Pleiotropically acting MicroRNA 10b regulates angiogenicity, invasion and growth of tumor cells resembling mesenchymal subgroup of glioblastoma multiforme

Pleiotropically-acting MicroRNA-10b Regulates Angiogenicity, Invasion and Growth of Tumor Cells Resembling Mesenchymal Subgroup of Glioblastoma Multiforme Lin Jiakai (B.Sc (Hons)), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2012 -1- Acknowledgements The work underlying this thesis was performed at the Institute of Bioengineering and Nanotechnology I would like to extend my gratitude to all who have encouraged me these years I would like to thank: My supervisor A/P Shu Wang, for his expert guidance and encouragement when times got tough Prof Jackie Ying and Ms Noreena, directors of the Institute of Bioengineering and Nanotechnology, for creating a challenging, exciting and stimulating working environment and support for my PhD studies My past and present lab mates for being fantastic people to work around with; Jerome, Jaana, Poonam, Daniel, Keryin, Yukti, Esther, Xiaoying, Seong Loong, Zhao Ying, Jieming, Chunxiao, Mohammad, Chrishan, Lam, Yovita, Timothy, Detu, Ghayathri My parents for being a strong pillar of support in my life My lovely wife, Huiling, and daughter, Rui En, for their unwavering support over the last few years I could not have achieved this without you -2- Publication Jiakai Lin, Shi Jia Teo, Jeyaseelan Kandiah, Shu Wang, MicroRNA-10b Pleiotropically Regulates Angiogenicity, Invasion and Growth of Tumor Cells Resembling Mesenchymal Subgroup of Glioblastoma Multiforme, Submitted Publication which I have contributed to whose work is not included in the thesis Chunxiao Wu*, Jiakai Lin*, Michelle Hong, Yukti Choudhury, Poonam Balani, Doreen Leung, Lam H Dang, Ying Zhao, Jieming Zeng and Shu Wang, Combinatorial Control of Suicide Gene Expression by Tissue-specific Promoter and microRNA Regulation for Cancer Therapy, Molecular Therapy 2009 December; 17(12): 2058–2066 *co-first authors -3- Table of Contents Table of Contents Summary List of Tables List of Figures List of Abbreviations 10 Chapter 1: Introduction 13 1.1 Discovery, biogenesis and mechanisms of action of microRNAs 13 1.2 MicroRNAs in glioblastoma multiforme (GBM) 15 1.3 MicroRNA loss-of-function studies 20 1.3.1 Considerations in microRNA sponge design 26 1.3.2 Advantages and limitations of microRNA sponge 34 1.3.3 Interrogating microRNA function via transient microRNA sponge expression 37 1.3.4 Interrogating microRNA function via stable microRNA sponge expression 39 1.3.5 Elucidation of microRNA function in cancer development 40 1.4 Key signaling pathways dysregulated in glioblastoma 44 multiforme 1.4.1 Genetic alterations occurring in the Receptor Tyrosine Kinase (RTK) pathways 1.4.2 Genetic alterations occurring in TP53 tumor suppressor 46 pathway 1.4.3 Genetic alterations occurring in RB1 tumor suppressor 48 pathway 44 Chapter 2: Aims and objectives 50 Chapter 3: Materials and methods 52 Chapter 4: Results 59 4.1 U87-2M1 is an invasive mesenchymal subline of U87 59 4.2 Inhibition of miR-10b decreases invasiveness of U87-2M1 68 4.3 miR-10b silencing decreases angiogenicity of U87-2M1 71 -4- 4.4 Inhibition of miR-10b increases apoptosis of U87-2M1 glioma cells and prolongs survival of U87-2M1-bearing mice 76 4.5 Pleiotropic miR-10b regulates a broad range of tumor suppressors 81 4.6 Perturbation of direct and indirect targets of miR-10b correlates with poorer patient survival 87 Chapter 5: Discussion 90 Chapter 6: Conclusion and future studies 94 Bibliography 96 -5- Summary Glioblastoma multiforme (GBM) is an extremely heterogeneous disease despite its seemingly uniform pathology Deconvolution of The Cancer Genome Atlas’s GBM gene expression data has unveiled the existence of distinct gene expression signatures underlying discrete GBM subgroups Recent conflicting findings proposed