Noncoding Rnas and Cancer January 8-11, 2012 • Eden Roc Renaissance Beach Resort and Spa • Miami Beach, FL

An AACR Special Conference Noncoding RNAs and Cancer January 8-11, 2012 • Eden Roc Renaissance Beach Resort and Spa • Miami Beach, FL

Conference Chairperson Phillip A. Sharp David H. Koch Institute for Integrative Cancer Research at MIT Cambridge, MA

Conference Co-Chairpersons Carlo M. Croce The Ohio State University Comprehensive Cancer Center Columbus, OH

William C. Hahn Dana-Farber Cancer Institute Boston, MA

Jeannie T. Lee Massachusetts General Hospital Boston, MA

Eric N. Olson University of Texas Southwestern Medical Center Dallas, TX

Table of Contents

Supporters 3 Awards 4 Conference Program 7 Proceedings Invited Presentations 13 Proffered Presentations 25 Poster Session A 29 Poster Session B 51 Participant List 69 Author Index 77 Subject Index 83

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 1 2 NONCODING RNAS AND CANCER The AACR thanks the following organizations for their generous support of this Noncoding RNAs and Cancer conference (as of 12/6/11). Major Supporter

Supporter

The AACR thanks the following organizations for their generous support of the travel awards provided at this conference. A complete list of awardees and their affiliations is available on pages 4 and 5.

Award Supporters

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 3 Scholar-in-Training Awards Four presenters of meritorious abstracts have been selected by the Conference Co- Chairpersons to receive awards to attend this conference. All graduate and medical students, postdoctoral fellows, and physicians-in-training were eligible for consideration. The names of the Scholar-in-Training awardees, their affiliations, and their poster or proffered presentation numbers are provided below. The AACR sincerely thanks Aflac, Inc., and AstraZeneca International for their support of these awards.

AACR-Aflac, Incorporated Scholar-in-Training Awards Ngai Na Chloe Co, The University of Texas MD Anderson Cancer Center, Houston, TX, A28 Christopher DeSevo, UT Southwestern Medical Center at Dallas, Dallas, TX, A2 David J. Drasin, University of Colorado, Anschutz Medical Campus, Aurora, CO, B1

AACR-AstraZeneca International Scholar-in-Training Award Catherine S.S. Enfield,BC Cancer Research Centre, Vancouver, BC, Canada, B21

4 NONCODING RNAS AND CANCER AACR Minority Scholar in Cancer Research Award The AACR is pleased to administer this important award program that is supported by a generous grant from the Center to Reduce Cancer Health Disparities (CRCHD) of the National Cancer Institute and provides funds for the participation of meritorious minority scientists in AACR conferences. These awards are available to predoctoral (graduate or medical) students or residents, clinical or postdoctoral fellows, and junior faculty who are making contributions to cancer research and are from minority groups considered to be underrepresented in cancer and biomedical research by the National Cancer Institute. The AACR Minority Scholar in Cancer Research awardee for this conference is:

Belinda R. Hauser, Howard University, Washington, DC, B30

AACR Minority-Serving Institution Faculty Scholar in Cancer Research Award The AACR MSI Faculty Scholar Awards program is supported by a generous grant from the Center to Reduce Cancer Health Disparities of the National Cancer Institute. The purposes of the award are to increase the scientific knowledge base of faculty members at MSIs, to encourage them in their research, and to assist in inspiring their students to pursue careers in cancer research. The AACR offers these awards for participation in its meetings and conferences to full-time faculty of minority-serving institutions (historically black colleges and universities [HBCUs], Hispanic-serving institutions [HSIs], Indian tribally controlled colleges and universities [ITCCUs], and other postsecondary institutions as defined by the U.S. Department of Education) who are scientists at the assistant professor level or above. Only citizens of the United States or Canada or scientists who are permanent residents in these countries may receive one of these awards. The AACR Minority-Serving Institution Faculty Scholar in Cancer Research awardees for this conference are:

Maria del Mar Gonzalez-Pons, University of Puerto Rico Comprehensive Cancer Center, San Juan, PR

Xinbin Gu, Howard University, Washington, DC, A13

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 5 UPCOMING CONFERENCES

Advances in Prostate Cancer Research Fifth AACR Conference on Tumor Invasion and Metastasis Chairpersons: Arul M. Chinnaiyan and The Science of Cancer Health Disparities in Co-Chairpersons: Bruce R. Zetter and Zena Charles L. Sawyers Racial/Ethnic Minorities and the Medically Werb February 6-9, 2012 Underserved January 20-23, 2013 Orlando, FL Chairperson: William G. Nelson San Diego, CA October 27-30, 2012 AACR 103rd Annual Meeting 2012 San Diego, CA Ninth AACR-JCA Joint Conference Chairperson: Benjamin G. Neel Co-Chairpersons: Tyler Jacks and Kohei March 31-April 4, 2012 AACR-NCI-EORTC International Miyazono Chicago, IL Conference on Molecular Targets and February 21-25, 2013 Cancer Therapeutics Maui, HI Molecularly Targeted Therapies: Scientific Committee Chairpersons: Stefan Mechanisms of Resistance Sleijfer, James H. Doroshow, and Kenneth C. Synthetic Lethality and Cancer Co-Chairpersons: Pasi A. Jänne, Mace L. Anderson Chairperson: William C. Hahn Rothenberg, and Carlos L. Arteaga November 6-9, 2012 Additional chairpersons and location May 9-12, 2012 Dublin, Ireland to be announced San Diego, CA May 2013 Post-GWAS Horizons in Molecular Pancreatic Cancer: Progress and Epidemiology: Digging Deeper into the Animal Models of Cancer Challenges Environment Co-Chairpersons: Thomas Look, Cory Co-Chairpersons: Daniel D. Von Hoff, David Co-Chairpersons: Shelley S. Tworoger and Abate-Shen, and Terry A. Van Dyke A. Tuveson, Dafna Bar-Sagi, and Chi Van Cornelia M. Ulrich Fall 2013 Dang November 11-14, 2012 June 18-21, 2012 Hollywood, FL Bioinformatics Lake Tahoe, NV Co-Chairpersons: Andrea Califano, Arul M. AACR-NCI Conference on Cancer Chinnaiyan, and Franziska Michor Chemical Systems Biology: Assembling Genomics: The Cancer Genome Atlas and Fall 2013 and Interrogating Computational Beyond Models of the Cancer Cell by Chemical Co-Chairpersons: Matthew L. Meyerson, AACR-NCI-EORTC Molecular Targets and Perturbations Arul M. Chinnaiyan, and Barbara Wold Cancer Therapeutics Chairperson: Andrea Califano November 2012 Chairpersons to be announced Additional chairpersons and location October 19-23, 2013 to be announced Tumor Immunology Boston, MA June/July 2012 Co-Chairpersons: Elizabeth M. Jaffee, Glenn Dranoff, and Stanley Riddell Also in development: Eleventh Annual International Conference December 2-5, 2012 Pediatric Cancer on Frontiers in Cancer Prevention Miami, FL Melanoma Research KRAS Chairperson to be announced AACR-SNM Joint Conference on State- Developmental Pathways in Cancer October 16-19, 2012 of-the-Art Molecular Imaging in Cancer Anaheim, CA Biology Chromatin Remodeling in Cancer Co-Chairpersons: David Piwnica-Worms Targeted Therapies in Hematological and Carolyn Anderson Malignancies Early 2013

Please visit www.aacr.org/meetingcalendar for additional conferences and program updates. Conference Program

Sunday, January 8

4:00 p.m.-8:00 p.m. Registration

8:00 p.m.-9:00 p.m. Keynote Presentation

Physiological and pathological functions of microRNAs David Baltimore, California Institute of Technology, Pasadena, CA

9:00 p.m.-10:30 p.m. Opening Reception

Monday, January 9

7:00 a.m.-8:00 a.m. Continental Breakfast

8:00 a.m.-10:00 a.m. Session 1: Long Noncoding RNAs Session Chairperson: Jeannie T. Lee, Massachusetts General Hospital, Boston, MA

Navigating the balance of INK4/ARF expression in cancer: ncRNAs as sensors and regulators of transcription Martin J. Walsh, Mount Sinai School of Medicine, New York, NY

Regulatory networks in onco-lncRNAomics: cis-regulation and nonconservation Leonard Lipovich, Wayne State University School of Medicine, Detroit, MI

Linking RNA to human health and disease John L. Rinn, Beth Israel Deaconess Medical Center, Boston, MA

BRAFV600E remodels the melanocyte transcriptome and induces BLNCR1 to regulate melanoma cell migration* Ross J. Flockhart, Stanford University, Stanford, CA

10:00 a.m.-10:15 a.m. Break

*Short talks from proffered papers

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 7 PROGRAM

10:15 a.m.-12:15 p.m. Session 2: Chromatin Modifications Session Chairperson: Danesh Moazed, Harvard Medical School, Boston, MA

Mechanism of RNAi-mediated heterochromatin assembly Danesh Moazed

Epigenetic genome control by heterochromatin machinery and noncoding RNAs Shiv Grewal, National Cancer Institute, Bethesda, MD

Elucidation of enhancer-like RNAs Ramin Shiekhattar, The Wistar Institute, Philadelphia, PA

12:15 p.m.-2:30 p.m. Lunch On Own/Free Time

2:30 p.m.-4:30 p.m. Session 3: miRNA Biology Session Chairperson: David P. Bartel, Massachusetts Institute of Technology, Cambridge, MA

microRNAs and their regulatory targets David P. Bartel

Unraveling the complexities of p63 in cancer and stem cells Elsa R. Flores, The University of Texas MD Anderson Cancer Center, Houston, TX

microRNAs and control of metabolism Markus Stoffel, Institute of Molecular Systems Biology, Zurich, Switzerland

4:30 p.m.-6:30 p.m. Poster Session A/Reception

6:30 p.m.- Dinner On Own/Evening Off

8 NONCODING RNAS AND CANCER PROGRAM

Tuesday, January 10

7:00 a.m.-8:00 a.m. Continental Breakfast

8:00 a.m.-10:00 a.m. Session 4: Biology of Long Noncoding RNAs Session Chairperson: Jeannie T. Lee, Massachusetts General Hospital, Boston, MA

Spreading of X-chromosome inactivation via a hierarchy of defined Polycomb stations Jeannie T. Lee

Control of gene expression in the nucleus by small RNAs David R. Corey, UT Southwestern Medical Center, Dallas, TX

Small RNA pathways in germline Alexei A. Aravin, California Institute of Technology, Pasadena, CA

Functional characterization of p53-regulated long intervening noncoding RNAs (lincRNAs)* Nadya M. Dimitrova, David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA

10:00 a.m.-10:15 a.m. Break

10:15 a.m.-12:15 p.m. Session 5: miRNA Regulation in Development Session Chairperson: Eric N. Olson, UT Southwestern Medical Center, Dallas, TX

microRNA control of muscle development and disease: From new biology to new therapeutics Eric N. Olson

Long-term, efficient inhibition of miRNA function in mice using Tough Decoys delivered by adeno-associated virus vectors Philip D. Zamore, University of Massachusetts Medical School, Worcester, MA

microRNA regulation in stem cells and cancer Richard I. Gregory, Children’s Hospital Boston/Harvard Medical School, Boston, MA

12:15 p.m.-2:30 p.m. Poster Session B/Lunch

*Short talks from proffered papers

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 9 PROGRAM

2:30 p.m.-4:30 p.m. Session 6: miRNA Regulation in Cancer Session Chairperson: Carlo M. Croce, The Ohio State University Comprehensive Cancer Center, Columbus, OH

microRNAs as therapeutic targets Carlo M. Croce

microRNA reprogramming in cancer: Mechanisms and consequences Joshua Mendell, UT Southwestern Medical Center, Dallas, TX

The ceRNA hypothesis and the noncoding revolution in cancer research and therapy Pier Paolo Pandolfi, Beth Israel Deaconess Medical Center, Boston, MA

Characterization of a novel pseudogene expressed antisense RNA that regulates PTEN expression* Per Johnsson, Karolinska Institutet, Stockholm, Sweden

4:30 p.m.- Dinner On Own/Evening Off

Wednesday, January 11

7:00 a.m.-8:00 a.m. Continental Breakfast

8:00 a.m.-10:00 a.m. Session 7: RNA in Cancer and Disease Session Chairperson: William C. Hahn, Dana-Farber Cancer Institute, Boston, MA

Functional genomics, small RNAs, and cancer William C. Hahn

Mechanisms of miRNA-mediated cellular reprogramming Edward E. Morrisey, University of Pennsylvania, Philadelphia, PA

New therapeutic strategies for lymphomas inspired by functional and structural genomics Louis M. Staudt, National Cancer Institute, Center for Cancer Research, Bethesda, MD

Molecular function of the RNA binding protein EWS in RNA processing* Bethsaida I. Nieves, Stanford University School of Medicine, Stanford, CA

10:00 a.m.-10:15 a.m. Break

*Short talks from proffered papers

10 NONCODING RNAS AND CANCER PROGRAM

10:15 a.m.-12:15 p.m. Session 8: RNA Therapeutics Session Chairperson: William C. Hahn, Dana-Farber Cancer Institute, Boston, MA

Targeting of microRNAs for therapeutics Sakari Kauppinen, Santaris Pharma, Horsholm, Denmark

Long noncoding RNAs as drug targets Claes R. Wahlestedt, University of Miami Miller School of Medicine, Miami, FL

Anti-miR therapeutics for cancer Eric Marcusson, Regulus Therapeutics, San Diego, CA

Treatment of ovarian cancer with targeted tumor-penetrating siRNA nanocomplexes* Yin Ren, David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA

12:15 p.m. Closing Remarks/Departure

*Short talks from proffered papers

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 11 PROGRAM

12 NONCODING RNAS AND CANCER Invited Abstracts

Keynote Presentation Session 1: Long Noncoding RNAs Physiological and pathological functions Navigating the balance of INK4/ARF of microRNAs. David Baltimore. California expression in cancer: ncRNAs as sensors Institute of Technology, Pasadena, CA. and regulators of transcription. Martin J. 1 1 1 microRNAs have emerged as major Walsh , Francesca Aguilo , SiDe Li , Kyoko 1 2 1 1 regulators of the functions of genes in Yap , Jesus Gil , Ming-Ming Zhou . Mount multicellular organisms. They generally Sinai School of Medicine, New York, NY, 2 and perhaps always act as reducers of the Clinical Sciences Centre, Hammersmith protein output from a given mRNA allotment Hospital Campus, Imperial College Faculty of generated from a particular gene. However, Medicine, London, United Kingdom. when acting on mRNAs that function in a The INK/ARF (CDKN2A) locus expresses regulated pathway, they can either inhibit p15Ink4B, p16Ink4A, p14Arf and is under control of or augment the output from that pathway Polycomb Repressive Complexes PRC1 and depending on whether they act on inhibitory PRC2 to impose transcriptional silencing to gene transcripts or activating gene transcripts. bypass cellular senescence upon oncogenic We have concentrated on miRs that function stress. Previously, we had identified a within the circuitry of the hematopoietic constituent member of human PRC1, known system, focusing on those that impinge on as the chromobox protein 7 (CBX7), is innate and adaptive immune responses. endowed the capacity to bind specifically We have found that miR-146a acts as a to elements of the long non-coding RNA classic negative regulator of inflammation identified as the non-coding anti-sense RNA while miR-155 acts as a positive regulator by of the INK locus (ANRIL) RNA transcript targeting inhibitory proteins. A miR that has as a mature processed polyadenylated particularly interested us recently is miR125b, RNA transcript. ANRIL represent a large which both acts during the development of heterogeneous population of individual hematopoietic cells from the hematopoietic transcripts and their isoforms arising from the stem cell and modulates the function of localized genomic region overlapping INK/ myeloid cells. In all of this work, we have ARF. These transcripts reveal independent relied on mouse knockouts, siRNA knockouts patterns of expression dependent on the state and overproducing constructs to probe miR of cell growth and differentiation. Moreover, function and have been impressed by the specific transcripts overlapping the ANRIL ease with which these genetic manipulations region of chromosome 9p21.3 provide can lead to hyperproliferation and cancer. strict correlation to human embryonic stem Overexpression of miR-125b leads to cell pluripotency and self-renewal versus particularly rapid induction of very aggressive differentiation. We recently identify specific tumor cells that can be of either the myeloid ANRIL transcripts and isoforms that are or lymphoid lineage. The developmental origin correlative with human prostate tumor stages of these tumors is under investigation. and states of malignancy. We had previously microRNAs have been seen as modulators shown that certain transcripts correlate with of gene expression. Because it is becoming PRC1 is organized on the INK/ARF locus clear that they are so powerful in their through the interaction between CBX7 and physiological and pathological properties, the nascent ANRIL transcript to help establish it seems more appropriate to now consider and maintain repressive histone H3K27 them as co-regulators of gene expression, methylation and H2AK119 monoubiquitination. partners with transcription factors in Both PRC1 and PRC2 can interact with determining the functional outcome of different ANRIL transcript regions and it developmental and regulatory pathways. remains unclear whether this activity is cooperative. This activity is abrogated in the absence of RNA polymerase II activity thereby

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 13 invited aBstracts

requiring nascent transcription of anti-sense exon-exon, cis-antisense overlaps. Through transcripts of INK/ARF by RNA polymerase their simultaneous localization in exons of II (RNA POLII). Interestingly, TAF15/ two overlapping genes, these SNPs may TAFII68 can be tethered to CBX7 in prostate contribute to multigenic or contiguous-gene carcinoma cells where the ANRIL ncRNA phenotypes. transcripts are elevated. Our recent finding 1,213 cis-antisense pairs had both sense is that specificANRIL transcripts directed by and antisense transcripts represented by RNAPOLII are influenced by RNA polymerase distinct reliable probesets on Affymetrix U133 III (RNA POLIII) as measured through the microarrays. To search for lncRNA regulators depletion of the core RNA POLIII factor BRF1. of breast cancer gene expression, we first This evidence suggests that RNA POLIII may interrogated lncRNA-mRNA co-expression populate RNA synthesis of shorter transcripts at these loci in a human Affymetrix breast to influence PRC1 and PRC2 binding thereby cancer dataset (GSE4922). Co-expression instructing the behavior of RNA POLII of cis-encoded lncRNAs and mRNAs was synthesis. This provides a rational mechanism observed in half of the cis-antisense pairs. whereby RNA POLIII with RNAPOLIII provide Simultaneous detection of sense and cis- the genomic RNA context to instruct PRC1 antisense RNAs for each antisense pair and PRC2 to exert antisense transcription at in cancer samples opens new avenues the expense of transcriptional activity of INK/ for expression-based diagnostic marker ARF. development and for functional investigations of cis-antisense regulation involving non- Regulatory networks in onco-lncRNAomics: conserved lncRNAs. Four co-expressed cis-regulation and nonconservation. lncRNA-mRNA cis-antisense pairs contained Leonard Lipovich. Wayne State University, lncRNAs which had positional equivalents Detroit, MI. (Engström et al 2006), but not conserved orthologs, in the mouse genome. BC042008, Global studies of the transcriptome reveal that one such non-conserved lncRNA, was co- approximately half of human transcriptional expressed in breast cancer with its partner units (genes) encode solely non-protein- ZEB1, a known repressor of IL-2 and coding RNAs (ncRNAs), which, in addition to plakophylin, the latter in cancer progression. microRNAs and other small RNAs, include This implies biomarker and even functional the far more numerous long non-coding RNAs potential for human cis-antisense lncRNAs (lncRNAs). While the lncRNAome (the total that are not conserved in mouse. set of all lncRNAs) is increasingly recognized as a rich repository of novel regulators, The abundance of lncRNAs in breast cancer functional information is still lacking for the cis-antisense data prompted us to interrogate majority of lncRNAs (Lipovich et al 2010). the complete human lncRNAome, including We used transcript-to-genome alignments though not limited to cis-antisense pairs, to identify and manually curate 4,511 human for breast cancer function. Recently, we cis-antisense gene pairs, each comprised generated a catalog of human lncRNAs of two distinct genes mapping to opposite (Jia et al 2010). By quantitative RTPCR, strands of the same locus with overlapping we identified estrogen-activated (DLEU1, exons. These pairs contained 3,769 lncRNA Deleted in Leukemia 1) and estrogen- genes, most of which (2,818) resided in repressed (BC038211) lncRNAs that are coding-noncoding (mRNA-lncRNA) pairs, endogenously expressed in human MCF7 suggesting that antisense lncRNAs may be cells. Among Affymetrix probesets for 786 cis-regulators of protein-coding genes. 803 genes significantly (p<0.01) associated with lncRNAs from cis-antisense loci contained, estrogen receptor (ER) status of clinical in addition to nonrepetitive exons, exonic breast tumor samples (Ivshina et al 2006), we observed 10 lncRNAs, including XIST. primate-specific Alu repeats, indicating that lncRNAs at cis-antisense loci have undergone To directly identify ERα-targeted lncRNAs, we intersected genomewide ChIP-PET gene structure modification specifically in primate evolution. We mapped 410 SNPs to ERα TFBS localization (Lin et al 2007) with

14 NONCODING RNAS AND CANCER invited aBstracts the MCF7 transcriptome. Results included positive feedback loop of the ES regulatory a novel lncRNA initiating at the TBX2 network (Sheik Mohamed et al 2010). We bidirectional promoter in the BCAS3 (breast also used a human cancer cell line system, carcinoma amplified sequence 3) locus. SH-SY5Y neuroblastoma cells, where we This non-conserved lncRNA with a nearby focused on the non-conserved, primate- ERα binding site has a positional equivalent specific lncRNA BDNFOS, which shares but not a conserved ortholog in mouse. a bidirectional promoter with LIN7C and a Having previously used our lncRNA catalog cis-antisense overlap with BDNF. Previously, to develop and implement custom human with microarrays and quantitative RTPCR lncRNA expression analysis microarrays in (Lipovich and Loeb, ms in prep), we multiple cancer cell lines, post-mortem brain, demonstrated reciprocal patterns of BDNF and surgically resected brain samples, we and BDNFOS transcription in highly active are currently deploying these arrays to find regions of human neocortex removed as additional estrogen-responsive lncRNAs in a treatment for intractable seizures. RNAi- the MCF7 transcriptome. mediated knockdown of BDNFOS increased To understand the regulatory network BDNF expression, suggesting that BDNFOS placement of lncRNAs, including those that directly downregulates BDNF. Therefore, lack conservation and reside at cis-antisense RNAi and overexpression of lncRNAs can gene pairs, we have addressed, in systems help establish both cis- (e.g. BDNF) and beyond breast cancer, two fundamental trans-regulatory (e.g. Miat) lncRNA network questions: Which transcription factors directly functions. regulate lncRNAs by binding directly at Finally, to investigate whether the human promoters or intragenic regions of lncRNA onco-lncRNAome may function through genes? And what are the downstream translation of the lncRNAs, rather than effects of altering the cellular levels of these purely at the RNA level, we performed lncRNAs? To address the first question, proteogenomic integration of tandem mass we annotated genomewide TFBS datasets spectrometry and RNAseq data from two for several transcription factors, including ENCODE cell lines, GM12878 and the mouse Oct4 and Nanog (key regulators of chronic myelogenous leukemia cell line K562 pluripotency in embryonic stem cells) and (ENCODE AWG). LncRNAs were ~13 fold human REST. By manual identification and less likely to produce detectable peptides than protein-coding capacity assessment of the mRNAs expressed at similar levels, indicating cDNA- or EST-supported transcriptional that ~92% of Gencode v7 lncRNAs are unit nearest to each TFBS, regardless of untranslated in these cell lines. Intersecting whether the unit corresponded to a known 9,640 lncRNA loci with 79,333 peptides gene, we found that 10% of mouse Oct4 yielded 85 unique peptides matching only 69 and Nanog proximal targets in ES cells Gencode lncRNAs, with the majority of these are lncRNAs, while 23% of human REST peptides derived from the few rare protein- binding sites proximally target lncRNAs. We coding transcripts that were mis-annotated validated specific lncRNA direct targets of as lncRNAs in Gencode and that were hence these transcription factors: perturbation of the absent from our independent, non-Gencode transcription factors altered the levels of the lncRNA catalog. We conclude that ectopic targeted lncRNAs (Johnson et al 2009). translation and cryptic mRNAs are rare in the To examine the second question, we lncRNAome of these cells. Summarily, our perturbed the lncRNAs Miat, a direct target results, including those from human clinical of Oct4, and AK141205, a direct target of breast cancer datasets and diverse cancer Nanog, in mouse ES cells by RNAi and cell line systems, indicate that numerous overexpression. These perturbations caused specific lncRNAs, including non-conserved distinct changes in Oct4 and Nanog mRNA and cis-antisense transcripts, exert cis- and trans-regulatory functions in regulatory levels, lineage-specific gene expression, and pluripotency, highlighting the lncRNA networks. Miat as a novel Oct4 co-activator in a

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 15 invited aBstracts

References: diverse biological pathways and diseases, Engström P et al. PLoS Genet. 2006 Apr; with a common theme of interfacing with and 2(4):e47. modulating protein regulatory complexes. Ivshina AV et al. Cancer Res. 2006 Nov 1; 66(21):10292-10301. Jia H et al. RNA. 2010 Aug; 16(8):1478-1487. Session 2: Chromatin Modifications Johnson R et al. RNA. 2009 Jan; 15(1):85-96. Epigenetic genome control by Lin CY et al. PLoS Genet. 2007 Jun; 3(6):e87. heterochromatin machinery and Lipovich L, Johnson R, Lin CY. Biochim noncoding RNAs. Shiv Grewal. National Biophys Acta. 2010 Oct; 1799:597-615. Cancer Institute, Bethesda, MD. Sheik Mohamed J et al. RNA. 2010 Feb; Heterochromatin assembly involving 16(2):324-337. posttranslational modifications of histones is Notes: A full listing of collaborators, with critical for various chromosomal processes funding acknowledgments, will be provided including the regulation of gene expression during the presentation. Due to time and the maintenance of genomic integrity. limitations, presentation of all results that are Defective heterochromatin formation has mentioned in this abstract is not guaranteed. been linked to cancer. Our previous work has shown that non-coding RNAs and the RNAi machinery, involved in the processing Linking RNA to human health and of non-coding RNAs, play prominent roles in 1 1 disease. Moran Cabili , Eric Lander , Pardis the assembly of heterochromatin structures. 1 1 1 Sabeti , Aviv Regev , John L. Rinn , John Indeed, the loss of factors involved in 2 3 1 L. Rinn , John L. Rinn , Cole Trapnell , RNAi such as Argonaute, Dicer and RNA- 2 1 2 Cole Trapnell , Loyal Goff , Loyal Goff , dependent RNA polymerase cause severe 1 2 Kate Broadbent , Kate Broadbent , Mitchell defects in centromeric heterochromatin 1 1 Guttman . Broad Institute of MIT and formation, leading to missegregation 2 Harvard, Cambridge, MA, Harvard University, of chromosomes during cell division. 3 Cambridge, MA, Harvard Medical School, An Argonaute-containing RNAi effector Boston, MA. complex named RITS has been identified In the 50 years since RNA was identified as that facilitates the loading of a conserved a central component in the flow of genetic histone methyltransferase Clr4/Suv39h, information, it has become increasingly clear which is essential for heterochromatin that RNA is more than a mere messenger assembly. We have recently discovered an and instead performs vast and diverse unexpected role for heterochromatin factors functions. Numerous studies have revealed in the RNA quality control. Heterochromatin that the mammalian genome is pervasively factors localize broadly across the genome transcribed, giving rise to many thousands and collaborate with RNAi machinery to of non-coding transcripts including a class of suppress potentially deleterious RNAs, the long intergenic noncoding RNAs (lincRNAs). uncontrolled accumulation of which can Raising the question of what do lincRNAs do? cause DNA damage and modify epigenetic To address this question we have developed genomic profiles. I will present our recent a ‘guilt by association’ to ‘predict’ lincRNA findings showing that non-coding RNAs functions leading to hypothesis driven and heterochromatin play important roles in experimentation. Our guilt by association dynamic regulation of genomes, which has method pointed to a clear connection of important implications for human health and lincRNAs and numerous cellular pathways disease. ranging from pluripotency, cancer, References: adipogenesis to parasitology. Experimental Grewal, S.I.S. and Jia, S. (2007). perturbation experiments have unraveled Heterochromatin revisited. Nat. Rev. Genet., a myriad of functional roles for lincRNAs in 8:35-46. these pathways. Together, these results point Chen, E.S., Zhang, K., Nicolas, E., Cam, to key regulatory roles for lincRNAs across H., Zofall, M., and Grewal, S.I.S. (2008)

