דוקטור לפילוסופיה Doctor of Philosophy

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

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

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

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

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

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, cell motility, tumor growth in vivo , and promotes the formation of liver metastases when the cells are injected into mice. L1 is an adhesion molecule with a large extra cellular domain (ECD) consisting of several regions. Previous studies in the nervous system have shown that the different L1 ECD regions are responsible for the binding of L1 to different partners, and thus mediate different L1 functions. The goal of this study was to determine the roles of the various extracellular interactions of L1 in L1- mediated CRC metastasis. By expressing in CRC cells L1 that contains ECD point mutations, we found that all tested ECD mutations abolish the ability of L1 to promote metastasis. Further investigation of two ECD mutations implied a more significant role for the homophilic interactions of L1 as compared to other types of L1 associations. By global gene expression analysis we identified L1-signaling targets that are affected uniquely by these two L1 ECD mutations. One of the genes that were affected solely by the homophilic binding deficient mutation in L1 was CD10. By additional investigations we found that suppressing CD10 levels in CRC cells overexpressing L1 abolished the metastatic capacity of these cells. Our studies demonstrated the importance of the ECD of L1 in human CRC metastasis, a subject that needs further investigation. Understanding the roles of the different regions in the extracellular domain of L1 and their effect on L1-mediated signaling inside the cell, will help in shedding more light on the role of L1 in CRC metastasis, and assist in achieving more effective therapeutic options in CRC that involve L1.

תקציר סרטן ה מעי הגס הוא הסרטן הרביעי הנפוץ בעולם המערבי והגורם השני ה נפוץ ביותר כגורם לתמותה ממחלת הסרטן . סימן ההיכר של סרטן ה מעי הגס אוה הפעי לות הלא המבוקרת של קומפלקס ה שעתוק β-catenin-TCF . מולקולת התאחיזה , L1 , היא תוצר מטרה של קומפלקס השעתוק β-catenin-TCF בסרטן ה מעי הגס ומבוטא בצורה בלעדית בחזית הפולשנית של רקמת הסרטן ב מעי הגס . ביטוי עודף של L1 יאבת סרטן ה מעי הגס גורם לעלייה בקצב ה חלוקה של התא , תנועתיות התא , וכן עלייה בקצב גדילת גדולים ויצירת גרורות נסרט יות בעכברים . ל L1- יש אזור חוץ - תאי ארוך המכיל מספר תת- אזורים שונים . מחקרים קודמים בתאי עצב הראו ש תת- אזורים שונים בחלק החוץ תאי של L1 אחראים לקשרים עם מולקולות שונות ובכך מקדמים את הפעילויות השונות של L1 . מטרת המחקר הזה הייתה להבין את התפקיד של תתי- האזורים החוץ תאיים השונים בקידום יצירת גרורות סרטניות בתאי מעי שמתווכ ים ע"י L1 . באמצעות ביטוי מולקולות L1 אשר מכילות מוטציות נקודתיות בחלק החוץ תאי ( מוטציות הידועות כגורמות לצורות שונות של פיגור שכלי בזמן ההתפתחות של המוח באדם , עקב פגיעה בתאי עצב) , גילינו שכל מוטציה נקודתית כזאת גרמה לאיבוד היכולת של L1 לתווך יצירת גרורות בכבד בניסוי בעכברים . בדיקה מעמיקה יותר של תאי סרטן ה מעי הגס המבטאים שתי מוטציות בחלק החוץ תאי של L1 ה עלתה את ההשערה שקשרים הומופיליים של מולקולת ה L1 , בעלי חשיבות רבה יותר מאשר הקשרים האחרים שהמולקולה מייצרת . בבדיקת ביטוי מערכי גנים זיהי נו גנים שהם מטרות של L1 אשר מושפעים בצורה בלעדית רק ע"י אחת המוטציות . אחד הגנים שהתגלו כמושפעים רק ע"י הקשרים ההומופיליים של L1 , היא CD10 . בחינה מעמיקה יותר גילתה שהשתקת הביטוי של CD10 , בתאים שמבטאים L1 , גורמת לאיבוד מוחלט של היכולת של התאים ליצור גר ורות . המחקר שלנו מדגים את ה חשיבות של החלק החוץ- תאי של L1 ביצירת גרורות בסרטן ה מעי הגס , נושא שטרם נחקר ביסודיות עד כה . הבנה מעמיקה יותר של תפקיד התת- אזורים החוץ תאיים של L1 וההשפעה שלהם על האותות שמועברים לתוך התא , יכולה לשפוך אור נוסף על מנגנון היווצרות גרורות בסרטן ה מעי הגס , ולסייע בפיתוח אופציות טיפוליות טובות יותר בעתיד.

Introduction The development of cancer is a multistage process driven by genetic changes, which first confer properties such as accelerated cell proliferation, resistance to apoptosis and induction of angiogenesis. In later stages, the tumor cells develop metastatic capacity that allows them to leave the primary tumor and establish new, secondary metastases in other organs in the patient's body. Metastasis is a complex process with many steps, each requiring a different set of cellular properties. In order to metastasize, tumor cells must lose their adherence to their neighbors and to detach from the primary tumor. Next, the cells become more motile, gain the ability to migrate through the stroma, and to intravasate into the blood or lymph vessels. While traveling though the blood and lymph vessels, the cells have to survive the mechanical stress involved and to escape from the immune system. Final steps include the ability to adhere to endothelial cells in the secondary organ, extravasate and establish contacts with the underlying ECM and regain cellular properties similar to those of tumor cells in the primary tumor. Human colorectal cancer (CRC) is the third most commonly diagnosed cancer. In CRC, 25% of the patients already have liver metastases when diagnosed, and an additional 25% will develop such metastases during the course of the disease. Only 10% of these patients will survive. Thus, metastasis is a dramatic contributor to mortality in CRC and as such, the mechanisms underlying the capacity of tumor cells to metastasize are of great interest [1-3].

