CHARACTERIZING ROLES OF ANNEXIN 1 IN BREAST CANCER DEVELOPMENT BY MASS SPECTROMETRY BASED QUANTITATIVE PROTEOMICS

CHARACTERIZING ROLES OF ANNEXIN-1 IN BREAST CANCER DEVELOPMENT BY MASS SPECTROMETRY-BASED QUANTITATIVE PROTEOMICS SWA LEE FOON HANNAH B Sc.(Merit), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 Acknowledgements This graduate journey is akin to a jigsaw puzzle Besides the science involved, completion of the full picture would not be possible without any of the following ‘jigsaw pieces’ I would like to acknowledge and express my gratitude to: A/P Lina Lim, my supervisor, for her patience and guidance I am also thankful for the freedom, help and support she has graciously given me in this journey Asst Prof Jayantha Gunaratne, my co-supervisor and mentor, for his grace, guidance and support His hard-working and passion in science greatly motivates me Prof Walter Blackstock, my ex-boss of the lab He was the one who cleared the obstacles-filled path for me, making the commencement of this graduate studies possible Past and present members of the Quantitative Proteomics Group (QPG), especially Suat Peng and Sheena for their friendship, support and hearing ears whenever I whine Siok Ghee, for her expertise in mass spectrometry and Rachel for her help in all the bioinformatics and visualization plots Past and present members of the Inflammation and Cancer Lab, especially Durkesh, Sunitha and Suruchi for their friendship, for always being there for me This graduate fellowship we have shall be a beautiful memory Institute of Molecular and Cell Biology, A*STAR, for funding part of this graduate programme My family members for their support, and especially my grandmother for her constant love and concern for me Above all, I present my heartfelt thanksgiving and gratitude to God, the One who loves me, never gives up on me and the One who holds the pieces of the jigsaw puzzle together ii Table of Contents DECLARATION i  Acknowledgements ii  Summary………… vii  List of Tables ix  List of Figures x  List of Abbreviations xiii  List of Symbols xvi  Chapter 1:  Introduction 1  1.1:  Breast cancer 1  1.2:  Annexin 2  1.2.1:  Structure of ANXA1 2  1.2.2:  Functions of ANXA1 3  1.2.2.1:  Anti-inflammatory role 3  1.2.2.2:  Regulator of Cellular Processes 4  1.2.3:  ANXA1 and Cancer 6  1.3:  Models for breast cancer 8  1.3.1:  Cell line models for breast cancer 8  1.3.2:  Mouse models for breast cancer 9  1.3.2.1:  Xenograft models 9  1.3.2.2:  Genetically-modified models 10  1.3.3:  Mammary gland development 10  1.3.4:  Hallmarks of cancer 12  1.4:  Proteomics 15  1.5:  MS-based proteomics 17  1.6:  Quantitative MS-based proteomics 22  1.6.1:  Label-free Quantification 25  1.6.2:  Chemical labeling 26  1.6.3:  Metabolic Labelling 32  iii 1.6.3.1:  Stable isotope labelling by amino acids in cell culture (SILAC) 32  1.6.3.2:  1.7:  Super-SILAC 35  Aims of Study 36  Chapter 2:  Methods and Materials 37  2.1:  Isolation and culture of primary mammary gland epithelial cells 37  2.2:  Adaptation of cell lines to SILAC media 37  2.3:  Filter-aided sample preparation (FASP) for SILAC incorporation check …………… 37  2.4:  Cell lysates preparation 38  2.5:  Murine mammary tumors lysates preparation 39  2.6:  In-Gel Dehydration, Reduction, Alkyation and Tryptic Digestion 39  2.7:  Super-SILAC mix preparation 40  2.8:  Protein quantification 40  2.9:  Western blot analysis 40  2.10:  RT-PCR analysis 42  2.11:  Heat treatment 43  2.12:  Comet assay 44  2.13:  Transfection experiments 44  2.14:  Cell adhesion assay 45  2.15:  Hydrogen peroxide (H2O2) treatment and Cell proliferation (WST-1) assay … 45  2.16:  Migration assay 46  2.17:  Wound healing assay 46  2.18:  Reactive oxygen species (ROS) assay 46  2.19:  Liquid Chromatography-Mass Spectrometry (LC-MS) 46  2.20:  Identification and Quantification of Peptides and Proteins 47  2.21:  Bioinformatics Analysis 47  Chapter 3:  Results 48  3.1:  Proteome profiling of ANXA1+/- and ANXA1-/- normal mammary gland cells 48  3.1.1:  SILAC workflow for mass