16 NONCODING RNAS AND CANCER invited aBstracts

Cell cycle control of centromeric repeat governing this are not clearly understood. We transcription and heterochromatin assembly. have generated conditional knockout mice Nature, 451: 734-747. that allow the functional separation of TAp63 Zofall, M., Fischer, T., Zhang, K., Zhou, M., and ΔNp63. Using the TAp63 conditional Cui, B., Veenstra, T., and Grewal, S.I.S. knock out mouse model, we found that TAp63 (2009) Histone H2A.Z cooperates with RNAi suppresses tumorigenesis and metastasis and heterochromatin factors to suppress through coordinate transcriptional regulation antisense RNAs. Nature, 461: 419-422. of Dicer and miR-130b. Interestingly, we Zhang, K., Fischer, T., Porter, R., have recently found that ΔNp63 also plays Dhakshnamoorthy, J., Zofall, M., Zhou, M., an important role in miRNA processing Veenstra, T., Grewal, S.I.S. (2011). Clr4/ by transcriptionally activating DGCR8. In Suv39 and RNA quality control factors turn, DGCR8 is required for processing of cooperate to trigger RNAi and suppress key miRNAs that repress genes required antisense RNA. Science 331: 1624-1627. for stem cell pluripotency, Oct4, Sox2, and Nanog. Strikingly, epidermal cells derived Elucidation of enhancer-like RNAs. Ramin from ΔNp63 deficient mice express markers Shiekhattar. The Wistar Institute, Philadelphia, of pluripotency at levels comparable to PA. mouse embryonic stem cells. Moreover, these cells exhibit characteristics of stem Spatial and temporal regulation of gene cells that can self-renew and can differentiate expression is achieved through the instruction into multiple cell fates in vitro and in vivo. provided by the distal transcriptional Our data reveal a novel role for ΔNp63 in regulatory elements known as enhancers. the commitment to epidermal differentiation How enhancers transmit such information to through transcriptional regulation of DGCR8 their targets has been a subject of intense and downstream modulation of key miRNAs investigation. Recent advances in high required to maintain stem cell pluripotency. throughput analysis of the mammalian Taken together, our data show that p63 plays transcriptome have revealed a surprising critical roles in regulating miRNAs in multiple result indicating that a large number of biological processes. enhancers are transcribed to non-coding RNAs. While long non-coding RNAs were initially shown to confer epigenetic microRNAs and control of metabolism. transcriptional repression, recent studies Markus Stoffel. Institute for Molecular have uncovered a role for a class of such Systems Biology, Zurich, Switzerland. transcripts in gene-specific activation often Obesity has been shown to be a strong risk from distal genomic regions. We will discuss factor for adult-onset diabetes. Importantly the recent findings on the role of long non- and less widely appreciated, there is also a coding RNAs in transcriptional regulation significant link between obesity and cancer. with an emphasis on new developments on Recent studies have clearly demonstrated the functional links between long non-coding that obesity is associated with the incidence RNAs and enhancers. and mortality of a number of malignancies, including colon, pancreatic, kidney, as Session 3: miRNA Biology well as aggressive prostate cancer in men and breast and endometrial cancer Unraveling the complexities of p63 in in women. Other studies have also found cancer and stem cells. Elsa R. Flores. The that individuals with various abnormalities University of Texas MD Anderson Cancer associated with the insulin resistance such as Center, Houston, TX. hypertriglyceridemia, hyperglycemia and high Aberrant expression of microRNAs (miRNAs) insulin levels are at increased risk of certain and the enzymes that control their processing cancers. Moreover, recent reports have have been reported in multiple biological suggested the possibility of an association processes including in cancer and the between use of synthetic insulin and cancer. regulation of stem cells, but the mechanisms Together, a body of evidence suggests that

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 17 invited aBstracts

a link between obesity and cancer that may Session 4: Biology of Long result from similar etiological mechanisms Noncoding RNAs common to cancer, diabetes and insulin use. Spreading of X-chromosome inactivation We are studying the role of microRNAs in the via a hierarchy of defined Polycomb control of glucose and lipid metabolism. Initial stations. Stefan F. Pinter1, Stefan F. Pinter2, studies conducted in our lab have revealed a Stefan F. Pinter3, Yesu Jeon1, Yesu Jeon2, role of miR-375, the most abundant microRNA Yesu Jeon3, Toshiro K. Ohsumi2, Toshiro K. in the pancreatic beta cell, in maintaining Ohsumi3, Mark Borowsky2, Mark Borowsky3, normal beta cell mass. We recently Jeannie T. Lee1, Jeannie T. Lee2, Jeannie broadened our investigations on the function T. Lee3, Ruslan I. Sadreyev1, Ruslan I. of other microRNAs that regulate pancreatic Sadreyev2, Ruslan I. Sadreyev3, Eda Yildirim1, b-cell homeostasis and insulin sensitivity. Two Eda Yildirim2, Eda Yildirim3. 1Howard Hughes miRNAs and their role in regulating glucose Medical Institute, Boston, MA, 2Massachusetts homeostasis will be discussed: General Hospital, Boston, MA, 3Harvard 1. miR141/200c cluster: Expression analysis Medical School, Boston, MA. of the miR-200 family cluster revealed X-chromosome inactivation (XCI) achieves a predominant localization in endocrine dosage balance in mammals by repressing organs, such as the pancreatic beta cells, one of two X-chromosomes in females. the pituitary and the gastrointestinal tract. During XCI, the long noncoding Xist RNA Specific overexpression of these microRNAs and Polycomb proteins spread along the in the pancreatic beta cell results in inactive X (Xi) to initiate chromosome-wide profound hyperglycemia, ketoacidosis and silencing. Although inactivation is known insulinopenia due to massive b-cell apoptosis. to commence at the X-inactivation center Conversely, mice lacking miR141/200c are (Xic), how it propagates remains unknown. protected from streptozotocin-induced b-cell We have examined allele-specific binding death. These results implicate this microRNA of Polycomb repressive complex 2 (PRC2) cluster as a key regulator of programmed cell and chromatin composition during XCI, and death in vivo. generate a chromosome-wide profile of Xi 2. miR-103/107: These two related miRNAs and Xa (active X) at nucleosome-resolution. are upregulated in livers of obese mice Initially, Polycomb proteins are localized to a and humans. We showed that silencing of limited number of sites along the X. As Xist miR-103/107 leads to improved glucose RNA spreads in cis during the XCI process, homeostasis and insulin sensitivity. In thousands of additional sites are recruited. contrast, gain of miR-103/107 function PRC2 and H3K27 methylation spread along a in either liver or fat is sufficient to induce gradient in an Xi-specific manner not seen on impaired glucose homeostasis. We identified autosomes. Our findings suggest that XCI is caveolin-1, a critical regulator of the insulin governed by a hierarchy of defined Polycomb receptor, as a direct target gene of miR- stations that spread H3K27 methylation in cis. 103/107 and demonstrated that caveolin-1 is upregulated upon miR-103/107 inactivation in adipocytes and that this is concomitant with Control of gene expression in the stabilization of the insulin receptor, enhanced nucleus by small RNAs. David Corey. UT insulin signaling, decreased adipocyte size Southwestern, Dallas, TX. and enhanced insulin-stimulated glucose Double-stranded RNAs are widely used to uptake. These findings demonstrate the control gene expression and are a promising central importance of miR-103/107 to insulin class of therapeutic agent. Most studies sensitivity and identify a new target for the have focused on recognition of mRNA in the treatment of type 2 diabetes and obesity. cytoplasm and inhibition of translation. My presentation focuses on the action of small RNAs in the nucleus to modulate transcription or redirect alternative splicing. In 2005, my laboratory observed that

18 NONCODING RNAS AND CANCER invited aBstracts synthetic RNAs complementary to the terminus of the gene to the promoter. promoter region at the transcriptional start site We have investigated whether RNAs of human progesterone receptor (PR) could can modulate other nuclear events. We block PR expression [1]. Inhibition was potent designed small RNAs to be complementary and robust, consistent with the suggestion to sequence within exons or introns near that the synthetic RNAs were exploiting an splice sites. These RNAs redirect alternative endogenous mechanism for controlling gene splicing and AGO2 was recruited to pre- expression. Promoter-targeted synthetic mRNA [7]. These data provide more support miRNAs were also capable of robust gene for the action of small RNA in cell nuclei and silencing [2], further supporting the existence for the conclusion that AGO2 may not always of endogenous regulatory pathways. cause RNA cleavage when it encounters Modulation required complete seed sequence complementary targets in the nucleus. complementarity to the PR promoter, consistent with a mechanism that involves Our data suggest that small RNAs can bind RNA-RNA recognition through Watson-Crick noncoding transcripts and modulate gene base-pairing. expression. Binding occurs in proximity to the gene promoter, in cis relative to the target In 2007, we observed that small RNAs could gene. The RNA-AGO complex acts like a also target the PR gene promoter and activate transcription factor to affect the transcription PR expression [3]. Activation was observed machinery and tip the balance of inducible in cells that had a low, but detectable, level genes like PR towards repression or of PR expression, while gene silencing had activation. been observed in cells with a high level of PR expression. Inactive and active RNAs References: compete for recognition, demonstrating that 1. Janowski, B. A. et al. Inhibiting gene small changes in target site location affect expression at transcription start sites in activity. These results are reminiscent of chromosomal DNA by antigene RNAs. Nat. protein transcription factors, which bind to Chem. Biol. 1, 216-222 (2005). specific sequences, are sensitive to target 2. Younger, S. T. and Corey, D. R. site location or small conformation changes, Transcriptional gene silencing in mammalian and can activate gene expression in some cells by miRNA mimics that target gene contexts but not others. promoters. Nucl. Acids Res. 39, 5682-5691 (2011). Our promoter-targeted RNAs bind to 3. Janowski, B. A., et al. Activating gene long noncoding RNA transcripts at expression in mammalian cells with promoter- the PR promoter [4]. Use of antisense targeted duplex RNAs. Nat. Chem. Biol. 3 oligonucleotides to reduce transcript levels 166-173 (2007). also reduces RNA-mediated modulation of 4. Schwartz, J. C., et al. Antisense transcripts gene expression. The small RNAs recruit are targets for small activating RNAs. Nat. AGO2 to the noncoding transcript, and Struct. Mol. Biol. 15, 842-848 (2008). inhibition of AGO2 expression reverses 5. Chu, Y. et al. Argonaute proteins and gene silencing or activation [5]. While modulation of progesterone receptor AGO2 is normally expected to cleave fully expression by promoter-targeted RNAs. Nucl. complementary targets in the cytoplasm, our Acids Res. 38, 7736-7748 (2010). data suggests that AGO2 does not cleave 6. Yue, X. et al. Regulation of transcription transcripts associated with gene promoters in by small RNAs complementary to sequences the nucleus. downstream from the 3’ termini of genes. Nat. A noncoding RNA transcript also overlaps the Chem. Biol. 6, 621-629 (2010). 3’ terminus of the PR gene [6]. Small RNAs 7. Liu, J. et al. (2011). Expanding the action complementary to this noncoding transcript of duplex RNAs into the nucleus: redirecting modulate PR expression, depending on the alternative splicing. Nucl. Acids Res. in Press. cell line used. The PR gene loops, juxtaposing its 3’ and 5’ termini and providing a direct path for a signal transmitting a signal from the 3’

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 19 invited aBstracts

Small RNA pathways in germline. Alexei Gain- and loss-of-function studies in mice A. Aravin. California Institute of Technology, have revealed striking functions for these Pasadena, CA. miRNAs in numerous processes, such as the In animals, a discrete class of small RNAs, control of muscle regeneration, nerve-muscle the piwi-interacting RNAs (piRNAs), guard interactions, myosin expression, fibrosis, germ cell genomes against the activity of hypoxia, myocyte survival, and metabolism. mobile genetic elements. Identification of miRNA targets has revealed new mechanisms and regulators of piRNAs are generated, via an unknown development and disease. Disease-inducing mechanism, from apparently single-stranded miRNAs can be persistently silenced in vivo precursors that arise from discrete genomic through systemic delivery of miRNA inhibitors, loci, termed piRNA clusters. Presently, little allowing for therapeutic modulation of disease is known about the signals that distinguish mechanisms. Opportunities for manipulating a locus as a source of piRNAs. It is also miRNA biology in the settings of muscle unknown how individual piRNAs are selected disease will be discussed. from long precursor transcripts. To address these questions, we inserted new artificial sequence information into piRNA clusters Long-term, efficient inhibition of miRNA and introduced these marked clusters as function in mice using Tough Decoys transgenes into heterologous genomic delivered by adeno-associated virus positions in mice and flies. Profiling of piRNA vectors. Phillip D. Zamore. University of from transgenic animals demonstrated that Massachusetts Medical School, Worcester, artificial sequences were incorporated into MA. the piRNA repertoire. Transgenic piRNA Understanding the function of individual clusters are functional in non-native genomic miRNA species in mice would require contexts in both mice and flies, indicating that the production of hundreds of loss-of- the signals that define piRNA generative loci function strains. To accelerate analysis of must lie within the clusters themselves rather miRNA biology in mammals, we combined than being implicit in their genomic position. recombinant adeno-associated virus (rAAV) We found that both local and long-range vectors with miRNA ‘Tough Decoys’ (TuDs) sequence environments inform the generation to inhibit specific miRNAs. Intravenous of individual piRNAs from precursor injection of rAAV9 expressing anti-miR-122 transcripts. or anti-let-7 TuD depleted the corresponding miRNA and increased its mRNA targets. Session 5: miRNA Regulation in rAAV producing anti-miR-122—but not anti- Development let-7—TuD reduced serum cholesterol by 40% for 18 weeks in wild-type mice. High microRNA control of muscle development throughput sequencing of liver miRNAs from and disease: From new biology to the treated mice confirmed that the targeted new therapeutics. Eric N. Olson. UT miRNAs were depleted and revealed that Southwestern Medical Center, Dallas, TX. TuD RNAs induce miRNA tailing and trimming We seek to define the gene regulatory in vivo. rAAV-mediated miRNA inhibition networks that govern muscle and thus provides a simple way to study miRNA cardiovascular development and disease. function in adult mammals and a potential The signaling pathways and transcriptional therapy for dyslipidemia and other diseases networks that control these processes are caused by miRNA deregulation. intertwined with a collection of microRNAs (miRNAs) that act as negative regulators of gene expression. We have identified miRNAs associated with muscular dystrophy and diverse cardiovascular disorders, including cardiac hypertrophy, heart failure, myocardial infarction, and angiogenesis.

20 NONCODING RNAS AND CANCER invited aBstracts

Session 6: miRNA Regulation in 34c are transactivated by tumor protein p53 Cancer (TP53)14 and that the miR-34b/miR-34c cluster maps at 17p suggests the possible microRNAs as therapeutic targets. Carlo M. Croce. The Ohio State University, Columbus, existence of a genetic link and significant molecular interactions between the 17p Ohio. and 11q chromosomal deletions in CLL. At Chronic lymphocytic leukemia (CLL) is the present, it is not known how the 13q, 11q, and most common leukemia among adults in the 17p deletions contribute to CLL pathogenesis Western world, with an annual incidence in and affect the outcome of patients with CLL. the United States of approximately 10 000 new cases. The clinical staging systems devised by Rai et al. and Binet et al. are microRNA reprogramming in cancer: useful for assessing the extent of CLL in a Mechanisms and consequences. Joshua patient, but they fail to differentiate between T. Mendell. UT Southwestern Medical Center, the indolent and aggressive forms of CLL. Dallas, TX. Most typically these forms are characterized miRNA gain- and loss-of-function can by low and high levels of zeta-chain (TCR)– potently influence cellular behavior in normal associated protein kinase 70kDa (ZAP70), physiologic states and in diseases such as respectively. Using fluorescence in situ cancer. The regulation of miRNA expression hybridization (FISH), Döhner et al. found and activity by cellular signaling cascades that chromosomal abnormalities occurred can therefore result in dramatic phenotypic in 82% of cases and included the 13q outputs. We previously demonstrated deletion (55%), 11q deletion (18%), and 17p extensive control of miRNA expression by deletion (7%). Patients with the 17p and 11q well-characterized oncogenic and tumor deletions experience the aggressive form of suppressor networks including the Myc, Kras, the disease, whereas patients with the 13q and p53 pathways. We are now employing deletion or with normal cytogenetic profiles novel mouse models with gain and loss of experience the indolent form. The occurrence miRNA function to investigate the roles of the of common and recurring chromosomal miRNAs embedded within these signaling abnormalities suggests that these deletions pathways and the pathologic consequences affect thus-far undefined pathways important when their functions are disrupted. Insights for the pathogenesis of CLL. gained from these functional studies have led to the development of novel therapeutic microRNAs are small, noncoding RNAs with strategies for cancer and other pathologic regulatory functions7; microRNA expression states based on miRNA delivery. I will present is frequently deregulated in tumors. In most CLLs, the expression of the microRNA 15a our latest findings related to miRNA regulation (miR-15a [GenBank 406948])/microRNA in cancer and exploitation of these findings for the development of novel therapeutic 16-1 (miR-16-1 [GenBank 406950]) cluster, approaches. which maps within a 30-kilobase region of loss at 13q14.3 (henceforth designated 13q14), is abolished or reduced. We have The ceRNA hypothesis and the noncoding previously shown that the expression of this revolution in cancer research and microRNA cluster is inversely correlated therapy. Pier Paolo Pandolfi. Beth Israel with the expression of B-cell CLL/lymphoma Deaconess Cancer Center and Harvard 2 (BCL2 [GenBank 596]), an anti apoptotic Medical School, Boston, MA. gene overexpressed in most CLLs. The loss The central dogma of molecular biology, as of the long arm of chromosome 11 involves proposed by Francis Crick, demonstrates the 11q23.1 region (henceforth designated that genetic information is transferred from 11q23), where the microRNA 34b (miR-34b DNA in our genomes to the generation of [GenBank 407041]) /microRNA 34c (miR-34c functional proteins, through a messenger RNA [GenBank 407042]) cluster is located.13 The (mRNA) intermediate. This suggests that the observation that microRNA 34a (miR-34a key function of each mRNA is to encode for [GenBank 407040]), miR-34b, and miR- protein.

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 21 invited aBstracts

We have demonstrated that mRNAs can difficult. In addition, the number and types exert a biological activity that is independent of candidate targets that require functional of the protein for which they encode. validation in vivo represent a significant Since this phenomenon is dependent on barrier to using this information as a starting sequences contained in RNA transcripts, point for drug development programs, since this phenomenon applies to protein-coding many do not represent targets amenable to genes, pseudogenes, of which 19,000 have traditional small molecule or antibody-based been predicted in the genome as well as long therapeutic approaches. To address this non-coding RNAs that have been recently problem, we have combined comprehensive identified to be pervasive in the cell and in analyses of cancer genomes with novel biology. materials science to develop a platform that This new function is brought about by the enables the ready in vivo validation of cancer ability of mRNAs—and RNAs in general—to targets. Specifically, we have developed a bind and sequester microRNA molecules. screening platform to perform loss of function screens at genome scale and have applied microRNAs specifically repress the expression levels of many genes and have this in a massively parallel manner to screen consequently been shown to play important several hundred full characterized cancer roles in diseases including cancer. We show cell lines. We have combined the outputs that any messenger RNA and RNA molecule of these screens with data emerging from can sequester microRNA molecules acting the study of cancer genomes to identify as a “competitive endogenous RNAs.” We candidate oncogenes. To credential these therefore term them ceRNAs. Given that targets in vivo, we have helped develop microRNAs can bind to multiple mRNAs, we novel nanocomplexes formed from siRNA argued that mRNAs compete for the binding bound to tandem peptides containing tumor- to a given microRNA. We originally tested this penetrating, endosomal escape and siRNA- hypothesis and proved its validity by studying binding domains that permit deep penetration the interaction between the mRNA encoding in tumor. We use these nanoparticles to for the PTEN tumor suppressor gene and target a hitherto undiscovered ovarian its closely related pseudogene, PTENP1, cancer oncogene, ID4, an undruggable which we show to act as a tumor suppressor transcriptional regulator, and show that these through this new mechanism. We therefore tumor-penetrating nanocomplexes induced identify PTENP1 as well as the several complete suppression of tumor growth and thousand uncharacterized RNA molecules as significant improvement of survival. Taken potential human disease genes. together, these studies provide a platform for the discovery and initial validation of We have now expanded this analysis to the candidate cancer targets. whole transcriptome. Our findings therefore defines a new biological dimension that will allow for the rapid identification and functional Mechanisms of miRNA-mediated cellular characterization of new disease genes. reprogramming. Edward Morrisey. University of Pennsylvania, Philadelphia, PA. Session 7: RNA in Cancer and Recent evidence has indicated that miRNAs Disease have the ability to promote dramatic changes in cell fate including pluripotent stem cell Functional genomics, small RNAs, and reprogramming. Certain miRNA clusters cancer. William C. Hahn. Dana-Farber including miR302/367 are highly expressed Cancer Institute, Boston, MA. in embryonic stem cells and when over- Efforts to sequence cancer genomes have expressed can convert fibroblasts to induced begun to uncover comprehensive lists of pluripotent stem cells. Moreover, recent genes altered in cancer. Unfortunately, publications have shown that certain miRNAs the complexity and sheer volume of data can directly convert fibroblasts to neurons that emerges from these efforts has made indicating that this technique may be widely dissecting the underlying biology of cancer applicable to other cell lineages. Despite

22 NONCODING RNAS AND CANCER invited aBstracts these intriguing results, little is understood Session 8: RNA Therapeutics about the molecular mechanism underlying Targeting of microRNAs for miRNA mediated reprogramming. Since therapeutics. Sakari Kauppinen. Santaris miRNAs can target hundreds of mRNAs, Pharma, Horsholm, Denmark. identification of specific targets as well as molecular pathways affected will require microRNAs act as important post- multiple approaches. Data will be presented transcriptional regulators of gene expression on how our lab is using systems biology by mediating mRNA degradation and and genomic approaches to decipher the translational repression. Moreover, there underlying mechanism of miRNA-mediated is ample evidence that perturbations in cellular reprogramming. the levels of individual or entire families of miRNAs are strongly associated with the pathogenesis of a wide range of human New therapeutic strategies for lymphomas diseases. Besides cancer, miRNAs have inspired by functional and structural also been implicated in viral infections, genomics. Louis M. Staudt. National Cancer cardiovascular and muscle diseases and CNS Institute, Bethesda, MD. disorders. Thus, disease-associated miRNAs Seeking an unbiased method to discover represent a potential new class of targets for therapeutic targets in cancer, we developed oligonucleotide-based therapeutics, which a loss-of-function genetic screen using may yield patient benefits unobtainable by genomic-scale libraries of small hairpin other therapeutic approaches. RNAs that mediate RNA interference. These LNA is a bicyclic high-affinity RNA analogue, “Achilles heel” screens are designed to reveal in which the ribose ring is locked in an RNA- genes essential for cancer cell proliferation like, N-type (C3’-endo) conformation by and survival. In a parallel structural genomics the introduction of a 2’-O,4’-C methylene approach, we are using RNA-seq to globally bridge. Transfection of LNA-modified identify somatic mutations and other structural antimiR oligonucleotides into cells results abnormalities in cancer. RNA-seq also in potent and specific inhibition of miRNA provides an unprecedented view of gene function with concomitant derepression expression that provides more accurate of direct target mRNAs. In addition, quantitative data than traditional microarray systemically delivered, unconjugated approaches. The intersection of the RNA LNA-antimiRs with a phosphorothioate interference and RNA-seq data sets has backbone show high metabolic stability and helped us to discover novel pathogenetic uptake in many tissues in mice, coinciding pathways that control the proliferation with long-term miRNA silencing in vivo. and survival of the aggressive lymphoma These findings support the utility of LNA- subtypes. The malignant phenotype is seen modified antimiR oligonucleotides in the as an aberrant regulatory network that development of therapeutic strategies aimed includes oncogene-induced microRNAs, at pharmacological inhibition of disease- deregulated transcription factors and aberrant associated miRNAs. We will describe recent signaling from cell surface receptors. progress in targeting of disease-implicated This view suggests several therapeutic miRNAs for therapeutics using LNA-antimiRs opportunities using targeted agents that might and present an update on the clinical initially augment and eventually supplant the development of antimiR-122 for treatment of more toxic chemotherapeutic regimens used hepatitis C virus infection. to treat these cancers today.

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 23 invited aBstracts

Long noncoding RNAs as drug targets. Claes Robert Wahlestedt. University of Miami Miller School of Medicine, Miami, FL. Much of the mammalian genome is transcribed into non-coding RNAs of different categories. This lecture will primarily be concerned with natural antisense transcripts (NATs) most of which are long noncoding RNAs. NATs are found in most gene loci and regulate gene expression through several distinct mechanisms including chromatin modifications. Inhibition/ perturbation of endogenous NATs by modified oligonucleotides called AntagoNATs, in vitro or in vivo, reveals concordant or discordant regulation and results in down- or upregulation of conventional (protein-coding) gene expression, respectively. Evidence will be presented that AntagoNATs can potently induce locus-specific and reversible modulation of gene expression associated with alterations in chromatin marks.

24 NONCODING RNAS AND CANCER Proffered Abstracts

PR1 BRAFV600E remodels the melanocyte PR2 Functional characterization of transcriptome and induces BLNCR1 to p53-regulated long intervening noncoding regulate melanoma cell migration. Ross J. RNAs (lincRNAs). Nadya M. Dimitrova, Tyler Flockhart, Dan E. Webster, Kun Qu, Nicholas Jacks. David H. Koch Institute for Integrative Mascarenhas, Joanna Kovalski, Markus Cancer Research at MIT, Cambridge, MA. Kretz, Paul A. Khavari. Stanford University, LincRNAs (long intervening noncoding Stanford, CA. RNAs) constitute a novel class of long, Aberrations of protein-coding genes are noncoding RNAs that have been implicated a focus of cancer genomics, however, the in regulating gene expression in diverse impact of oncogenes on expression of the biological processes by modulating the ~50% of transcripts without protein-coding activity of chromatin-modifying complexes. potential, including long non-coding RNAs In collaboration with the Rinn laboratory (lncRNAs), is largely uncharacterized. at the Broad Institute, we have identified Activating mutations in the BRAF oncogene a set of lincRNAs that appear to be direct are present in 60% of melanomas, 90% of transcriptional targets of p53. These which produce active mutant BRAFV600E. To lincRNAs contain p53 binding elements define the impacts of oncogenic BRAF on in their promoter regions and are strongly the melanocyte transcriptome, massively upregulated in a p53-dependent manner parallel cDNA sequencing (RNA-Seq) upon DNA damage and in response to was performed on genetically matched oncogenic stress. We have hypothesized that normal human melanocytes with and these lincRNAs may be new components without BRAFV600E expression. To enhance of the p53 network and may contribute to potential disease relevance by verifying p53 tumor suppressor function by regulating expression of altered genes in BRAF- the expression of genes in cancer-relevant driven cancer tissue, parallel RNA-Seq was pathways. Using a combination of molecular also undertaken on two BRAFV600E-mutant genetic studies, cell biology, biochemistry, as human melanomas. BRAFV600E regulated well as gene targeting in the mouse, our aim expression of 1,027 protein-coding transcripts is to dissect the biological function of these and 39 annotated lncRNAs, as well as 70 lincRNAs in vivo and in isolated cells. un-annotated, potentially novel, intergenic To date, we have generated a conditional transcripts. Coding potential analysis of knockout mutant for lincRNA-p21, one of these 70 transcripts suggested that most the p53-regulated lincRNAs, which was may represent newly identified non-coding recently shown to mediate gene repression RNAs. BRAF-regulated long non-coding RNA in the p53 transcriptional network. Using 1 (BLNCR1) was identified as a recurrently, this genetic tool, we plan to characterize highly expressed, previously un-annotated the consequences of lincRNA-p21 loss 693 bp transcript on chromosome 9 with a in mice and in isolated mouse embryonic potential functional role in melanoma cell fibroblasts. Our goal is to identify genes that migration. Combining RNA-Seq of oncogene- are regulated by lincRNA-p21 and to map expressing normal cells with RNA-Seq of their out interactions between lincRNA-p21 and corresponding human cancers may represent the p53 transcriptional network. We will also a useful approach to discover new oncogene- focus on dissecting the mechanism by which regulated RNA transcripts of potential clinical lincRNA-p21 may regulate the expression of relevance to cancer. target genes. This abstract is also presented as Poster A11. We have also identified a lincRNA, linc9, which is induced by DNA damage in a dose-dependent manner. Even though the expression of linc9 appears to be p53-