The WNT pathway in CRC Secreted Wnt proteins propagate an array of cellular responses that influence the fate and function of both embryonic and adult tissues [4]. After Wnt proteins bind to receptors of the Frizzled (Fz) family, the Wnt signal is transmitted to the primarily cytoplasmic protein Disheveled (Dvl) that determines which Wnt signaling pathway will be activated by functioning as a switch between canonical and non-canonical Wnt signaling [5]. The canonical pathway is also known as the Wnt/ β-catenin pathway (Fig 1). In unstimulated cells, β-catenin is phosphorylated by a complex of proteins that includes axin and APC, by the kinases glycogen synthase kinase 3 β (GSK3 β) and casein kinase 1 (CK1). Following phosphorylation of β-catenin on N-terminal serines and a threonine residue, β-catenin is recognized by β-TrCP, a component of the E3 ubiquitination complex and after

Figure 1. The dual role of β-catenin in Wnt signaling and in cell-cell adhesion. β-catenin ( β) and plakoglobin (Pg) bind to the cytoplasmic domain of cadherin transmembrane adhesion receptors and, via α- catenin ( α), associate with the actin cytoskeleton to form adherens junctions (AJ). When the Wnt signaling pathway is inactive, the cytoplamsic pool of β-catenin is directed to degradation by a molecular complex including APC and Axin, and the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK), that phosphorylate β-catenin (PPPP) on N-terminal residues. Phosphorylated β-catenin is recognized by the E3 ubiquitin ligase component β-TrCP, which together with Skp1, Cul1, and the E1 and E2 ubiquitination complexes mediates the ubiquitination of β-catenin (Ub) that directs β-catenin to degradation by the 26S proteasome. The binding of Wnt to Frizzled (Frz) and the co-receptor LRP activates Wnt signaling. This induces a disheveled (Dsh)-mediated inhibition of β-catenin phosphorylation by GSK thereby blocking β- catenin degradation, and its accumulation and complexing, in the nucleus, with T-cell factor (TCF). The β- catenin-TCF complex transactivates target genes such as cyclin D1, L1 and Nr-CAM and many other genes (β-catenin signaling in biological control and cancer. Gavert N, Ben-Ze'ev A. J Cell Biochem. 2007 1;102(4):820-8). polyubiquitination, β-catenin is targeted for proteolytic degradation by the proteasome [6]. Activation of the Wnt pathway results in the disruption of the CK1-GSK3 β-axin-APC complex, inhibition of GSK3 β activity, and the stabilization of β-catenin against degradation. This leads to a significant increase in both the cytoplasmic and nuclear β- catenin levels. In the nucleus, β-catenin binds to members of the TCF/LEF family of transcription factors and functions as a co-activator of target gene transcription. In CRC,

9 aberrant activation of the Wnt signaling pathway is a crucial oncogenic driver in 90% of patients [7]. Aberrant Wnt signaling prevents the normal degradation of β-catenin, a key component of the pathway. Primarily, mutations in the adenomatous polyposis coli (APC) gene inhibit the degradation of β-catenin by the ubiquitin-proteasome system and lead to its accumulation and nuclear translocation. In the nucleus, β-catenin forms transcriptionally active complexes with LEF/TCF factors [8], that induce target gene transcription, including a set of growth promoting genes, described by our lab and others, such as Cyclin D1 [9] and c-myc [10], known to promote tumor progression in the early stages of the disease. Inappropriate activation of β-catenin signaling can also contribute to later stages of tumorigenesis by inducing genes that confer invasive and metastatic capacities. These include metalloproteinases [11-14], ECM components [15, 16] and cell adhesion receptors [17, 18]. In recent studies, our laboratory identified genes coding for members of the immunoglobulin (Ig)-like cell adhesion receptor superfamily (L1 and NrCAM) as targets of β-catenin/TCF signaling in colon cancer cells and studied their role in CRC metastasis [19, 20].

L1 Cell Adhesion Molecule L1 (or L1CAM - L1 Cell Adhesion Molecule) is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily of cell adhesion molecules (IgCAM), with an extracellular domain (ECD) that consists of 6 Ig-like domains and 5 fibronectin type III (FNIII) repeats and a short, highly conserved, cytoplasmatic tail. L1 was originally described to function in the development of the nervous system, where it has an important role in neurite elongation, axon fasciculation and migration of neuronal precursors [21-23]. L1 is found mostly in postmitotic neurons of the central and peripheral nervous systems and in non-myelinating Schwann cells of peripheral nerves [24, 25]. L1 has a pivotal role in the development of the nervous system during embryogenesis as revealed in studies of L1 knock out (KO) mice that are viable, but are smaller, with a reduced corticospinal tract and reduced brain volume compared to wild type mice [26, 27]. Inherited mutations in the human L1 gene were correlated with several brain disorders, such as X-linked hydrocephalus, MASA syndrome, and L1 syndrome [28-30]. L1 was shown to promote homophilic cell-cell adhesion by binding to L1 molecules on neighboring cells [31]. In

10 addition, L1 is capable of binding to numerous other partners (reviewed by Brümmendorf and Rathjen [31] and Haspel and Grumet [32]) including other members of the L1 family (axonin-1 [33], F3/F11 contactin [34]), extracellular matrix proteins (chondroitin sulfate proteoglycans, phosphocan, neurocan and laminin [35, 36]), growth factor receptors (Neuropilin-1, FGFR [37, 38]) and various integrins [39-44]. The heterophilic associations of L1 were also shown to be important for promoting L1-mediated neurite outgrowth [33, 45, 46], while binding to integrins were found to be involved in cell adhesion and motility during regeneration of the neuronal tissue [47].

