עבודת גמר (תזה) לתואר Thesis for the degree דוקטור לפילוסופיה Doctor of Philosophy מוגשת למועצה המדעית של Submitted to the Scientific Council of the מכון ויצמן למדע Weizmann Institute of Science רחובות, ישראל Rehovot, Israel מאת By Gal Haase גל הזה תרומתו של האזור החוץ תאי של מולקולת האדהזיה L1 ביצירת גרורות של תאי סרטן המעי הגס The roles of the L1 adhesion molecule extracellular domain in human colon cancer cell metastasis מנח :ה :Advisor פרופ' אברי בן- זאב Prof’ Avri Ben-Ze’ev חשוון, תשע"ו November. 2015 1 Table of Contents Abbreviations ............................................................................................................................................... 4 Abstract ........................................................................................................................................................ 6 7 ............................................................................................................................................................. תקציר Introduction .................................................................................................................................................. 8 The WNT pathway in CRC .................................................................................................8 L1 Cell Adhesion Molecule ..............................................................................................10 L1 in human cancers .........................................................................................................11 Mechanisms of L1 action in human CRC .........................................................................12 The extracellular domain of L1 in cancer .........................................................................13 Materials and Methods ............................................................................................................................... 17 Cell lines and tissue culture ...............................................................................................17 Plasmids and expression vectors .......................................................................................17 Transfections .....................................................................................................................17 Establishment of stable clones ..........................................................................................17 Western blot analysis ........................................................................................................18 Reverse transcriptase polymerase chain reaction (RT-PCR) ............................................19 Quantitative real time polymerase chain reaction (qPCR) ................................................20 Immunofluorescence staining ...........................................................................................20 Antibodies .........................................................................................................................20 Growth assays ...................................................................................................................21 Wound healing assays .......................................................................................................21 Tumor formation and metastasis assays ............................................................................21 Statistics ............................................................................................................................21 Aim of the research ..................................................................................................................................... 22 2 Results ........................................................................................................................................................ 23 The role of L1 shedding in the L1-mediated effect in CRC cells .....................................23 CRC cells expressing wtL1 bind to wt and mutant L1-Fc particles, while L1/H210Q expressing cells can only bind to wtL1-Fc. ................................................................................................................................. 23 Binding of L1-Fc to either wtL1 or mutant L1 expressing cells did not alter cell behavior ........................ 25 Activation of cellular signals by H210Q and D598N mutations ......................................25 Characterizing D598N, a second point mutation in the ECD Ig-6 domain of L1 ........................................ 25 Activation of NF-κB by L1 expression is lost in cells expressing the L1/H210Q mutant ............................ 28 Interactions of the ECD mutants with ezrin ............................................................................................... 28 The transcription of L1-ezrin-NF-κB target genes is altered in cells expressing L1/H210Q ....................... 28 Comparing gene expression patterns of cells expressing L1/H210Q and L1/D598N mutations ...........................................................................................................................31 L1/H210Q cells have more genes whose expression is altered compared to wtL1-expressing cells ........ 31 Validation of the gene array results by RT-PCR ......................................................................................... 32 Clusterin expression is downregulated only in L1/H210Q expressing cells ............................................... 33 The role of CD10 in L1-mediated metastasis....................................................................35 The level of CD10 is altered in L1/H210Q expressing CRC cells ................................................................. 35 Silencing CD10 in L1 expressing cells abolishes L1-mediated cellular properties ..................................... 36 CD10 is a target of L1-NF-κB signaling ....................................................................................................... 39 Discussion ................................................................................................................................................... 40 The importance of the Ig-2 and Ig-6 domains in L1-mediated metastasis ........................40 Homophilic interactions, mediated by the Ig-2 domain are crucial for ezrin activation ...42 The role for L1 shedding in promoting metastasis ............................................................42 The “bigger role” of the Ig-2 domain for L1 functions .....................................................43 Conclusions .......................................................................................................................45 Supplementary tables ................................................................................................................................. 46 References .................................................................................................................................................. 53 3 Abbreviations APC Adenomatous polyposis coli BCA Bicinchoninic acid BCS Bovine calf serum BSA Bovine serum albumin CAPN6 Calpain 6 cDNA Complementary deoxyribonucleic acid CDH17 Liver-intestine cadherin 17 CHO Chinese hamster ovary CLU Clusterin CRC Colorectal cancer DAPI 4',6-diamidino-2-phenylindole dH2O Double distilled water DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethylsulfoxide DTT Dithiothreitol ECD Extracellular domain ECL Enhanced chemiluminescent light (reaction) ECM Extracellular matrix EDTA Ethylene diamine tetra acetic acid ELISA Enzyme-linked immune absorbent assay FBS Fetal bovine serum FITC Fluorescein isothiocyanate FN1 Fibronectin 1 GAPDH Glyceraldehyde 3-phosphate dehydrogenase HRP Horseradish peroxidase HEK Human embryonic kidney IGFBP2 Insulin-like growth factor binding protein 2 IgG Imnunoglobolin G IκB Inhibitor of κB ISG15 Ubiquitin-like protein ISG15 4 KLK6 Kallikrein 6 MME Neutral endopeptidase, Neprilisyn, CD10 MTT 3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide MUC2 Mucin 2 NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells PBS Phosphate buffered saline PBST Phosphate buffered saline + Tween-20 PFA Paraformaldehyde qPCR Quantitative polymerase chain reaction RIPA Radio-immunoprecipitation assay RPMI-1640 Roswell Park Memorial Institute-1640 ROCK Rho-associated protein kinase RT-PCR Reverse transcription polymerase chain reaction SDS Sodium dodecyl sulfate SDS-PAGE Polyacrylamide gel electrophoresis in the presence of SDS shRNA Small hairpin ribonucleic acid siRNA Small interfering ribonucleic acid SMOC2 SPARC related modular calcium binding protein 2 ST6 ST6GAL1, ST6 beta-galactosamide alpha-2,6- sialyltranferase 1 STC1 Stanniocalcin 1 TBS Tris buffered saline TBST Tris buffered saline + Tween-20 TEMED N,N,N’,N’,-Tetra methyl ethylene diamine TGF β1 Transforming growth factor beta 1 Tris Tris(Hydroxymethyl)aminomethane Tween-20 Polyoxyethylene-(20)-sorbitanmonolaureate VCAN Versican XTT 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5- Carboxanilide 5 Abstract Colorectal cancer (CRC) is the fourth most common form of cancer and the second leading cause of cancer-related death in the Western world. Hyperactivation of β-catenin-T-cell- factor (TCF)-regulated gene transcription is a hallmark of CRC. The neural cell adhesion molecule L1CAM (L1), a target gene of β-catenin/TCF signaling in CRC cells, is exclusively expressed at the invasive front of CRC tissue. L1 overexpression in CRC cell lines increases cell growth,