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 25 ProFFered aBstracts

independent, our preliminary experiments transcript as well as its potential to sequester indicate that linc9 may influence the outcome microRNAs and PTEN mRNA translation. of p53 activation by promoting apoptosis in In summary, we have characterized a novel response to high levels of DNA damage. PTENpg1 encoded asRNA that acts on the In sum, by studying the function and PTEN gene by recruitment of chromatin- mechanism of p53-regulated and DNA remodeling factors such as DNMT3a and damage-induced lincRNAs, we hope to EZH2 to the PTEN promoter. We also achieve a better understanding of the role demonstrate that PTENpg1 asRNA functions of long noncoding RNAs in modulating the as a dual-type regulator of both PTEN p53 pathway and more broadly in regulating transcription and translation. Knowledge of cancer-related processes. this molecular pathway may prove useful This abstract is also presented as Poster B19. in understanding epigenetic and asRNA modulation of gene expression in human cells, and in the case of PTEN may prove PR3 Characterization of a novel beneficial in developing targeted approaches pseudogene expressed antisense RNA to induce PTEN transcription. that regulates PTEN expression. Per This abstract is also presented as Poster B25. Johnsson1, Amanda Ackley2, Dan Grandér1, Kevin V. Morris2. 1Karolinska Institutet, Stockholm, Sweden, 2The Scripps Research PR4 Molecular function of the Institute, La Jolla, CA. RNA binding protein EWS in RNA PTEN is a tumor suppressor gene that is processing. Bethsaida I. Nieves, Shuang dysregulated in several different forms of Niu, Dedeepya Vaka, Julia Salzman, Patrick cancer. PTEN was recently reported to be Brown, E. Alejandro Sweet-Cordero. Stanford involved in a regulatory microRNA network University School of Medicine, Stanford, CA. where the PTEN pseudogene (PTENpg1) The Ewing sarcoma (EWS) protein is a sequesters microRNAs and thus affects the member of the FET family of RNA-binding translation of the PTEN mRNA. proteins and is involved in chromosomal Here, we show that the pseudogene-based translocations that generate oncogenic regulation of PTEN is more complex than fusion genes found in a variety of sarcomas. previously appreciated. We report that the EWS has been recently reported to be PTENpg1 locus also encodes an antisense involved in regulating transcription, pre- RNA (asRNA), which directly regulates mRNA processing, translation and DNA PTEN on the transcriptional level in trans. repair. However, the precise physiological We show that the PTENpg1 asRNA localizes role of EWS in RNA processing remains to the PTEN promoter and also binds to largely unknown. Using cross-linking the DNA methyltransferase 3a (DNMT3a). immunopercipitation coupled with high- throughput sequencing (CLIP-Seq), we Overexpression of the PTENpg1 asRNA suppresses the expression of PTEN while identified the full complement of RNAs bound depletion of the PTENpg1 asRNA decreases to EWS in Hela cells. Analysis of RNA binding DNMT3a and EZH2 mediated epigenetic by EWS revealed that the greatest increase silencing of the PTEN promoter, resulting in in binding was to A-I editing enzymes increased transcriptional activity of PTEN. ADAR1, ADARB1 and ADARB2 as well as long non-coding RNAs (lncRNAs) NEAT1 We further show that specific targeting of the PTENpg1 asRNA induces G0/G1 cell cycle and MALAT1. Interestingly, these lncRNAs arrest and sensitizes relatively chemoresistant localize to specific subnuclear structures cells to DNA damaging doxorubicin treatment. and have been implicated in regulating A-I editing and RNA splicing respectively. We We also find that the PTENpg1 asRNA hypothesize that EWS could play a role in functions as a concordant regulator of the regulating the subnuclear localization of PTENpg1 sense by stabilizing this transcript other proteins involved in editing and splicing via an RNA:RNA interaction. This RNA:RNA by virtue of its association with NEAT1 and interaction stabilizes the PTENpg1 sense

26 NONCODING RNAS AND CANCER ProFFered aBstracts

MALAT1. We also found that binding of EWS Here we describe a tumor penetrating to pre-mRNAs influenced alternative splicing. nanocomplex (TPN) comprised of siRNA Paired-end RNA-Seq of the transcriptome complexed to a tandem tumor-penetrating revealed that knock down of EWS resulted and membrane-translocating peptide, which in the over expression of 116 genes and enables the homing of siRNA deep into the down regulation of 68 genes, including tumor parenchyma. Upon complexation EWS itself. Further analysis identified 16 with siRNA, the resulting nanocomplex is alternatively spliced genes following EWS stable, non-immunostimulatory, displays knock down across two independent RNA homing peptides in a multivalent fashion that seq experiments. Of these genes, 6 were increases their binding avidity and delivers also shown to bind EWS by CLIP-seq. These siRNA to the cytosol of tumor cells through genes include, CENPA, CSTF1, RBBP6, receptor-specific interactions and membrane RAC1, FANC1 and ANAPC5. These studies translocation. Upon systemic administration highlight a potential role for EWS as a critical into mice, this nanocomplex penetrates into regulator of alternative splicing and A-I the parenchyma of metastatic peritoneal editing. tumors and silences target genes in cells of This abstract is also presented as Poster B8. interest in a receptor-specific manner. We employed TPNs in vivo to evaluate ID4, a novel candidate oncogene in ovarian cancer, PR5 Treatment of ovarian cancer which we identified by combining genome- with targeted tumor-penetrating siRNA scale RNAi screening of cancer cell lines nanocomplexes. Yin Ren1, Hiu Wing with genome-scale sequence analysis of Cheung2, Ronny Drapkin3, David Root2, patient tumors. We show that treatment of Justin Lo4, Valentina Fogal5, Erkki Ruoslahti5, tumor-bearing mice with ID4-specific TPNs William Hahn2, Sangeeta Bhatia4, Geoffrey suppresses tumor growth and significantly von Maltzahn1, Amit Agrawal1, Glenn improved survival. These findings provide a Cowley2, Barbara Weir2, Jesse Boehm2, framework for the identification, credentialing, Pablo Tamayo2, Jill Mesirov2, Alison and understanding of novel cancer targets as Karst3. 1Harvard-MIT Division of Health well as validating a specific therapeutic target Sciences and Technology, Cambridge, in ovarian cancer. 2 MA, Broad Institute of Harvard and MIT, This abstract is also presented as Poster B2. Cambridge, MA, 3Dana-Farber Cancer Institute, Boston, MA, 4Massachusetts Institute of Technology, Cambridge, MA, 5Burnham Institute for Medical Research, Santa Barbara, CA. Whole-genome analysis of cancer samples is identifying many potential therapeutic targets, by virtue of their being frequently mutated or functionally essential in specific types of cancer. However, we lack efficient ways to test the therapeutic benefit of modulating targets in vivo. RNAi offers one potential solution; however, approaches to deliver siRNA in vivo have been challenging due to their susceptibility to serum nucleases, endosomal entrapment, and stimulation of innate immunity. Furthermore, nanoparticle- and antibody-based siRNA delivery approaches have historically suffered from limited tumor penetration and low transvascular transit, thereby limiting the applicability of parenchymal siRNA targets.

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 27 ProFFered aBstracts

28 NONCODING RNAS AND CANCER Poster Session A

A1 Long stress-induced noncoding protein structures formed by the interaction transcripts (LSINCTs) and cancer. David of NEAT1 and members of the Drosophila I. Smith1, Silva M. Jessica2. 1Mayo Clinic, Behavior Human Splicing family of proteins Rochester, MN, 2Washington University, St. including PSPC1. Paraspeckles appear to Louis, MO. be critical to the control of gene expression We previously utilized a whole genome through the nuclear retention of certain tiling array to identify novel transcripts which species of RNA. It is though that paraspeckles had altered expression in response to the and their components play a role in controlling genotoxic effects of the tobacco carcinogen gene expression during a number of different NNK. Our goal was to identify novel long cellular processes including differentiation, stress-induced non-coding transcripts viral infection and stress responses. Using that could play important regulatory roles RNA fluorescent in situ hybridization, we within cells and be important targets of found LSINCT5 to co-localize with NEAT1 alteration during cancer formation. Utilizing within the nucleus. Hence, this novel very stringent criteria, we examined all stress-induced transcript may be another transcriptionally active regions that had important long non-coding transcript that is altered expression in response to NNK involved in the normal cellular function of the paraspeckles. treatment and identified a group of 12 transcripts, termed long stress-induced noncoding trancripts (LSINCTs). These novel A2 miR-10a regulation of PIK3CA and long non-coding transcripts are between 2 response to Taxol in non-small cell lung and 4 Kb in length and do not contain any cancer. Chris DeSevo1, Liqin Du2, Carmen open reading frames or have homology to Behrens3, Ignacio I. Wistuba3, John Minna1, any mRNAs. 11 LSINCTs are intergenically Alexander Pertsemlidis2. 1UT Southwestern located and one LSINCT is intragenically Medical Center at Dallas, Dallas, TX, 2UT located within the genome. We found Health Science Center at San Antonio, San LSINCTs to be more highly expressed in Antonio, TX, 3UT MD Anderson Cancer more proliferative normal human tissues Center, Houston, TX. and to be frequently over-expressed in a number of different cancers including breast Phosphatidylinositol 3-kinases (PI3Ks) are a cancer. We studied one of these transcripts in family of enzymes involved in diverse cellular greater detail, LSINCT5. We found LSINCT5 functions including cell growth, proliferation, to be a polyadenylated nuclear transcript differentiation, motility, survival and of exactly 2,647 base pairs. It is highly apoptosis. PI3K allows for the transmission of over-expressed in both breast and ovarian extracellular cues to an intracellular response cancers and knocking down its’ expression via its activation through receptor tyrosine in either a breast or ovarian cancer cell line kinase or k-ras. Activation of this pathway resulted in decreased cellular proliferation. is antagonized by the phosphatase PTEN. We further compared gene expression in the This signaling pathway is under extremely breast cancer cell line, MCF7, before and tight regulation and even slight perturbations after knocking down LSINCT5 expression can lead to aberrant pathway activation, as and identified a number of genes whose is the case in a majority of cancers. We are expression changed more than 2-fold in interested in whether PIK3CA is regulated by response. Two of the genes identified were one or more microRNAs and whether those the gene for the major paraspeckle protein miRNAs have a therapeutic effect on cancer PSPC1, and another long non-coding cell viability and drug response in PI3K-driven transcript NEAT1. Paraspeckles are irregularly oncogenesis. shaped compartments found in the nucleus’ In a systematic approach to identifying miRNA interchromatin space. Paraspeckles are RNA- inhibitors with significant effects on cell

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 29 POSTER SESSION A

viability and response to paclitaxel in NSCLC, A3 miR-31 and its host gene lncRNA we performed a high-throughput screen and LOC554202 are regulated by promoter identified several candidate microRNAs. hypermethylation in triple-negative Regulatory targets of candidate miRNAs breast cancer. Katarzyna Augoff, Brian were identified through a combination of McCue, Edward F. Plow, Khalid Sossey- in vitro and in silico approaches. Targets Alaoui. Cleveland Clinic Lerner Research were then validated using qRT-PCR, protein Institute, Cleveland, OH. quantification, and luciferase reporter assays. Background: microRNAs have been The response of cancer cells to perturbations established as powerful regulators of gene of the candidate miRNA was assessed expression in normal physiological as well as through flow cytometric analysis of cell cycle in pathological conditions, including cancer phase distribution, colony formation and progression and metastasis. Recent studies caspase activation assays. have demonstrated a key role of miR-31 in The inhibitor screen revealed that inhibition the progression and metastasis of breast of miR-10a increased cellular growth rate and cancer. Downregulation of miR-31 enhances resistance to paclitaxel. Further validation several steps of the invasion-metastasis demonstrated a 10-fold increase in cell cascade in breast cancer, i.e., local invasion, viability in the presence of miR-10a inhibitor, extravasation and survival in the circulation and a 10-fold decrease in the presence of system, and metastatic colonization of distant miR-10a mimic. Manipulation of miR-10a sites. miR-31 exerts its metastasis-suppressor levels in either direction resulted in significant activity by targeting a cohort of pro-metastatic changes in both mRNA and protein levels genes, including RhoA and WAVE3. The of its predicted target, the catalytic subunit molecular mechanisms that lead to the loss of of phosphatidylinositol 3-kinase (PI3K), miR-31 and the activation of its pro-metastatic which was confirmed to be a direct target by target genes during these specific steps of luciferase reporter assay. Increasing miR-10a the invasion-metastasis cascade are however levels inhibited the ability to form colonies, unknown. while decreasing miR-10a levels increased Results: In the present report, we identify colony formation. To assess the prognostic promoter hypermethylation as one of the value of miR-10a we assessed expression major mechanisms for silencing miR-31 in in NSCLC patients and observed that high breast cancer, and in the triple-negative miR-10a levels correlate with longer overall breast cancer (TNBC) cell lines of basal survival. subtype, in particular. miR-31 maps to the The identification of a miR-10a as a modulator intronic sequence of a novel long non-coding of cellular response to taxanes, and PI3K (lnc)RNA, LOC554202 and the regulation of as a regulatory target which mediates its transcriptional activity is under control of that response, define a novel regulatory LOC554202. Both miR-31 and the host gene pathway modulating paclitaxel sensitivity LOC554202 are down-regulated in the TNBC in lung cancer, which may provide novel cell lines of basal subtype and over-expressed adjuvant strategies along with paclitaxel in in the luminal counterparts. Treatment of the the treatment of lung cancer and may also TNBC cell lines with either a de-methylating provide biomarkers for predicting paclitaxel agent alone or in combination with a de- response in NSCLC. Ultimately this study will acetylating agent resulted in a significant provide new insights into the molecular basis increase of both miR-31 and its host gene, of lung cancer and a new set of potential suggesting an epigenetic mechanism for the therapeutic targets for this devastating silencing of these two genes by promoter disease. hypermethylation. Finally, both methylation- specific PCR and sequencing of bisulfite- converted DNA demonstrated that the LOC554202 promoter-associated CpG island is heavily methylated in the TNBC cell lines and hypomethylated in the luminal subtypes.

30 NONCODING RNAS AND CANCER POSTER SESSION A

Conclusion: Loss of miR-31 expression of the PCA3 transcript revealed a number of in TNBC cell lines is attributed to stable secondary RNA structures that may hypermethylation of its promoter-associated be precursors for microRNA biogenesis. On CpG island. Together, our results provide the Northern blots, one small PCA3-derived RNA initial evidence for a mechanism by which could be detected, and this small PCA3 RNA miR-31, an important determinant of the was found to be in complex with the AGO2 invasion metastasis cascade, is regulated in protein. MiRNA qPCR analysis revealed that breast cancer. this PCA3-derived miRNA was up-regulated in prostate cancer. A4 The functional role of the PCA3 Discussion: Our results demonstrate noncoding RNA in human prostate that PCA3 contributes to prostate cancer cancer. Gerald W. Verhaegh, Kelly van den proliferation and survival. The formation Oudenalder, Frank Smit, Martijn A. Huynen, of specific PCA3 RNA-protein complexes Jack A. Schalken. Radboud University and the processing of the PCA3 transcript Nijmegen Medical Centre, Nijmegen, The into a microRNA may contribute to the Netherlands. observed PCA3-mediated growth stimulation. Identification of thePCA3 RNA-binding Introduction: PCA3 is a prostate-specific proteins and PCA3 microRNA target genes is gene that is highly over-expressed in prostate ongoing. cancer. The PCA3 gene lacks an extensive open reading frame and no in vitro translation products could be identified, suggesting that A5 Cytotoxic extracts of ethnomedicinal PCA3 acts as a non-coding RNA (ncRNA). plants: Podophyllum peltatum (mayapple) The aim of this study is to reveal the function and Echinacea angustifolia. Olena T. of the PCA3 ncRNA and its mechanism. James, E. Lewis Myles. Tennessee State Material and Methods: To study the University, Nashville, TN. phenotypic effects of PCA3, prostate cancer Phytochemical and other natural compounds cell lines were transfected with PCA3 are historically established as cures for expression vectors. Anchorage-independent many ailments in rural America and other cell growth was measured by colony developing countries. Our lab is testing plants formation in soft agar. PCA3 RNA-protein that show potential for inhibiting the growth interactions were studied by sucrose gradient of cancer cells. The family Asteraceae and centrifugation and by protein-RNA UV-cross- the family Berberidacae are very well known linking assays. In silico PCA3 RNA secondary for their values as medicinal plants. More structure modeling was performed using specifically, the genus species Echinacea the Vienna RNA Package (v.1.6). MicroRNA angustifolia and genus species Podophyllum processing and expression were determined peltatum commonly known as the Mayapple, by Northern blotting, AGO2 RNA co-IP, and both are noted for their unique medicinal qPCR analysis. purposes. The crude methanol extracts of Results: PCA3 over-expressing prostate these plants were tested against human cancer cells displayed an enhanced capacity metastatic cell lines representing breast to form colonies in soft agar (i.e., more and (MCF-7), (BT-549) and colon (SW620) tissues. Trypan blue exclusion is used bigger colonies were formed). This finding indicates that PCA3 plays a role in prostate to determine viable cell counts. Alamar cancer cell proliferation and survival. After BlueTM and florescent analysis were used sucrose centrifugation of total cell extracts, to evaluate the cytoxicity of the extracts. PCA3 was present in fractions of higher Analysis of the crude extracts of Echinacea density compared to naked RNA, indicating angustifolia has shown on MCF-7, BT- that PCA3 RNA-protein binding occurs. 549, and SW620 that most of them exhibit Indeed, in UV cross-linking assays, the PCA3 anticancer activity against the cell lines at RNA specifically interacted with several low the maximum concentration of 200 μg /mL. molecular-weight proteins. In silico analysis In particular, less diluted concentrations have demonstrated exceptional inhibitory effects

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 31 POSTER SESSION A

with IC50 values ranging between 0.10-15 μg A7 Roles of miRNAs in the switch /mL, respectively. On the other hand, analysis of TGF-β from tumor suppressor of the crude extracts of Podophyllum peltatum to pro-oncogenic factor in cancer (Mayapple) has shown on MCF-7, BT-549 progression. Yoshiko Nagano, Maxwell P. and SW620 some anticancer activity against Lee, Lalage M. Wakefield. National Cancer cell lines at the maximum concentration Institute, Bethesda, MD. of 200 μg /mL. More specifically, the more TGF-β is a widely expressed pleiotropic diluted concentrations have demonstrated growth factor that plays complex roles in exceptional growth inhibition with IC50 values tumorigenesis. TGF-β switches from tumor ranging between 2-8 μg /mL suppressor to pro-oncogenic factor during breast cancer progression. Our goal is A6 microRNA sequencing of AKXD to identify molecular determinants of the recombinant inbred panel identifies TGF-β switch. Since miRNA expression is miR-216b as a candidate metastasis deregulated in cancer, and TGF-β is reported suppressor in a murine model of breast to regulate expression and biogenesis of cancer. Farhoud Faraji1, Ying Hu1, Natalie miRNAs, we hypothesize that miRNAs Goldberger1, Gang Wu2, Ken H. Buetow1, may contribute to this switch. To model the Jinghui Zhang2, Kent W. Hunter1. 1National TGF-β switch, we used a series of human Cancer Institute, Bethesda, MD, 2St. Jude breast cancer cell lines derived from the Children’s Research Hospital, Memphis, TN. spontaneously immortalized MCF10A human breast epithelial cell line. The model Metastatic disease continues to be the consists of 4 cell lines. In the MCF10A (M- primary cause of mortality in cancer. I; normal), MCF10AT (M-II; premalignant) Using a combination of complex genetics, and MCF10CA1hcl4 (M-IIIcl4; low-grade mouse models of breast cancer, and tumor) lines, TGF-β is a tumor suppressor. In human epidemiology, our laboratory has contrast, in the MCF10CA1a cell line (M-IV), demonstrated that germline polymorphism which gives rise to aggressive metastatic contributes to an individual’s susceptibility tumors, the tumor suppressor activity is lost to metastasis. In the current study, we and TGF-β promotes metastasis. To test the have used the AKXD recombinant inbred possible involvement of miRNAs in the TGF-β panel, which is derived from inbred mice of switch, we compared expression profiles of metastasis resistant, DBA, and metastasis- miRNAs by Next Gen miRNA sequencing 1) prone, AKR, genetic backgrounds, to identify basally in cultures of all four cell lines and 2) microRNAs whose expression is associated in M-IIIcl4 and M-IV with and without TGF-β with metastasis. microRNA sequencing of treatment. A variety of patterns of change tumor tissue from the progeny of AKXD x in basal miRNA expression with malignant Polyoma Middle-T mice demonstrated that progression were seen. We focused on the expression of the miR-216/217 cluster miRNAs whose expression level differs is significantly negatively correlated with basally between M-IIIcl4 and M-IV, since pulmonary metastases in the AKXD panel. the switch occurs between these two cell Linkage analysis demonstrated a significant lines. 46 miRNAs were expressed > 1.5 fold association between the miR-216/217 locus higher in M-IIIcl4 than in M-IV, whereas 29 and the DBA allele, suggesting that the miRNAs were expressed > 1.5 fold higher in difference in expression is inherited rather M-IV than in M-IIIcl4. We selected several than somatically acquired. Finally, we show miRNAs whose expression was most different that ectopic expression of miR-216b in murine between the two cell lines, and examined mammary tumor cells suppresses pulmonary whether these miRNAs regulate cellular metastasis without significantly impacting responses to TGF-β. As an example, let-7i primary tumor growth in vivo. These findings and let-7g were expressed higher in M-IV suggest that miR-216b is an inherited modifier than in M-IIIcl4. Forced expression of these of metastasis with metastasis suppressing miRNAs partially reversed TGF-β-induced activity in a mouse model of breast cancer. growth inhibition in M-IIIcl4. A similar strategy was applied to miRNAs whose regulation

32 NONCODING RNAS AND CANCER POSTER SESSION A by TGF-β is different between M-IIIcl4 and A9 De novo sequencing of circulating M-IV. The results of these experiments will be microRNAs identifies novel markers discussed. predicting clinical outcome of locally advanced breast cancer. Xiwei Wu, George Somlo, Melanie Palomares, Yun Yen, John A8 Defining specific microRNAs that Rossi, Harry Gao, Shizhen Emily Wang. City cooperatively regulate GRP78, an ER of Hope Beckman Research Institute and chaperone and signaling regulator, in Medical Center, Duarte, CA. cancer. Sheng-Fang Su, Amy S. Lee, Purpose: MicroRNAs (miRNAs) have been Gangning Liang. USC/Norris Comprehensive Cancer Center, Los Angeles, CA. recently detected in the circulation of cancer patients, where they are associated with GRP78, a major endoplasmic reticulum clinical parameters. Discovery profiling of (ER) chaperone and signaling regulator, circulating small RNAs has not been reported is commonly over-expressed in cancer in breast cancer (BC), and was carried out in and contributes to cancer cell proliferation, this study to identify blood-based small RNA survival and tumorigenesis. Moreover, markers of BC clinical outcome. induction of GRP78 by a variety of anticancer drugs, including histone deacetylase Patients and Methods: The pre-treatment inhibitors, confers chemo-resistance to sera of 42 stage II–III locally advanced and cancer. Thus, targeting GRP78 to inhibit inflammatory BC patients who received tumor survival and proliferation, sensitize neoadjuvant chemotherapy (NCT) followed tumor cells to chemotherapeutic drugs and by surgical tumor resection were analyzed consequently induce apoptosis serves as a for marker identification by deep sequencing promising avenue in anti-cancer therapy. As all circulating small RNAs. An independent endogenous regulators, microRNAs (miRNAs) validation cohort of 26 stage II–III BC patients play important roles in modulating gene was used to assess the power of identified expression; therefore, we sought to identify miRNA markers. miRNA(s) that target GRP78. We report Results: More than 800 miRNA species were that multiple miRNAs (miR-30d, miR-181a, detected in the circulation, and exhibited miR-199-5p) commonly down-regulated in patterns associated with the histopathological human cancer cell lines can repress GRP78 profiles of BC. Groups of circulating miRNAs and lead to increased apoptosis in C42B differentially associated with ER/PR/HER2 prostate cancer cells. Luciferase reporter status and inflammatory BC were identified. assays indicate that these miRNAs act as The relative levels of selected miRNAs negative regulators of GRP78 by directly measured by PCR showed consistency targeting the 3’ untranslated region of GRP78. with their abundances determined by deep Importantly, the combination of multiple sequencing. Two circulating miRNAs, miRNAs cooperated resulting in greater miR-375 and miR-122, exhibited strong inhibition on GRP78 than individual miRNAs, correlations with clinical outcomes, including suggesting the efficacy of tumor suppression NCT response and relapse with metastatic by combined miRNAs. In addition, we showed disease. In the validation cohort, higher levels that transfection of the multiple miRNAs in of circulating miR-122 specifically predicted C42B cells increased sensitivity of cancer metastatic recurrence in stage II–III BC cells to trichostatin A (TSA), a histone patients. deacetylase inhibitor, induced apoptosis, Conclusion: Our study indicates that certain and inhibited cell proliferation and colony miRNAs can serve as potential blood-based formation. Therefore, we suggest that delivery biomarkers for NCT response, and that miR- of combinations of specific miRNAs could 122 prevalence in the circulation predicts BC be a novel anti-cancer approach capable of metastasis in early-stage patients. These suppressing GRP78 and in turn inhibiting results may allow optimized chemotherapy GRP78-mediated tumorigenesis. treatments and preventive anti-metastasis interventions in future clinical applications.

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 33 POSTER SESSION A

A10 microRNA 27a is downregulated expression of a number of genes involved in cisplatin-resistant bladder cancer cells with the synthesis of GSH was then measured and contributes to resistance through the by rt-pcr and western blotting, and several key targeting of xCT, a cystine transporter proteins involved with GSH biosynthesis were involved with glutathione production. Ross found to be upregulated in resistant cells, M. Drayton, Ewa Dudziec, James W.F. Catto, suggesting increased levels of intracelluar Helen E. Bryant. Institute for Cancer Studies, GSH could be responsible for the observed Sheffield University Medical School, Sheffield, decrease in the levels of cisplatin-DNA South Yorkshire, United Kingdom. adducts observed in resistant cells. Cisplatin resistance represents a serious The 3’ untranslated regions (3’ UTRs) of these obstacle to the chemotherapeutic treatment mRNAs were examined using TargetScan, of many tumors. The purpose of this study an online miR target prediction program, to was to create a cell culture model of cisplatin find potential sites of miR regulation. The resistant bladder cancer (CRBC), then most upregulated protein, xCT (SLC7A11), use this model to identify changes in the responsible for cystine import (known to be expression of microRNAs (miRs) that may the rate-limiting step in GSH synthesis) was influence resistance. found to contain target sites for three miRs Single cell clones resistant to cisplatin were which had been shown to be significantly generated from parental bladder cancer cell downregulated in our model of CRBC, namely lines by continuous culture in increasing miRs 25, 27a and 32. concentrations of cisplatin for several months. Upregulation of miRs 25, 27a and 32 by The resultant CRBC cell lines were able to transfection of resistant cells with the pri-miR tolerate high doses of cisplatin (typically > 10 precursor molecules revealed that restoring μM). MicroRNA expression in parental and expression of miR-27a resensitized CRBC CRBC cell lines was measured using q-pcr cells to the cytotoxic effects of cisplatin. based low-density tiling arrays and a panel Restoration of miR-27a expression in of miRs which were consistently altered in all CRBC cells was also shown to result in a CRBC cell lines was established. In addition downregulation of the expression of xCT at the induction and repair of specific cisplatin- both the protein and mRNA level. DNA adducts in parental and CRBC cell In summary, we have identified a panel of lines was assessed using antibodies against microRNAs that are consistently dysregulated the guanine–guanine (Pt-[GG]) intrastrand in CRBC, and implicated one of these (miR- crosslink. Four fold more cisplatin was 27a) in the modulation of cisplatin resistance required to produce the equivalent damage via increased glutathione production. We in CRBC cell lines compared to the parental also provide evidence that reversal of this control, while the rate of repair, as assessed change in miR expression results in a by measurement of the removal of cisplatin- reversal of the effects on downstream target, DNA adducts over time, remained broadly and critically, a reversal of the cisplatin similar between parental and resistant lines. resistant phenotype. The contribution made This demonstrated that resistance was due by miR-27a to the development of cisplatin to reduced adduct formation rather than any resistance might represent a potential site for defect in cross-link repair. therapeutic intervention in the treatment of Glutathione (GSH) inactivates cisplatin by CRBC. Measurement of miR-27a expression

binding with it irreversibly to form Pt(SG)2 in tumor material or urine may also serve adducts, thus preventing the drug from as a predictive biomarker for patients likely forming cytotoxic adducts with DNA. One response to cisplatin chemotherapy. cause of decreased adduct formation is therefore deregulation of the GSH biosynthesis pathway. Upon examination intracellular levels of GSH and its oxidized form glutathione disulfide (GSSG) were found to be increased in resistant cells. The

34 NONCODING RNAS AND CANCER POSTER SESSION A

A11 BRAFV600E remodels the miRNA profiling approach is not sufficient to melanocyte transcriptome and induces identify the respective role of each miRNA BLNCR1 to regulate melanoma cell gene during the tumorigenesis. migration. Ross J. Flockhart, Dan E. Here, to assess the individual role of each Webster, Kun Qu, Nicholas Mascarenhas, miRNA gene and its different isomirs Joanna Kovalski, Markus Kretz, Paul A. in a specific cell environment, we have Khavari. Stanford University, Stanford, CA. constructed a lentiviral miRNA expression This abstract is being presented as a short library containing more than 1100 human talk in the scientific program. A full abstract known and novel miRNA precursors. The is printed in the Proffered Abstracts section arrayed layout of our library allowed high- (PR1) of the conference Proceedings. throughput screens with a large spectrum of functional read-outs using either normal or tumor cells. To exemplify this approach, A12 Identification of microRNA-based the results of three different screens will therapeutic candidates using a unique be presented; i.e. identification of miRNAs lentiviral microRNA overexpression that inhibit the BRAF pathway, miRNAs that 1 library. Paula I. van Noort , Negar inhibit tumor angiogenesis and miRNAs 2 3 Babae , Gerald W. Verhaegh , Willemijn that stimulate the mesenchymal to epithelial 1 1 M. Gommans , Francesco Cerisoli , Mark transition. In addition, beyond this hit Verheul1, Raymond M. Schiffelers2, Arjan 4 3 identification step, we will present detailed W. Griffioen , Jack A. Schalken , Eugene characterization of the role of the identified 5 5 Berezikov , Edwin Cuppen , Roel Q. J. miRNAs in tumor progression by means of 1 5 Schaapveld , Jos B. Poell , Gregoire P. molecular and cellular functional assays. Prevost1, Meriem Bourajjaj1, Suzanna Vidic1, Judy R. van Beijnum4, Rick J. van Haastert1, Combining our unique miRNA expression Iman Schultz1, Thijs de Gunt1, Onno van library with a functional screening platform Hooij3. 1InteRNA Technologies, Utrecht, has allowed the identification and the further The Netherlands, 2Utrecht Institute for characterization of several miRNAs able Pharmaceutical Sciences, Utrecht University, to significantly impact on tumor behavior The Netherlands, 3Department of Urology, supporting the therapeutic interest of some Radbout University Medical Center, Centre candidates. for Molecular Life Sciences, Nijmegen, The 4 Netherlands, Vrije Universiteit Amsterdam A13 The microRNA-200 family Medical Center, Amsterdam, The Netherlands, overcomes cisplatin resistance by 5 Hubrecht Insitute, Cancer Genomics Center, targeting ceramide glycosylation. Chunxiao University Medical Center, Utrecht, The Cai1, Hassan Ashktorab2, Xiaowu Pang2, Netherlands. Yuan Zhao2, Wei Sha2, Yulan Liu1, Xinbin microRNA (miRNA) genes transcribed by Gu2. 1Peking University, Beijing, China, RNA polymerase II generate small noncoding 2Howard University, Washington, DC. miRNAs of 18 to 24 nucleotides after Background: Chromodomain-helicase- maturation process. The mature miRNAs DNA-binding protein 5 (CHD5) is a newly and their associated isomirs specifically identified tumor suppressor that is frequently bind to different mRNA transcripts, resulting downregulated in a variety of human in down regulation of multiple genes within cancers. Our previous work revealed that the cell in a highly multiplexed way. miRNA the low expression of CHD5 in colorectal expression profiles differ between human cell cancer is correlated with CHD5 promoter types suggesting cell-specific impacts of each CpG island hypermethylation. In this study, miRNA on the regulation of different biological we investigated the effect of microRNA-211 processes. Comparison of miRNA profiles of (miR-211)-regulated CHD5 expression on tumor samples and adjacent normal tissues colorectal tumorigenesis. Methodology/ showed that some miRNAs are up- or down- Principal Findings: miR-211 was predicted to regulated and suggested their implication target CHD5 by TargetScan software analysis. during tumor progression. However, such a