L1 in human cancers In recent years, several studies have showed that L1 is involved in cancer progression, especially in the induction of metastasis. In human CRC, L1 expression positively correlates with poor prognosis and with the presence of micro-metastases [48, 49]. In a study from our lab, L1 was exclusively observed in cells at the invasive front of CRC tissue samples [50], implying a possible role for L1 in developing invasive capacities. As in CRC, L1 expression in ovarian cancer correlates with poor prognosis and metastasis. L1 was also observed at the invasive front of epithelial ovarian carcinoma tissue and, in more advanced stages of the disease, in metastatic lesions [51, 52]. L1 expression was suggested as a marker for advanced tumor stages and metastasis in renal cell carcinoma (RCC), cutaneous malignant melanoma, pancreatic adenocarcinoma and in breast cancer [53-56]. While the role of L1 in cancer progression is supported by numerous studies, the mechanism/s by which L1 activates downstream signaling inside the cell remains unclear. Previous studies have shown that L1 expression both in normal and cancer cells results in enhanced cell motility and invasiveness via activation of the ERK pathway. ERK activation results from the association of L1 with several growth factor receptors including FGFR, EGFR and HGFR, depending on the cell type [52, 57, 58]. In ovarian cancer cell lines, L1 was shown to promote cell proliferation and protection from apoptosis [58]. In melanoma cells, L1 can bind to αvβ3 integrin, and when this binding is blocked, haptotactic cell migration is significantly reduced [40]. In pancreatic cancer cells, L1 was suggested to have a role in conferring chemoresistance by promoting increased IL-1β secretion [54]. In MCF7, a breast cancer cell line, overexpression of L1 leads to disruptions of adherens junctions,

11 cell scattering and increased β-catenin-TCF transcriptional activity. When endogenous L1 was suppressed by siRNA, MCF7 cells became less motile and formed more compact colonies with less scattering [59].

Mechanisms of L1 action in human CRC Studies from our lab showed that the expression of L1 in CRC cell lines increased cell motility and proliferation under starvation conditions [20]. In addition, L1 expression induced metastasis to the liver when the cells were injected into the spleen of nude mice [50]. More importantly, this study showed that full length L1 is required for liver metastasis, since mutant L1 lacking either the extracellular domain or the cytoplasmatic tail are unable to confer metastasis, indicating that both internal signaling and extracellular interactions of L1 are required for the ability to induce metastasis [50]. A more recent study from our lab showed that L1-induced metastasis depends on the activation of nuclear factor κB (NF-κB) signaling [60]. L1 recruits I κB, a NF-κB inhibitor, mediates its phosphorylation and directs it for proteasomal degradation. Degradation of I κB releases NF-κB that enters into the nucleus and affects the transcription of target genes (such as c-Kit [61], IGFBP-2 [62] and SMOC2 [63]). We also found that NF-κB activation by L1 requires the cytoskeletal crosslinking protein ezrin. Ezrin is a member of the ERM (ezrin, radixin, moesin) family of actin-associated proteins that have a key role in the formation of microvilli and other membrane protrusions [64]. Several studies implicated the involvement of ezrin in the metastatic spread of various neoplasms [65, 66]. In CRC cells L1 recruits ezrin from filopodia to cell-cell contacts to form a complex together with I κB allowing IκB phosphorylation and subsequent degradation. Recruitment of ezrin is essential for the L1- induced metastasis since when ezrin levels were suppressed by shRNA the cells became less motile and failed to metastasize. Moreover, the suppression of ezrin reduced NF-κB activation by blocking the phosphorylation of I κB [60].

At the invasive front of CRC tissue, L1 is co-expressed with the disintegrin and metalloproteinase 10 (ADAM10) that is known to cleave the L1 ECD [20, 67]. This co- expression implies that cleavage and shedding of the L1 ECD may be important for gaining invasive and motile capacities. Moreover, overexpression of ADAM10 enhances the metastatic capacity of L1 overexpressing CRC cells [50]. In addition, in ovarian cancer cells

12 the shed ECD of L1 was found in the serum of patients and their ascitic fluid [68]. In CHO cells, de novo L1 expression stimulates a drastic increase in cell migration on fibronectin or laminin. This increase was blocked when cells were treated with a metalloproteinase inhibitor, but reoccurred when the metalloproteinase-inhibited cells were treated with soluble L1-Fc [69]. These findings demonstrate the importance of L1 shedding in the migration of these cells.

The extracellular domain of L1 in cancer The extracellular interactions of L1 facilitate the different functions of L1 in the nervous system [45, 46]. One fundamental question about the L1 ECD is by what mechanism(s) does it modulate and regulate the binding to its many different partners? One possibility could be that in order to bind to a certain molecule, specific domains of the L1 ECD must be involved. Point mutations and deletions of L1 shed light on the role of each of its molecular domains, establishing a "binding-map" of the L1 ECD. For example, homophilic trans-binding (between two neighboring cells) requires the participation of Ig1-4 domains, but for homophilic cis-binding (on the surface of the same cell) the 2nd FNIII domain alone is sufficient. Binding to integrins requires mostly the Ig-6 domain, while interactions with Neuropilin-1 require the Ig-1 domain (reviewed by Haspel and Grumet [32]).

In the human nervous system, over sixty point mutations in the L1 gene, mostly in the ECD of L1 ( http://www.l1cammutationdatabase.info ) have been identified and shown to be responsible for congenital brain diseases (such as MASA syndrome). Studies of these mutations in nerve cells demonstrated the importance of the ECD integrity and of the extracellular interactions to the functions of L1 in nerve cells. Moreover, these experiments also pointed to the various (eleven) domains of the ECD that have a role in interactions with the different binding partners. In previous studies in our lab, we characterized some of these point mutations in the L1-ECD (see Fig. 2) by expressing them in a CRC cell line, Ls174T that lacks endogenous L1 expression. The mutations are as follows:

H210Q – A mutation in the Ig-2 domain, the main domain responsible for homophilic-trans interactions. In a biochemical assays by De Angelis et al. [70, 71] this mutation was shown

13 to strongly interfere with the ability of L1 to bind another L1 molecule, while having almost no effect on binding to other types of molecules.