spectrometer analysis 48  3.1.2:  MS data analysis 52  3.1.2.1:  Clustering proteins into up- and down-regulated with ANXA1-/-………… 55  3.2:  Analysis of down-regulated proteins with ANXA1-/- 57  3.2.1:  Pathway analysis 57  iv 3.2.1.1:  Identification of proteins in DNA-damage response pathway…………………………… 57  3.2.2:  Biochemical validation of selected down-regulated proteins 59  3.2.3:  Functional validation of pathway analysis 60  3.2.3.1:  Comet assay analyses of thermal-stressed mammary gland cells…………… 61  3.2.3.2:  Reactive oxygen species (ROS) assay of mammary gland cells……… 63  3.2.3.3:  Cell proliferation assays of oxidative-stressed mammary gland cells……… 65  3.2.3.4:  Over-expression of down-regulated protein reverses DNAdamage response in ANXA1-/- mammary gland cells 66  3.3:  Analysis of up-regulated proteins with ANXA1-/- 69  3.3.1:  Pathway analysis 69  3.3.1.1:  Identification of proteins in cell adhesion/motility pathway69  3.3.2:  Biochemical validation of selected up-regulated proteins 72  3.3.3:  Functional validation of pathway analysis 72  3.3.3.1:  Migration assay of the mammary gland cells 73  3.3.3.2:  Wound healing assay of mammary gland cells 74  3.3.3.3:  Silencing of up-regulated protein reverses adhesive phenotype in ANXA1-/- mammary gland cells 76  3.4:  Proteome profiling of ANXA1+/- and ANXA1-/- mammary tumors 82  3.4.1:  Western blot analysis of the mammary tumors 82  3.4.2:  Workflow for Super-SILAC mass spectrometry analysis 83  3.4.3:  Relative quantification of proteins 88  3.4.4:  Analysis of the Super-SILAC mix 89  3.5:  Categorization of up- and down-regulated proteins with ANXA1-/- 91  3.6:  Bioinformatics analyses of down- and up-regulated proteins with ANXA1-/- 92  3.6.1:  Down-regulated proteins with ANXA1-/- 92  3.6.2:  Up-regulated proteins with ANXA1-/- 93  3.6.3:  Cancer-Associated Proteins (CAPs) and Cancer Driver Mutations (CDMs) 94  3.6.3.1:  Mapping up- and down-regulated CAPs to CDMs 99  3.6.3.2:  Network with the up-regulated proteins 102  3.6.3.3:  Network with the down-regulated proteins 102  3.7:  Comparison of normal mammary gland cells (SILAC-based) and mammary tumors (Super-SILAC based) 102  3.7.1:  Up-regulated in both mammary gland cells and mammary tumors103  v 3.7.2:  Down-regulated in both mammary gland cells and mammary tumors 104  3.7.3:  Inverse correlation between up- and down-regulated proteins in mammary gland cells and mammary tumors 105  3.7.4:  Up- or down-regulated proteins in mammary gland cells but no change in mammary tumors 107  3.7.5:  Up- or down-regulated protein in mammary tumors but not detected in mammary gland cells 108  Chapter 4:  4.1:  Discussion 110  Characterization of ANXA1 from SILAC experiments 110  4.2:  Possible roles of ANXA1 in tumorigenesis revealed by Super-SILAC experiments 118  4.3:  Distinct differential regulation by ANXA1 between normal mammary gland cells and mammary tumors 125  4.4:  Use of SILAC, Super-SILAC and the mouse models 128  4.5:  Conclusion 129  4.6:  Future directions 130  Bibliography 132  Appendix 153  Tables………… 153  Publications 190  Supplementary CD 191  vi Summary………… Annexin-1 (ANXA1) has been reported to be involved in important physiopathological implications including cell proliferation, apoptosis, cancer and metastasis However, with controversies in ANXA1 expression in breast carcinomas, its role in breast cancer initiation and progression remains unclear The study presented here seeks to characterize ANXA1 in initiation and tumorigenesis of breast cancer by understanding the dysregulated pathways involved Through quantitative mass spectrometry-based proteomics, SILAC (stable isotope labelling by amino acids in cell culture) and Super-SILAC were applied to normal mammary gland cells and mammary tumors respectively from ANXA1-heterozygous (ANXA1+/-) and null (ANXA1-/-) mice to study their changes at the proteome level Quantitative comparison of the proteomes of normal mammary gland cells using SILAC quantified over 4000 proteins with 214 up-regulated