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A ThesisThesis entitled Identification and characterization of RhoGAPs involved in the regulation of invadopodia by Kyle Lee Snyder Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Cellular and Molecular Biology _________________________________________ Dr. Rafael Garcia-Mata, Committee Chair _________________________________________ Dr. Deborah Chadee, Committee Member _________________________________________ Dr. Song-Tao Liu, Committee Member _________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo April, 2016 Copyright 2016, Kyle Lee Snyder This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Identification and characterization of RhoGAPs involved in the regulation of invadopodia by Kyle Lee Snyder Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Cellular and Molecular Biology The University of Toledo April, 2016 Invadopodia are actin rich structures that enhance a cancer cells ability to degrade the extracellular matrix (ECM) and promote metastasis. Formation of invadopodia is regulated by Rho GTPases, a family of small G proteins that regulate actin rearrangement, cellular migration, and invasion. These proteins exist in two states, inactive GDP-bound, and active GTP-bound conformations. Activation is regulated by GEFs (guanine nucleotide exchange factors), whereas inactivation is modulated by GAPs (GTPase activating proteins). In our preliminary studies we screened 18 members of the RhoGAP family to identify if any were involved in signaling events contributing to invadopodia formation. We identified three candidates, TCGAP, CHN1 and ARHGAP12. We have confirmed that the knockdown of each of these genes is sufficient to increase invadopodia formation, and validated these results with over expression studies. We have also been able to identify associated Rho proteins for CHN1 and ARHGAP12. Future work will include further characterization of the activity of these RhoGAPs. This work adds to the current knowledge available regarding invadopodia and will contribute to future intervention strategies targeting metastatic events. iii This body of work is dedicated to those that were lost along the way. Table of Contents Abstract iii Table of Contents v List of Tables vii List of Figures viii List of Abbreviations ix List of Symbols x 1. Introduction 1 1.1 Cancer and Metastasis 2 1.2 Invadopodia 4 1.3 Rho Family GTPases 6 1.4 RhoGAPs 9 1.5 TCGAP 13 1.6 CHN1 15 1.7 ARHGAP12 18 1.8 Sum159 Cells 21 2. Materials and Methods 23 2.1 General Cell Maintenance 23 2.2 Antibodies and other reagents 23 2.3 Cell lysis and immunoblotting 23 2.4 Plasmids and other reagents 24 2.5 shRNA mediated knockdown 24 2.6 Immunofluorescence microscopy 25 v 2.7 High throughput image acquisition and analysis 25 2.8 Immunofluorescence invadopodia quantification 26 2.9 Preparation of cDNA 26 2.10 qRT-PCR 27 2.11 Cell transfection 29 2.12 Rho GTPase activity assay 29 2.13 RhoGAP-GTPase binding assay 30 3. Results 32 3.1 RhoGAP Screen 32 3.2 TCGAP 39 3.3 CHN1 43 3.4 ARHGAP12 50 4. Discussion 54 4.1 RhoGAP Screen 54 4.1.1 Future Aims 55 4.2 TCGAP 56 4.2.1 Future Aims 58 4.3 CHN1 60 4.3.1 Future Aims 62 4.4 ARHGAP12 63 4.4.1 Future Aims 65 References 66 vi List of Tables Table 1 Primers used for qRT-PCR analysis of gene knockdown. Forward (sense) and Reverse (anti-sense) used to amplify a 100-150 bp region of the targeted gene. ................................................................................................................28 Table 2 A list representing the RhoGAPs assessed in the initial screening performed. 18 GAPs were chosen, numbered in the left hand column. The middle column contains a reference number from the GAP shRNA library used and the right column identifies the RhoGAP tested by the most common reference name. ...............................................................................................33 vii List of Figures Figure 1 Rho GTPase Cycle ............................................................................................7 Figure 2 The RhoGAP Family ......................................................................................11 Figure 3 TCGAP domain structure ................................................................................14 Figure 4 CHN1 domain structure ...................................................................................17 Figure 5 ARHGAP12 domain structure .........................................................................20 Figure 6 96 well plate layout ..........................................................................................35 Figure 7 GAP screen ......................................................................................................37 Figure 8 RhoGAP screen (images) .................................................................................38 Figure 9 TCGAP is involved in invadopodia formation ................................................40 Figure 10 TCGAP knock down increases RhoG activity.................................................42 Figure 11 CHN1 is involved in invadopodia formation ...................................................44 Figure 12 Punctate invadopodia form in CHN1 knock down ..........................................45 Figure 13 CHN1 suppresses invadopodia and binds Rac1, RhoG ...................................47 Figure 14 CHN1 knock down increases GTPase activity ................................................49 Figure 15 ARHGAP12 is involved in invadopodia formation .........................................51 Figure 16 ARHGAP12 suppresses invadopodia and binds Rac1.....................................53 viii List of Abbreviations aa ................................amino acid bp(s) ...........................base pairs cDNA .........................Complementary DNA DAG ...........................Diacylglycerol DAPI ..........................4',6-Diamidino-2-Phenylindole, Dihydrochloride DMEM .......................Dulbecco’s Modified Eagle Medium DNA ...........................deoxyribonucleic acid ECM ...........................Extracellular Matrix FBS ............................Fetal Bovine Serum FITC ...........................Fluorescein isothiocyanate GAPs ..........................GTPase Activating Proteins GDI ............................GDP-dissociation inhibitor GEFs ..........................Guanine Nucleotide Exchange Factor GST ............................Glutathione S-Transferase HGF............................Hepatocyte Growth Factor HRP ............................Horseradish Peroxidase KD ..............................Knock Down kDa .............................kilodalton MMPs .........................Matrix Metalloproteases mRNA ........................Messenger RNA N-WASP ....................Neuronal Wiskott-Aldrich Syndrome Protein P/S ..............................Penicillin/Streptomycin PBD ............................PAK Binding Domain PBS ............................Phosphate Buffered Saline PDBu ..........................Phorbol 12,13– dibutyrate PH ..............................Pleckstrin Homology PRR ............................Proline rich region(s) PX ..............................Phox Homology Q61L ..........................glutamine to leucine substitution at amino acid residue 61 Q63L ..........................glutamine to leucine substitution at amino acid residue 63 qRT-PCR....................quantitative real time polymerase chain reaction Rho .............................Ras Homology RNA ...........................Ribonucleic Acid ROCK ........................Rho-Associated Protein Kinase ROS ............................Reactive Oxygen Species RSV-CEF ...................Rous Sarcoma Virus Transformed Chicken Embryo Fibroblasts SH2 ............................Src Homology 2 SH3 ............................Src Homology 3 shRNA........................Short Hairpin RNA TKS4 ..........................Tyrosine Kinase Substrate with four Src homology 3 domains TKS5 ..........................Tyrosine Kinase Substrate with five Src homology 3 domains TNBC .........................Triple Negative Breast Cancer(s) TRITC ........................Tetramethylrhodamine WW ............................Domain containing two conserved Tryptophan (W) amino acids ix List of Symbols º ..........degree C .........Celsius α .........alpha β .........beta µ .........Mu [micro] x Chapter One 1. Introduction Understanding the molecular mechanisms controlling carcinogenic cell invasion has the potential to reveal novel targets for therapeutic intervention. Cancer cells invade other tissues and enter the bloodstream by forming actin rich membrane protrusions called invadopodia. Invadopodia enhance the cells’ ability to degrade the extracellular matrix (ECM) and promote metastasis (Baldassarre et al., 2006). Formation of invadopodia is regulated by Rho GTPases, a family of small G proteins that has roles involving actin rearrangement, cellular migration, and invasion (Ridley, 2015). These proteins exist in two states; inactive GDP-bound, and active GTP-bound conformations. Activation is regulated by RhoGEFs (Guanine nucleotide exchange factors) and inactivation is modulated by RhoGAPs (GTPase activating proteins) (Bishop and Hall, 2000). Interestingly, only a small number of

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