E309K – A mutation in the Ig-3 domain. This mutation was shown to interfere mostly with interactions of L1 with other membrane molecules, while having almost no effect on the homophilic binding ability of L1 [70, 71].

S542P – A mutation in the Ig-6 domain. This domain appears to be responsible for interactions with integrins, mainly via an RGD sequence in this domain [40, 42].

Figure 2. Scheme of the L1 molecule. The different ECD mutations are marked with colored rectangles.

Ls174T cells overexpressing L1 form liver metastases when injected into the distal tip of the spleen of nude mice [50]. In a previous study in our lab, we have observed that all three mutant forms of L1 mentioned above lack the capacity to mediate metastasis when expressed in Ls174T cells (Fig. 4C). By looking at immunoflourescent images of mutant L1 expressing cells we found that in L1/S542P cells the proper transportation of the mutant molecule to the membrane is affected, therefore making it useless in attempts to examine the role/s of the Ig-6 ECD domain (Fig. 3). When examining in vitro properties (such as cell motility and proliferation), we observed that the H210Q mutant has a more prominent effect than the E309K mutation (Fig. 4A, B), suggesting the importance of homophilic L1-L1

interactions over the interaction of L1 with other molecules. In the current study, we aimed to investigate the roles of the extracellular interactions of L1 in its ability to mediate metastasis, by focusing on two ECD pivotal domains – the Ig-2 (responsible for homophilic interactions) and the Ig-6 (integrins).

Figure 3. Subcellular localization of mutant L1 forms in CRC cells. A, Ls174T CRC cells stably transfected with L1/H210Q (B), L1/S542P (C) and L1/E309K (D) were immunostained for L1 to determine protein localization (red, left image in each pair). The cells were co-stained with DAPI to visualize nuclei (right image in each pair). For control, L1 (wtL1) and empty vector (pcDNA3) expressing cells were stained (A).

Figure 4. Properties of Ls174T cells expressing mutant L1 forms. A, Artificial wounds were introduced into monolayers of wtL1, control (con), L1/H210Q, L1/E309K and L1/S542P transfected cells, images were taken immediately after wounds were introduced and after 24 hours. B, Cell proliferation rate was determined using MTT in triplicates. MTT activity was measured after 24 hours and after 6 days. Each cell line was normalized to its day 1 measurement. C, Ls174T expressing wtL1, L1/H210Q, L1/E309K and L1/S542P mutants, were injected into the spleen of nude mice and tumor growth at the injection site (spleen) and the formation of metastases (liver) were determined. Arrows point to tumors formed in the spleen.

Materials and Methods

Cell lines and tissue culture 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum (BCS) and 1% Pen-Strep solution. Ls174T cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% pen-strep solution. Ls174T-L1, Ls174174T-L1/H210Q, Ls174174T-L1/D598N, Ls174174T-L1/S542P, Ls174174T-L1/E309K, LsS174174T-CD10 cells were maintained in medium containing neomycin (800 µg/ml), and Ls174T L1+shCD10 cells were cultured in medium containing both neomycin (800 µg/ml) and puromycin (10 µg/ml). Ls174T cells expressing the Tet-repressor were obtained from M. van de Wetering and H. Clevers (Hubrecht Laboratory, Utrecht, The Netherlands), and were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% Pen-Strep solution and blasticidine (10 µg/ml).

Plasmids and expression vectors

The L1/H210Q, L1/D598N, L1/S542P, L1/E309K, L1/W1036P expression vectors were obtained from M. Shtutman. The CD10 expression vector was obtained from N. Iwata (Nagasaki University, Japan). ShCD10 shRNA was prepared in pSUPER.puro according to the manufacturer's instructions (pSUPER.puro RNAi System, OligoEngine, Seattle, WA) using the target sequences 5 ′-CAGGCAATTTCAGGATTAT-3′, 5′- GGCCAGATTGATTCGTCAGGAA-3′.

Transfections Transient transfection of 293T cells was performed using the calcium phosphate/DNA precipitation method. Transfection of Ls174T cells was performed using the Lipofectamine TM 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. Transfection of Ls174T-L1cells with shCD10 was performed using the Dharamafect transfection reagent (DharmaCon) according to the manufacturer’s instructions.

Establishment of stable clones Ls174T cells stably expressing L1/H210Q, L1/D598N, L1/S542P, L1/E309K, L1/W1036P or CD10 were obtained by transfecting the cells with 5 µg DNA using the

Lipofectamine TM 2000 transfection reagent, followed by selection with 800µg/ml neomycin. Ls174T cells stably expressing both L1 and shCD10 were obtained by transfecting Ls174T- L1 cells (Ls174T-L1 clone 30) with 5 µg SUPER.puro.shCD10 expression plasmid using Dharmafect transfection reagent followed by selection with 800 µg/ml neomycin and 10µg/ml puromycin. The selective concentration of the antibiotics was previously determined as the lowest concentration that leads to 100% cell death. Positive clones that express the desired protein at the highest level were analyzed by Western blot analysis and were used for further studies.