and 169 down-regulated in ANXA1-/- Bioinformatics analysis of the up- and downregulated proteins revealed that ANXA1 is potentially implicated in DNAdamage response and cell adhesion/motility pathways Relevant functional assays showed accumulation of more DNA damage with slower recovery on heat stress and an impaired oxidative damage response in ANXA1-/- cells in comparison to ANXA1+/- cells Over-expressing Yes-associated protein (Yap1), the most down-regulated protein in DNA-damage response pathway cluster, reversed the proliferative response in ANXA1-/- cells when insulted, indicating the protective role ANXA1 plays via regulating a group of proteins involved in the DNA-damage response pathway Both migration and wound healing assays showed that ANXA1+/- cells possess higher motility with better wound closure capability than ANXA1-/- Silencing of β-parvin in ANXA1-/-, the protein with the highest fold change in the cell adhesion protein cluster reversed its motility phenotype This indicated that the pro-migratory role ANXA1 plays via regulating a group of proteins that are involved in cell adhesion/motility pathway vii After establishing roles of ANXA1 in DNA-damage response and cell adhesion/motility that could contribute to breast cancer initiation, its role in breast cancer progression was investigated Super-SILAC analysis on mammary tumors derived from PyMT+ANXA1+/- and PyMT+ANXA1-/- mice quantified over 5000 proteins with 369 up- and 365 down-regulated proteins observed in PyMT+ANXA1-/- Bioinformatics analysis of up-regulated proteins showed that ANXA1 may be involved in cell cycle regulation pathways, whereas the analysis of down-regulated proteins showed its possible roles in cystic fibrosis and cytoskeletal remodelling during tumorigenesis As these outcomes are different from pathway analyses performed for the regulated protein clusters from the normal mammary gland cell experiments, overlapping up- and down-regulated protein clusters between mammary cells and tumors were closely compared Interestingly, the non-overlapped up- and down-regulated clusters were enriched in cell adhesion/migration and DNAdamage response pathway respectively, further indicating that ANXA1 plays a different role in tumorigenesis Out of the differentially-regulated proteins, 32 from the up-regulated and 25 from the down-regulated were identified to be cancer-associated Together with functional interaction network mapping, ANXA1 is proposed to play a modulatory role in several pathways in tumorigenesis Altogether the study here suggests that ANXA1 plays different, yet important roles in breast cancer initiation and progression viii List of Tables Table 1: The status of ANXA1 in clinical cancer tissues Table 2: Antibodies used for western blotting Table 3: Primers used for RT-PCR analysis Table 4: Down-regulated proteins involved in DNA-damage response pathway Table 5: Up-regulated proteins involved in cell adhesion and motility Table 6: Cell lines used in the Super-SILAC mix Table 7: Up-regulated proteins (from Super-SILAC experiments) known to be CAPs Table 8: Down-regulated proteins (from Super-SILAC experiments) known to be CAPs Table 9: Overlapping up-regulated proteins in SILAC and Super-SILAC experiments Table 10: Overlapping down-regulated proteins in SILAC and SuperSILAC experiments Table 11: Proteins with inverse regulations between SILAC and SuperSILAC experiments ix aldehyde dehydrogenase 16 family, member A1 component of oligomeric golgi complex dymeclin S-adenosylhomocysteine hydrolase-like MAD1 mitotic arrest deficient 1-like adaptor-related protein complex 3, beta subunit eukaryotic translation initiation factor 2-alpha kinase fatty acid synthase interferon regulatory factor binding protein pyridoxal-dependent decarboxylase domain containing immunoglobulin (CD79A) binding protein motile sperm domain containing glucosidase, alpha, acid X-ray repair complementing defective repair in Chinese hamster cells platelet-activating factor acetylhydrolase peptidylprolyl isomerase (cyclophilin)-like