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 35 POSTER SESSION A

A stably expressing exogenous miR-211 suggesting that these interactions mediate colorectal cancer cell line (HCT-116miR-211) crosstalk between canonical oncogenic was generated using lentiviral transduction pathways. RNA silencing of 13 microRNA- and used as a model for in vitro and in vivo mediated PTEN regulators, whose locus studies. The expression level of miR-211 deletions are predictive of PTEN expression in HCT-116miR-211 cells was upregulated variability, was sufficient to downregulate by 16-fold compared to vector control cells PTEN in a 3’ UTR-dependent manner and (HCT-116vector). Exogenous miR-211 directly to increase tumor-cell growth rates. Thus, binds to the 3’ untranslated region (3’ UTR) of this microRNA-mediated network provides CHD5 mRNA, resulting in a 50% decrease in a mechanistic, experimentally validated CHD5 protein level in HCT-116miR-211 cells. rationale for the loss of PTEN expression in The levels of cell proliferation, tumor growth, a large number of glioma samples with an and cell migration of HCT-116miR-211 cells intact PTEN locus. Recently, we extended this were significantly higher than HCT-116vector work to three other tumor types, showing that cells under both in vitro and in vivo conditions, microRNA-mediated interactions propagate as determined using the methods of MTT, the effects of genetic alterations to regulate colony formation, flow cytometry, tumor distal oncogenes and tumor suppressors in xenografts, and scratch assay, respectively. In multiple cancers. Our results suggest that, addition, we found that enforced expression although implemented by context-specific of miR-211 in HCT-116 cells was able to factors, hundreds of thousands of these alter p53 pathway-associated regulatory interactions are context-independent. proteins, such as MDM2, Bcl-2, Bcl-xL, and Bax. Conclusion/Significance: Our results demonstrate that CHD5 is a direct target of A15 Somatic mutations of KEAP1 gene miR-211 regulation. Enforced expression of in common solid cancers. An Changhyeok, miR-211 promotes tumor cell growth at least Yoo Namjin, Kim Hyung Ran, Lee Sug in part by downregulating the expression Hyung. Catholic University Medical College, level of the CHD5 tumor suppressor. Our Seoul, Republic of Korea. results provide a better understanding of the Aims: KEAP1 inhibits NRF2-induced association of between miR-211-regulated cytoprotection and is considered to be CHD5 expression and CHD5 function in a candidate tumor suppressor. Somatic colorectal tumorigenesis. mutation of NRF2 has been analyzed in wide types of human cancer, whereas somatic mutation of KEAP1 has been reported only A14 microRNA-mediated interactions in lung and gall bladder cancers. The aim of regulate established oncongenic pathways our study was to address whether KEAP1 in cancer. Pavel Sumazin, Hua-Sheng Chiu, mutation is widespread in human cancers. Andrea Califano. Columbia University, New York, NY. Methods: We analyzed 499 cancer tissues from lung, breast, colon, stomach, liver, larynx By analyzing gene expression data in and prostate, and leukemias by single-strand gliobastoma in combination with matched conformation polymorphism. microRNA profiles, we have uncovered a post-transcriptional regulation layer of Results: We detected somatic mutations of surprising magnitude, comprising hundreds the KEAP1 in gastric (11.1%), hepatocellular of thousands of microRNA -mediated (8.9%), colorectal (7.8%), lung (4.6%), breast interactions. These include ~7,000 genes (2.0%) and prostate (1.3%) carcinomas. whose transcripts act as microRNA ‘sponges’ Allelic losses of KEAP1 locus were identified and 148 genes that act through alternative, in 42.9% of cancers with the KEAP1 mutation, non-sponge interactions. Biochemical but no NRF2 mutation was detected in these analyses in cell lines confirmed that this cancers. NRF2-activated cytoprotective network regulates established drivers of proteins (NQO1 and GCLC) were expressed tumor initiation and subtype, including PTEN, in all of the cancers with the KEAP1 mutation. PDGFRA, RB1, VEGFA, STAT3, and RUNX1, Conclusions: Our data show that that KEAP1

36 NONCODING RNAS AND CANCER POSTER SESSION A mutation occurs widely in solid cancers if MeCP2 would have increased binding to irrespective of histologic types. Biallelic the identified methylated CpG sites. Second, inactivation of KEAP1 gene and increased quantitative methylation specific PCR (qMSP) cytoprotective proteins in the cancers suggest will be used to measure the methylation levels that KEAP1 mutations might protect cancer of miR-22 genes in oral cancer tissues and cells from oxidative insults and play a role in their normal control using SYBR Green– the development of solid cancers. based detection technology. Third, in vitro methylation and promoter-driven luciferase assay will be used to investigate whether DNA A16 miR-22 downregulation via methylation plays a direct role in regulating epigenetic control in oral cancer cells. Li- miR-22 promoter activity and whether CpG Wha Wu. National Cheng Kung University, island deletion affects the relative luciferase Tainan, Taiwan. activity. Together, this study would implicate The deregulation of certain microRNAs has that the expression of miR-22 is controlled been associated with the progression and by epigenetic control in oral cancer cells. metastasis of various cancer types. DNA However, the exact nature of miR-22 in oral methylation in the CpG islands plays a carcinogenesis remains to be characterized. crucial role in the down-regulation of tumor suppressor genes, including miRNAs. MiR- 22 has been shown to be deregulated in A17 miR-15, miR-16, and miR-21 are several cancer types, including breast cancer direct transcriptional targets of E2F1 and multiple myeloma. Using real-time RT- that limit E2F-induced proliferation and PCR analysis, we also found that miR22 apoptosis. Matan Ofir, Berta Ben-Shachar, was down-regulated in the majority of tested Dalia Hacohen, Doron Ginsberg. Bar-Ilan oral cancer cell lines and 70% of the clinical University, Ramat Gan, Israel. specimens when compared with their normal microRNAs (miRs) are small non-coding counterparts. Consistent with the notion that RNA molecules that have recently emerged DNA methylation might play a role in the as critical regulators of gene expression gene silencing, we did detect a putative CpG and are often deregulated in cancer. In island (~ 1.2 kb in length) located upstream particular, miRs encoded by the miR-15a, from the putative miR-22 transcription start miR-16-1 cluster are frequently deleted in site (+1) using two independent CpG island certain cancers and appear to act as tumor prediction softwares. Two oral cancer lines suppressors. Furthermore, miR-21 is over- with low miR-22 expression were treated for expressed in a wide variety of human tumors 3-5 days with the indicated dose of 5’-azaC and functions as an oncogene. Here, we together with or without trichostatin A. Total provide evidence that miR-21, the miR-15a, RNA was isolated from the treated cells for miR-16-1 cluster and related miR-15b, miR- real-time quantitative PCR analysis of miR- 16-2 cluster comprise miRs regulated by 22 expression. The expression of miR-22 E2F1, a pivotal transcription factor that can was significantly induced by either inhibitor. induce both proliferation and cell death. E2F1 Combined treatment further enhanced the is a critical downstream target of the tumor expression, suggesting the involvement suppressor RB. The retinoblastoma pathway of epigenetic control in the decreased is often inactivated in human tumors resulting expression of miR-22 in oral cancer cells. in deregulated E2F activity. We show that We first divided the putative CpG island into expression levels of the five mature miRs, 5 regions and designed nested PCR primers miR-15a, miR-16-1, miR-15b, miR-16-2 for bisulfate sequencing PCR. We found and miR-21 are elevated upon activation that only regions 3 to 5 in the putative CpG of ectopic E2F1. Moreover, activation of island were subjected to be influenced by endogenous E2Fs up-regulates expression of methylation in certain oral cancer lines. Since these miRs and endogenous E2F1 binds their MeCP2 selectively binds to methylated CpG respective promoters. dinucleotides, chromatin immunoprecipition Importantly, we corroborate that miR-15a/b together with qPCR will be used to examine inhibits expression of cyclin E, a key direct

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 37 POSTER SESSION A

transcriptional target of E2F pivotal for the A19 Profiling long noncoding RNA G1/S transition. Similarly, miR-21 inhibits expression in cancer cells by real-time the expression of the pro-apoptotic tumor PCR arrays. Fangting Wu1, Yin-Yuan suppressor gene maspin, another direct Mo2. 1System Biosciences, Mountain View, transcriptional target of E2F. Therefore, CA, 2Southern Illinois University, Springfield, our data suggest the existence of feed IL. forward loops that modulate E2F activity Long noncoding RNAs (lncRNAs), as a new and consist of E2F1; E2F1-regulated miRs class of transcripts, have been recently -miR-15 and miR-21; and E2F1-regulated shown to be pervasively transcribed in the genes-cyclin E and maspin. In support of genomes of human and mouse. In particular, this, ectopic expression of miR-15 inhibits accumulating evidence link the expression G1/S transition and, conversely, inhibition of of lncRNAs to diverse human diseases, miR-15 expression enhances E2F1-induced highlighting their potential as biomarkers G1/S transition and up-regulation of cyclin E and therapeutic targets. However, there levels. Also, inhibition of miR-21 expression are still many missing dots in our current enhances E2F1-induced up regulation of understanding of lncRNA function. Thus, maspin, as well as E2F1-mediated apoptosis. investigation of the expression patterns of In summary, our data identify miR-15, miR-16 lncRNAs is a crucial step to understanding and miR-21 as novel transcriptional targets of of their roles in many model systems. To E2F that, in turn, modulate E2F activity. this end, we developed a qPCR based disease-related human lncRNA profiler, which allows for the quantification of differential A18 p53 regulates epithelial- expression of 83 individual lncRNAs among mesenchymal transition through various experimental RNA samples. All 83 microRNAs targeting ZEB1 and lncRNAs chosen for the array are based 1 1 ZEB2. Taewan Kim , Tae Jin Lee , Young- on publications and they are implicated in 1 2 2 Jun Jeon , Pascal Pineau , Anne Dejean , diseases ranging from neurodegeneration 1 1 Carlo Maria Croce . Ohio State University, to cancer. The array plate also includes 2 Columbus, OH, Institut Pasteur, Paris, the house keeping genes and small RNA France. transcripts for normalization purposes. To p53 suppresses tumor progression and determine the utility of this lncRNA profiler, metastasis. Epithelial-mesenchymal transition we profiled their expression in breast cancer (EMT) is a key process in tumor progression specimens compared to matched normal and metastasis. The transcription factors tissue and identified several lncRNAs are ZEB1 and ZEB2 promote EMT. Here we dysregulated, suggesting that lncRNAs may show that p53 suppresses EMT by repressing function as oncogenes or tumor suppressors. expression of ZEB1 and ZEB2. By profiling Gene expression profiling with this lncRNA 92 primary hepatocellular carcinomas (HCCs) profiler in a series of breast cell lines such and 9 HCC cell lines, we found that p53 as MCF10A, MCF7 and MDA-MB-231 cells upregulates microRNAs including miR-200 identified differential expression patterns and miR-192 family members. The miR-200 unique to each cell line, which may provide a family members transactivated by p53 then basis for future analysis of these differentially repress ZEB1/2 expression. p53-regulated expressed lncRNAs. Furthermore, we miR-192 family members also repress ZEB2 determined the effect of p53 on lncRNA expression. Inhibition or over-expression of expression in HCT-116 and MCF-7 cells. the microRNAs affects p53-regulated EMT Initial testing indicated that at least three by altering ZEB1 and ZEB2 expression. Our lncRNAs are induced by p53 in both cell findings indicate that p53 can regulate EMT, lines. Experiments are underway to further and that p53-regulated microRNAs are critical characterize these p53 inducible lncRNAs. mediators of p53-regulated EMT. Together, these results suggest that disease- related human lncRNA profiler is a valuable research tool that can be tailored for specific needs in different research laboratories. The

38 NONCODING RNAS AND CANCER POSTER SESSION A features of this profiler are easy, convenient, been implicated in the impairment of TGF- sensitive and specific. Accordingly it serves β-mediated growth suppression through the first step toward the understanding of the repression of the cell cycle inhibitor, p21, and role of lncRNAs in human diseases. pro-apoptotic factor, Bim. These data suggest that Six1-induced upregulation of these miRNA may mediate the switch in TGF-β A20 The Six1-regulated miR-106b-25 signaling from tumor suppressive to tumor cluster is a mediator of the tumor promotional. Surprinsingly, bioinformatic promotional effects of TGF-β signaling analysis revealed that the miR-106b-25 in human breast cancer. Anna L. Smith, cluster may also contribute to the activation Ritsuko Iwanaga, David J. Drasin, Douglas of TGFβ signaling through repression of the S. Micalizzi, Rebecca L. Vartuli, Heide L. TGF-β signaling inhibitor, Smad7, which Ford. University of Colorado Anshutz Medical mediates the degradation of TβRI. Indeed, Campus, Aurora, CO. overexpression of the miR-106b-25 cluster Recent studies have highlighted the results in repression of the Smad7 protein, developmental transcription factor Six1 with concominant upregulation of TβRI. as an important mediator of breast cancer Furthermore, activation of TGF-β signaling is progression and metastasis. Six1 is observed with miR-106b-25 overexpression, overexpressed in a striking 90% of metastatic as demonstrated by an increase in breast cancer lesions, and patients whose phosphorylation of the downstream effector tumors overexpress Six1 have a decreased of the TGF-β pathway, Smad3, and by time to metastasis and relapse, as well as an an upregulation of downstream TGF-β overall decrease in survival. Six1 mediates transcriptional targets. miRNA inhibition also metastasis via multiple mechanisms, demonstrates that miR-106b and miR-93 are including its ability to induce an epithelial- necessary for Six1 induced TGF-β activation. to-mesenchymal transition (EMT) and tumor Additionally, like Six1, the miR-106b-25 initiating cell (TIC) characteristics, both of cluster is sufficient to induce features of which are dependent on an upregulation of EMT, including a redistribution of E-cadherin, TGF-β signaling. Indeed, the Six1-induced and increased β-catenin transcriptional increase in TGF-β signaling is critical for the activation. Furthermore, the miR-106b-25 ability of Six1 to induce late stage metastasis. cluster is also sufficient to increase the TIC Interestingly, Six1 not only activates TGF-β population as measured by flow cytometry, signaling, but it also enables the switch of mammosphere formation, and in vivo serial TGF-β signaling from tumor suppressive dilution assays. Finally, we demonstrate a to tumor promotional, a phenomenon significant correlation between miR-106b, of considerable importance in cancer Six1, and activated TGF-β signaling in human pathogenesis. This event, coined the “TGF-β breast cancers, and further show that high paradox,” has been an area of extensive levels of miR-106b and miR-93 in breast research, but remains largely elusive. tumors significantly predicts shortened time to To further investigate the mechanism by relapse. These findings expand the spectrum of oncogenic functions of miR-106b-25, and which Six1 mediates the switch in TGF-β may provide a novel molecular explanation, signaling, we performed a miRNA microarray through the Six1 regulated miR-106b-25 screen and identified a cluster of miRNAs, the miR-106b-25 cluster, that is upregulated in cluster, by which TGF-β signaling shifts from tumor suppressive to tumor promotional. response to Six1 overexpression. The miR- 106b-25 cluster consists of three miRNAs, miR-106b, miR-93, and miR-25, which reside together in the intron of the MCM7 gene. Importantly, overexpression and knockdown experiments demonstrate that Six1 regulates all three miRNAs within the cluster. Interestingly, these miRNA have previously

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 39 POSTER SESSION A

A21 MALAT-1 is essential for lung Exon microarrays of the WT and KO cell cancer metastasis in a novel human lines have revealed multiple migration- or knockout model. Tony Gutschner1, metastasis-associated transcripts deregulated Moritz Eißmann2, Monika Hämmerle1, by loss of MALAT1 and hence potential target Marion Stentrup1, Catherina Hildenbrandt1, genes. These analyses also uncovered, Matthias Groß1, Martin Zörnig2, Sven that splicing is likely not the only functional Diederichs1. 1German Cancer Research mechanism of MALAT1 in the nucleus. Center (DKFZ) & Institute of Pathology, Taken together, we have developed a novel, Heidelberg, Germany, 2Georg-Speyer-Haus, highly effective approach for the knockout Frankfurt, Germany. of genes that can be used for non-coding as The highly conserved long non-coding RNA well as coding RNAs in human tumor cells as MALAT-1 (Metastasis-Associated in Lung well as cells from other species. Knockout of Adenocarcinoma Transcript 1) had been MALAT1 in human lung cancer cells revealed discovered as a prognostic marker associated its essential function in metastasis as well with poor survival and development of distant as genes directly and indirectly targeted by metastasis in lung adenocarcinoma. Since MALAT1. then, it has been found to be deregulated in numerous tumor entities and has been linked to splicing. However, its functional relevance A22 Genome-wide miRNA screens in tumor cells remains to be elucidated. revealed miRNAs that regulate MGMT Knockdown models for MALAT1 have been expression. Deepa Kushwaha1, Valya described but suffer from insufficient silencing Ramakrishnan2, Bob Carter2, Clark C. efficiency of the highly abundant, nuclear non- Chen2. 1Dana-Farber Cancer Institute, Boston, coding RNA (ncRNA). MA, 2Moores Cancer Center, University of In this project, we have developed a novel California, San Diego, CA. strategy to create ncRNA knockouts in human DNA alkylating agents remain the critical cancer cell lines. We have successfully used chemotherapeutic agents for a wide spectrum a synthetic Zinc Finger Nuclease engineered of cancers, including glioblastoma. The to target the 5’-region of MALAT1 to stably tumoricidal activity of DNA alkylating agents and biallelically integrate RNA-destabilizing is largely derived from the induction of O6- elements into the genome of human lung alkyl-guanine which is the major carcinogenic cancer cells (A549). This approach resulted in lesion in DNA. A major mechanism of a specific and more than 1000-fold silencing therapeutic resistance involves the DNA of MALAT1 in individual clones compared to a repair enzyme O6- methylguanine-DNA less than 5-fold silencing using siRNAs. Thus, methyltransferase (MGMT). The primary this approach can be used to create functional role of this enzyme is to remove the alkyl knockouts of coding as well as non-coding group from the modified guanine and restore genes also in human tumor cell lines allowing the DNA to its undamaged form. This loss-of-function studies also of non-conserved enzyme plays a critical role in modulating ncRNAs in the future. the therapeutic efficiency of DNA alkylating Phenotypically, the MALAT1-Knockout chemotherapies, including temozolomide, cells (KO) greatly differ from their parental in the treatment of glioblastomas. In cell line and wildtype clones (WT): Next to glioblastoma, low levels of MGMT expression morphological changes, the migration of are associated with favorable response to the KO cells is largely impaired as shown in temozolomide therapy. scratch assays. In xenograft assays after i.v. In an analysis of tumor specimens derived injection, the KO cells form significantly fewer from glioblastoma patients, we found and smaller lung metastases than their WT counterparts. Since no large difference was significant disparity in the expression levels of MGMT mRNA and protein in a large observed after subcutaneous injection of the number of specimens. The MGMT mRNA WT and the KO cells, this indicates a specific, levels correlated poorly with MGMT protein active and essential function of MALAT1 in levels by Western blotting (R2=0.04, p=0.34) metastasis.

40 NONCODING RNAS AND CANCER POSTER SESSION A or by ImmunoHistoChemical (IHC) stain reduced after transfection of miR-603 relative quantitation (R2=0.02, p=0.50). To exclude to a control miRNA. Mutating the putative the possibility that the poor correlation miRNA binding sites abolished these effects. was due to substandard specimens, we Additionally, MGMT mRNA preferentially determined the mRNA and protein levels of immunoprecipitated with biotinylated miR- Colony Stimulating Factor 1 (CSF1), a gene 603. Finally, we demonstrated that the previously shown to exhibit excellent mRNA/ high expression of miR-603 correlated with protein correlation. In contrast to MGMT, the improved clinical response to temozolomide in mRNA expression of CSF1 correlated well two independent clinical datasets. 2 with protein (R =0.47, p=0.001). Overall, our data suggest that MGMT We found that a major contributor to this expression is regulated by multiple miRNAs. disparity involved the expression of MGMT Further, the expression level of such regulating microRNA. We hypothesized that miRNAs correlate with clinical response the increased expression of such microRNAs to temozolomide treatment. An exhaustive would be associated with favorable response identification of such miRNAs may harbor to temozolomide and looked for such miRNAs implications in terms of personalizing by a genome-wide microRNA profiling oncologic care as it relates to alkylating screen using specimens derived from 87 chemotherapies. glioblastoma patients and correlating these findings with temozolomide response. We identified miR-181d as one such candidate. A23 A noncoding RNA panel Indeed, transfection of miR-181d down- predicts intermediate-risk childhood regulated MGMT mRNA and protein rhabdomyosarcoma prognosis better expression. Further, luciferase reporter than standard pathologic criteria and 1 assays and co-precipitation studies revealed coding genes. Anirban P. Mitra , Jonathan 1 2 3 direct interactions between miR-181d and D. Buckley , Sheetal A. Mitra , Elai Davicioni , 4 5 the MGMT 3’ UnTranslated Region (UTR). James R. Anderson , Philipp Kapranov , 6 7 Finally, MGMT expression inversely correlated Stephen X. Skapek , Douglas S. Hawkins , 2 1 with miR-181d expression in independent Timothy J. Triche . University of Southern 2 glioblastoma collections. However, this California, Los Angeles, CA, Children’s correlation was modest. Hospital Los Angeles, Los Angeles, CA, 3GenomeDx Biosciences Inc., Vancouver, BC, The modest correlation between miR-181d Canada, 4University of Nebraska, Omaha, NE, and MGMT suggests the existence of other 5The St. Laurent Institute, Cambridge, MA, MGMT regulating miRNAs. We thus carried 6UT Southwestern Medical Center, Dallas, TX, out a biochemical screen transfecting 885 7Seattle Children’s Hospital, Seattle, WA. known miRNAs into MGMT expressing T98 glioblastoma cells to identify miRNAs that Introduction: Rhabdomyosarcoma (RMS) down-regulated MGMT expression upon is the most common pediatric soft-tissue transfection. The top 15 candidates identified sarcoma, stratified by the Children’s Oncology were then tested in A1207, LN18 and LN340 Group (COG) into low/intermediate/high risk glioblastoma cells, three independent MGMT based on clinical outcomes. However, most expressing cell lines. We found that miR- patients are categorized as intermediate-risk 603 consistently down-regulated MGMT where survival is highly heterogeneous, thus expression on both the protein and mRNA suggesting an inability to accurately stratify a level. The effect was observed in both majority of patients. We profiled intermediate- conventional and neurosphere glioblastoma risk RMS’s for levels of coding and non- lines. Importantly, the suppression of MGMT coding transcripts to construct prognostic expression and induction of temozolomide signatures. The goal was to identify panels sensitivity by miR-603 could be reversed of RNAs that reflect underlying tumor biology by the expression of miR-603 antagomirs. and provide better risk stratification than Further, the luciferase activity from the routine clinicopathologic parameters. construct containing the MGMT 3’UTR was

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 41 POSTER SESSION A

Methods: Transcriptomes from 79 A24 Interaction of miR-106a~363Xpcl1 prospectively-obtained primary tumors and p27Kip1 in thymopoiesis and from intermediate-risk RMS patients under lymphomagenesis. Daniel Kuppers, Thomas COG clinical trial protocols were profiled on Schmitt, Bruce Clurman, Harry Hwang, Affymetrix Human Exon 1.0 ST microarrays. Matthew L. Fero. Fred Hutchinson Cancer Expressions of 1,400,033 probe sets Research Center, Seattle, WA. representing annotated and unannotated The Xpcl1 (Kis2) gene encodes the miR- transcripts were analyzed using Genetrix suite 106a~363 miRNA cluster in mice. Although of microarray analysis tools. Cox regression it was first identified as common integration and leave-n-out cross validation were used to site of the M-MuLV retrovirus in tumors from derive and finalize the expression signatures. p27Kip1 (Cdkn1b) knockout mice, the nature An effort was made compare individual of its interaction with cell cycle genes is prognostic potentials of the coding and non- uncertain. The miR-106a~363 miRNAs are coding signatures, and that of a signature that highly conserved across species, implying combined both features. an important role in normal biological Results: Standard pathologic prognosticators processes. However, in the absence of a such as histologic subtype classification reported phenotype in knockout mice, its (alveolar versus embryonal) and PAX-FKHR normal function is unclear. We examined fusion gene status were unable to predict miR-106a~363 expression in developing outcome in this cohort (p=0.40 and 0.45, T lymphocytes and found that it is highly respectively). Cox regression analysis on expressed in immature T-cells but is 17,049 coding transcripts created a 42-gene repressed at the CD4+/CD8+ double positive meta-feature that was able to predict survival (DP) stage of thymopoiesis. Using a T cell (p=0.00024). Leave-n-out cross validation specific Xpcl1 transgene, we found that forced of this meta-feature upheld its prognostic expression of miR-106a~363 altered the ability (p=0.00030). Analysis of probe set distribution of T cell subsets. Xpcl1 repressed regions (PSRs) corresponding to unannotated T-cell receptor expression and directly “dark matter” transcripts identified a 32-PSR targeted the CD69 cell surface protein. This meta-feature that also predicted survival with causes accumulation of DP thymocytes greater significance than PSRs corresponding through complementary mechanisms. to coding transcripts (p<0.00001). To We show that, in developing T-cells, p27Kip1 reduce feature redundancy, multiple PSRs is differentially regulated due to variation interrogating the same genomic locus were in expression of FoxO family transcription replaced by a representative PSR that shrunk factors. Likewise, p27 is upregulated at the meta-feature size to 24 PSRs, which the transcriptional level by Xpcl1, which is was still able to predict survival better than surprising since this should have a tumor the coding gene meta-feature (p<0.00001). suppressor effect. Nonetheless, miR- A meta-feature that combined coding and 106a~363 transgenic (Lx) mice developed non-coding RNA features retained its ability to spontaneous high grade T cell lymphomas. predict outcome (p=0.00002), with non-coding The combination of p27Kip1 deletion with RNA features contributing towards the bulk of miR-106a~363 overexpression, markedly its prognostic potential. accelerated lymphoma development, Conclusions: A more concise non-coding confirming cooperation of these two RNA meta-feature was able to better predict mutations. We conclude that deletion of outcome than a larger coding gene meta- p27Kip1 directly overcomes a tumor-suppressor feature in intermediate-risk RMS, where activity of miR-106a~363, whereas the standard pathologic prognosticators failed. principal oncogenic function of the miRNA This suggests the role of non-coding cluster remains unknown. transcripts in regulating and determining RMS biology and aggressiveness, and their potential to serve as novel prognostic indicators.