Western blot analysis Cells were washed once and then harvested with ice-cold PBS, pelleted by centrifugation (5 minutes at 2000 rpm at 4ºC) and lysed with RIPA buffer (150mM NaCl, 50mM Tris pH 7.5, 1mM EDTA, 1% sodium deoxycholate, 1% NP-40, and 0.1% sodium dodecyl sulfate (SDS) supplemented with the Complete Protease Inhibitor Cocktail (Roche). When expression of phosphorylated proteins was analyzed, phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich) were added to the lysis buffer. Cell lysates were incubated on ice for 15 minutes during which they were vortexed every five minutes, and then centrifuged for 20 minutes at 14000 rpm at 4ºC. Protein concentrations were determined using the BCA TM Protein Assay Kit (Thermo Scientific). For whole cell lysates, cells were collected in boiling Laemmli’s buffer (20% glycerol, 3% DTT, 6% SDS, 0.1% Bromophenol Blue, 62.5mM Tris pH 6.8). The volumes of the Laemmli buffer were normalized according to protein BCA measurements done in a duplicate dish that was lysed using RIPA buffer. Proteins were separated by 8-12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a 0.2 µm nitrocellulose transfer membrane (Schleicher & Schuell). The membrane was first incubated for 30 seconds in Ponceau S solution (Sigma-Aldrich) to visualize total protein expression, washed in TBS-T and then incubated with blocking solution (5% BSA in TBS-T) for 1 hour and then with primary antibodies for 20-60 minutes at room temperature or overnight at 4ºC. After washing three times with TBS-T for 5 minutes, the membrane was incubated with secondary antibodies against rabbit, mouse or goat IgG conjugated to horse radish peroxidase (HRP, Jackson ImmunoResearch Laboratories) diluted 1:10,000 for 45 minutes at room temperature, washed again three times with TBS-T for 5 minutes and the signals were visualized by ECL. Expression of

tubulin was used as an internal loading control using mouse anti-tubulin specific antibody. Primary antibodies were diluted in red solution (5% BSA in TBS-T, 0.02% sodium azide, phenol-red, pH 7.4).

Reverse transcriptase polymerase chain reaction (RT-PCR) Total RNA was isolated from cells using the EZ-RNA II Total RNA isolation kit (Biological Industries) according to the manufacturer’s protocol. First strand cDNA was synthesized from 2µg total RNA using random primers (Promega) by the Superscript TM First Strand Synthesis System (Life Technologies) and amplified by PCR using Red PCR Master Mix (Larova) with specific primers (see table). The PCR conditions were as follows: 3 minutes at 94 °C, followed by 26-32 cycles of 30 seconds at 94 °C, 30 seconds at 55 °C and 30 seconds at 72 °C. GAPDH was amplified as a control to verify equal amounts of cDNA in the samples. PCR products were analyzed by 2% agarose gel electrophoresis. Gene Forward Reverse L1 5'-GTGAGTGCCATCATCCTCCT-3' 5'-CCTTCTCCTTCTTGCCACTG-3' GAPDH 5'-ACCACAGTCCATGCCATCAC-3' 5'-TCCACCCTGTTGCTGTA-3' c-Kit 5'-CCACACCCTGTTCACTCCTT -3' 5'-TGTACTTCATACATGGGTTTCTG-3' IGFBP2 5'-GAGCAGGTTGCAGACAATGG-3' 5'-CGGCCAGCTCCTTCATAC-3' SMOC2 5′-AAGGAAGTATACCCAGGAGCAA-3′ 5′-GTGTAGCTGTGACACTGGACCT-3′ CD10 5'-ACAGTCCAGGCAATTTCAGG-3' 5'-AAACCCGGCACTTCTTTTCT-3' CDH17 5'-GCAAGAGTCTCGACCACTGAA-3' 5'-GGCCATATCCAGTTGCCAAATAAA-3' CLU 5′-CAGGCCATGGACATCCACTT-3′ 5′-GTCATCGTCGCCTTCTCGTA-3′ KLK6 5'-TGTGGTGACACACGCTGTAG-3' 5'-CTTGAGTCGGGGGAAGGAAC-3' ST6 5'-ACTACTGAGGGACAGCGACA-3' 5'-CACAGGAGGATGTAGACGGC-3' CAPN6 5'-TCGTGGCTACCCGAAAGTAG-3' 5'-TAGAAAATGGCCTGGGTGTC-3' TGF β1 5'-GGAAATTGAGGGCTTTCGCC-3' 5'-CCGGTAGTGAACCCGTTGAT-3' STC1 5'-CACTCAGGGAAAAGCATTCGT-3' 5'-GAAAGTGGAGCACCTCCGAA-3' VCAN 5'-ACTGTGGGGATGAATGGAAA-3' 5'-ACTTGGCCTTCAGTGCTTGT-3' FN1 5'-ACAAGCATGTCTCTCTGCCA-3' 5'-TCAGGAAACTCCCAGGGTGA-3' MUC2 5'-GAGTGTACCAAAGAGGGGGC-3' 5'-GCGGAGGGTTGTAGTAGTCG-3' IGS15 5'-GAGAGGCAGCGAACTCATCT-3' 5'-AGCATCTTCACCGTCAGGTC-3'

Quantitative real time polymerase chain reaction (qRT-PCR) Relative gene expression was calculated using the quantitative real-time PCR (qPCR) method. qPCR was performed on the Applied Biosystems (AB) thermocycler platform with the Fast SYBR Green Master mix (AB). Triplicate samples of 2ng of cDNA template and 500nM gene specific primers (see table). The GAPDH gene was used as endogenous control. Primers were examined for efficiency, displaying an amplification slope of -3.33±0.3 and r 2 ≥ 0.98. The PCR reaction was started by incubating the mixture at 95°C for 15 minutes and followed by conventional qPCR amplification cycles (95°C for 20 seconds and 60°C for 1 minute, 40 cycles). Data analysis was performed using the ∆∆ CT method with the StepOne software v2.3 (AB).