RNA binding motif protein general transcription factor II I nudix (nucleotide diphosphate linked moiety X)-type motif acyl-CoA synthetase family member muscleblind-like carboxypeptidase D F11 receptor legumain SEC16 homolog A (S cerevisiae) catalase retinol binding protein 1, cellular cyclin-dependent kinase inhibitor 1B ADP-ribosylation factor GTPase activating protein AT rich interactive domain 1A (SWI-like) G protein-coupled receptor kinase-interactor abhydrolase domain containing kinesin family member 1B glyoxylate reductase homolog (Arabidopsis) selenium binding protein TBC1 domain family, member ERGIC and golgi ribosome binding protein NHL repeat containing methylcrotonoyl-Coenzyme A carboxylase (beta) 3'-phosphoadenosine 5'-phosphosulfate synthase von Willebrand factor A domain containing 5A hexosaminidase B 177 Aldh16a1 Cog7 Dym Ahcyl2 Mad1l1 Ap3b1 Eif2ak2 Fasn Irf2bp2 Pdxdc1 Igbp1 Mospd2 Gaa Xrcc1 Pafah2 Ppil2 Rbm5 Gtf2i Nudt3 Acsf2 Mbnl2 Cpd F11r Lgmn Sec16a Cat Rbp1 Cdkn1b Arfgap2 Arid1a Git2 Abhd5 Kif1b Glyr1 Selenbp1 Tbc1d4 Ergic2 Rrbp1 Nhlrc3 Mccc2 Papss1 Vwa5a Hexb laminin, alpha phospholipase D family, member milk fat globule-EGF factor protein interferon regulatory factor binding protein intercellular adhesion molecule toll-like receptor N-acetyl galactosaminidase, alpha Huntingtin interacting protein related PDZ and LIM domain (elfin) DEP domain containing MTOR-interacting protein phosphatidylcholine transfer protein transcobalamin PYD and CARD domain containing interferon gamma inducible protein 30 cytidine monophospho-N-acetylneuraminic acid synthetase sortilin-related receptor, LDLR class A repeats-containing solute carrier family 28 (sodium-coupled nucleoside transporter), member Keratin-76 coagulation factor II Keratin, type I cytoskeletal 17 adaptor protein complex AP-1, mu subunit (Bos taurus) Apolipoprotein A-I precursor stabilin lactate dehydrogenase B liver glycogen phosphorylase laminin, alpha asparaginase like desmoplakin NLR family member X1 crystallin, lambda ATPase, Ca++ transporting, ubiquitous UDP-N-acetyl-alpha-D-galactosamine:polypeptide Nacetylgalactosaminyltransferase guanine deaminase serine (or cysteine) peptidase inhibitor, clade A, member 1D aldhehyde dehydrogenase family 5, subfamily A1 insulin-like growth factor binding protein, acid labile subunit lymphocyte antigen complex, locus E immunoglobulin heavy constant mu serine (or cysteine) peptidase inhibitor, clade A, member 3M actin, alpha 1, skeletal muscle 178 Lama2 Pld3 Mfge8 Irf2bp1 Icam1 Tlr3 Naga Hip1r Pdlim1 Deptor Pctp Tcn2 Pycard Ifi30 Cmas Sorl1 Slc28a3 KRT76 F2 Krt1-17 Ap1m2 Stab1 Ldhb Pygl Lama1 Asrgl1 Dsp Nlrx1 Cryl1 Atp2a3 Galnt3 Gda Serpina1d Aldh5a1 Igfals Ly6e Ighm Serpina3m Acta1 serine (or cysteine) peptidase inhibitor, clade A, member and PZP-like alpha-2-macroglobulin domain-containing protein spectrin beta sortilin epidermal growth factor receptor collagen, type I, alpha keratin 14 58 kDa cytokeratin FXYD domain-containing ion transport regulator serine (or cysteine) peptidase inhibitor, clade F, member ectonucleotide pyrophosphatase/phosphodiesterase complement component factor h sphingomyelin phosphodiesterase, acid-like 3A group specific component lactotransferrin Yamaguchi sarcoma viral (v-yes-1) oncogene homolog androgen binding protein alpha hemoglobin alpha, adult chain leucine-rich alpha-2-glycoprotein serine (or cysteine) peptidase inhibitor, clade A, member 3K collagen, type I, alpha N-acetylneuraminate pyruvate lyase tropomyosin 1, alpha thymus cell antigen 1, theta claudin complement factor D (adipsin) calponin lactalbumin, alpha myosin, light polypeptide 9, regulatory gamma-glutamyltransferase transferrin chymase 1, mast cell cytoglobin sialic acid acetylesterase fructose bisphosphatase neuroguidin, EIF4E binding protein spectrin beta mitogen-activated protein kinase peptidoglycan recognition protein HD domain containing GLE1 RNA export mediator (yeast) mortality factor like 179 Serpina6 CPAMD6 Spnb3 Sort1 Egfr Col1a2 Krt14 KRT5 Fxyd3 Serpinf2 