42 NONCODING RNAS AND CANCER POSTER SESSION A

A25 Differences in microRNA DICER. Enrichment ontology analysis of expression patterns in breast cancer the microRNAs differentially expressed in subtypes. Sandra L. Romero-Cordoba1, the TN tumors, detected pathways like p53 Rosa G. Rebollar-Vega1, Valeria Quintanar- and focal adhesion whose role in cancer Jurado1, Veronica Bautista-Pina2, Sergio development, invasion and metastasis might Rodriguez-Cuevas2, Antonio Maffuz-Aziz2, be crucial; and MAPK, which has been related Alfredo Hidalgo-Miranda1. 1Instituto Nacional to recurrence of TN tumors. Regarding the de Medicina Genomica, Mexico, 2Instituto de expression of Ago2 and DICER, we detected Enfermedades de la mama FUCAM, Mexico. down-regulation (approximately 20% less) of both proteins, mainly in TN tumors. Down- Breast cancer can be classified according to the gene expression signatures of the regulation in the triple negative tumors was tumors into four clinically relevant sub-types, further validated at the mRNA level by RT- luminal A, luminal B, Her2-like and basal- qPCR assays. Our data identified the altered like. Additionally microRNA expression expression of several microRNAs whose patterns can separate normal tissue from aberrant expression might have an important breast tumors, but there is currently limited impact on cancer-related cellular pathways information about the potential differences and whose role in breast cancer has not been previously described. microRNAs expression in microRNA expression profiles between is also capable to discriminate between the different tumor subtypes, defined by gene expression. In order to determine the different tumor subtypes and the expression of proteins involved in microRNA biogenesis microRNA expression profiles in breast tumors, and explore the differences in the might play a role in breast carcinogenesis, expression patterns of these molecules specifically in the triple negative subtype. between different cancer subtypes, we analyzed the expression of 664 microRNAs A26 DNA copy number alterations with the TaqMan low-density array platform in deregulate expression of miRNAs in lung 40 breast tumors from different subtypes, and adenocarcinoma. Greg L. Stewart, Katey in 21 adjacent normal breast tissues. Tumor SS Enfield, Stephen Lam, Wan Lam. British sub-typing was carried out with the PAM50 Columbia Cancer Research Centre, algorithm in expression data obtained with Vancouver, BC, Canada. the Affymetrix Human Gene ST 1.0 array, obtaining 8 Luminal A tumors, 9 luminal B, Background: Lung cancer represents an 8 Her2-like and 3 Triple negative basal-like enormous health burden, representing tumors. Given the low number of basal-like the most common cause of cancer death tumors, for the microRNA expression analysis, worldwide, with a five-year survival of less we included 12 additional triple-negative than 15%. microRNAs (miRNAs) have tumors defined by immunohistochemistry. emerged as major players in lung cancer Finally, we analyzed the expression of oncogenesis, displaying both oncogenic DICER and Ago2, involved in the biogenesis and tumor suppressive functions. DNA copy of microRNAs in the breast tumors. 131 number (CN) amplification of oncogenes is a microRNAs showed significant differential major molecular mechanism driving cancer, expression (adjusted P value=0.05, Fold and like protein coding genes, CN alterations Change=2) in breast tumors compared to the can influence miRNA expression levels. normal adjacent tissue. The role of 25% of We hypothesize that DNA copy number these microRNAs has not been previously gain modulates the expression of miRNAs reported in breast cancer. 10 microRNAs important to cancer cell growth, and that showed differential expression between integrative analysis can identify these cancer basal/triple negative tumors compared to driving miRNAs. hormone receptor and HER2 positive tumors. Methods: Global gene dosage profiles for 46 Transcriptional targets of these microRNAs lung adenocarcinoma and paired adjacent include genes involved in the carcinogenesis non-malignant tissues were generated by of triple negative tumors, like PARP1, or in array comparative genomic hybridization. the microRNA biogenesis machinery, like miRNA sequencing analysis was performed

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 43 POSTER SESSION A

on this same panel of tumors and matched worldwide. As of 2010, approximately non-malignant tissues using Illumina GAXII one-third of U.S. adults are obese, while small RNA sequencing technologies. CN and another one-third of adults are overweight. expression were correlated for each miRNA Obesity is one of the risk factors for ovarian (Spearman’s correlation >0.3, p<0.05) and cancer. Greater than 75% of ovarian cancer expression of each significant miRNA was patients present with advanced stage compared between tumors with and without disease. Tumors preferentially spread to CN gain (U-test p<0.05). To select for miRNAs the omentum, a major site of abdominal fat, that most likely play a role in cancer we where ovarian cancer cells infiltrate omental compared the expression between tumor and adipose tissue. Omental adipocytes may matched normal and selected for miRNAs interact directly with cancer cells to create a with a fold change of at least 2 in tumors as microenvironment niche that supports cancer compared to normal tissues. growth. Recent studies have shown that cells can communicate through the exchange Results: We identified a panel of miRNAs in lung adenocarcinoma whose expression of bioactive molecules via microvesicles deregulation was associated with CN (exosomes and ectosomes). Microvesicles alteration. These miRNAs were found to be fuse with target cell membranes and transfer gained in >15% of tumors and underwent at proteins, lipids, mRNAs and non-coding RNAs least a 2 fold greater increase in expression in from donor to recipient cells. We hypothesize tumors compared to matched normal tissues. that adipocytes secrete microvesicles containing tumor-supporting factors that This study identified several miRNAs known to play a role in cancer including miR-141, promote ovarian cancer progression. known to be highly upregulated in lung and Ion Torrent RNA sequencing and subsequent ovarian cancer, and miR-301a, upregulated in qRT-PCR analyses were used to identify invasive early cervical cancer. In addition we non-coding microvesicle RNAs secreted identified several novel miRNAs that have not by primary cultures of cancer associated yet been implicated in cancer. adipocytes (CAA), cancer associated Conclusion: Here we identify a panel of fibroblasts (CAF), normal adipocytes, normal miRNAs that are selectively gained at the fibroblasts and ovarian cancer cells. Of the genomic level to alter expression in lung non-coding microvesicle RNAs identified, adenocarcinoma. Several of these miRNAs microRNA 21 (miR21) showed significantly have been previously shown to have higher expression in microvesicles secreted oncogenic properties in many cancer types from CAA when compared to those from CAF including lung cancer and may be potentially and normal omental adipocytes, fibroblasts, useful as therapeutic targets. In addition we and ovarian cancer cells. Using qRT- identify several miRNAs that have not been PCR of RNA isolated from microdissected previously reported in lung cancer. Future frozen tissue and In-situ hybridization on characterization of these miRNAs may lead FFPE tissue sections, CAA demonstrated to increased knowledge of adenocarcinoma significant higher miR21 expression levels oncogenesis. compared to the adjacent cancer cells and normal adipocytes. MiR21 was up-regulated in metastatic ovarian cancer cells in the A27 microRNA 21 in adipocyte- omentum as compared to those in the derived microvesicles confers Taxol- primary tumor site. Moreover, strong miR21 resistance of ovarian cancer cells through staining was observed in omental cancer cells downregulation of apoptotic protease particularly at the invasion front, suggesting activating factor 1. Ngai Na Co, Rosemarie that miR21 may be transferred from CAA to Schmandt, Karen Lu, Samuel Mok. The their neighboring ovarian cancer cells. University of Texas MD Anderson Cancer To test this hypothesis, SKOV3 ovarian Center, Houston, TX. cancer cells were co-cultured with adipocytes Over the past 30 years, there has been a transfected with miR21-FAM or incubated dramatic increase in the incidence of obesity with fluorescently labeled adipocyte-derived

44 NONCODING RNAS AND CANCER POSTER SESSION A microvesicles. Confocal microscopy confirmed A28 DD3/PCA3 noncoding RNA the delivery of miR21 by adipocyte-derived regulates prostate cancer cell survival microvesicles to cancer cells. Since miR21 and modulates AR signaling. Luciana expression in tumor cells has been linked with Ferreira1, Antonio Palumbo2, Kivvi Mello1, resistance to a variety of chemotherapeutic Felipe Leite2, Cinthya Sternberg1, Mauricio agents, we further delineated the direct Caetano1, Adriana Neves3, Luiz Ricardo effect of miR21 on the chemoresistance of Goulart3, Etel Gimba4. 1Instituto Nacional de ovarian cancer cell. The ovarian cancer cell Câncer, Rio De Janeiro, Brazil, 2Universidade lines, OVCA432 and SKOV3, transfected Federal do Rio de Janeiro, Rio De Janeiro, with miR21 precursor showed an increase Brazil, 3Universidade Federal de Uberlândia, in cell survival and a decrease in apoptosis Uberlândia, Brazil, 4Universidade Federal in the presence of taxol. Furthermore, Fluminense, Rio Das Ostras, Brazil. miR21 was significantly up-regulated in The prostate cancer antigen 3 (DD3/ taxol-resistant HeyA8-MDR and SKOV3- PCA3) is a non-coding RNA (ncRNA) TR cells as compared to the parental cell specifically expressed in prostate tissues lines, HeyA8 and SKOV3, respectively. and overexpressed in prostate cancer These data strongly suggest that miR21 (PCa) tumors. Although widely applied as a may confer taxol resistance to ovarian diagnostic marker for PCa, to date nothing cancer cells. Using microarray analysis of has described about its role in PCa biology. RNA isolated from SKOV3 cells transfected We used herein small interfering RNA (siRNA) with the miR21 precursor, we identified in order to knockdown DD3 mRNA message a set of chemoresistance-related genes as an approach to elucidate DD3 functional associated with miR21 expression. One of roles in PCa cells. LNCaP cell line was used the most significantly down-regulated genes herein as an in vitro model for DD3 functional is Apoptotic Protease Activating Factor 1 assays. siRNA sequences were specifically (APAF1). By qRT-PCR, we confirmed that designed for DD3 exon 4 mRNA sequences miR21 over-expression decreased APAF1 (siDD3), as well an scrambled siRNA (siScr), mRNA expression levels in OVCA432 and as negative control. LNCaP cells were SKOV3 cells. A strong miR21 binding site is transiently transfected with siDD3 or siScr predicted on the 3’-UTR of APAF1, suggesting and DD3 expression was analysed by real APAF1 is the direct target of miR21. time PCR (qRT-PCR) using DD3 specific In summary, up-regulation of miR21 in oligonucleotides. LNCaP cells transfected with omental ovarian cancer cells is mediated siDD3 demonstrated a marked decrease in through transport of miR21 in adipocyte- cell proliferation and viability, as compared to derived microvesicles from CAA in the siScr transfected cells. This growth inhibition omentum. Further, miR21 confers taxol- was specific for DD3 expressing cells. Further, resistance in ovarian cancer cells by down- LNCaP cells in which DD3 was knocked-down regulating APAF1 expression in ovarian presented a significant increase in proportion cancer cells to decrease taxol-induced of cells in SubG0/G1 phase of cell cycle and apoptosis. presenting pyknotic nuclei, indicative of cells undergoing apoptosis. In order to investigate the putative mechanisms underlying the decrease of LNCaP cell survival as a result of DD3 knockdown, we then evaluated the involvement of DD3 on androgen receptor (AR) pro-survival signaling. DD3 expression was significantly upregulated as a result of LNCaP treatment with dihydrotestosterone (DHT), the active androgen metabolite. This effect was reverted by the addition of the AR antagonist, flutamide. Consistent to an AR activation by DHT treatment, LNCaP cells presented a significant upregulation

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 45 POSTER SESSION A

of AR target genes. Notably, siDD3/LNCaP cell lines that showed the appearance of transfected cells significantly inhibited the multinucleated structures indicative of skeletal expression of tested AR responsive genes. muscle-like myotubes in vitro. Moreover, Besides, DD3 knockdown was able to Notch3 knockdown impaired tumor growth in counteract DHT stimulatory effects over AR vivo. Here we expand this study evaluating, target gene expression. Despite negatively through microarrays profiling, the expression modulating the transcription of AR target of microRNAs 48 hours after Notch3 siRNA genes, DD3 knockdown did not alter Akt treatment compared to that of control siRNA. and ERK phosphorylation, suggesting that Several up- or down-regulated microRNAs DD3 is mainly controlling the expression were detected in both RH30 alveolar and of signaling pathways downstream to AR RD embryonal cells. The up-regulation of activation. Besides, DD3 mRNA expression muscle-specific microRNAs such as miR-1, was mainly detected in nuclei and ribosome miR-133 and miR-206 was confirmed 24 and cell compartments, indicating that in this cell 48 hours post-Notch3 depletion by RT-qPCR. niches DD3 ncRNA exert its main functional Moreover, non-muscle-specific microRNAs roles. In summary, our findings indicate that such as miR-26a and miR-214 that cooperate DD3 is a ncRNA whose expression is AR to myogenesis were up-regulated as well. regulated and is involved on the control of Altogether, these preliminary results indicate PCa cell survival and proliferation, in part by that Notch3 prevents the differentiation of modulating the AR signaling pathway and RMS cells also through down-regulation of its target genes. These findings correspond pro-myogenic microRNAs. More deep studies to the first description of DD3 roles on PCa are in course to dissect the Notch3-dependent cells and could provide new insights into modulation of these small non-coding RNAs understanding prostate carcinogenesis, in this soft tissue sarcoma. besides opening new prospects to use DD3 not only as a biomarker for PCa, but also as an specific target for therapeutic approaches A30 miRNA regulators of ovarian aiming to inhibit PCa growth through cancer metastasis that predict patient 1 negatively modulating AR pro-survival signal outcomes. Alexander S. Brodsky , Hsin-Ta 1 1 1 and their target genes. Wu , Souriya Vang , Benjamin Raphael , Laurent Brard2. 1Brown University, Providence, RI, 2Southern Illinois University A29 Notch-dependent modulation School of Medicine, Springfield, IL. of microRNAs in rhabdomyosarcoma. Serous ovarian cancer patients often 1 1 Rossella Rota, Sr. , Roberta Ciarapica, Jr. , succumb to aggressive metastatic disease, 1 1 Letizia Da Sacco, Jr. , Maria De Salvo, Jr. , yet little is known about the behavior and 1 2 Andrea Masotti, Sr. , Lucio Miele, Sr. , Franco genetics of ovarian cancer metastases. We 1 1 Locatelli, Sr. . Ospedale Pediatrico Bambino selected 12 matched primary and omental 2 Gesu’, Rome, Italy, University of Mississippi metastatic serous epithelial ovarian tumors Medical Center, Jackson, MS. to identify the copy number, mRNA and Rhabdomyosarcoma (RMS) is a pediatric miRNA expression differences. No significant tumor that originates from skeletal muscle recurring copy number changes were precursors. Therapeutic strategies aimed detected. In contrast, significant recurrence at restoring the differentiation potential with the same expression changes in of RMS tumor cells are accepted as anti- all or the large majority of patients was cancer approaches. Therefore, pathways observed for many mRNAs and miRNAs. We involved in skeletal muscle determination identify almost 1,000 genes with recurring are good potential targets. We have recently significant expression differences across demonstrated that Notch3 signaling is over- the patient cohort suggesting common activated in RMS cell lines of both embryonal phenotypic features are selected from and alveolar subtype. Down-regulation of heterogeneous mixture of primary tumors. Notch3 expression resulted in cell cycle Genes differentially expressed between inhibition of RMS alveolar and embryonal metastatic and primary tumors identify a

46 NONCODING RNAS AND CANCER POSTER SESSION A poor prognosis subtype in primary tumors that many of the metastatic miRNAs mediate that robustly distinguishes high risk patients colony formation, mobility, and/or spheroid across multiple platforms in two large size, but do not significantly affect monolayer independent data sets. Multivariate analysis culture. Predicted targets negatively correlate suggests that the expression signature is with miRNA expression better than sets of independent of residual disease, a common random permutations in the tumors, and clinical risk factor. To gain insight into the some of these targets negatively correlate regulation of these mRNAs, we measured with miRNAs in spheroids vs. monolayers. the expression of 377 miRNAs using Taqman These metastatic miRNAs appear to qPCR in matched primary and metastatic promote metastasis in part by enhancing serous ovarian tumors. We identified 17 β-catenin signaling through repression of miRNAs with significant differential expression APC and suppressing apoptosis. Using in primary and metastatic ovarian human miRNA expression profiles and functional tumors including miR-21, miR-31, and novel studies, we have established the utility of miRNAs previously not associated with spheroid cultures to examine these clinically metastasis. Many of these miRNAs have >10 relevant metastatic miRNAs. In sum, we fold expression differences across multiple have identified metastatic miRNAs, from one patients. We confirmed that the expression of the first miRNA profiles of metastases, differences originate from cancer cells for critical for aggressive disease in ovarian, and many of the miRNAs by in situ hybridization perhaps other, cancers, with potential for and laser capture microdissection of cancer biomarker and therapeutic development. We cells from tumors followed by qPCR. We find that multiple miRNAs, many expressed identify combinations of metastatic miRNAs in metastases and not in primary tumors in with significantly stronger predictions of some patients, are likely important to drive patient outcomes than random combinations metastasis by regulating multiple gene of miRNAs in The Cancer Genome Atlas networks. data. Some of these same miRNAs show the same expression changes in liver metastases compared to colorectal primary tumors. A31 Differential processing of let- Ovarian cancer cells form multicellular 7a precursors influences gemcitabine aggregates, or spheroids, as they disseminate chemosensitivity in pancreatic cancer: throughout the peritoneal cavity and we find Role of ribonucleotide reductase subunit that these metastatic miRNAs affect spheroid M2. Rajgopal Govindarajan. University of formation and growth. All 7 of the metastatic Georgia, Athens, GA. miRNAs expressed in two ovarian cancer cell Ribonucleotide reductase subunit M2 (RRM2) lines are up-regulated in spheroids compared involved in deoxyribonucleotide synthesis acts to monolayers, recapitulating the observations as a key determinant of chemoresistance to in human metastases compared to primary nucleoside drugs (e.g., gemcitabine) used in tumors, suggesting that similar adaptations treating pancreatic cancer. While silencing required for 3D culture are needed to RRM2 expression by synthetic means (e.g., establish metastases. Interestingly, we find siRNA) improves the chemosensitivity of miR-31 promotes metastasis in ovarian pancreatic cancer cells to gemcitabine, cancer suggesting a context dependent targeting endogenous molecules to advance function compared to breast cancer. miR-31 gemcitabine chemotherapeutic responses is up-regulated in metastases by qPCR and in has never been explored. Based on existing situ hybridization, up-regulated in spheroids evidence and computational predictions, we compared to monolayers and inhibition hypothesized that the let-7 tumor suppressor reduces spheroid size and viability using microRNAs will inhibit RRM2-mediated three different inhibitors without significant gemcitabine chemoresistance in pancreatic effects on monolayer growth. We have tested cancer. Reduced expression of the majority the function of 6 other metastatic miRNAs, of let-7 miRNAs with an inverse relationship which predict patient survival, using both gain to RRM2 expression was identified in and loss of function experiments. We find inherently gemcitabine-resistant pancreatic

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 47 POSTER SESSION A

cancer cells. Direct binding of let-7 miRNA ncRNA sequences, we evaluated the effects to the 3’-UTR of RRM2 transcripts identified of target-specific knockdown via RNAi on post-transcriptional repression of RRM2 as a various signal transduction pathways within a novel phenomenon influencing gemcitabine panel of CellSensor® reporter-gene assays. chemosensitivity. Intriguingly, overexpression This has enabled us to create an activity of human precursor-let-7 miRNAs led to profile for these ncRNAs by analyzing them differential RRM2 expression and gemcitabine with the same assay readout in different chemosensitization in a poorly differentiated cell backgrounds (e.g., analysis of Wnt gemcitabine-resistant pancreatic cancer cell signaling in HCT-116 vs. SW480 cells), as line MIA PaCa-2. Defective processing of the well as comparing their effects on different let-7a precursors to mature forms explained, signaling pathways within the same cell in part, the discrepancies observed with let-7a background (e.g., Notch signaling vs. heat expressional outcomes. Screening putative shock response). The positive hits from regulators of let-7a biogenesis in MIA PaCa-2 these screens were evaluated further using cells identified LIN-28 and SET oncoprotein TaqMan® non-coding RNA assays and to differentially modulate let-7 biogenesis related cell-based assays. Many interesting and chemosensitivity in gemcitabine- findings have surfaced and we are currently sensitive versus-resistant pancreatic cancer sorting out what the actual roles are (or cells. Additionally, acquired gemcitabine might be) for many of these transcripts. chemoresistance was found to be This discovery-based approach that uses independent of let-7 alterations. These data tools from our company – providing a novel demonstrate an intricate post-transcriptional offering that is unavailable anywhere in the regulation of RRM2 expression by let-7 and world today – should greatly advance the that the manipulation of let-7 processing understanding of how ncRNAs impact cell machinery may provide a mechanism for regulatory and disease mechanisms. improving gemcitabine chemosensitivity in “For Research Use Only. Not for human or innately-resistant pancreatic cancer cells. animal therapeutic or diagnostic use.”

A32 Decoding the functional role of A33 The role of microRNA-181a noncoding RNAs. Coby B. Carlson1, Lesslie 2 2 in TGF-β-mediated breast cancer Beauchamp , Susan Magdaleno , David progression. Molly A. Taylor1, William 1 1 2 Piper . Life Technologies, Madison, WI, Life P. Schiemann2. 1Case Western Reserve Technologies, Austin, TX. University, Cleveland, OH, 2Case Less than 2% of the human genome Comprehensive Cancer Center, Cleveland, actually encodes protein sequences. The OH. remaining untranslated or non-coding RNA Transforming growth factor-beta (TGF-β) (ncRNA) has been historically disregarded is a multifunctional cytokine that functions as transcriptional “noise”. There is increasing to inhibit breast cancer development evidence to suggest, however, that the in the normal mammary epithelium by importance of ncRNAs might mirror that inducing cell cycle arrest, and maintaining of protein-coding genes. And although the microenvironmental homeostasis through precise function of these molecules is still extracellular matrix (ECM) deposition and largely unknown, it may not be surprising remodeling. Mammary tumorigenesis causes that the dysregulation of ncRNA expression a loss in TGF-β-mediated cytostasis and appears to be a primary feature of many leads to a switch in TGF-β function, whereby complex human diseases. TGF-β promotes epithelial-mesenchymal In an effort to better understand the functional transition (EMT), proliferation, invasion and role of various ncRNAs, we have employed metastasis. This switch in TGF-β function, a complimentary set of genomic tools and from tumor suppressor to tumor promoter, cellular assay research technologies. Using is referred to as the “TGF-β paradox” and a library of Silencer® Select siRNA oligos occurs through molecular mechanisms that designed against a validated set of 45 known remain incompletely understood. Moreover,

48 NONCODING RNAS AND CANCER POSTER SESSION A this switch in TGF-β function is often accompanied by desmoplastic and fibrotic reactions, leading to the formation of a more rigid microenvironment, which in and of itself has been associated with tumor progression. We have shown that tissue compliance and extracellular matrix rigidity mediate how cells sense and respond to TGF-β. Indeed, we can recapitulate the “TGF-β paradox” by exposing metastatic breast cancer cells to compliant ECM signals, which reinstates the cytostatic activities of TGF-β. As a possible mechanism for the influence of matrix rigidity on TGF-β signaling, we sought to define the miRNA expression profiles induced by TGF-β in metastatic cells as compared to their non- metastatic counterparts when grown in either compliant or rigid conditions. We show that ECM rigidity regulates the miRNA expression signature induced by TGF-β in breast cancer cells. From this miRNA signature, we have identified the miR-181 family as a miR family that is highly upregulated in metastatic cells in response to TGF-β. Additionally, overexpression of a miR-181a mimic enhances TGF-β-mediated invasion. Conversely, treating cells with a hairpin inhibitor directed against miR-181a inhibits TGF-β-mediated invasion, EMT, and survival signaling. Furthermore, inhibition of miR-181a abrogates pulmonary outgrowth and enhances overall survival in vivo. Collectively, our findings identify the miR-181a family as a novel effector of TGF-β-driven tumor progression and suggest that measures capable of diminishing miR-181a levels may alleviate breast cancer progression. [FY09-DOD-BCRP-Predoctoral Traineeship Award].

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 49 POSTER SESSION A

50 NONCODING RNAS AND CANCER Poster Session B

B1 microRNA-424 as a potential mammary stem cell population in vitro. These regulator of mammary cell epithelial- data suggest that microRNA-424 may play to-mesenchymal transition and breast an important role in both the mesenchymal cancer stem-like cell properties. David programming and increased tumor-initiating J. Drasin, Deepika Neelakantan, Heide cell formation that occur during an oncogenic L. Ford. University of Colorado Anschutz EMT. Further investigation of microRNA-424 Medical Campus, Aurora, CO. may yield new targets for inhibiting EMT and/ The epithelial-to-mesenchymal transition or tumor-initiating cell formation during breast (EMT) is a cellular program in which epithelial cancer. cells lose epithelial characteristics and gain mesenchymal characteristics, resulting in a B2 Treatment of ovarian cancer with range of acquired traits such as increased targeted tumor-penetrating siRNA plasticity that allow stationary epithelial nanocomplexes. Yin Ren1, Hiu Wing cells to become motile. This process is Cheung2, Ronny Drapkin3, David Root2, necessary for proper development during Justin Lo4, Valentina Fogal5, Erkki Ruoslahti5, embryogenesis and for wound healing William Hahn2, Sangeeta Bhatia4, Geoffrey later in life. A similar aberrant, and more von Maltzahn1, Amit Agrawal1, Glenn transient, EMT program can contribute to the Cowley2, Barbara Weir2, Jesse Boehm2, metastatic dissemination of epithelial cancer Pablo Tamayo2, Jill Mesirov2, Alison cells, and is referred to as an oncogenic Karst3. 1Harvard-MIT Division of Health EMT. Recent breast cancer studies have Sciences and Technology, Cambridge, identified components of the microRNA-200 MA, 2Broad Institute of Harvard and MIT, family as major players in maintaining the Cambridge, MA, 3Dana Farber Cancer epithelial phenotype of cancer cells; the Institute, Boston, MA, 4MIT, Cambridge, MA, loss of the microRNA-200 family coincides 5Burnham Institute for Medical Research, with more aggressive breast cancers in Santa Barbara, CA. humans. We find that through the course of an induced EMT in human mammary cells, This abstract is being presented as a short mesenchymal programming is activated prior talk in the scientific program. A full abstract to epithelial gene repression and prior to loss is printed in the Proffered Abstracts section of members of the microRNA-200 family; (PR5) of the conference Proceedings. however, epithelial genes are ultimately decreased. This simultaneous expression B3 Reprogramming transcription of mesenchymal and epithelial proteins at by distinct classes of enhancers this early time point, as opposed to an all or Ivan Garcia- none phenotype, may more closely resemble functionally defined by eRNA. Bassets. University of California, San Diego, changes seen in human carcinomas. To La Jolla, CA. identify microRNAs important during this early mesenchymal-like transition, we performed a Mammalian genomes are populated with microRNA microarray screen. We identified thousands of transcriptional enhancers that multiple oncogenic EMT inducers as up- orchestrate cell-type-specific gene expression regulating microRNA-424 concomitantly programs, but how those enhancers are with the induction of a mesenchymal-like exploited to institute alternative, signal- phenotype, and before the repression of dependent transcriptional responses remains epithelial markers and the microRNA-200 poorly understood. Here we present evidence family. Overexpression of microRNA-424 in that cell-lineage-specific factors, such as non-transformed human mammary epithelial FoxA1, can simultaneously facilitate and cells is sufficient to produce a mesenchymal- restrict key regulated transcription factors, like phenotype, as well as increase the exemplified by the androgen receptor (AR),

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 51 Poster session B

to act on structurally and functionally distinct ovarian cancer patients. Further studies with classes of enhancer. Consequently, FoxA1 larger sample sizes are warranted to validate downregulation, an unfavorable prognostic our results. sign in certain advanced prostate tumors, triggers dramatic reprogramming of the hormonal response by causing a massive B5 A comprehensive noncoding RNA switch in AR binding to a distinct cohort of pre- map in lung, liver, and breast cancer established enhancers. These enhancers are uncovers novel players in tumorigenesis. 1 functional, as evidenced by the production of Maria Polycarpou-Schwarz , Sebastian enhancer-templated non-coding RNA (eRNA) Aulmann2, Thomas Longerich2, Joachim 3 3 based on global nuclear run-on sequencing Rom2, Thomas Muley , Michael Meister , 3 (GRO-seq) analysis, with a unique class Heike Zabeck , Peter Sinn2, Philipp 3 apparently requiring no nucleosome A. Schnabel2, Hans Hoffmann , Peter Schirmacher2, Tony Gutschner1, Sven remodeling to induce specific enhancer- 1 1 promoter looping and gene activation. Diederichs , Stefanie Grund , Monika 1 1 1 GRO-seq data also suggest that liganded Hämmerle , Anna Roth , Julia Neugebauer , 1 1 AR induces both transcription initiation and Evgenij Fiskin , Catherina Hildenbrandt , Arne 2 1 elongation. Together, these findings reveal a Warth . German Cancer Research Center large repository of active enhancers that can (DKFZ) & Institute of Pathology, Heidelberg, be dynamically tuned to elicit alternative gene Germany, 2University Hospital, Heidelberg, 3 expression programs, which may underlie Germany, Thoraxklinik, Heidelberg, many sequential gene expression events in Germany. development, cell differentiation and disease Beyond proteins, the human genome progression. contains many more functionally important and clinically informative entities: the noncoding RNAs (ncRNA). However, for most B4 microRNAs differentially expressed long ncRNAs, neither their function nor their in preoperative human serum of ovarian expression patterns in health and disease are 1 1 cancer. Angel Chao , Tzu Hao Wang , known. Hua-Chien Chen2, Shu-Jen Chen2, Chyong- Huey Lai1. 1Chang Gung Memorial Hospital, Here, we have discovered novel, important Guishan, Taiwan, 2Chang Gung University, ncRNAs in three major tumor entities that Guishan, Taiwan. could be active players in tumorigenesis or serve as tumor biomarkers. We profiled the MicroRNAs (miRNAs) have been reported expression of 17000 lncRNAs in lung, breast to play important regulatory roles in many and liver cancer as well as normal tissues biological processes and tumorigenesis. To from the respective organs (in total: N=150) investigate the roles of serum miRNAs in using a microarray platform. This expression patients before tumor bulk was surgically landscape was complemented with a profiling removed from ovarian cancer patients, we study of a cell line model for chemotherapy utilized miRNA real-time QPCR profiling response: lung and liver cancer cells were technology to characterize the miRNAs treated with the DNA-damaging cytotoxic in pre-operative and postoperative sera. agents cisplatin and etoposide and ncRNA Six pairs of sera were analyzed using 270 expression patterns were established. miRNA SyberGreen probes. Upregulation of miR187, miR499-3p, miR548c-5p, miR1, This comprehensive expression map miR107, miR125a-5p, miR18b were identified revealed significantly regulated ncRNAs in the preoperative sera. Targetscan was for all parameters tested: besides tissue- utilized to identify Dab2 as the target gene specific ncRNAs, these studies also identified of miR187. In vitro studies of SKOV3 cells prognostic signatures and tumor-specific showed that ectopic expression of miR187 ncRNAs which were validated in two suppressed Dab2 and resulted in increased independent patient cohorts. In addition, cell proliferation. We concluded that miR187 ncRNAs associated with the DNA damage may be a potential biomarker in sera of response were unraveled that affect tumor cell viability.