Immunofluorescence staining Cells cultured on glass coverslips were permeabilized with 0.5% Triton X-100 in 4% paraformaldehyde (PFA) in PBS for 2 minutes and then fixed with 4% PFA in PBS for 20 minutes. Fixed cells blocked in 5% BSA in PBS for 1 hour and then stained with primary antibody for 1 hour. The cells were then washed with PBS for 15 minutes and incubated with secondary antibodies (FITC goat anti-rabbit/mouse IgG, or Cy3-labeled goat anti- rabbit/mouse IgG, Jackson ImmunoResearch Laboratories) diluted 1:1,000 in PBS and 5µg/ml 4’-6-diamidino-2-phenylindole (DAPI, Sigma) for 1 hour. Stained cells were washed with PBS for 15 minutes and mounted on glass slides using Elvanol.

Antibodies Antibody Company Dilution Rabbit anti-L1 Gift from V. Lemmon 1:8,000 Rabbit anti-phospho-ezrin Cell Signaling #3141 1:5,000 Mouse anti-ezrin Sigma-Aldrich E8897 1:1,000 Mouse anti-IκBα/MAD-3 BD Biosciences 610690 1:1,000 Rabbit anti-phospho-IκBα Cell Signaling #2859 1:1,000 Mouse anti-CD10 Abcam 56C6 1:500 Mouse anti-Clusterin Santa Cruz sc-5289 1:1,000 Goat-CDH17 Santa Cruz sc-6978 1:1,000 Mouse anti-β-Tubulin Sigma T9026 1:100,000

Growth assays Cell growth was determined by MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assay, a total number of 5x10 3 cells were seeded in 96- well plates in the presence of medium containing 10% or 0.5% serum. Cell viability was determined every day by addition of 10µl MTT solution (5 µg/ml, Sigma) for 2 hours followed by addition of 100µl stop solution (hydrochloric acid diluted 1:100 in isopropanol) to dissolve insoluble purple formazan products. OD was determined using the ELISA reader at 620nm and 570nm. Results were presented as the relative ratio between readings at 570nm and 620nm.

Wound healing assays An artificial wound was introduced into a confluent cell culture using a micropipette. The cell culture medium was replaced immediately after introducing the wound with fresh medium containing 0.35µg/ml Mitomycin C to inhibit cell proliferation. Pictures were taken with a Delta Vision microscope at 0 and 20-24 hours after introducing the wound.

Tumor formation and metastasis assays Tumor formation assays were carried out by injecting 5x10 6 cells subcutaneously into both flanks of 4-weeks old male CD1-nude mice. Experimental cells on one side and control cells on the other side. Mice were sacrificed after 15 days and tumor weight was determined. The ability of cells to metastasize was determined by injecting 1x10 6 cells in PBS in a total volume of 20µl into the distal tip of the spleen of 7 weeks old male CD-1 nude mice using a Hamilton syringe. Mice were anesthetized by peritoneal injection of 1µl/mg Xylazine (20mg/ml) and 1 µl/mg Ketamine (100mg/ml). Animals were sacrificed after 7 weeks, and primary tumor formation in the spleen and metastasis formation in the liver were examined.

Statistics Statistical significance in wound closure, proliferation rate and PCR assays was determined by t-test, using GraphPad online tool. A P-value of <0.05 was considered significant and was marked by an asterisk. A P-value of <0.005 was considered very significant and was marked by two asterisks. A P-value of <0.0005 was considered extremely significant and was marked by three asterisks.

Aim of the research

The aim of this research was to understand the mechanisms by which L1 confers metastatic capacities in CRC cells, focusing on the role of the extracellular domain (ECD) of L1 in the initiation and subsequent activation of downstream signaling pathways. We investigated the importance of two different L1 ECD sequences: Ig-2 and Ig-6 in the propagation of metastasis, and identified intracellular signals that are activated by these domains. By expressing point mutant L1 molecules that have been implicated in human neural developmental syndromes which are known to affect L1 interactions, we determined the role of each of the domains (and the extracellular interactions that are promoted by these domains) in the propagation of metastasis, and identified cellular signals that are activated by each of the domains. Determining the effects of these L1 mutations on CRC cell properties should help us identify the ECD domains that are important for the induction of L1-medidated metastasis and identify possible L1 partners that participate in CRC progression.

Results

The role of L1 shedding in the L1-mediated effect in CRC cells

CRC cells expressing wtL1 bind to wt and mutant L1-Fc particles, while L1/H210Q expressing cells can only bind to wtL1-Fc. As previously described, the cleavage of L1 and its shedding into the culture medium can affect various cellular properties, including cell migration and invasiveness. In a previous study, we wished to determine whether the changes induced by L1 require its cleavage and shedding. We treated Ls174T cells with a general metalloproteinase inhibitor (GM6001) and examined cell motility. L1 expressing cells exhibited a significant decrease in wound closure, while control cells remained unaffected. Next, we added conditioned medium from different cells to GM6001-treated L1 expressing cells and determined the rate of wound closure. Cells treated with both GM6001 and conditioned medium from L1 expressing cells displayed a higher rate of wound closure compared to cells treated with GM6001 and conditioned medium from control cells, or from cells expressing the H210Q mutant, supporting the hypothesis that soluble L1 might be an important determinant of L1- mediated activities in CRC cells.

To determine whether the shed L1 ECD is the factor in the conditioned medium that induces the increase in cell motility, we added soluble Fc-L1 conjugated particles to cells expressing L1 (wt and the H210Q mutant). It was reported that Fc particles, conjugated to wtL1 (Fc-L1) can bind to wt and mutant L1 molecules, but that mutant Fc-L1 (Fc-H210Q) cannot bind to L1/H210Q molecules. This was only shown in biochemical assays with purified proteins [70, 71]. We wished to determine whether this observation occurs also in cell culture. We immunostained cells expressing wtL1, or L1/H210Q, after allowing them to interact with Fc particles added to the culture medium. Both cell lines displayed a strong interaction with Fc-L1 particles (Fig. 5A, middle row). However, when incubated with Fc- H210Q particles, the amount of bound Fc particles decreased dramatically only in cells expressing the L1/H210Q (Fig. 5A, bottom row), confirming that the mutant protein cannot bind to itself as efficiently as to the wtL1 protein.