Enpp3 Cfh Smpdl3a Gc Ltf Lyn Abpa Hba-a2 Lrg1 Serpina3k Col1a1 Npl Tpm1 Thy1 Cldn7 Cfd Cnn1 Lalba Myl9 Ggt5 Trf Cma1 Cygb Siae Fbp2 Ngdn Spnb2 Mapk9 Pglyrp1 Hddc3 Gle1 Morf4l1 DDRGK domain containing general transcription factor II E, polypeptide (beta subunit) carboxylesterase 1D transmembrane protein 176B glucuronidase, beta ATPase, H+ transporting, lysosomal V1 subunit C2 PDZK1 interacting protein dihydrodiol dehydrogenase (dimeric) leucyl-tRNA synthetase gamma-glutamyl hydrolase heat shock protein 1-like PHD finger protein 2-oxoglutarate and iron-dependent oxygenase domain containing synaptojanin binding protein polyamine-modulated factor papillary renal cell carcinoma (translocation-associated) GINS complex subunit (Psf3 homolog) NIMA (never in mitosis gene a)-related expressed kinase B cell CLL/lymphoma 7C translocase of inner mitochondrial membrane 17b polymerase (DNA directed), alpha lysosomal trafficking regulator CUGBP, Elav-like family member Profilin-1 pleckstrin homology-like domain, family A, member translocase of inner mitochondrial membrane 17a expressed sequence AI462493 non-SMC condensin I complex, subunit D2 murinoglobulin BCLl2-like 15 ubiquitin related modifier homolog (S cerevisiae) cyclin-dependent kinase 15 cytochrome c oxidase subunit VIIa polypeptide 2-like Ddrgk1 Gtf2e2 Ces1d Tmem176b Gusb Atp6v1c2 Pdzk1ip1 DHDH Lars Ggh Hspa1l Phf6 Ogfod1 Synj2bp Pmf1 Prcc Gins3 Nek6 Bcl7c Timm17b Pola2 Lyst Celf2 Pfn1 Phlda3 Timm17a AI462493 Ncapd2 Mug2 Bcl2l15 Urm1 Cdk15 Cox7a2l Table 5: List of the 328 differentially-regulated proteins from the SILAC experiments that are unregulated in the Super-SILAC experiments Protein Names Yap1 protein UPF0609 protein C4orf27 homolog Gene Names Yap1 UPF0609 180 SILAC up/down Down Down UBX domain-containing protein Ubiquitin-like domain-containing CTD phosphatase Ubiquitin-conjugating enzyme E2L Ubiquitin-associated protein 2-like Ubiquitin carboxyl-terminal hydrolase Triosephosphate isomerase Syne2 protein SWI/SNF complex subunit SMARCC1 Sterile alpha motif domain-containing protein 9like StAR-related lipid transfer protein SNW domain-containing protein SH3 and PX domain-containing protein 2B Serine hydroxymethyltransferase Schlafen RNA-binding protein 27 RNA polymerase II subunit A C-terminal domain phosphatase RelA-associated inhibitor Receptor-transporting protein Ras and Rab interactor Rapamycin-insensitive companion of mTOR Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative deoxyribose-phosphate aldolase Psme2 protein Protein TANC1 Protein SDA1 homolog Protein FAM114A2 Protein FAM107B Proteasome subunit beta type-8 Proteasome activator complex subunit Prostaglandin G/H synthase Prostacyclin synthase Probable RNA-binding protein EIF1AD 181 Ubxn1 Down Ublcp1 Ube2l6 Ubap2l Usp18 Tpi1 Syne2 Smarcc1 Down Down Down Down Down Down Down Samd9l Stard5 Snw1 Sh3pxd2b Shmt1 RP23392I13.10001 Rbm27 Down Down Down Down Down Ctdp1 Ppp1r13l Rtp4 Rin1 Rictor Stat2 Ripk3 Mov10 Oas1a Ifit1 Mthfd1 Anxa4 Oasl1 Rasa1 Dera Psme2 Tanc1 Sdad1 Fam114a2 Fam107b Psmb8 Psme1 Ptgs2 Ptgis Eif1ad Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Probable ATP-dependent RNA helicase DDX58 Plasminogen activator inhibitor 2, macrophage Phosphoglycerate mutase Phosphoglycerate kinase Opioid growth factor receptor NudC domain-containing protein Nuclear receptor corepressor Nuclear factor NF-kappa-B p100 subunit Nuclear autoantigenic sperm protein (Histonebinding) Novel protein, possible orthologue of human peroxisomal proliferator-activated receptor A interacting complex 285 PRIC285 Nicotinamide phosphoribosyltransferase Myosin-Ic Mtap1b protein Mpa2l protein Mixed lineage kinase domain-like protein Microtubule-associated protein 1A MFLJ00316 protein MARCKS-related protein Macrophage migration inhibitory factor Low molecular weight phosphotyrosine protein phosphatase Low molecular weight phosphotyrosine protein phosphatase L-lactate dehydrogenase LIM domain-containing protein Inverted formin-2 Interferon-related developmental regulator Interferon-inducible