52 NONCODING RNAS AND CANCER Poster session B

As one functional example, a novel transcript B6 Human genome contains a large that we called “Lung Cancer intergenic RNA number of very long intergenic transcribed 1” (LuCaiR1) is strongly induced in lung regions, some of which are associated adenocarcinoma and its loss impacts the with cancer outcome. Anirban P. Mitra1, viability of lung cancer cells. LuCaiR1 is also Timothy J. Triche2, Sheetal A. Mitra2, downregulated upon DNA damage in lung Jonathan D. Buckley1, Poul H. B. Sorensen3, cancer cells reversing the tumor-associated C Patrick Reynolds4, Robert J. Arceci5, deregulation. To study loss-of-function Patrice Milos6, Georges St. Laurent7, Philipp phenotypes in human lung cancer cells, we Kapranov7. 1University of Southern California, have developed a novel technique to create Los Angeles, CA, 2Children’s Hospital Los functional knockouts of ncRNAs in human cell Angeles, Los Angeles, CA, 3University of lines using Zinc Finger Nucleases (ZFN). British Columbia, Vancouver, BC, Canada, 4 5 To elucidate the molecular functions of the Texas Tech University, Lubbock, TX, Johns 6 novel ncRNAs, we have used RNA affinity Hopkins University, Baltimore, MD, Pfizer purification to identify the protein interaction Center for Therapeutic Innovation, Boston, partners of the tumor-associated ncRNAs. MA, 7The St. Laurent Institute, Cambridge, In one such network, we have identified MA. regulators of the Actin cytoskeleton as major Introduction: Transcriptional output of interaction partners for a novel, cytoplasmic human genome is far more complex than breast cancer-induced ncRNA MaCaiR1 predicted by the current set of protein-coding (Mamma Carcinoma intergenic RNA 1). annotations. However, the fraction of the In summary, we provide the first global genome that is utilized to produce cellular comprehensive map of long ncRNA RNA whose function is hitherto not completely expression in a broad range of human tumor defined, the so called “dark matter RNA”, and normal tissue samples and discovered and its role in regulating cancer progression many new lncRNAs associated with three remains an open issue. Furthermore, our major tumor entities. Initial functional understanding of the repertoire of human characterization has already revealed their RNAs is far from complete, and almost functional importance at the cellular level as all RNA-Seq studies have missed this well as provided hints towards their molecular complexity due to the limited view obtained functions. when only interrogating the polyA+ RNA fraction confounded by biases due to PCR amplifications. This study aimed to assess the complexity of RNAs produced by cancerous and normal human tissues in an unbiased fashion, and examine any potential roles of such transcripts in modulating cancer outcome using pediatric rhabdomyosarcoma as a model malignancy. Methods: Total RNA was profiled from a number of tissues using single-molecule sequencing (SMS) to examine the complexity of transcriptome in the human genome in an unbiased fashion. To minimize methodological biases, no PCR amplification, ligation, or size selection were used. In parallel, to examine the relevance of different RNAs in predicting cancer outcome, expression profiles on 79 intermediate-risk childhood rhabdomyosarcoma primary tumors were generated on Affymetrix Human Exon 1.0 ST microarrays and compared to prospectively-

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 53 Poster session B

obtained outcome information. B7 microRNA regulation of cell viability Results: SMS analysis revealed that “dark and drug sensitivity in lung cancer. Liqin 1 2 2 matter RNAs” comprised up to two-thirds Du , Christopher DeSevo , Borkowski Robert , 2 2 of total non-ribosomal, non-mitochondrial Baker Michael , Adi F. Gazdar , John D. 2 1 1 RNA in human cells. PolyA+ RNA fraction Minna , Alexander Pertsemlidis . UT Health Science Center at San Antonio, San Antonio, had a significantly lower complexity then the 2 total RNA, especially in non-exonic regions. TX, UT Southwestern Medical Center, Dallas, Strikingly, several hundreds of very long TX. (100’s of kbs) abundant intergenic transcribed Introduction: Lung cancer is the leading regions (vlinc’s) were identified in areas of the cause of cancer-related deaths, with the genome that were devoid of protein-coding majority of deaths due to failed therapy from annotations. Most (~80%) of the genomic tumor drug resistance. Third-generation sequences covered by vlincs did not overlap chemotherapeutic agents represent the with previously documented long intergenic standard first-line treatment for advanced non-coding (linc) RNAs, suggesting large small cell (SCLC) and non-small cell (NSCLC) number of RNAs in intergenic space are yet lung cancer patients. Response rates are to be uncovered. To address the question poor (20-40%) with a median survival of 8–10 whether vlincs may be associated with months. prognosis in cancer, specifically childhood Methods: In an unbiased and comprehensive rhabdomyosarcoma, array-based whole- approach, we have combined a high- genome expression profiles were analyzed. throughput screening platform with a library Interestingly, probe sets that were the best of chemically synthesized microRNA mimics predictors of cancer outcome were found to and inhibitors. We have used this platform to be outside boundaries of exons of protein- identify mimics and inhibitors that reduce cell coding transcripts. Strikingly, most of the viability in general, and those that specifically top-ranked probe sets were found to cluster sensitize cells to taxanes. together and defined a >230 kb vlinc region on chromosome 2, previously found by the Results: We have identified several miRNAs SMS analysis. This vlinc region was singularly for which over-expression or inhibition has a able to predict survival for the entire cohort dramatic and selective effect on cell viability (p=0.007). or drug response. We have demonstrated that miR-337-3p mimic sensitizes NSCLC cells to Conclusions: Our data suggest that many taxanes. By combining in vitro and in silico genomic regions currently defined as approaches, we identified STAT3 and RAP1A intergenic give rise to very long transcribed as direct targets that mediate the effect of regions that can modulate ultimate cancer miR-337-3p by enhancing taxane-induced behavior. This indicates that a great number arrest in the G2 phase of the cell cycle. We of the “dark matter” RNAs uncharacterized have also identified an inhibitor of miR-139-5p thus far may be involved in tumorigenesis, as a potent and selective regulator of SCLC and can be used as diagnostic, prognostic cell viability. Inhibiting miR-139-5p decreases and therapeutic-response indicators. In turn, SCLC cell viability by over 80%, but has a this argues for functional importance of the minimal cytotoxic effect on that of NSCLCs or genome’s “dark matter”. immortalized human bronchial epithelial cells. We are currently investigating the targets of miR-139-5p that mediate its effect on SCLC cell viability. Conclusion: We have identified miR-337-3p as a regulator of taxane sensitivity in NSCLC, and miR-139-5p as a modulator of viability in SCLC. Increasing levels of miR-337-3p and inhibiting miR-139-5p may therefore provide novel therapeutic tools for the treatment of NSCLC and SCLC lung tumors, respectively.

54 NONCODING RNAS AND CANCER Poster session B

B8 Molecular function of the continuously administered by replacing the RNA binding protein EWS in RNA medium every 24 hrs. MiRNA expression processing. Bethsaida I. Nieves, Shuang profiling was carried out using TaqMan Low Niu, Dedeepya Vaka, Julia Salzman, Patrick Density Arrays from Applied BioSystems Brown, E. Alejandro Sweet-Cordero. Stanford (Carlsbad, CA). University School of Medicine, Stanford, CA. Results: miR-10a, -34a, -125b, -28-5p, This abstract is being presented as a short and -183 were consistently up-regulated talk in the scientific program. A full abstract and miR-450-5p, -151-3p and -138-2 were is printed in the Proffered Abstracts section consistently down-regulated >2 fold (p < (PR4) of the conference Proceedings. 0.05) in all three ATRA sensitive cell lines at three hours post-ATRA. At 7 days post-ATRA, there were more differentially expressed B9 Critical early microRNA-mediated miRNAs, with 13 miRNAs being up-regulated signaling events during ATRA induced and 18 down-regulated > 2 fold (p < 0.05) neuroblastoma cell differentiation. Niamh across all three cell lines. Among the miRNAs H. Foley, Isabella M. Bray, Raquel Fernandez, differentially expressed at 3 hours post-ATRA, Raymond L. Stallings. Royal College of miR-10a, miR-125b and miR-34a remained Surgeons in Ireland, Dublin, Ireland. differentially expressed at 7 days post- Introduction: Neuroblastoma (NB) is a ATRA. Remarkably, none of the differentially childhood cancer arising from precursor expressed miRNAs in the ATRA sensitive cells of the sympathetic nervous system cell lines were differentially expressed in the with highly heterogeneous clinical behavior, ATRA resistant cell lines at the three hour ranging from spontaneous regression to time point, and very few displayed altered rapid progression and death due to disease. expression in the ATRA resistant cell lines Amplification and over-expression of the even after a 7 day exposure to ATRA. qPCR MYCN transcription factor in neuroblastoma and Western blot analysis of the critical miR- tumors is highly correlated with poor survival 10a target, NCOR2, revealed that this gene of patients. Children with high risk NB are is down regulated at both mRNA and protein given the synthetic retinoic acid, 13-cis- level at the 3 hour time point. In addition retinoic acid, to reduce minimal residual to the targeting of NCOR2 by miR-10a, disease, and exposure of a number of we noted that two additional up-regulated NB cell lines to the related compound, all- miRNAs, miR-125b and miR-132 (significantly trans-retinoic acid (ATRA), induces neural up-regulated within 7 days post-ATRA) are differentiation along with the down-regulation also predicted to target NCOR2, indicating of MYCN. We previously determined that substantial redundancy in miRNA mediated miRNA-10a/b plays a critical role in the signaling. Direct targeting of NCOR2 by process of ATRA induced neuroblastoma these additional miRNAs was experimentally cell differentiation through direct targeting of validated by luciferase reporter assays. This nuclear co-repressor receptor 2 (NCOR2) redundancy in miRNA targeting explains why (Foley et al Cell Death & Differentiation 2011: antagomir inhibition of single miRNAs prior 18:1089). Here, we have profiled three ATRA to ATRA treatment only delays, but does sensitive (SK-N-BE, LAN5 and SHSY-5Y) not block the induction of differentiation. and two ATRA resistant (Kelly and SK-N-AS) Finally, we present evidence that an initial neuroblastoma cell lines for the expression early decrease in MYCN levels, which is well of 750 miRNA loci at early (3 hours) and known to occur in neuroblastoma cells treated late (7 days) time points following ATRA with ATRA, is miRNA mediated. treatment. The purpose of our experiments Conclusions: We have identified critical and was to identify critical early miRNA signaling redundant early miRNA signaling events events, as well as miRNAs that might mediate that take place during the process of ATRA sensitivity versus resistance to ATRA. induced neuroblastoma cell differentiation. Methods: All cells were treated with 5 μmol The absence of these events in ATRA ATRA (Sigma, St. Louis, MO) which was resistant cell lines suggests new avenues of

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 55 Poster session B

investigation for elucidating potential miRNA all differentially methylated regions at putative mediated mechanisms involved with ATRA miRNA promoters. Hypomethylated regions therapy failure. are limited to strictly defined sequence stretches, predominantly found outside of CpG islands (89%). B10 Genome-wide profiling reveals hyper- and hypomethylation at microRNA Correlating promoter methylation with miRNA promoters in chronic lymphocytic expression, we observed loss of methylation leukemia. Constance Baer1, Michael Rehli2, and strong transcriptional upregulation of John C. Byrd3, Clemens-Martin Wendtner4, mir-155 in CLL. Similarly, the promoter Christoph Plass1, Rainer Claus1, Lukas of mir-1204 showed significantly reduced Frenzel4, Manuela Zucknick1, Yoon Jung methylation in CLL associated with increased Park1, Lei Gu1, Dieter Weichenhan1, Martina expression. Mir-129-2, a well known tumor Fischer1, Christian Pallasch4. 1German suppressor in solid tumors, could be detected Cancer Research Center (DKFZ), Heidelberg, among the epigenetically silenced miRNAs. Germany, 2University Hospital Regensburg, Furthermore mir-551b, which has not yet Regensburg, Germany, 3The Ohio State been described in the context of CLL, was University, Columbus, OH, 4University found to be downregulated and a target of Hospital of Cologne, Cologne, Germany. promoter hypermethylation in CLL cells. Chronic lymphocytic leukemia (CLL) is the In conclusion, a combination of DNA most common leukemia in the Western methylation profiling and comprehensive world. Dysregulation of microRNA (miRNA) miRNA promoter identification has allowed expression contributes to CLL pathogenesis. us to identified novel aberrantly regulated Abnormal promoter methylation is involved in miRNAs in CLL. Hypomethylation, which aberrant gene expression in leukemic cells. has previously been underestimated, is Here, we investigate the role of epigenetic determined to be a major mechanism for mechanisms in the global regulation of miRNA miRNA activation in CLL. expression in CLL. In order to characterize disease-specific B11 Expression of microRNAs- aberrant methylation upstream of miRNAs, 135b and -196b in response to cellular methylated DNA from 24 CLL patients stress in leukemia cells. Tsui-Ting Ho, and 10 healthy age-matched controls was Xiaolong He, Ahmet Dirim Arslan, William enriched by Methyl-CpG immunoprecipitation. T. Beck. University of Illinois at Chicago, Differentially methylated genomic loci were Chicago, IL. identified using a custom tiling array covering MicroRNAs are a class of non-coding small the genomic sequences 35 kb upstream and RNA molecules that have roles in important 5 kb downstream of 939 annotated miRNAs. biological processes. Multidrug resistance is We identified putative promoter sequences a key obstacle to successful cancer therapy for 781 miRNAs by determining regions of in human cancers. To determine whether enriched trimethylated histone 3 lysine 4 microRNAs have a role in the development (H3K4me3) in CLL samples, healthy B cells or or maintenance of multidrug resistance, we cancer cell lines. used the TaqManTM Human MicroRNA Array The methylation patterns upstream of miRNAs to investigate the expression of microRNAs in clearly distinguished CLL cells from healthy multidrug resistant (MDR) CCRF-CEM human B cells. Differentially methylated sequences T-cell leukemia cells (CEM/VM-1-5) and drug- are enriched in H3K4me3, suggesting that sensitive parental line, CCRF-CEM (CEM). miRNA promoters are non-randomly targeted The MDR line was selected for resistance to for epigenetic dysregulation. 458 miRNAs teniposide and is cross-resistant to etoposide display differential methylation in at least and other drugs. Our profiling study of 365 5 out of the 24 CLL samples. Noteworthy, microRNAs revealed that certain microRNAs hypomethylation accounted for two thirds of are differentially expressed between CEM/ VM-1-5 and CEM cells, and we focused our

56 NONCODING RNAS AND CANCER Poster session B subsequent work on two of these: miR-135b B12 The miR-15 family and miR-155 and -196b. Since MDR cells were selected mediate WEE1 and CHEK1 effects on and developed by stepwise increases in cisplatin resistance in cancer cells. Lynn concentrations of teniposide, we therefore M. Pouliot1, Jennifer Bai1, Matthew asked whether these altered microRNAs D. Hall1, Scott E. Martin2, Michael M. in MDR CEM/VM-1-5 cells are associated Gottesman1. 1National Cancer Institute, with cellular responses to genotoxic agents. Bethesda, MD, 2National Human Genome We observed substantial increases in miR- Research Institute, Rockville, MD. 135b and -196b expression in drug-sensitive A major challenge in the successful CEM cells following their short exposure treatment of cancer is the ability of cancer (3 to 48 hours) with etoposide, doxorubicin cells to acquire a multi-drug resistant and topotecan, but not in cells treated with (MDR) phenotype, preventing response vinblastine or paclitaxel, suggesting that the to chemotherapeutic agents. Cisplatin upregulation of these two microRNAs might (cis-diamminedichloroplatinum(II)) is a be the consequence of DNA damage. We chemotherapeutic drug commonly used also found similar increases in miR-135b against malignancies including ovarian, and -196b in other leukemia cell lines (RPMI and cervical tumors. Cisplatin resistance 8226 [myeloma], HL-60 [acute promyelocytic (CP-r) studied in cultured cancer cells often leukemia], Jurkat and MOLT-4 [both acute results from a cellular defense mechanism T cell leukemia]), but not in solid tumor- that results in a highly complex pleiotropic derived cell lines (MCF7 breast cancer and phenotype that confers resistance by reducing A2780 ovarian cancer). This suggests that apoptosis, up-regulating DNA damage repair induction of expression of these microRNAs mechanisms, altering cell cycle checkpoints, is histiotype-specific. To further confirm that altering protein localization, redirecting cell upregulation of these two microRNAs is a surface transporters, and disrupting assembly consequence of DNA damage, we examined of the cytoskeleton thereby changing protein expression of miR-135b and -196b following distribution in the cell. This results in cells that ionizing radiation. When we treated CEM are permanently resistant not only to cisplatin pairs with ionizing radiation, MDR CEM/ but also show cross-resistance to multiple VM-1-5 cells were more radioresistant toxic compounds that piggyback into cells on compared to CEM cells. As expected, higher existing transporters. The highly pleiotropic doses of radiation induced miR-135b and CP-r phenotype occurs in a single step that -196b expression in MDR CEM/VM-1-5 cells has yet to be identified. compared to CEM cells, in accordance with what we observed in cells treated with the miRNAs have crucial roles in diverse DNA damaging agent etoposide. Moreover, biological processes such as phenotypic time-course and dose-response studies stabilization, and have been shown to also showed that the elevation of miR-135b and be involved in tumor growth and response to -196b correlated positively with the exposure chemotherapy. We hypothesized that miRNAs time and radiation dosage. Together, our may play a role in mediating the phenotypic results suggest that microRNAs might play changes associated with a pleiotropic cisplatin a significant role in cellular response to resistance mechanism. genotoxic agents and upregulation of miR- KB-CP.5 (resistant to 0.5ug/ml CP) cells were 135b and -196b appears to be a marker of screened with whole kinome siRNA (Ambion DNA damage in leukemia cells. SilencerSelect, 3 siRNAs per gene) and (Supported in part by RO1CA40570 from NCI miRNA mimics (Sanger miRBase 13.0). Cells to WTB, and in part by UIC.) were then challenged with cisplatin at IC5 and IC30 doses (normally causing minimal toxicity to CP-r cells). Cell viability was determined using the Promega CellTiter-Glo luminescent viability assay, and conditions that sensitized the CP-r cells were examined. The miR- 15/16/195/424/497 family miRNA mimics were

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 57 Poster session B

found to confer >2-fold increased sensitivity assay to measure miRNA expression levels to CP.5 cells. miR-16 gave a 5-fold increase at single cell resolution and to extract the in sensitivity and its family member miR-15a contextual information embedded within both caused a 3.4-fold increase, accounting for cancer and non-cancer cell compartments of two of the top three hits in the primary screen. the tumor microenvironment. Follow-up dose-response analysis indicated In this on-going clinical study, we are that miR-15a and miR-16 mimics yielded a characterizing the cell type-specific altered 20- and 10-fold shift in the cisplatin sensitivity expression of a panel of breast cancer- of CP.5 cells, respectively. The two strongest associated miRNAs on large and independent kinome effects were elicited by silencing cohorts of breast cancer patients. To WEE1 and CHEK1. Using TargetScanHuman maximize the number of independent patient (available at www.targetscan.org), an cases per slide and to minimize the number algorithm program for the prediction of miRNA of independent experiments and slides for targets, we found that the miR-15 family is data analysis, we are interrogating miRNA predicted to target WEE1 and CHEK1. For expression on high-density tissue microarrays the miR-15 family, RT-PCR shows a 2 to (TMAs). TMAs designed by NCI Cooperative 5-fold decrease in expression in the CP-r cells Breast Cancer Tissue Resources to find compared to KB-3-1 parental cells. In addition statistically significant associations between miR-155 has a 3-fold decrease in expression an investigative biomarker and disease in KB-CP.5 compared to KB-3-1 parental progression and/or disease outcome will cells. TargetScan analysis of WEE1 indicates serve as discovery set. Our institutional that miR-155 is also a potential regulator of TMAs will serve as validation set and will the WEE1 kinase. We confirmed the ability of also enable us to assess the potential utility the miR-15 family and miR-155 to sensitize of these miRNA contextual signatures to resistant cells to cisplatin by targeting WEE1 predict treatment response in different and CHEK1 by performing loss-of -function adjuvant and neoadjuvant therapy settings and gain-of-function experiments. Using using recurrence-free survival and complete the MTT cytotoxicity assay and full cisplatin pathological response, respectively, as the dose response curves, miRNA inhibitors of clinical endpoint marker. the miR15 family increased resistance while mimics sensitized resistant cells. miR-155 Our preliminary results indicate that mimics resulted in sensitivity returning to inflammation and desmoplasia present as co- parental levels and inhibitors of WEE1 and diseases or co-morbidities in breast cancer, CHEK1 sensitized KB-CP.5 cells to CP. and they are both major confounding factors in the correct interpretation of expression In conclusion, we have identified a miRNA profiling experiments and the assignment family responsible for regulating the cell- of etiological roles to leukocyte-expressed cycle checkpoint kinases WEE1 and CHEK1 miRNAs such as miR-155 and reactive associated with cisplatin resistance. fibroblast-expressed miRNAs such as miR-21. Intriguingly, we have identified a subgroup of B13 Automated multiplex assay for HER2-overexpressing cases in which miR-21 diagnostic application of contextual expression is mainly upregulated with cancer microRNA signatures in breast cells, while miR-21 expression predominantly cancer. Wendy Wells, Sung-hee Choi, Gary accumulates within reactive fibroblasts in the Schwartz, Lorenzo F. Sempere. Dartmouth- rest of HER2-overexpresssing cases and the Hitchcock Medical Center, Lebanon, NH. immense majority of cases from other breast cancer subtypes. The clinical implications MicroRNAs (miRNAs) are rapidly emerging of cell source and expression levels of miR- as a novel class of biomarkers for theranostic 21 and other miRNAs under study will be applications in cancer. Since solid tumor presented at this meeting. lesions are complex and heterogeneous mixtures of different cell types, we previously We will also discuss innovative strategies developed a multiplex tissue slide-based that we are implementing for integration of translational miRNA and well-established

58 NONCODING RNAS AND CANCER Poster session B protein biomarkers to increase molecular of two major pathways in the upstream characterization of breast tumors and activation of miR-135b: APC-β-Catenin and for pipeline automatization from assay SRC-PI3K. MiR-135b up-regulation resulted performance to whole slide image acquisition in reduced apoptosis and increased invasion and computer-assisted analysis to enhance and metastasis due to the down-regulation diagnostic power of current clinical assays. of TGFRB2, DAPK1, APC and HIF1AN. Silencing of miR-135b in vivo reduced tumor multiplicity and tumor load in the AOM/DSS B14 Anti-miR-135b in colon cancer CRC model. Mice treated with anti-miR-135b 1 treatment. Nicola Valeri , Roberta showed well differentiated tumors and acinar 1 Gafa’2, Gerard Nuovo , Giovanni Lanza2, pattern while tumors in the control groups 1 3 Wendy Frankel , Peter K. Vogt , Joanna showed low differentiation and adenomatous 1 4 1 Groden , Michael Karin , Carlo M. Croce , pattern. Conclusions: Our data suggest 1 1 Chiara Braconi , Pierluigi Gasparini , that miR-135b is a key molecule whose 4 3 Sergei Grivennikov , Jonathan R. Hart , activation is downstream of oncogenes and 1 1 Alessio Paone , Francesca Lovat , Muller oncosuppressor genes frequently altered in 1 1 Fabbri . The Ohio State University, CRC. Our study defines specific pathways Columbus, Ohio, 2University of Ferrara, that converge on the activation of the same 3 Ferrara, Italy, The Scripps Research Institute, microrna. The “in vivo” silencing of miR-135 4 La Jolla, CA, UCSD, La Jolla, CA. shows preclinical efficacy with low toxicity and Background: MicroRNAs (miRs) are small non represents the first in vivo study for the use of coding RNAs involved in cell homeostasis. antimiRs in CRC treatment miRs are deregulated in colorectal cancer (CRC). Our study aimed at identifying miRs with a driver role in carcinogenesis altered B15 Repression of PDGFRA-targeting by similar mechanisms in both human and miR-34a promotes tumorigenesis in mouse CRC. Goal of the study was to use proneural malignant gliomas. Joachim CRC mouse models for the pre-clinical Silber, Anders Jacobsen, Tatsuya Ozawa, development of anti-miRs as therapeutic Girish Harinath, Eric C. Holland, Chris drugs. Methods: Azoximetane (AOM)/ Sander, Jason T. Huse. Memorial Sloan- Dextran-Sulfate (DSS) treated mice or Kettering Cancer Center, New York, NY. CDX2Cre-APC f/wt mice were used to study Malignant gliomas - particularly glioblastoma inflammation-associated and sporadic APC- (GBM) - continue to cause a disproportionate related CRC. Human Inflammatory Bowel degree of morbidity and mortality within Disease associated (n=30), and sporadic human oncology. Recent integrated genomics (n=90) CRC with their matched normal has demonstrated biologically distinct tissues were collected according to Good subclasses within malignant glioma that Clinical Practice recommendation and transcend conventional histopathological subjected to RNA extraction using Trizol. miR boundaries. Perhaps the broadest of these and gene expression profiling was assessed subclassess has been termed “proneural” by nCounter technology (Nanostring Seattle). and includes both a large fraction of AntimiR-135b and scrambled probes for in GBMs along with most of their lower-grade vivo studies were synthesized by Girindus. astrocytic and oligodendroglial counterparts. Results: miRs profiling from AOM/DSS and The pathogenesis of proneural gliomas CDX2Cre-APC f/wt CRC. revealed that has been strongly linked to dysregulated miR-135b is one of the most up-regulated PDGF signaling; but although genomic miRs in both models. In humans miR-135b amplification or activating mutations involving over-expression was found in both IBD and the PDGF receptor (PDGFRA) characterize sporadic CRC and was associated with a significant subset of proneural GBMs, the reduced Progression Free Survival and mechanisms driving dysregulated PDGF Overall Survival in CRC patients. Molecular signaling and downstream oncogenic studies in Mouse Embryo Fibroblast and networks in non-amplified/mutated tumors human CRC cell lines highlighted the role remain unclear. MicroRNAs (miRNAs) are a

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 59 Poster session B

class of small, noncoding RNAs that regulate stem cells (iPSCs) and is likely involved in gene expression on a pre-translational malignant stem cell biology. level by binding loosely to complimentary Results: MicroRNA expression profiling of sequences in target mRNAs. The role of FACS purified sub-populations was performed miRNA biology in numerous cancer variants by small RNA seq and confirmed by Q-PCR is well established. In an analysis of miRNA assays. We demonstrate a highly consistent involvement in the phenotypic expression signature of 25 up-regulated microRNAs in and regulation of oncogenic PDGF signaling, the CSC-like population. In silico pathway we found that expression of miR-34a is analysis tools demonstrate that 15 of these responsive to PDGF pathway activation in microRNAs target 21 components of the vitro. Similarly, analysis of data from the p53 signaling pathway including 8 apoptosis Cancer Genome Atlas (TCGA) revealed that genes and 6 cell cycle genes with multiple expression of miR-34a is highly negatively microRNAs targeting the same genes. correlated in proneural gliomas and in gliomas Importantly, 6 of these microRNAs target harboring amplified PDGFRA compared p21(CIP/Waf) , a major regulator of the G1/S to other tumor subtypes. Using primary checkpoint implicated in the regulation of GBM cells maintained under neurosphere neural stem cell differentiation and down conditions, we then demonstrated that miR- regulated during iPSC regeneration. We 34a specifically affects growth of proneural demonstrate markedly reduced levels of GBM cells in vitro and in vivo. Using data p21 as well as remarkably consistent stem from TCGA, we identified PDGFRA as cell-like cell cycle in neuroblastoma CSC a direct target of miR-34a and validated sub-populations. We propose that differential this interaction experimentally. Finally, we regulation of microRNAs targeting p21 provided evidence for a p53-independent disables the G1/S checkpoint and promotes mechanism mediated by PDGF signaling for the maintenance of a distinct CSC sup- negative regulation of miR-34a in proneural population in neuroblastoma. tumors. Taken together, our data suggest reciprocal negative feedback regulation of miR-34 and PDGFRA expression in proneural B17 Inhibition of lung tumor gliomas and, as such, identify a subtype progression in a metastatic mouse specific therapeutic potential for miR-34a. model following intravenous delivery of siRNA nanocomplexes. Jason G. Fewell, Khursheed Anwer, Kevin Polach, Majed B16 The microRNA signature Matar, Jennifer Rice, Angela Rea-Ramsey, characterizing neuroblastoma cancer Jeff Sparks, Diane McClure, Casey Pence, stem cells collectively targets the p53 Elaine Brunhoeber, Leslie Wilkinson. EGEN, 1 2 pathway. Danielle Hsu , Ashley Benham , Inc., Huntsville, AL. Zaowen Zaowen1, Cristian Coarfa1, Eugene Kim1, Preethi Gunaratne3, Jason Exploiting the RNAi pathway offers the M. Shohet1. 1Baylor College of Medicine, potential to advance the treatment of many Houston, TX, 2Methodist Hospital, Houston, diseases through highly specific and efficient TX, 3University of Houston, Houston, TX. silencing of gene products. Unfortunately, the requisite safe and efficient delivery We have recently isolated a novel sub- of nucleic acids to target cells remains a population of neuroblastoma tumor cells fundamental problem for the development of with phenotypic and molecular similarities to RNA and DNA based therapeutics. We have induced pluripotent stem cells. In addition, developed a versatile lipopolyamine based we demonstrate that this population is delivery system that has been optimized for highly tumorigenic, can self-renew, has a in vivo delivery by incorporating functional stem cell like cell cycle, and recapitulates groups onto the core cationic lipopolyamine entire heterogeneous tumors composed of structure. The direct modification of the core progeny and a small CSC-like population. structure (named Staramine) allows for the Disabled p53 function is a common feature of generation of nanoparticle formulations embryonic stem cells and induced pluripotent