Figure 5. Binding of Ls174T cells expressing wtL1 and L1/H210Q to Fc-L1 particles. A, Cells expressing wtL1 and L1/H210Q were incubated for 24 hours with Fc particles conjugated to ECD of wtL1 (Fc-L1) or L1/H210Q (Fc-H210Q), then immunostained for L1 on cells (with ab against the cytosolic tail of L1, green), and for Fc (red). B, Images of wtL1 expressing cells treated with Fc-L1, demonstrating the localization of L1 at cell-cell adhesions, and the Fc particles binding only to the available (“free”) areas on the cell membrane.

Binding of L1-Fc to either wtL1 or mutant L1 expressing cells did not alter cell behavior Next, we examined the functional effects of adding Fc particles to cells. L1 is known to promote cell-cell adhesion by homophilic interactions [72]. To determine whether in our model system, L1 expressing cells can adhere to L1 presented to them on a dish, we coated dishes with soluble Fc-L1 particles and then seeded cells, allowing them to adhere to the plate for several hours. We observed that cells expressing both wt and mutant L1 adhered strongly to plates coated with Fc-L1 (compared to control Fc – Fig. 6A). The adhesion of L1 and L1/H210Q expressing cells to Fc-L1/H210Q particles was also examined, but the results were inconsistent and we were unable to conclude whether the mutant cells can adhere to mutant Fc as well as to the wtL1 expressing cells. Next, we conducted functional assays to examine the effect of adding Fc-L1 particles to L1 (wt or mutant) expressing cells (i.e. wound healing – Fig. 6B, ezrin phosphorylation levels, expression of L1-NF-κB target genes – Fig. 6C), but did not detect any effects in these assays, even though we confirmed the presences of the Fc particles in the medium of L1 and control cells after 24 hours of incubation (Fig. 6D).

Activation of cellular signals by H210Q and D598N mutations

Characterizing D598N, a second point mutation in the ECD Ig-6 domain of L1 Because the Ig-6 mutation S542P, previously characterized (see the Introduction) was found to affect the proper post-translational modification (or transport) of the L1 molecule to the membrane, we characterized a second point mutation, also found in the Ig-6 domain – the D598N mutation. The D598N mutant L1 was properly expressed and localized on the cell membrane and at cell-cell contacts (Fig 7A,B). In addition, L1-mediated cell motility was partially lost in the cells expressing D598N (Fig. 7C) and the cells failed to form liver metastases when injected into mice (Fig. 7D). Taken together these results suggested that the L1/D598N molecule is properly localized in the membrane and the effects observed in the motility and metastasis assays are due to the loss of function of the Ig-6 domain.

Figure 6. Binding of L1 expressing cells to Fc-L1 particles did not altered cell behavior. A, wtL1, L1//H210Q expressing and control (con) Ls174T cells were incubated for 4 hours on Fc-L1 and Fc-H210Q coated dishes. Cells were then fixed and stained with crystal violet. The number of cells was measured by the intensity of the staining and each cell line was normalized to the empty Fc sample. B, wound closure of monolayers of wtL1 expressing and control (con) cells treated with GM6001 and Fc-L1. Images were taken after wound introduction and after 24 hours. C, RNA levels of c-Kit were determined by semi- quantitative PCR for cells with and without treatment of Fc-L1. D, Fc (e), Fc-L1 (L1) and Fc-L1/H210Q

(H) particles levels in the culture media of cells after 24 hours of treatment were measured by western blotting for L1 (with polyclonal ab that recognizes both cellular and Fc-L1) and for Fc.

Figure 7. Properties of Ls174T cells expressing L1/D598N mutant A, L1/D598N expressing cells were immune-stained for L1 to determine protein localization (upper row), the cells were co-stained with DAPI. B, L1 protein levels in L1/D598N expressing cells was determined by western blotting. Tubulin was used to measure equal loading of protein. C, Artificial wound closure assay with monolayers of L1/D598N expressing clones and compared to wtL1 and control (con) cells. D, Control cells (con), wtL1, L1/D598N expressing Ls174T cells were injected into the spleen of nude mice and the formation of metastases (in the liver) was determined. Arrows point to tumors formed in the spleen.

Activation of NF-κB by L1 expression is lost in cells expressing the L1/H210Q mutant Our lab has characterized the L1-ezrin-NF-κB signaling pathway [60] in which L1 recruits both ezrin and I κB to the cell membrane, induces both ezrin and I κB phosphorylation in this complex and leads to the subsequent degradation of I κB. This releases NF-κB from inhibition and enables its nuclear translocation followed by alterations in the transcription of various genes including IGFBP2, c-Kit and SMOC2 [61, 63, 73]. By examining the levels of phosphorylated I κB (pI κB) in cells (after proteasomal degradation inhibition) we can assess the level of NF-κB in the cells. When examining the amount of pI κB in cells expressing L1/H210Q, we observed a reduction in the amount of pI κB (Fig. 8A), suggesting that this ECD mutation affects the proper activation of the NF-κB pathway. On the other hand, cells expressing L1/D598N, displayed high levels of pI κB, similar to that seen in wtL1 expressing cells (Fig. 8B).