GTPase Interferon-induced very large GTPase Interferon-induced protein with tetratricopeptide repeats Interferon-induced helicase C domain-containing protein Interferon-activable protein 205-B Interferon-activable protein 204 Inositol-3-phosphate synthase Histidine triad nucleotide-binding protein Hippocalcin-like protein High mobility group protein B3 High mobility group protein B2 Heat shock protein HSP 90-alpha Heat shock 70 kDa protein Guanylate binding protein 4.1 182 Ddx58 Serpinb2 Pgam1 Pgk1 Ogfr Nudcd2 Ncor2 Nfkb2 Down Down Down Down Down Down Down Down Nasp Down BC006779 Nampt Myo1c Mtap1b Mpa2l Mlkl Map1a Gbp6 Marcksl1 Mif Down Down Down Down Down Down Down Down Down Down Acp1 Down Acp1 Ldha Limd2 Inf2 Ifrd1 Iigp1 Gvin1 Down Down Down Down Down Down Down Ifit3 Down Ifih1 Ifi205b Ifi204 Isyna1 Hint1 Hpcal1 Hmgb3 Hmgb2 Hsp90aa1 Hspa4 Gbp4 Down Down Down Down Down Down Down Down Down Down Down Growth arrest-specific protein Golgi membrane protein Glycylpeptide N-tetradecanoyltransferase Glutathione S-transferase P Glutathione reductase, mitochondrial Glucose-6-phosphate isomerase Gem-associated protein Galactokinase Fumarylacetoacetase Fructose-bisphosphate aldolase Four and a half LIM domains Four and a half LIM domains Formin-1 FK506-binding protein Ezrin Eyes absent homolog Endoplasmic reticulum metallopeptidase Ectonucleotide pyrophosphatase/phosphodiesterase family member Dual specificity mitogen-activated protein kinase kinase DNA ligase DNA excision repair protein ERCC-6-like DEXH (Asp-Glu-X-His) box polypeptide 58 Developmentally-regulated GTP-binding protein Deoxyhypusine synthase Deoxyguanosine kinase, mitochondrial Dedicator of cytokinesis protein 11 Ddx60 protein DCN1-like protein Cytosolic non-specific dipeptidase Cyclin-dependent kinase inhibitor 2A, isoform Copine-1 Centrosomal protein of 170 kDa Cellular tumor antigen p53 Cellular retinoic acid-binding protein CD166 antigen Caspase-3 cAMP-dependent protein kinase type II-beta regulatory subunit Calpastatin Calpastatin Breast cancer anti-estrogen resistance protein 183 Gas2 Golm1 Nmt2 Gstp1 Gsr Gpi Gemin5 Galk1 Fah Aldoa Fhl1 Fhl1 Fmn1 Fkbp3 Ezr Eya3 Ermp1 Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Down Enpp1 Down Map2k3 Lig1 Ercc6l Dhx58 Down Down Down Down Drg2 Dhps Dguok Dock11 Ddx60 Dcun1d5 Cndp2 Cdkn2a Cpne1 Cep170 Tp53 Crabp1 Alcam Casp3 Down Down Down Down Down Down Down Down Down Down Down Down Down Down Prkar2b Cast Cast Bcar1 Down Down Down Down Bone marrow stromal antigen BH3-interacting domain death agonist Beta-enolase Aspartate aminotransferase Apol9a protein AP2-associated protein kinase Anamorsin Allograft inflammatory factor 1-like Aldose reductase Alcohol dehydrogenase class mu/sigma chain ADP-sugar pyrophosphatase Adenine phosphoribosyl transferase Activator of 90 kDa heat shock protein ATPase homolog Activated RNA polymerase II transcriptional coactivator p15 54 kDa 2'-5'-oligoadenylate synthetase-like protein 40 kDa peptidyl-prolyl cis-trans isomerase 2'-5' oligoadenylate synthetase 2'-5' oligoadenylate synthetase 15-hydroxyprostaglandin dehydrogenase [NAD+] 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial 184 Bst2 Bid Eno3 Got1 Apol9a Aak1 Ciapin1 Aif1l Akr1b1 Adh7 Nudt5 Aprt Down Down Down Down Down Down Down Down Down Down Down Down Ahsa1 Down Sub1 Down Oasl2 Ppid Oas3 Oas2 Down Down Down Down Hpgd Down Adi1 Down Pdk1 similar to Glyceraldehy de-3phosphate dehydrogenas e (GAPDH) isoform 13 kDa protein similar to interferon activated gene 204 similar to very large inducible GTPase similar to Glyceraldehy de-3phosphate Down Down Down Down Down Down Zinc transporter ZIP11 Vitamin K epoxide reductase complex subunit Vascular endothelial growth factor D Uncharacterized protein KIAA0564 homolog Uncharacterized glycosyltransferase AER61 UDP-glucuronosyltransferase 1-6 Ubiquitin-conjugating enzyme E2 J1 Ubiquitin carboxyl-terminal hydrolase isozyme L1 Tyrosine-protein phosphatase non-receptor type substrate Tyrosine-protein kinase Twisted gastrulation protein homolog