uncontrolled proliferation and inappropriate survival of damaged cells in the body. Interestingly, many processes used to direct the proper growth, differentiation, and cell death of tissues in the developing embryo, are identical to the genetic pathways that are perturbed in the cancerous state. Recent studies have shown that microRNAs (miRNAs) control important developmental events in animals and can directly function as tumor suppressor genes and oncogenes. In C. elegans, the let-7 miRNA controls the timing of seam cell terminal differentiation during epidermal cell development. However, in let-7 mutant animals, cells frequently fail to terminally differentiate, and instead elect to divide again, a hallmark of cancer. We have shown that the let-7 family targets let-60, the nematode RAS gene and found that this genetic interaction is conserved in humans, where it has been linked to cancer. Human let-7 is poorly expressed or deleted in lung cancer, and over-expression of let-7 in lung cancer cells inhibits their growth, demonstrating a role for let-7 as a tumor suppressor gene in lung tissue. We have also shown that human let-7 is expressed in the developing mammalian lung and regulates the expression of the RAS oncogene as well as other genes associated with cellular proliferation in human cells. We are currently investigating the role of let-7 in regulating proto-oncogene expression during development and cancer, and on using miRNAs to suppress tumorigenesis. These studies promise to reveal miRNAs as a novel class of cancer prevention genes with immense therapeutic potential.

10:10 microRNA Profiling of Breast Cancer Using a Novel Locked Nucleic Acid (LNA)-Based Microarray Søren Møller, Ph.D., Vice President, Research & Development, Exiqon Abnormal expression of microRNAs (miRNAs) in cancer implies that these small ~22-nucleotide molecules play a role in oncogenesis Therefore miRNAs may comprise a novel class of diagnostic and prognostic signatures. Here, we study the global expression profiles of miRNAs in breast cancer and normal adjacent tissue in order to identify possible new biomarkers for breast cancer. Recently, we have developed a novel microarray platform for detection of miRNA based on locked nucleic acid (LNA) modified capture probes. We used this platform for analysis of miRNA expression patterns from tumor and normal breast tissue, and found numerous differentially expressed miRNAs, including those previously reported to be associated with breast cancer, such as let-7a/d/f, miR-125a/b, miR-21, miR-32, and miR-136. The differential expression has been confirmed using miRCURY LNA qPCR. In addition, we have identified several novel miRNA candidates by 454 sequencing that have not previously been connected with breast cancer. Some of these novel miRNA signatures could have diagnostic and prognostic potential for breast cancer patients.

Sponsored by:

10:30 Coffee Break with Exhibit and Poster Viewing

11:15 microRNA Expression Profiling Using a Bead-Based Multiplex System Keld Sorensen, Ph.D., Director of R&D, Luminex Bioscience Group As microRNA research becomes more prevalent and additional microRNAs and microRNA patterns are identified, researchers will need technology that allows for both high density and high throughput screening. Many traditional technologies that allow for high-throughput applications cannot multiplex many tests at once, while many technologies that enable high-density screening cannot maintain the reproducibility required in high-throughput applications. FlexmiR microRNA assays are based on Luminex xMAP® technology which is an open-architecture platform that can be configured to perform a wide variety of bioassays quickly, cost-effectively and accurately. The favorable reaction kinetics of the liquid bead array delivered by xMAP technology give faster, more reproducible results than solid, planar arrays. This liquid array approach also offers excellent manufacturing and assay standardization due to the nature of the microspheres when compared to competing flat arrays, which are limited by solid phase kinetics. The flexibility of this bead-based microRNA system provides a universal assay platform for conducting broad screenings as well as target validations with standard panels or custom tailored panels.