that microRNA10b exclusively regulates glioma growth or invasion but not both We showed that silencing of microRNA-10b by baculoviral decoy vectors in a glioma cell line resembling the mesenchymal subgroup of GBM reduces its growth, invasion and angiogenesis whilst promoting apoptosis in vitro In an orthotopic human glioma mouse model, inhibition of microRNA-10b diminishes the invasiveness, angiogenicity and growth of mesenchymal glioma cells in the brain and significantly prolonged survival of glioma-bearing mice We demonstrated that the pleiotropic nature of microRNA-10b was due to its suppression of multiple tumor suppressors including TP53, FOXO3, CYLD, PAX6, PTCH1 and NOTCH1 By interrogation of the REMBRANDT database, we noted that dysregulation of many direct targets of microRNA-10b was associated with significantly poorer patient survival Thus, our studies uncovered a novel role for microRNA-10b in regulating angiogenesis and suggest that microRNA-10b may be a pleiotropic regulator of gliomagenesis -6- List of Tables Table 1…………………………………………………………… 21 Table 2…………………………………………………………… 43 Table 3…………………………………………………………… 60 Table 4…………………………………………………………… 67 -7- List of Figures Figure Principle of using microRNA sponge to inhibit endogenous microRNA function……………………………… Figure Design of an expression cassette harboring a microRNA sponge……………………………………………… Figure Illustration of the sequence of a microRNA-10b binding site ……………………………………………………… Figure Genomic sequence alterations and copy number changes for components of the RTK/Ras/PI(3)K network of genes ………… ………………………………………………… Figure Genomic sequence alterations and copy number changes for components of the TP53 network of genes…… Figure Genomic sequence alterations and copy number changes for components of the RB1 network of genes……… Figure U87 and U87-2M1 glioma cells were xenografted intracranially in Balb c/nude mice for three weeks…………… Figure U87-2M1 showed higher endogenous protein expression of N-cadherin, fibronectin, vimentin, Twist, Stat3, MMP13, FOXM1, HGF, PLAUR and PLAU compared to U87 cells………………………………………………………………… Figure Quantification of miR-10b expression in glioma cell lines………………………………………………………………… Figure 10 Construction of control sponge or miR-10 sponge in baculoviral vectors and the transduction efficiency in U87-2M1 cells…………………………………………………………… Figure 11 MicroRNA-10b decoy vector, but not the control decoy vector, relieves the suppression of luciferase expression by endogenous miR-10b in U87-2M1 glioma cells Figure 12 MicroRNA-10b decoy vector decreases the level of detectable miR-10b in U87-2M1 glioma cells………………… Figure 13 Inhibition of miR-10b in U87-2M1 cells diminishes its capacity to invade through a transwell membrane coated with basement membrane matrix……………………………… Figure 14 Prior silencing of miR-10b in U87-2M1 cells in vitro reduces growth of orthotopic tumor with no evident signs of localized invasion………………………………………………… Figure 15 MicroRNA-10b silencing reduces protein expression of β-catenin, MMP13, RhoC, PLAUR, PLAU and HGF and upregulates HOXD10 protein expression…………… Figure 16 Over-expression of miR-10b promotes invasiveness of U87-2M1………………………………………… Figure 17 Inhibition of miR-10b suppresses angiogenesis by U87-2M1 glioma cells…………………………………………… 25 28 32 46 47 49 59 61 62 63 65 65 68 69 70 71 72 -8- Figure 18 Angiogenic potential of U87-2M1 cells were determined by an in vitro endothelial cell tube formation 73 assay………………………………………………………………… Figure 19 MicroRNA-10b likely reduces angiogenic potential of U87-2M1 by decreasing expression of pro-angiogenic 74 proteins such as VEGF, IL8, TGFβ2, CTGF and THBS1…… Figure 20 A panel of 13 angiogenic genes down-regulated by miR-10b silencing is frequently over-expressed in the Mesenchymal subgroup of glioblastoma multiforme (GBM)… Figure 21 MicroRNA-10b silencing promotes apoptosis of