Tubulin beta-3 chain Tropomyosin beta chain Transmembrane protein 65 Transmembrane protein 59 Transmembrane protein 106B Transforming growth factor beta-1-induced transcript protein Tln2 protein Tissue factor TGF-beta receptor type-2 TGF-beta receptor type III Syndecan-4 Synaptonemal complex protein SC65 Stromal cell-derived factor Steryl-sulfatase STE20/SPS1-related proline-alanine-rich protein kinase Slc4a4 protein Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial SH3 domain-containing kinase-binding protein Ribonuclease Retinol dehydrogenase 10 Ras-related protein R-Ras2 Ras-related protein Rab-7L1 Ras-related protein Rab-32 Ras-related protein Rab-30 Ras-related protein M-Ras RAB9, member RAS oncogene family Pyruvate kinase isozymes M1/M2 Putative uncharacterized protein 185 dehydrogenas e Slc39a11 Vkorc1 Figf Kiaa0564 Aer61 Ugt1a6 Ube2j1 Up Up Up Up Up Up Up Uchl1 Up Sirpa Jak1 Twsg1 Tubb3 Tpm2 Tmem65 Tmem59 Tmem106b Up Up Up Up Up Up Up Up Tgfb1i1 Tln2 F3 Tgfbr2 Tgfbr3 Sdc4 Sc65 Sdf2 Sts Up Up Up Up Up Up Up Up Up Stk39 Slc4a4 Up Up Acadsb Sh3kbp1 Rnase4 Rdh10 Rras2 Rab7l1 Rab32 Rab30 Mras Rab9 Pkm2 Tspan3 Up Up Up Up Up Up Up Up Up Up Up Up Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Putative uncharacterized protein Protein S100-A13 Protein phosphatase 2C, magnesium dependent, catalytic subunit Protein NipSnap homolog Protein disulfide-isomerase Prostaglandin E synthase Prolyl 4-hydroxylase subunit alpha-3 Progesterone receptor Procollagen-lysine,2-oxoglutarate 5-dioxygenase PRKC apoptosis WT1 regulator protein Pre-B-cell leukemia transcription factorinteracting protein Polymerase delta-interacting protein Podoplanin Plcd1 protein Plasminogen activator inhibitor Placenta-specific gene protein Pituitary tumor-transforming gene proteininteracting protein Phosphatidylinositide phosphatase SAC1 Peroxiredoxin-6 Peroxiredoxin-4 PDZ and LIM domain protein Pccb protein p21 (CDKN1A)-activated kinase Ornithine aminotransferase, mitochondrial Nucleobindin-2 Neural cell adhesion molecule Neural cell adhesion molecule Neudesin NADH-cytochrome b5 reductase N-acylethanolamine-hydrolyzing acid amidase N-acetylglucosamine-6-sulfatase Myristoylated alanine-rich C-kinase substrate Myosin-Id Multiple coagulation factor deficiency protein homolog 186 Rps4y2 Lamb2 Hk1 Gpc6 Cd81 S100a16 Kdelc2 S100a13 Up Up Up Up Up Up Up Up Ppm2c Nipsnap1 P4hb Ptges P4ha3 Pgr Up Up Up Up Up Up Plod2 Pawr Up Up Pbxip1 Poldip3 Pdpn Plcd1 Serpine1 Plac8 Up Up Up Up Up Up Pttg1ip Sacm1l Prdx6 Prdx4 Pdlim5 Pccb Pak3 Oat Nucb2 Ncam1 Ncam1 Nenf Cyb5r3 Naaa Gns Marcks Myo1d Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Mcfd2 Up Microtubule-associated protein Microsomal glutathione S-transferase Methylmalonate-semialdehyde dehydrogenase [acylating], mitochondrial Methylglutaconyl-CoA hydratase, mitochondrial Methionine-R-sulfoxide reductase B3, mitochondrial Metalloreductase STEAP3 Metalloproteinase inhibitor Metalloproteinase inhibitor Matrix-remodeling-associated protein Matrix-remodeling-associated protein Matrix metalloproteinase-14 Mannosyl-oligosaccharide 1,2-alphamannosidase IA Mannosidase 2, alpha Lysosome membrane protein Lymphocyte antigen 6A-2/6E-1 Lymphocyte antigen complex, locus C1 LRRN4 C-terminal-like protein Lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex, mitochondrial Lipid phosphate phosphohydrolase Lipid phosphate phosphohydrolase Leukocyte elastase inhibitor A Lectin, galactoside-binding, soluble, binding protein Large neutral amino acids transporter small subunit Laminin subunit alpha-4 Isochorismatase domain-containing protein 2A, mitochondrial Interleukin-6 receptor subunit beta Integrin alpha-11 Integral membrane protein 2C Insulin-like growth factor-binding protein Insulin-like growth factor-binding protein Immunoglobulin superfamily member Histone H1.3 Histone H1.2 Histone H1.1 Histone H1.0 Heat shock 70 kDa protein 12A Guanine nucleotide-binding protein subunit alpha-11 187 Map6 Mgst1 Up Up Aldh6a1 Auh Up Up Msrb3 Steap3 Timp3 Timp2 Mxra8 Mxra7 Mmp14 Up Up Up Up