Sponsored by:

11:35 microRNAs in Leukemic and Normal Hematopoietic Stem Cells
Christopher Park, M.D., Ph.D., Instructor, Pathology, Stanford University School of Medicine
Comparing miRNA expression in human acute myeloid leukemia stem cells (LSC) to their normal counterparts - hematopoietic stem cells (HSCs) and committed progenitors - is likely to identify miRNAs that are important in regulating LSC function and/or leukemogenesis. In addition, such data may identify miRNA targets for LSC-specific therapy. We have conducted a detailed analysis of miRNA expression in highly purified human AML LSC and normal bone marrow-derived HSC and progenitors. Using a highly sensitive TaqMan-based real-time PCR strategy, we have identified miRNAs that are preferentially expressed in human AML LSC, human HSC, and myeloid progenitor populations. Ectopic expression of one miRNA that is highly-expressed in normal HSC and LSC results in a chronic myeloproliferative disease when transduced into immature mouse progenitor cells. These studies show that miRNAs likely regulate normal HSC function and mediate early steps in leukemogenesis.

microRNA TARGETS FOR DIAGNOSTIC
BIOMARKERS AND THERAPEUTIC DEVELOPMENT

1:45 Chairpersons' Remarks
Michael Liebman, Ph.D., Executive Director, Windber Research Institute

1:50 Her2/neu, microRNA’s and Herceptin
Michael Liebman, Ph.D., Executive Director, Windber Research Institute
Herceptin treatment in breast cancer requires the observance of overexpression of Her2/neu in the patient, as measured by FISH and/or IHC. Only 25% of all patients overexpress her2/neu, and only 40% of these patients respond to Herceptin. In collaboration with BIOBASE, we have pursued upstream analysis of the observed gene expression differences in patients where FISH and IHC present different results and have determined that a microRNA appears to function as a switch in determining the differential response. This has been analyzed in terms of its potential use as a diagnostic and/or therapeutic target to improve decision-making for treatment in breast cancer patients.

2:20 microRNA-directed Therapy for Pancreatic Cancer
Thomas Schmittgen, Ph.D., Associate Professor, College of Pharmacy, Ohio State University
Our prior gene expression profiling studies has identified a large number of miRNAs with increased expression in pancreatic adenocarcinoma tissues including miR-21, miR-221, miR-212 and miR-301. The purpose of this study is to inhibit the expression of these miRNAs in pancreatic cancer cell lines and normal pancreas ductal epithelial cells using chemically modified antisense oligonucleotides. Our results demonstrate that low nM concentrations of antisense to miR-21 and miR-221 is toxic to the pancreatic cancer cell lines but not to normal pancreas epithelial cells. These results suggest that inhibition of miRNA is selectively toxic to pancreas cancer cells. Future studies will study the role of antisense oligonucleotides in pancreas cancer using a transgenic mouse model of pancreatic adenocarcinoma.

2:50 Refreshment Break with Exhibit and Poster Viewing3:30 MicroRNA-based Diagnostic Tools for Cancer
Nitzan Rosenfeld, Ph.D., Head, Computational Biology, Rosetta Genomics, Rehovot, Israel
We have developed an integrative platform for profiling known and novel microRNAs, and protocols for the extraction of microRNAs from formalin-fixed, paraffin-embedded archival tissues, from fresh/frozen tissues and various body fluids. We collected and profiled hundreds of primary and metastatic tumor samples from a range of tumor sites and histological sub-types. Expression levels of specific microRNAs provide highly accurate information at critical diagnostic decision points, allowing correct classification of cancers of squamous histology or of metastases at common cancer sites. Diagnostic tests are being developed for the identification of squamous cell carcinoma vs other lung cancers, and of mesothelioma vs adenocarcinomas. The microRNA-based decisions are integrated into a binary tree classification algorithm that identifies tumor origin with high accuracy using a small number of microRNA biomarkers.

4:00 miRNA Expression Profiles Associated with Colorectal Cancer and Derived from FFPE Tissues
Mike Wilson, Ph.D., Senior Scientist, Array R&D Manager, Asuragen, Inc.

4:30 Therapeutic Targeting of MicroRNAs with Antisense Oligonucleotides
Juergen Soutschek, Ph.D., Program Project Leader, Regulus Therapeutics
Regulus develops microRNA therapeutics that have the ability to modulate multiple genes in relevant disease pathways. Using antisense oligonucleotides we previously showed beneficial effect on lipid metabolism in a couple of relevant mouse models by in vivo inhibition of the liver-expressed microRNA miR-122, which is also a host factor for HCV. We’ll report significantly improved chemical designs for in vivo miR-122 inhibition in metabolic disease and HCV.

5:00 Close of Conference