U87-2M1 cells……………………………………………………… Figure 22 MTS assay shows a decrease in cell viability after inhibition of miR-10b……………………………………………… Figure 23 Elevated caspase activity after miR-10b silencing was detected by the use of a caspase-sensitive Casp-Glo reagent……………………………………………………………… Figure 24 Western blotting confirms increased protein expression of cleaved caspase-3 and cleaved caspase-7…… Figure 25 Delivery of miR-10b decoy vector, but not the control decoy vector, into a subcutaneous U87-2M1 tumor results in enhanced TUNEL-positive staining………………… Figure 26 Mice bearing U87-2M1 tumors that arose from prior treatment in vitro with PBS, control decoy vector, or miR-10b decoy vector showed significantly different survival trends…… Figure 27 Predicted miR-10b binding sites in TP53, FOXO3, PAX6, CYLD, PTCH1 and NOTCH1…………………………… Figure 28 Identification of TP53, FOXO3, CYLD, NOTCH1, PTCH1 and PAX6 as targets of miR-10b……………………… Figure 29 Silencing of miR-10b up-regulates target proteins TP53, FOXO3, CYLD, PTCH1, NOTCH1 and PAX6………… Figure 30 NFkB transcriptional activity is significantly lower in miR-10b-silenced U87-2M1 cells………………………………… Figure 31 A mechanistic model summarizing the pleiotropic actions of miR-10b………………………………………………… Figure 32 Perturbed expression of direct targets of miR-10b are associated with poor patient survival……………………… Figure 33 Upregulation of indirect oncogenic targets of miR10b significantly correlates with poor patient survival………… 75 76 77 77 78 79 80 82 83 84 85 87 88 89 -9- List of Abbreviations AGO AKT AMO AMPK ANGPT1 ANXA2 BCL2 BIM CAB39 CCND1 CDK4 CDKN2A CMV COL1A2 CTGF CTNNB1 CYLD DAVID DVL E2F1 EGFP EGFR ELM EMT ERK EZH2 FN1 FOXM1 FOXO3 GADD45A GADD45B GBM GDP GTP HGF HOXD10 HUVEC IKB-Α IKB-Β IL8 ITGA4 JAG1 Argonaute Protein kinase B Anti-miRNA oligonucleotides 5' adenosine monophosphate- activated protein kinase Angiopoietin1 Annexin A2 B-cell CLL/lymphoma BCL2-like 11 (apoptosis facilitator) Calcium-binding protein 39 Cyclin D1 Cyclin dependent kinase Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) Cytomegalovirus Collagen 1A2 Connective tissue growth factor Catenin (cadherin-associated protein), beta Cylindromatosis (turban tumor syndrome) Database for Annotation, Visualization, and Integrated Discovery Dishevelled E2F transcription factor Enhanced green fluorescent protein Epidermal growth factor receptor Experimental lung metastasis Epithelial-mesenchymal-transition Extracellular signal-regulated kinase Enhancer of zeste homolog Fibronectin1 Forkhead box protein M1 Forkhead box O3 Growth arrest and DNA-damage-inducible, alpha Growth arrest and DNA-damage-inducible, beta Glioblastoma multiforme Guanosine diphosphate Guanosine triphosphate Hepatocyte growth factor Homeobox D10 Human umbilical vein endothelial cells I-kappa-B-kinase-alpha I-kappa-B-kinase-beta Interleukin Integrin, alpha Jagged - 10 - 80 Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, et al Silencing of microRNAs in vivo with 'antagomirs' Nature 2005 Dec 1;438(7068):685-9 81 Elmen J, Lindow M, Schutz S, Lawrence M, Petri A, Obad S, et al LNA-mediated microRNA silencing in non-human primates Nature 2008 Apr 17;452(7189):896-9 82 Davis S, Lollo B, 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Jia Teo, Jeyaseelan Kandiah, Shu Wang, MicroRNA- 10b Pleiotropically Regulates Angiogenicity, Invasion and Growth of Tumor Cells Resembling Mesenchymal Subgroup of Glioblastoma Multiforme, Submitted... Inhibition of miR -10b decreases invasiveness of U87-2M1 68 4.3 miR -10b silencing decreases angiogenicity of U87-2M1 71 -4- 4.4 Inhibition of miR -10b increases apoptosis of U87-2M1 glioma cells and prolongs... both We showed that silencing of microRNA- 10b by baculoviral decoy vectors in a glioma cell line resembling the mesenchymal subgroup of GBM reduces its growth, invasion and angiogenesis whilst promoting