Up Up Up Man1a1 Man2a1 Scarb2 Ly6a Ly6c1 Lrrn4cl Up Up Up Up Up Up Dbt Ppap2b Ppap2a Serpinb1a Up Up Up Up Lgals3bp Up Slc7a5 Lama4 Up Up Isoc2a Il6st Itga11 Itm2c Igfbp7 Igfbp6 Igsf8 Hist1h1d Hist1h1c Hist1h1a H1f0 Hspa12a Up Up Up Up Up Up Up Up Up Up Up Up Gna11 Up Growth factor receptor-bound protein 10 Growth arrest-specific protein Growth arrest-specific protein Golgi SNAP receptor complex member Glypican-4 Glutathione S-transferase, theta Glutathione S-transferase Mu Glutamate dehydrogenase 1, mitochondrial Gamma-adducin Follistatin-related protein FK506-binding protein FK506-binding protein 14 Filamin-B Fibulin-2 Extracellular superoxide dismutase [Cu-Zn] Estradiol 17-beta-dehydrogenase 11 ES1 protein homolog, mitochondrial Epoxide hydrolase 1, microsomal Ephrin-B1 Endosialin Electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial Early endosome antigen Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Cytochrome P450 1B1 Cysteine and glycine-rich protein CTP synthase Creatine kinase B-type Complement C1s-A subcomponent Complement C1q tumor necrosis factor-related protein Collagen alpha-1(III) chain Collagen alpha chain type VI Coactosin-like protein Clathrin light chain B CDGSH iron sulfur domain-containing protein CD9 antigen CD302 antigen CD151 antigen CB1 cannabinoid receptor-interacting protein Cathepsin H Carbonyl reductase Carbonic anhydrase 5B, mitochondrial 188 Grb10 Gas6 Gas1 Gosr2 Gpc4 Gstt3 Gstm2 Glud1 Add3 Fstl1 Fkbp7 Fkbp14 Flnb Fbln2 Sod3 Hsd17b11 D10Jhu81e Ephx1 Efnb1 Cd248 Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Etfdh Eea1 Up Up Dlat Cyp1b1 Csrp2 Ctps2 Ckb C1sa Up Up Up Up Up Up C1qtnf3 Col3a1 Col6a3 Cotl1 Cltb Cisd1 Cd9 Cd302 Cd151 Cnrip1 Ctsh Cbr3 Ca5b Up Up Up Up Up Up Up Up Up Up Up Up Up Calcium-binding protein 39-like Calcium-binding atopy-related autoantigen Bifunctional 3'-phosphoadenosine 5'phosphosulfate synthetase ATP synthase mitochondrial F1 complex assembly factor Anoctamin-6 Aminopeptidase N Alpha-type platelet-derived growth factor receptor Alpha-crystallin B chain Alpha-catulin Alpha-1-syntrophin Advillin ADP-ribosylation factor Acyl-CoA synthetase long-chain family member Actin, aortic smooth muscle Actin, alpha cardiac muscle Acetyl-CoA acetyltransferase, mitochondrial Acetolactate synthase-like protein AAM-B protein beta-hydroxysteroid dehydrogenase type 17beta-hydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase 189 Cab39l Cbara1 Up Up Papss2 Up Atpaf1 Ano6 Anpep Up Up Up Pdgfra Cryab Ctnnal1 Snta1 Avil Arf2 Up Up Up Up Up Up Acsl4 Acta2 Actc1 Acat1 Ilvbl Mettl7a1 Hsd3b7 Up Up Up Up Up Up Up Hsd17b10 similar to collagen prolyl 4hydroxylase alpha III subunit similar to IKK interacting protein 89 kDa protein leucine rich repeat containing 32 Up Up Up Up Up Publications Swa, H.L., Blackstock, W.P., Lim, L.H., and Gunaratne, J (2012) Quantitative proteomics profiling of murine mammary gland cells unravels impact of annexin-1 on DNA damage response, cell adhesion, and migration Mol Cell Proteomics 11, 381-393 Gunaratne, J., Goh, M.X., Swa, H.L., Lee, F.Y., Sanford, E., Wong, L.M., Hogue, K.A., Blackstock, W.P., and Okumura, K (2011) Protein interactions of phosphatase and tensin homologue (PTEN) and its cancer-associated G20E mutant compared by using stable isotope labeling by amino acids in cell culture-based parallel affinity purification J Biol Chem 286, 18093-18103 190 Supplementary CD 191 ... tagging for relative and absolute quantification IAA Iodoacetamide ICAT Isotope-coded affinity tagging K0R0 12 C 614 N2-lysine and 12 C 614 N4-arginine K8R10 13 C 615 N2-lysine and 13 C 615 N4-arginine... Annexin 1. 2 .1: Structure of ANXA1 ANXA1 is one of the members of the annexin superfamily of 13 members (A1-A13) All members of this superfamily have the same principal property of binding to negatively-charged... System, Singapore on 30 January 2 013 This work was presented as a poster in Proteomics Forum held in Berlin, Germany from 17 – 21 March 2 013 xvii Chapter 1: Introduction 1. 1: Breast cancer Breast cancer