60 NONCODING RNAS AND CANCER Poster session B with a wide range of physicochemical B18 The transcribed noncoding genome properties and does not require the co- modulates the biology of Ewing sarcoma formulation of commercial helper or pegylated family of tumors. Sheetal A. Mitra1, Daniel H. lipids. We have previously reported safe, Wai1, William A. May1, Jonathan D. Buckley2, efficient and persistent siRNA mediated Philipp Kapranov3, Robert J. Arceci4, Timothy transcript knockdown specifically in the lung J. Triche1. 1Children’s Hospital Los Angeles, endothelium using the Starmaine delivery Los Angeles, CA, 2University of Southern system. Here we extend those observations California, Los Angeles, CA, 3The St. Laurent by administering a siRNA that targets Institute, Cambridge, MA, 4Johns Hopkins vascular endothelial growth factor receptor University, Baltimore, MD. 2 (VEGFR-2; KDR/Flk-1) in an animal model Introduction: Ewing sarcoma family of of metastatic lung cancer. VEGFR-2, a tumors (EFT) is the second most common receptor tyrosine kinase, has been shown primary malignant bone tumor in children to be mainly expressed on endothelial cells and adolescents. It features a characteristic and plays a critical role in cell proliferation chromosomal translocation; the most common and differentiation and consequently is also being (t11;22)(q24;q12), which encodes important in angiogenesis associated tumor for the chimeric EWS-FLI1 oncoprotein. growth and metastasis. Repeated intravenous The disease is highly invasive with a administration of nanocomplexes comprised 30% 5-year survival rate for patients with of Staramine formulated VEGFR-2 siRNA metastatic disease. This study identifies (~2 mg/kg/dose) resulted in a significant and characterizes a non-coding locus that (40%) reduction of VEGFR-2 transcript is unique to EFTs and mediates at least in levels in isolated tumors from the lungs of part the role of the chimeric oncogene in mice compared to control injected animals. EFT pathogenesis. This particular locus Similar levels of transcript knockdown were suggests that better understanding of the not achieved in tumors when using siRNAs functional non-coding transcriptome of this against targets not having the high level of developmental cancer can potentially assist endothelial cell restriction that is seen with in discovering biomarkers that improve the VEGFR-2. Immunohistopathology of the lungs diagnosis and therapeutic management of the of tumor bearing mice administered VEGFR-2 disease. siRNA indicated a significant decrease in vascular density in lung tumors and was Methods: Expression profiles from a panel of consistent with an overall reduction in tumor 500 tissue samples including over 100 EFTs, burden in the mouse lungs. These results other primary tumors, and normal tissues support the continued evaluation of using were generated using Affymetrix Human Exon Staramine to deliver siRNAs and other similar 1.0 ST microarrays. Data were analyzed using molecules as potential therapies for diseases institutional software, Genetrix. RNAseq data of the lung where the vascular endothelium was generated on two EFT cell lines derived may be involved. from a primary tumor and its metastatic derivative. Data was validated by PCR on 33 cell lines including EFTs and other tumors. Functional studies on EFT cell lines were performed using various RNAi techniques and viral transductions. Additional characterization of the RNA was done by cellular fractionation studies and northern blots. Results: Expression microarray profiles showed unannotated transcripts to be the strongest discriminators of EFTs from all other tumor types. One such transcript, AK057037, was the most highly expressed RNA (1.07 x 10-20) in EFTs. In silico modeling and RNASeq analyses revealed that this

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 61 Poster session B

transcript is a multi-exonic, presumably, long mice were crossed with 25 AKXD (AKR/J non-coding RNA (lncRNA) with a maximum x DBA/2J) recombinant inbred strains to predicted open reading frame of 137 amino produce F1 progeny with varying metastatic acids that had no homology, protein domains, indices. All mammary tumors in the F1 or characteristic motifs. Additionally, multiple progeny were evaluated by miRNA microarray variants of the transcript exist in both the and correlated with metastatic index to nucleus and the cytoplasm. Validation studies produce miR-290 (containing mature miR- confirmed that this lncRNA was exclusively 290-3p and miR-290-5p) as one of the top expressed in EFTs. The EFT-specific candidates. Further analysis showed miR- oncoprotein, EWS-FLI1, regulates expression 290-3p was downregulated in normal lung and of this transcript by directly binding to its breast tissue from highly metastatic AKR/J promoter region. Furthermore, functional data strains versus less metastatic DBA/2J strains, based on knocking down and overexpressing thereby supporting a germline component to the transcript shows that AK057037 acts miR-290 regulation. Next, when miR-290 was as an oncogene in EFT and enhances ectopically expressed in Mvt-1 and 6DT1, tumorigenicity by increasing the metastatic and orthotopically injected into FVB/N mice, potential of the tumor cell lines. a significant reduction in both mammary Conclusion: The study established tumor burden and the number of pulmonary that lncRNA AK057037 promotes EFT metastases was observed. Computational pathogenesis. Its exclusive presence in EFT analysis identified breast cancer biomarker suggests that expression of this lncRNA is Arid4b as a top target of miR-290-3p, tissue-specific and highly regulated. Targeting which was confirmed by luciferase reporter AK057037 in these tumors may help improve assay. In conclusion, these results suggest outcomes in children with metastatic disease. germline genetic changes may reduce miR- Further work on the non-coding transcriptome 290 expression, in turn increasing Arid4b may reveal additional functional RNAs that expression, to create a predisposition towards contribute to the biology of this disease. breast cancer progression.

B19 Functional characterization of B21 The paralogous microRNA clusters p53-regulated long intervening noncoding miR-17-92 and miR-106-25 are specifically RNAs (lincRNAs). Nadya M. Dimitrova, Tyler overexpressed in metastatic non-small cell Jacks. David H. Koch Institute for Integrative lung carcinomas. Katey SS Enfield, Stephen Cancer Research at MIT, Cambridge, MA. Lam, Wan L. Lam. BC Cancer Research Centre, Vancouver, BC, Canada. This abstract is being presented as a short Background: Lung cancer is the cause of the talk in the scientific program. A full abstract is printed in the Proffered Abstracts section most cancer-related deaths worldwide, with poor survival being largely attributed to late (PR2) of the conference Proceedings. stage of disease at diagnosis and frequent metastasis. Understanding the mechanisms B20 Breast cancer progression by which lung tumors metastasize could suppressor miR-290 targets breast cancer enable the development of anti-metastatic biomarker Arid4b . Natalie Goldberger, interventions or more specific therapeutics Renard Walker, Chang Hee Kim, Kent for metastatic disease. MicroRNAs (miRNAs) Hunter. National Cancer Institute, Bethesda, are major regulators of gene expression and MD. control a wide range of cellular processes Several microRNAs (miRNAs) have been involved in metastasis, including apoptosis and cell cycle progression. Recently, classified as metastasis regulators in breast cancer, yet few studies have examined how increased expression of two paralogous germline variations may promote metastasis miRNA clusters, miR-17-92 and miR- via miRNA dysregulation. To explore this 106b-25, was tied to control of these concept, highly metastatic MMTV-PyMT functions through antagonizing transforming growth factor-β (TGFβ) signaling. A tumor

62 NONCODING RNAS AND CANCER Poster session B suppressive cytokine, TGFβ regulates cell B22 miR-34c is a putative tumor cycle progression and apoptosis through suppressor microRNA in Dicer-Pten activation of p21 and BIM, respectively. double knockout high-grade serous However, evidence suggests this regulation carcinomas. Zhifeng Yu, Jaeyeon Kim, Chad can be repressed by the overexpression of Creighton, Shannon Hawkins, Martin Matthew these microRNA clusters to promote tumor Matzuk. Baylor College of Medicine, Houston, progression. To this end, we sought to TX. determine whether or not expression of these MicroRNAs (miRNAs) are short non-coding clusters was increased in non-small cell lung RNAs that could have large-scale biological cancer (NSCLC) cases positive for nodal or effects by directing gene regulation through distant metastases compared to those with translational repression and degradation only locally invasive disease. of multiple complementary target mRNAs. Methods: A panel of 41 non-metastatic Like other regulatory molecules, altered NSCLCs and a panel of 28 NSCLCs with miRNA expression has been suggested nodal or distant metastases were collected to be involved in the formation of many along with paired adjacent non-malignant human diseases, including ovarian cancer. tissues. Expression analysis of miRNAs was Our laboratory created a mouse model conducted in these specimens using Illumina with high-grade serous carcinomas by GaXII small RNA sequencing technologies. conditionally deleting both Dicer (essential for Matched tumor and normal miRNA normalized miRNA biosynthesis) and Pten (a negative read count comparisons were performed regulator of the PI3K pathway) in the female for each miRNA in the miR-17-92 (miR-17, reproductive tract. Due to the fact that Pten miR-18a, miR-19a, miR-20a, miR-19b-1, miR- knockout alone did not result in serous 92a-1) and miR-106b-25 (miR-106b, miR-93, ovarian cancer, the significance of the miR-25) clusters (Wilcoxon Signed-Rank impaired mature miRNA biosynthesis should test p<0.05). Only those miRNAs that were be highly emphasized in this Dicer-Pten significantly overexpressed and displayed a double knockout (DKO) high grade-serous minimum average expression fold change of 2 carcinomas. To define the specific miRNAs were further investigated. in this DKO mouse model, we created cell lines derived from primary ovarian tumors Results: The significant overexpression of in the Dicer-Pten DKO mice. Lack of Dicer several miRNAs occurred specifically in the metastatic cohort, and included miR-20a, makes the cell lines generated from these miR-92a-1, miR-106b and miR-93. miR-18a mice to be a valuable platform on functionally and miR-19a were significantly overexpressed evaluating the significance of miRNAs in this in both tumor cohorts as compared to model. We delivered control miRNA, miR-31, matched normal tissue; however, expression miR-100, let-7b, and miR-34c mimic to Dicer- levels were substantially higher in the Pten DKO cell lines by transient transfection, due to our previous studies of these miRNAs metastatic cohort and increased by 9.5 and 3 fold, respectively. miR-17 and miR-19b-1 in human ovarian cancer or the abundance were upregulated in both cohorts to a similar of these miRNAs in mouse normal fallopian level, while expression of miR-25 was not tubes. We observed a growth inhibitory effect of miR-34c that is accompanied with cell significantly altered. cycle arrest at G1 phase and induction of Conclusion: miRNAs in the miR-17-92 apoptosis. miR-34c is a direct transcriptional and miR-106b-25 clusters, save miR- target of p53 whose mutation is the most 25, were significantly overexpressed in frequent in human ovarian cancers. Using NSCLC cases positive for nodal or distant quantitative real-time PCR, miR-34c levels metastases. There was an overall trend of were found to be extremely low in human increasing involvement of miRNAs from serous adenocarcinomas compared with these clusters moving from NSCLC cases normal fallopian tube. Enforced expression of without metastases to those with metastases, miR-34c in a human serous ovarian cancer suggesting upregulation of these miRNAs is cell line induced cell growth arrest further involved in the metastatic process.

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 63 Poster session B

indicating that our data on miR-34c in our was also clearly visualized by the in situ mouse model could be translated to women. ‘view RNA’ method. Furthermore, MALAT1 ASO significantly delayed tumor growth in C26 colon cancer and reduced tumor size B23 Selective inhibition of a long in TRAMP mouse model of prostate cancer, noncoding RNA (lncRNA), MALAT1 by where the target RNA was decreased by 80% antisense oligonucleotides results in in tumor cells. In a human NSCLC patient- significant antitumor effects in a variety derived xenograft model, both significant of preclinical cancer models.. Jeff Hsu, MALAT1 RNA reduction and a delay in Guobin He, Gourab Bhattacharjee, Tianyuan tumor growth were achieved after MALAT1 Zhou, Chris May, Xiaokun Xiao, Gene Hung, ASO treatment. The effects of MALAT1 Brett P. Monia, A. Robert MacLeod, Youngsoo downregulation by ASO were not limited to Kim. Isis Pharmaceuticals Inc., Carlsbad, CA. the inhibition of tumor growth alone. MALAT1 An increasing body of evidence supports ASO treatment not only inhibited the growth the notion that long noncoding RNAs of primary tumors in the EBC-1 human (lncRNAs) play important roles in diseases NSCLC xenograft model, but also resulted in including cancer. MALAT1 (also called a decrease in lung metastasis as measured NEAT2) was originally identified in tumors of by micro CT scanning. Furthermore, cross- highly metastatic non-small cell lung cancer species MALAT1 ASOs greatly improved the (NSCLC), however, the functional role of survival of animals bearing Hep3B human MALAT1 in carcinogenesis has remained hepatocellular carcinoma (HCC) tumor largely unknown. However, MALAT1 is highly orthotopically (48.5 days with control ASO expressed in various types of cancer and vs 88 days with MALAT1 ASO, p=0.005). is implicated in the regulation of alternative Taken together, these results demonstrate splicing. To determine if MALAT1 constitutes previously undiscovered roles of MALAT1 as an important driver of tumor growth in an important regulator in vivo tumor growth vivo, we developed potent antisense and metastasis and suggest that selective oligonucleotides (ASOs) that were able to inhibition of MALAT1 by ASO could have achieve strong inhibition of MALAT1 in the therapeutic value for the cancer treatment. tumor cells of a variety of preclinical models following systemic delivery. MALAT1 ASOs were well tolerated in rodents at doses B24 New views on microRNAs involvement in melanoma. Eyal Greenberg1, leading to >95% inhibition of MALAT1 RNA 2 2 in the liver. Inhibition of MALAT1 expression Eran Eyal , Gideon Rechavi , Noam 1 2 2 in tumor and tumor-associated stromal cells Shomron , Jacob Schachter , Gal Markel , 1 2 2 was determined by both q-RT-PCR using Yael Nemlich , Steven Hajdu , Orit Itzhaki , Liat Edry1, Oz Solomon2, Ninette Amariglio2, species-specific probe/primer sets and/or 2 1 1 in situ ‘viewRNA’ technology, where target Michal J. Besser , Yona Keisari . Tel Aviv 2 knockdown can be visualized on a cell by University, Tel Aviv, Israel, Sheba Medical cell basis. Systemic administration of mouse Center, Ramat Gan, Israel. MALAT1 ASOs resulted in a decrease in the MicroRNAs (miRNAs) are small non-coding numbers of polyps and proliferation index RNAs with regulatory roles, which are (BrdU (+)) in the small intestine of Apcmin involved in a broad spectrum of physiological mouse model of colon cancer and correlated and pathological processes, including cancer. well with MALAT1 inhibition in the polyps. Most published data is focused on miRNAs Mouse MALAT1 ASOs were also effective that are differentially expressed between in a DEN-induced HCC model, where ASO melanocytes and melanoma cells. These treatment reduced the target RNA ~ 90% in experiments lead to the discovery of a tumor cells with a concomitant decrease in plurality of new miRNA-based mechanisms. tumor numbers, while control ASO had no However, this widely common approach effects on either measure. In addition, higher fails to address at least two important expression of MALAT1 in non-treated tumors issues: a) the effect of genomic mutations compared to adjacent normal hepatocytes on target gene regulation by miRNAs; b) the

64 NONCODING RNAS AND CANCER Poster session B role of miRNAs in shaping the phenotypic B25 Characterization of a novel heterogeneous nature of metastatic disease. pseudogene expressed antisense RNA Here we address these issues to provide new that regulates PTEN expression. Per insights on the pathogenesis of melanoma Johnsson1, Amanda Ackley2, Dan Grandér1, and facilitate the development of innovative Kevin V. Morris2. 1Karolinska Institutet, diagnostic and therapeutic implementations. Stockholm, Sweden, 2The Scripps Research Melanoma cells acquire tens of thousands Institute, La Jolla, CA. of genomic mutations, most of which This abstract is being presented as a short are defined as “passengers” and few as talk in the scientific program. A full abstract “drivers”. We analyzed a fully sequenced is printed in the Proffered Abstracts section genome of melanoma tumor and found that (PR3) of the conference Proceedings. >200 mutations hit the 3’UTR of genes, the main target area of miRNAs (out of 499 that hit transcribed genes). Three different B26 microRNA-181a regulates ovarian computational algorithms (PITA, miRanda cancer progression and is associated and our in-house tool, MirHB) concur and with poor outcome in late-stage epithelial predict that these somatic mutations globally ovarian cancer. Aditya Parikh, Analisa DiFeo, reduce the binding of miRNAs to the mutated Christine Lee, Alessia Baccarini, Valentin 3’UTRs. This phenomenon is not genome- Kolev, Jamal Rahaman, Peter Dottino, specific and proved to reflects the nature Tamara Kalir, Brian D. Brown, Goutham of the characteristic UV-induced mutation, Narla. Mount Sinai School of Medicine, New C-to-T. Accordingly, mutation patterns in other York, NY. malignancies, which are not primarily induced Epithelial ovarian cancer (EOC) is the fifth by UV-radiation, such as lung cancer and leading cause of cancer death in women AML, do not yield similar predictions. This and the most lethal gynecologic malignancy. indicates that miRNA-based regulation can The poor overall survival of this disease is progressively perturb even with no apparent due to both late stage diagnosis and the alterations in the expression of miRNAs. We development of chemotherapy resistance. define these mutations as “dormant drivers”, Although numerous studies have investigated as each mutation is expected to have a the mechanisms of drug resistance, effective moderate effect, but the cumulative effect is targets to overcome chemotherapy resistance significant. and methods to predict an individual’s We next identified miRNAs that regulate response to a specific therapy are still the aggressive phenotype of established lacking due to the variety and complexity of melanoma cells. For that purpose, we molecules and pathways involved in drug tested two isogenic human melanoma resistance. Deciphering the mechanisms cell lines (derived from the same patient) that lead to chemotherapy resistance is an that dramatically differ in net proliferation, important challenge and ideally, a regulatory invasion, tube formation and tumorigenicity molecule, such as microRNAs (miRNAs), in vivo. Comparative screening revealed that can regulate the expression and a large cohort of differentially expressed function of multiple genes associated with a miRNAs. Molecular manipulation of highly chemoresistant phenotype would be the ideal ranked miRNAs (miR-31, -34a, -184, -185 and target for the development of therapeutics -204 and miR-17-5p) resulted in significant and/or biomarker for chemotherapeutic changes of the aggressive phenotype in response. Here, we show that miR-181, a vitro and in vivo. Remarkably, none of these microRNA that is induced by MYCN and miRNAs has been studied in cutaneous TGF-β and upregulated during differentiation, melanoma before. In conclusion, this study promotes epithelial-to-mesenchymal transition might lead to the development of novel lines (EMT) and correlates with poor patient of therapy. outcome in late-stage epithelial ovarian cancer (25 months vs. 66 months; p=0.002). Additionally, our results suggest that Smad7

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 65 Poster session B

is a direct and functional target of miR-181 We found that cAuNPs10-miR1-PEG0.5 and enforced expression of miR-181a alone conjugates, which had the highest payload was sufficient to activate TGF-β signaling. were non-toxic, taken up by 90% of cells in 20 Furthermore, ectopic overexpression of miR- minutes without the need for electroporation 181a resulted in increased cellular survival, or lipofection. In addition they exhibited migration and in vivo tumor burden as well as rapid endosomal escape and retained their drug resistance. In contrast, stable inhibition specificity with respect to gene targets and of miR-181 using a lentivirus-based sponge phenotypic impact up to 5 days following decoy vector inhibited TGF-β –dependent delivery. miR-31-AuNP conjugates down- transcriptional responses and diminished regulated predicted targets E2F2, STK40 and cellular survival, migration and in vivo tumor CEBPA and decreased proliferation of NGP dissemination. This data suggests that miR- and SY5Y revealing a new tumor suppressor 181a represents a genetic determinant of microRNA for NB. cellular response to TGF-β through the direct Surprisingly, miR-1323-AuNP conjugates regulation of the inhibitory Smad, Smad7, and significantly increased proliferation, introduces a novel biomarker and therapeutic clonigenicity and in vivo tumor growth of target for late-stage ovarian cancer. neuroblastoma lines SY5Y and CHLA255. As siRNAs to MYCN and ALK decrease B27 Novel nanoparticle conjugated viability of NB cell lines, this suggested microRNA delivery reveals new tumor that the oncogenic function of miR-1323 suppressors for neuroblastoma. Rajib is likely to result from the repression of Ghosh1, Jayarathne Lalithya1, Chen Zaowen2, a tumor suppressor for NB. A search for Hsu Danielle2, Jason M. Shohet2, Preethi H. putative tumor suppressive miR-1323 target Gunaratne1. 1University of Houston, Houston, genes uncovered Cbp/PAG1 as novel tumor TX, 2Texas Children’s Cancer Center, suppressor for NB that is potently down- Houston, TX. regulated by miR-1323. The transmembrane adaptor protein Cbp/PAG strongly suppresses High-risk neuroblastoma (NB) represents a proliferation driven by the Src family of major clinical challenge in pediatric oncology. kinases (SFKs) by sequestering them on A decade after the identification of MYCN lipid rafts of the cell membrane. Src inhibitors as the key driver of NB, the five-year overall such as pyrazolo[3,4-d]pyrimidine derivatives, survival is less than 40% suggesting that PP2, and Dasatinib have been demonstrated signaling pathways downstream of MYCN to substantially suppress proliferation and need to be explored urgently. The future increase anti-tumor activity in vitro and in vivo promise of microRNA-based therapeutics is mouse models of NB. Importantly we found a based on targeting or replacement of these strong correlation of low Cbp/PAG1expression ~22 nucleotide RNAs that are commonly with a striking decrease in overall survival deregulated in many of the known diseases. compared to patients carrying tumors with The large size and polyanionic nature of high levels of Cbp/PAG1 (overall survival p < naked miRNAs hamper cellular uptake. Viral 0.001). Our studies of microRNA function in delivery requires cloning, miRNA mimics/ neuroblastoma have uncovered Cbp/PAG1as inhibitors need expensive modifications and a novel tumor suppressor and therapeutic tool both require electroporation or lipofection. for high risk neuroblastoma. We have developed and tested 35 unique formulations of amine functionalized gold nanoparticle (AuNP), unmodified miRNA (miR) and polyethylene glycol (PEG) conjugates. Using these conjugates we have tested two tumor suppressor candidates miR-31 (Creighton et al. 2010) and miR- 1323 predicted to target sites in both MYCN and ALK (key oncogenes driving NB) in the neuroblastoma cell lines NGP and SY5Y.

66 NONCODING RNAS AND CANCER Poster session B

B28 microRNA-641 activates MAPK is characterized by a specific cluster of by targeting NF1 and cooperates with its regulated miRNAs. host gene AKT2 in human cancer. Edward More specifically, we found that miR-125b, J. Richards, Marc Coppola, Jianping Guo, which is overexpressed in the set of murine William Kong, Jin Q. Cheng. H. Lee Moffitt tumors that are capable of generating Cancer Center, Tampa, FL. metastasis, increases cell invasion in AKT2 is frequently up-regulated in human vitro and formation of metastasis in vivo. cancer. MiR-641 is an uncharacterized Furthermore, miR-125b expression is microRNA that locates at intron-1 of the significantly associated to metastatic outcome AKT2 gene. We hypothesize that miR-641 in two independent series of human breast and AKT2 are co-regulated, and cooperate tumors. In addition, we identified several to promote tumor development. Here we miR-125b target genes, including BRMS1 demonstrated that the transcription factors (Breast Metastasis suppressor 1) by which induce AKT2 also increase miR-641 immunoprecipitation of Argonaute protein- expression. Further, we identified miR-641 containing RNA-induced silencing complexes activation of the MAPK pathway by targeting (RISCs) followed by microarray analysis. tumor suppressor NF1, a GTPase-activating Importantly, siRNA-mediated depletion of protein that inhibits RAS. Upon ectopic BRMS1 limits the effects of miR-125b on expression of miR-641, NF1 expression was invasion in vitro. Since we found that BRMS1 reduced. As a result, pERK was elevated. also represses miR-125b, we demonstrate the Whereas knockdown of miR-641 exhibits existence of a BRMS1/miR-125b regulatory opposite effects. In addition, depletion of miR- feedback loop linked to tumor cell invasion 641 sensitizes tumor cells to cisplatin-induced and metastasis. apoptosis. Knockdown of miR-641 synergized with AKT2 inhibition to induce cell death. These data suggest that miR-641 functions as B30 Exogenous miR-128 enforced an oncomiR that co-regulates and cooperates HNSCC inhibits proliferation. Belinda 1 2 with AKT2 in human cancer. Hauser , Yubin Hao, Joseph Califano , Xinbin Gu1. 1Howard University, Washington, DC; 2Johns Hopkins University, Baltimore, MD. B29 A feedback regulatory loop between Head and neck squamous cell carcinoma miR-125b and BRMS1 is linked to breast (HNSCC) is a heterogeneous disease with 1 tumor progression. Delphine Allard , Magali complex molecular abnormalities. There are Lacroix-Triki2, Henri Roche2, Fabrice André1, more than 65,000 Americans diagnosed with Stephan Vagner1, Gerard Maillot2, Loubna head and neck cancer and 25,000 die from Mhamdi2, Sandra Pierredon2, Florine Obrist1, 1 2 this disease. microRNAs are 22~nucleotide- Aicha Goubar , Thomas Filleron , Margaux long endogenously expressed, highly 2 1 1 Roques , Véronique Scott . INSERM U981, conserved noncoding RNAs with important Institut Gustave Roussy, Villejuif, France, regulatory functions in proliferation, apoptosis, 2INSERM U563, Institut Claudius Regaud, and metastasis. miR-128 a brain-enriched Toulouse, France. microRNA most commonly in gliomas that A plethora of studies showed altered have shown to function as a tumor suppressor microRNA (miRNA) expression in cancer. which decreases cell proliferation and cell However, few reports investigated the role of invasiveness in prostate cancer. miR-128 has miRNAs in the metastatic process. Here we been shown to target BMI-1 in squamous used a mouse mammary tumor model (the cell carcinoma; however we believe miR- 4T1 model), in which a set of four distinct 128 functions by targeting multiple targets in tumor cell populations are able to complete HNSCC. distinct steps of metastasis when implanted Methods: We established HNSCC cell lines into the mammary glands of BALB/c mice. that stably expressed individual members of MiRNA profiling by microarrays allowed us the miR-128 using a lentiviral delivery system. to show that each of the four tumor types The levels of miR-128 and their targeted

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 67 Poster session B

proteins were analyzed by qRT-PCR and Western blot. miR-128 affinity was elucidated by MTT, colony formation, flow cytometry, and a tumor xenograft model. Results: Enforced transfection of miR-128 in HNSCC (JHU-22) expression was stable in vitro and in vivo. We identified five specific targets of miR-128 (BMI-1, BAG2, BAX, H3f3b, and Paip2). BMI-1 and H3f3b were shown to be a more dominant targeted of miR-128. Overexpression of miR-128, lead to the down regulation of these targets including decrease cell viability (30%), decrease of proliferation (55%), and significantly reduced solid tumor formation in tumor xenografts models. Conclusions: miR-128 exhibits anti- proliferative effects and phenotypic alteration in HNSCC by targeting multiple predicted conserved targets activity function in apoptosis, cell cycle, transcription, and translation. Therefore, screening for differentially expressed targets of miR-128 help elucidate the possible mechanisms underlining the function of miR-128.Our results indicate that miR-128 has the potential to serve as a therapeutic approach.

68 NONCODING RNAS AND CANCER Participant List As of December 6, 2011

Aravin Alexei Alexander Brodsky California Institute of Technology Brown University Pasadena, CA Providence, RI Phone: (626) 395-4952 Phone: (401) 863-2936 E-mail: [email protected] E-mail: [email protected] Ahanghyeok An Kirk Brown Catholic University Medical College Caris Life Sciences, Inc. Uijeongbu, Korea (Rep.) Phoenix, AZ Phone: 82-31-820-3998 Phone: (602) 464-7521 E-mail: [email protected] E-mail: [email protected] Chantal Autexier Coby Carlson McGill University Life Technologies Montreal, QC, Canada Madison, WI Phone: (514) 340-8222 Phone: (608) 204-5108 E-mail: [email protected] E-mail: [email protected] Constance Baer Linette Castillo-Pichardo German Cancer Research Center (DKFZ) Universidad Central del Caribe Heidelberg, Germany Toa Alta, PR Phone: 49 (0) 622-142-4325 Phone: (787) 379-8270 E-mail: [email protected] E-mail: [email protected] David Baltimore Angel Chao California Institute of Technology Chang Gung Memorial Hospital Pasadena, CA Taoyuan, Taiwan Phone: (626) 395-3581 Phone: 886 (3) 328-1200 x8983 E-mail: [email protected] E-mail: [email protected] Charles Bankhead Ngai Na Co MedPage Today University of Texas MD Anderson Cancer Center Houston, TX Houston, TX Phone: (973) 890-0985 Phone: (281) 650-8630 E-mail: [email protected] E-mail: [email protected] David Bartel David Corey Massachusetts Institute of Technology UT Southwestern Medical Center Cambridge, MA Dallas, TX Phone: (617) 258-5287 Phone: (214) 648-5096 E-mail: [email protected] E-mail: [email protected] William Beck Carlo Croce University of Illinois at Chicago Ohio State University Comp. Cancer Center Chicago, IL Columbus, OH Phone: (312) 996-0888 Phone: (614) 292-4930 E-mail: [email protected] E-mail: [email protected] Kirsten Block John Daniels Johns Hopkins University USC Keck School of Medicine Baltimore, MD Pacific Palisades, CA Phone: (410) 955-8506 Phone: (323) 865-3900 E-mail: [email protected] E-mail: [email protected]

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 69 ParticiPant List

Swadesh Das Catherine Enfield Virginia Commonwealth University BC Cancer Research Center Richmond, VA Vancouver, BC, Canada Phone: (804) 245-6944 Phone: (604) 675-7702 E-mail: [email protected] E-mail: [email protected] Christopher Desevo Xing Fan UT Southwestern Medical Center University of Michigan Dallas, TX Ann Arbor, MI Phone: (214) 648-7729 Phone: (734) 615-7266 E-mail: [email protected] E-mail: [email protected] Sven Diederichs Farhoud Faraji German Cancer Research Center National Cancer Institute Heidelberg, Germany Bethesda, MD Phone: 49 (6221) 42-4383 Phone: (301) 402-6121 E-mail: [email protected] E-mail: [email protected] Analisa Difeo Matthew Fero Mount Sinai School of Medicine Fred Hutchinson Cancer Research Center New York, NY Seattle, WA Phone: (212) 659-6742 Phone: (206) 667-5065 E-mail: [email protected] E-mail: [email protected] Nadya Dimitrova Stephen Fesik Koch Institute at MIT Vanderbilt University School of Medicine Cambridge, MA Nashville, TN Phone: (617) 253-0264 Phone: (615) 322-6303 E-mail: [email protected] E-mail: [email protected] David Drasin Jason Fewell University of Colorado, Anschutz Medical Campus EGEN, Inc. Aurora, CO Huntsville, AL Phone: (303) 724-3510 Phone: (256) 327-0532 E-mail: [email protected] E-mail: [email protected] Ross Drayton Surangani Dharmawardhane Flanagan University of Sheffield Medical School University of Puerto Rico Medical Science Sheffield, United Kingdom Campus Phone: 44 (0) 114-271-3165 San Juan, PR E-mail: [email protected] Phone: (787) 758-2525 x1630 E-mail: [email protected] Alexey Efanov Ohio State University Comp. Cancer Center Ross Flockhart Columbus, OH Stanford University Phone: (614) 688-3105 Palo Alto, CA E-mail: [email protected] Phone: (650) 804-4616 E-mail: [email protected] Hans Petter Eikesdal University of Bergen Elsa Flores Bergen, Norway University of Texas MD Anderson Cancer Center Phone: 00-479-205-1962 Houston, TX E-mail: [email protected] Phone: (713) 792-0413 E-mail: [email protected]

70 NONCODING RNAS AND CANCER ParticiPant List

Galina Gabriely Richard I. Gregory Brigham & Women’s Hospital Children’s Hospital Boston/ Brookline, MA Harvard Medical School Phone: (617) 525-5540 Boston, MA E-mail: [email protected] Phone: (617) 919-2273 E-mail: [email protected] Ivan Garcia-Bassets University of California San Diego Shiv Grewal La Jolla, CA National Cancer Institute Phone: (858) 534-6936 Bethesda, MD E-mail: [email protected] Phone: (301) 594-6389 E-mail: [email protected] Joseph Gera University of California Xinbin Gu Los Angeles, CA Howard University College of Dentistry Phone: (818) 895-9416 Washington, DC E-mail: [email protected] Phone: (202) 806-0345 E-mail: [email protected] Etel Gimba Instituto Nacional de Câncer Preethi Gunaratne Rio de Janeiro, Brazil University of Houston Phone: 55 (21) 3-233-1493 Houston, TX E-mail: [email protected] Phone: (713) 503-2148 E-mail: [email protected] Natalie Goldberger National Cancer Institute William Hahn Bethesda, MD Dana-Farber Cancer Institute Phone: (301) 496-8347 Boston, MA E-mail: [email protected] Phone: (617) 632-2641 E-mail: [email protected] María del Mar González-Pons University of Puerto Rico Comp. Cancer Center Belinda R. Hauser San Juan, PR Howard University Phone: (787) 772-8300 Washington, DC E-mail: [email protected] E-mail: [email protected]

Anton Gorbachev Tsui-Ting Ho The Cleveland Clinic University of Illinois at Chicago Cleveland, OH Chicago, IL Phone: (216) 444-9440 Phone: (312) 355-0839 E-mail: [email protected] E-mail: [email protected] Rajgopal Govindarajan Marina Holz University of Georgia College of Pharmacy Yeshiva University Athens, GA New York, NY Phone: (706) 542-5759 Phone: (212) 340-7838 E-mail: [email protected] E-mail: [email protected] Eyal Greenberg Olena James Tel Aviv University Tennessee State University Even Yehuda, Israel Nashville, TN Phone: 972 (3) 530-4591 Phone: (615) 963-5789 E-mail: [email protected] E-mail: [email protected]

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 71 ParticiPant List

Per Johnsson Gangning Liang Karolinska Institutet USC/Norris Comprehensive Cancer Center Stockholm, Sweden Los Angeles, CA Phone: 46 85-177-9394 Phone: (323) 865-0470 E-mail: [email protected] E-mail: [email protected] Sakari Kauppinen Biaoyang Lin Santaris Pharma SMC and UW Horsholm, Denmark Bellvue, WA Phone: 45 2-047-7994 Phone: (206) 291-8297 E-mail: [email protected] E-mail: [email protected] Youngsoo Kim Leonard Lipovich Isis Pharmaceuticals, Inc. Wayne State University School of Medicine Carlsbad, CA Detroit, MI Phone: (760) 603-3890 Phone: (313) 577-9683 E-mail: [email protected] E-mail: [email protected] Taewan Kim Shawn Lupold Ohio State University Johns Hopkins University School of Medicine Columbus, OH Baltimore, MD E-mail: [email protected] Phone: (410) 502-4822 E-mail: [email protected] Gu Kong Hanyang University College of Medicine Li Ma Seoul, Korea (Rep.) University of Texas MD Anderson Cancer Center Phone: 82 (2) 2-290-8251 Houston, TX E-mail: [email protected] Phone: (713) 792-6590 E-mail: [email protected] Hyun Kyung Kong Sookmyung Women’s University Marco Magistri Seoul, Korea (Rep.) University of Miami E-mail: [email protected] Miami Beach, FL Phone: 3-052-436-4684 Suet Ying Kwan E-mail: [email protected] University of Texas MD Anderson Cancer Center Houston, TX Eric Marcusson Phone: (713) 792-2897 Regulus Therapeutics E-mail: [email protected] San Diego, CA Phone: (858) 202-6308 Tae Jin Lee E-mail: [email protected] Ohio State University Comp. Cancer Center Columbus, OH Joshua Mendell Phone: (614) 292-4354 UT Southwestern Medical Center E-mail: [email protected] Dallas, TX Phone: (214) 648-5183 Jeannie Lee E-mail: Joshua.Mendell@UTSouthwestern. Massachusetts General Hospital edu Boston, MA Phone: (617) 726-5943 Sheetal Mitra E-mail: [email protected] Children’s Hospital Los Angeles Los Angeles, CA Jeong-Yeon Lee Phone: (323) 361-5607 Hanyang University E-mail: [email protected] Seoul, Korea (Rep.) Phone: 82 (2) 2-220-0632 E-mail: [email protected]

72 NONCODING RNAS AND CANCER ParticiPant List

Anirban Mitra Jong Hoon Park University of Southern California Sookmyung Women’s University Los Angeles, CA Seoul, Korea (Rep.) Phone: (323) 361-5607 Phone: (822) 710-9414 E-mail: [email protected] E-mail: [email protected] Danesh Moazed Chiara Pastori Harvard Medical School Miami University Boston, MA Miami, FL Phone: (617) 432-1258 Phone: (305) 243-4684 E-mail: [email protected] E-mail: [email protected] Samuel Mok Alexander Pertsemlidis University of Texas MD Anderson Cancer Center University of Texas Health Science Center at Houston, TX San Antonio Phone: (713) 792-1442 San Antonio, TX E-mail: [email protected] Phone: (210) 562-9062 E-mail: [email protected] Edward Morrisey University of Pennsylvania Lynn Pouliot Philadelphia, PA National Cancer Institute Phone: (215) 573-3010 Bethesda, MD E-mail: [email protected] Phone: (301) 435-6308 E-mail: [email protected] Yoshiko Nagano NCI-CCR Valya Ramakrishnan Bethesda, MD University of California Phone: (301) 594-1054 San Diego, CA E-mail: [email protected] Phone: (858) 534-8776 E-mail: [email protected] Bethsaida Nieves Stanford University School of Medicine Yin Ren Stanford, CA Koch Institute for Integrative Cancer Phone: (650) 736-2753 Research at MIT E-mail: [email protected] Cambridge, MA Phone: (734) 819-0957 Matan Ofir E-mail: [email protected] Bar-Ilan University Ramat Gan, Israel Penny Riggs Phone: 972 (3) 531-8805 Texas A&M University E-mail: [email protected] College Station, TX Phone: (979) 845-2616 Eric Olson E-mail: [email protected] UT Southwestern Medical Center Dallas, TX John Rinn Phone: (214) 648-1187 Beth Israel Deaconess Medical Center E-mail: [email protected] Cambridge, MA Phone: (617) 735-0535 Pier Paolo Pandolfi E-mail: [email protected] Beth Israel Deaconess Medical Center Boston, MA Sandra Romero Cordoba Phone: (617) 735-2121 National Institute of Genomic Medicine E-mail: [email protected] Mexico City, Mexico Phone: 52 (55) 5-350-1975 E-mail: [email protected]

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 73 ParticiPant List

Sharanjot Saini Raymond Stallings UCSF VA Medical Center Royal College of Surgeons in Ireland San Francisco, CA Dublin, Ireland Phone: (415) 221-4810 x3510 Phone: 353 (1) 402-8533 E-mail: [email protected] E-mail: [email protected] Eric Sawey Louis Staudt The Journal of Experimental Medicine NCI-CCR West Babylon, NY Bethesda, MD E-mail: [email protected] Phone: (301) 402-1892 E-mail: [email protected] Lorenzo Sempere Dartmouth Norris Cotton Cancer Center Greg Stewart Lebanon, NH British Columbia Cancer Research Center Phone: (603) 653-3646 Vancouver, BC, Canada E-mail: [email protected] Phone: (604) 675-8111 E-mail: [email protected] Ramin Shiekhattar The Wistar Institute Markus Stoffel Philadelphia, PA Institute of Molecular Systems Biology Phone: (215) 898-3896 Zurich, Switzerland E-mail: [email protected] Phone: 4-144-633-4560 E-mail: [email protected] Jason Shohet Baylor College of Medicine Gerald Sufrin Houston, TX State University of New York Phone: (832) 824-4200 Buffalo, NY E-mail: [email protected] Phone: (716) 859-2212 E-mail: [email protected] Joachim Silber Memorial Sloan-Kettering Cancer Center Pavel Sumazin New York, NY Columbia University Phone: (646) 888-3198 New York, NY E-mail: [email protected] Phone: (212) 851-5469 E-mail: [email protected] David Smith Mayo Clinic College of Medicine Yutong Sun Rochester, MN University of Texas MD Anderson Cancer Center Phone: (507) 266-0311 Houston, TX E-mail: [email protected] Phone: (713) 792-3677 E-mail: [email protected] Anna Smith University of Colorado Anshutz Medical Pankaj Trivedi Campus La Sapienza University Aurora, CO Rome, Italy Phone: (303) 724-3510 Phone: 39-064-997-3015 E-mail: [email protected] E-mail: [email protected] Khalid Sossey-Alaoui Christopher Umbricht The Cleveland Clinic Lerner Research Johns Hopkins University Institute Baltimore, MD Cleveland, OH Phone: (410) 614-2662 Phone: (216) 444-4314 E-mail: [email protected] E-mail: [email protected]

74 NONCODING RNAS AND CANCER ParticiPant List

Nicola Valeri Zhifeng Yu Ohio State University Baylor College of Medicine Columbus, OH Houston, TX Phone: (614) 688-3418 Phone: (713) 798-4924 E-mail: [email protected] E-mail: [email protected] Paula van Noort Philip Zamore InteRNA Technologies University of Massachusetts Medical School Utrecht, The Netherlands Worcester, MA Phone: 3-164-116-0728 Phone: (508) 856-2191 E-mail: [email protected] E-mail: [email protected] Gerald Verhaegh Ruiwen Zhang Radboud University Nijmegen Medical Centre Texas Tech University Health Sciences Center Nijmegen, The Netherlands Amarillo, TX Phone: 31-24-361-0510 Phone: (806) 356-4015 E-mail: [email protected] E-mail: [email protected] Claes Wahlestedt University of Miami Miller School of Medicine Miami, FL Phone: (305) 243-1367 E-mail: [email protected] Martin Walsh Mount Sinai School of Medicine New York, NY Phone: (212) 241-9714 E-mail: [email protected] Shizhen Wang City of Hope Beckman Research Institute Duarte, CA Phone: (626) 256-4673 x63118 E-mail: [email protected] Fangting Wu System Biosciences Mountain View, CA Phone: (650) 968-2200 x111 E-mail: [email protected] Li-Wha Wu National Cheng Kung University Tainan, Taiwan Phone: 886 (6) 235-3535 x3618 E-mail: [email protected]

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 75 ParticiPant List

76 NONCODING RNAS AND CANCER Author Index Poster Presentations A C Ackley, Amanda B25, PR3 Caetano, Mauricio A29 Agrawal, Amit B2, PR5 Cai, Chunxiao A13 Allard, Delphine B29 Califano, Andrea A14 Amariglio, Ninette B24 Carlson, Coby B. A33 Anderson, James R. A23 Carter, Bob A22 André, Fabrice B29 Catto, James W.F. A10 Anwer, Khursheed B17 Cerisoli, Francesco A12 Arceci, Robert J. B6, B18 Chang, Inik A27 Arora, Sumit A27 Changhyeok, An A15 Arslan, Ahmet Dirim B11 Chao, Angel B4 Ashktorab, Hassan A13 Chen, Clark C. A22 Augoff, Katarzyna A3 Chen, Hua-Chien B4 Aulmann, Sebastian B5 Chen, Shu-Jen B4 B Cheng, Jin Q. B28 Babae, Negar A12 Cheung, Hiu Wing B2, PR5 Baccarini, Alessia B26 Chiu, Hua-Sheng A14 Baer, Constance B10 Choi, Sung-hee B13 Bai, Jennifer B12 Ciarapica, Roberta A30 Bautista-Pina, Veronica A25 Claus, Rainer B10 Beauchamp, Lesslie A33 Clurman, Bruce A24 Beck, William T. B11 Co, Ngai Na A28 Behrens, Carmen A2 Coarfa, Cristian B16 Benham, Ashley B16 Coppola, Marc B28 Ben-Shachar, Berta A17 Cowley, Glenn B2, PR5 Berezikov, Eugene A12 Creighton, Chad B22 Besser, Michal J. B24 Croce, Carlo M. A18, B14 Bhatia, Sangeeta B2, PR5 Cuppen, Edwin A12 Bhattacharjee, Gourab B23 Boehm, Jesse B2, PR5 D Bourajjaj, Meriem A12 Da Sacco, Letizia A30 Braconi, Chiara B14 Dahiya, Rajvir A27 Brard, Laurent A31 Danielle, Hsu B27 Bray, Isabella M. B9 Davicioni, Elai A23 Brodsky, Alexander S. A31 de Gunt, Thijs A12 Brown, Brian D. B26 De Salvo, Maria A30 Brown, Patrick B8, PR4 Dejean, Anne A18 Brunhoeber, Elaine B17 Deng, Guoren A27 Bryant, Helen E. A10 DeSevo, Chris A2 Buckley, Jonathan D. A23, B6, DeSevo, Christopher B7 B18 Diederichs, Sven A21, B5 Buetow, Ken H. A6 DiFeo, Analisa B26 Byrd, John C. B10 Dimitrova, Nadya M B19, PR2 Dottino, Peter B26 Drapkin, Ronny B2, PR5 Drasin, David J. A20, B1 Drayton, Ross M A10 Du, Liqin A2, B7 Dudziec, Ewa A10

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 77 E H Edry, Liat B24 Hacohen, Dalia A17 Eißmann, Moritz A21 Hahn, William B2, PR5 Enfield, Katey S.S. A26, B21 Hajdu, Steven B24 Eyal, Eran B24 Hall, Matthew D. B12 Hämmerle, Monika A21, B5 F Harinath, Girish B15 Fabbri, Muller B14 Faraji, Farhoud A6 Hart, Jonathan R. B14 Fernandez, Raquel B9 Hauser, Belinda R. B30 Fero, Matthew L. A24 Hawkins, Douglas S. A23 Ferreira, Luciana A29 Hawkins, Shannon B22 Fewell, Jason G. B17 He, Guobin B23 Filleron, Thomas B29 He, Xiaolong B11 Fischer, Martina B10 Hidalgo-Miranda, Alfredo A25 Fiskin, Evgenij B5 Hildenbrandt, Catherina A21, B5 Flockhart, Ross J. A11, PR1 Ho, Tsui-Ting B11 Fogal, Valentina B2, PR5 Hoffmann, Hans B5 Foley, Niamh H. B9 Holland, Eric C. B15 Ford, Heide L. A20, B1 Hsu, Danielle B16 Frankel, Wendy B14 Hsu, Jeff B23 Frenzel, Lukas B10 Hu, Ying A6 Hung, Gene B23 G Hunter, Kent W A6, B20 Gafa’, Roberta B14 Huse, Jason T. B15 Gao, Harry A9 Huynen, Martijn A. A4 Garcia-Bassets, Ivan B3 Hwang, Harry A24 Gasparini, Pierluigi B14 Hyung Ran, Kim A15 Gazdar, Adi F. B7 Ghosh, Rajib B27 I Gimba, Etel A29 Itzhaki, Orit B24 Ginsberg, Doron A17 Iwanaga, Ritsuko A20 Goldberger, Natalie A6, B20 Gommans, Willemijn M. A12 J Gottesman, Michael M. B12 Jacks, Tyler B19, PR2 Goubar, Aicha B29 Jacobsen, Anders B15 Goulart, Luiz Ricardo A29 James, Olena T. A5 Govindarajan, Rajgopal A32 Jeon, Young-Jun A18 Grandér, Dan B25, PR3 Jessica, Silva M A1 Greenberg, Eyal B24 Johnsson, Per B25, PR3 Griffioen , Arjan W. A12 Grivennikov, Sergei B14 K Groden, Joanna B14 Kalir, Tamara B26 Groß, Matthias A21 Kapranov, Philipp A23, B6, Grund, Stefanie B5 B18 Gu, Lei B10 Karin, Michael B14 Gu, Xinbin A13 Karst, Alison B2, PR5 Gunaratne, Preethi B16, B27 Keisari, Yona B24 Guo, Jianping B28 Khavari, Paul A. A11, PR1 Gutschner, Tony A21, B5 Kim, Chang Hee B20 Kim, Eugene B16 Kim, Jaeyeon B22 Kim, Taewan A18

78 NONCODING RNAS AND CANCER Kim, Youngsoo B23 Mitra, Anirban P. A23, B6 Kolev, Valentin B26 Mitra, Sheetal A. A23, B6, Kong, William B28 B18 Kovalski, Joanna A11, PR1 Mo, Yin-Yuan A19 Kretz, Markus A11, PR1 Mok, Samuel A28 Kuppers, Daniel A24 Monia, Brett P. B23 Kushwaha, Deepa A22 Morris, Kevin V. B25, PR3 Muley, Thomas B5 L Myles, E. Lewis A5 Lacroix-Triki, Magali B29 Lai, Chyong-Huey B4 N Lalithya, Jayarathne B27 Nagano, Yoshiko A7 Lam, Stephen A26, B21 Namjin, Yoo A15 Lam, Wan L. A26, B21 Narla, Goutham B26 Lanza, Giovanni B14 Neelakantan, Deepika B1 Lee, Amy S A8 Nemlich, Yael B24 Lee, Christine B26 Neugebauer, Julia B5 Lee, Maxwell P. A7 Neves, Adriana A29 Lee, Tae Jin A18 Nieves, Bethsaida I. B8, PR4 Leite, Felipe A29 Niu, Shuang B8, PR4 Liang, Gangning A8 Nuovo, Gerard B14 Liu, Yulan A13 Lo, Justin B2, PR5 O Locatelli, Franco A30 Obrist, Florine B29 Longerich, Thomas B5 Ofir, Matan A17 Lovat, Francesca B14 Ozawa, Tatsuya B15 Lu, Karen A28 P M Pallasch, Christian B10 MacLeod, A. Robert B23 Palomares, Melanie A9 Maffuz-Aziz, Antonio A25 Palumbo, Antonio A29 Magdaleno, Susan A33 Pang, Xiaowu A13 Maillot, Gerard B29 Paone, Alessio B14 Majid, Shahana A27 Parikh, Aditya B26 Markel, Gal B24 Park, Yoon Jung B10 Martin, Scott E. B12 Pence, Casey B17 Mascarenhas, Nicholas A11, PR1 Pertsemlidis, Alexander A2, B7 Masotti, Andrea A30 Pierredon, Sandra B29 Matar, Majed B17 Pineau, Pascal A18 Matzuk, Martin Matthew B22 Piper, David A33 May, Chris B23 Plass, Christoph B10 May, William A. B18 Plow, Edward F. A3 McClure, Diane B17 Poell, Jos B. A12 McCue, Brian A3 Polach, Kevin B17 Meister, Michael B5 Polycarpou-Schwarz, Maria B5 Mello, Kivvi A29 Pouliot, Lynn M. B12 Mesirov, Jill B2, PR5 Prevost, Gregoire P. A12 Mhamdi, Loubna B29 Micalizzi, Douglas S. A20 Q Michael, Baker B7 Qu, Kun A11, PR1 Miele, Lucio A30 Quintanar-Jurado, Valeria A25 Milos, Patrice B6 Minna, John D. A2, B7

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 79 Somlo, George A9 R Sorensen, Poul H. B. B6 Rahaman, Jamal B26 Sossey-Alaoui, Khalid A3 Ramakrishnan, Valya A22 Sparks, Jeff B17 Raphael, Benjamin A31 St. Laurent, Georges B6 Rea-Ramsey, Angela B17 Stallings, Raymond L. B9 Rebollar-Vega, Rosa G. A25 Stentrup, Marion A21 Rechavi, Gideon B24 Sternberg, Cinthya A29 Rehli, Michael B10 Stewart, Greg L. A26 Ren, Yin B2, PR5 Su, Sheng-Fang A8 Reynolds, C. Patrick B6 Sug Hyung, Lee A15 Rice, Jennifer B17 Sumazin, Pavel A14 Richards, Edward J. B28 Sweet-Cordero, E. Alejandro B8, PR4 Robert, Borkowski B7 Roche, Henri B29 T Rodriguez-Cuevas, Sergio A25 Tamayo, Pablo B2, PR5 Rom, Joachim B5 Tanaka, Yuichiro A27 Romero-Cordoba, Sandra L. A25 Taylor, Molly A. A34 Root, David B2, PR5 Triche, Timothy J. A23, B6, Roques, Margaux B29 B18 Rossi, John A9 Rota, Rossella A30 V Roth, Anna B5 Vagner, Stephan B29 Ruoslahti, Erkki B2, PR5 Vaka, Dedeepya B8, PR4 Valeri, Nicola B14 S van Beijnum, Judy R. A12 Saini, Sharanjot A27 van den Oudenalder, Kelly A4 Salzman, Julia B8, PR4 van Haastert, Rick J. A12 Sander, Chris B15 van Hooij, Onno A12 Schaapveld, Roel Q.J. A12 van Noort, Paula I. A12 Schachter, Jacob B24 Vang, Souriya A31 Schalken, Jack A. A4, A12 Vartuli, Rebecca L. A20 Schiemann, William P. A34 Verhaegh, Gerald W. A4, A12 Schiffelers, Raymond M. A12 Verheul, Mark A12 Schirmacher, Peter B5 Vidic, Suzanna A12 Schmandt, Rosemarie A28 Vogt, Peter K. B14 Schmitt, Thomas A24 von Maltzahn, Geoffrey B2, PR5 Schnabel, Philipp A B5 Schultz, Iman A12 W Schwartz, Gary B13 Wai, Daniel H. B18 Scott, Véronique B29 Wakefield, Lalage M. A7 Sempere, Lorenzo F B13 Walker, Renard B20 Sha, Wei A13 Wang, Shizhen Emily A9 Shahryari, Varahram A27 Wang, Tzu Hao B4 Shohet, Jason M. B16, B27 Warth, Arne B5 Shomron, Noam B24 Webster, Dan E A11, PR1 Silber, Joachim B15 Weichenhan, Dieter B10 Sinn, Peter B5 Weir, Barbara B2, PR5 Skapek, Stephen X. A23 Wells, Wendy B13 Smit, Frank A4 Wendtner, Clemens-Martin B10 Smith, Anna L. A20 Wilkinson, Leslie B17 Smith, David I. A1 Wistuba, Ignacio I. A2 Solomon, Oz B24 Wu, Fangting A19

80 NONCODING RNAS AND CANCER Wu, Gang A6 Wu, Hsin-Ta A31 Wu, Li-Wha A16 Wu, Xiwei A9

X Xiao, Xiaokun B23

Y Yamamura, Soichiro A27 Yen, Yun A9 Yu, Zhifeng B22

Z Zabeck, Heike B5 Zaowen, Chen B27 Zaowen, Zaowen B16 Zhang, Jinghui A6 Zhao, Yuan A13 Zhou, Tianyuan B23 Zörnig, Martin A21 Zucknick, Manuela B10

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 81 82 NONCODING RNAS AND CANCER Subject Index

A-I editing B8 Lung cancer A21, A26, B5, Adipocytes A28 B7, B17 AKT2 B28 Lymphoma A24 Alternative splicing B8 MALAT-1 A21, B23 Androgen receptor B3 Malignant glioma B15 Androgen signaling A29 Melanoma A11, B24 Antisense oligonucleotides B23 Metastasis A6, A31, B18, Antitumor effects B23 B21, B29 Arid4b B20 MGMT A22 Biomarker A9 microRNA A7, A8, A10, Bladder cancer A10 A13, A14, A16, BRAF A11 A18, A20, A26, Breast cancer A6, A7, A9, A30, A31, A34, A25, B1, B13, B4, B7, B10, B20 B11, B12, B13, Cell cycle inhibitor A24 B15, B21, B22, CHD5 A13 B24, B27, B28 Circulating miRNA A9 microRNA expression profile A25 Cisplatin B12 Microvesicles A28 Cisplatin resistance A10 miR-10a B9 CLL B10 miR-135b B14 Colon cancer A13, B14 miR-21 A28 Cytotoxic A5 miR-216 A6 DD3/PCA3 A29 miR-290 B20 Differentiation B9 miR-31 A3 DNA damage B11 miR-34c B22 Drug resistance A2, B7 miR-708 A27 E2F1 A17 miRNA 15, 16, and 21 A17 Epigenetics A16, B10, B25 Multidrug resistance B11, B12 Epithelial-mesenchymal ncRNA profiling B5 transition A18, A34, B1 Neuroblastoma B9 eRNAs B3 NF1 B28 Ewing’s sarcoma B8 Noncoding RNA A4 Expression profiling A23, B6 Non-small cell lung cancer B21 Extracts A5 Notch A30, A33 Functional genomics B2 Oral cancer A16 G1/S transition A17 Outcome A23 Glioblastoma A22 Ovarian cancer A31, B2, B4, Gold nanoparticle delivery B27 B22, B26 GRO-seq B3 p53 A18, B16, B19 GRP78 A8 Pancreatic cancer A32 High-throughput assay A33 Paraspeckles A1 Human knockout model A21 PCA3 A4 In situ hybridization B13 PCR B4 KEAP1 gene A15 Pediatric oncology B16 Lentivirus A12 PI3K/AKT/PTEN pathway A2 LET-7 A32 Profiling A19 Library A12 Prognosis B6 Long noncoding RNAs A1, A11, A19, Promoter methylation A3 B18, B19 Proneural B15 Prostate cancer A4, A27, A29

PROGRAM AND PROCEEDINGS • January 8-11, 2012 • MiaMi Beach, FL 83 Pseudogene B25 PTEN B25 Regulation A14 Rhabdomyosarcoma A23, A30 Ribonucleotide reductase A32 RNA affinity purification B5 RNAi A33 RNAseq B18 Screening platform A12 Single-molecule sequencing B6 siRNA B2, B17 Six1 A20 Solid cancers A15 Stem cells B16 Stress-induced A1 T-cell development A24 TGF-beta A7, A20, A34, B26 Therapeutic resistance A22 Therapeutic target A8 Therapeutics B14 Triple negative A3, A25 Tumor suppressor A14, A27, B27 UV B24 VEGFr2 B17

84 NONCODING RNAS AND CANCER