Interactions of the ECD mutants with ezrin When examining the amount of phosphorylated ezrin (pEzrin), cells expressing L1/H210Q showed a reduction compared to wtL1 expressing cells (Fig. 8C), but immunofluorescent staining for ezrin (total, not phosphorylated), did not detect a difference between both mutant L1 expressing cells and cells expressing wtL1 (Fig. 8D, third and fourth columns). This suggests that even though the L1/H210Q mutation does not affect the protein’s ability to recruit ezrin from filopodia and bind to it, it is unable to activate it by phosphorylation, thereby inhibiting L1-ezrin-NF-κB signaling that leads to metastasis. This result implies that recruitment of ezrin in a complex with L1 is insufficient to promote its phosphorylation and activation and that these two steps are mediated by different signals from L1.

The transcription of L1-ezrin-NF-κB target genes is altered in cells expressing L1/H210Q During the last several years our lab identified several target genes of the L1-ezrin- NF-κB signaling and examined the importance of these molecules in L1-mediated metastasis. Among these were c-Kit [61], IGFBP2 [73] and SMOC2 [63]. We examined the expression of these genes in ECD mutant L1-expressing cells and found that in the L1/H210Q expressing cells a different level of transcription in all three L1 “target” genes

Figure 8. The H210Q mutation interferes with L1-induced activation of NF-κB, but exhibits ezrin localization similar to wtL1. wtL1, control (con), L1/H210Q (A) and L1/D598N (B) expressing Ls174T cells were treated with the proteasomal inhibitor MG132 for 24 hours and then lysed, protein levels where determined by western blotting for pI κB. C, Phosphorylated ezrin (pEzrin) levels in L1/H210Q expressing cells were determined by western blotting with pERM ab. D, Ezrin localization in wtL1, control (con), L1/H210Q and L1/D598N expressing cells was determined by immunostaining for ezrin (upper row – red) and L1 (middle row – green).

(Fig. 9A, C, D) when compared to wtL1 expressing CRC cells. In contrast to L1/H210Q, the RNA level of c-Kit and SMOC2 in L1/D598N expressing cells were similar to those in cells expressing wtL1 (Fig. 9B,D), while IGFBP2 RNA levels were reduced as in L1/H210Q expressing cells. The changes in RNA levels of SMOC2 and c-Kit in L1/H210Q cells support the hypothesis that the H210Q L1 mutant, and therefore the Ig-2 domain, affects the L1-ezrin-NF-κB signaling in these cells.

Figure 9. RNA levels for L1-ezrin-NF-κB signaling target genes is altered in L1/H210Q expressing cells . RNA levels of c-Kit in L1/H210Q (A) and in L1/D598N (B) expressing cells and for IGFBP2 in L1/D98N expressing cells were determined by semi-quantitative PCR. RNA levels of IGFBP2 in L1/H210Q expressing cells (C) and SMOC2 in both mutants were determined by real-time PCR analysis. wtL1 and empty vector (con) expressing cell were used as positive and negative control.

Comparing gene expression patterns of cells expressing L1/H210Q and L1/D598N mutations

L1/H210Q cells have more genes whose expression is altered compared to wtL1-expressing cells To get further insight into the possible mechanisms whereby the two ECD L1 mutations (H210Q and D598N) affect the metastatic capacities of human CRC cells, we conducted DNA microarray gene expression analyses using Affymetrix GeneChips from cells expressing these mutant L1 forms. We searched for genes whose transcription was altered in Ls174T cells overexpressing each of the two mutations compared to cells overexpressing the wtL1. We defined three groups of interest:

Group 1: Genes that mostly change in L1/H210Q as compared to wtL1 and L1/D598N (see Supplementary Tables 2 and 3). We included only genes that show at least a 2-fold difference.

Group 2: Genes that mostly change in L1/D598N as compared to wtL1 and L1/H210Q (Supplementary Tables 4 and 5). We included only genes that show at least a 2-fold difference.

Group 3: Genes that were affected by both L1 mutations (Supplementary Tables 6 and 7). Here we have chosen only genes where both mutations displayed at least a 2-fold difference from wtL1, while the difference between the two mutants was lower than 2-fold.

As summarized in Figure 10, the group of genes whose expression is altered exclusively (by the criteria mentioned above) in L1/H210Q expressing cells included a significantly larger number of genes (83 genes) than genes in the L1/D598N expressing cells (30 genes), and there were 59 genes that were affected by both mutations. The result of this gene array suggested that the L1/H210Q mutation has a “broader” effect on L1 activity than the D598N mutation, implying a more important role for the homophilic interactions of L1.

Figure 10. A scheme representing the number of genes affected by each mutation and by both mutations together.

Validation of the gene array results by RT-PCR From the list of genes we have chosen to focus on 12 genes. These genes were chosen because they are known to have a role (or a correlation) with the progression of cancer, metastasis, cancer cell migration, or invasion. The genes are: CAPN6, MME, KLK6, ST6, CLU and CDH17 from group 1, STC1, TGF β1 from group 2 and FN1, VCAN, ISG15 and MUC2 from group 3.

We validated the results from the gene array of the chosen genes by RT-PCR. In the PCR reactions we used as negative control the empty vector-transfected cells, to ensure that the changes we observed between the mutations and wtL1 correspond to a difference between wtL1 and the control cells. For example, we would expect from a gene that was downregulated by the mutated L1, to be downregulated in the control cells when as compared to wtL1 expressing cells. From the 6 genes chosen from group 1 (that displayed changes in expression by the H210Q mutation), 5 could be validated and confirmed the pattern observed in the gene array – meaning that a change in expression was detected mostly in L1/H210Q cells (CDH17, KLK6, CD10, CLU, and ST6). The sixth gene in the group, CAPN6, showed a dramatic effect in both mutations, unlike in the gene array. Of the 6 genes we observed, 4 showed a significant change between wtL1 and control, (CDH17, CD10, KLK6 and CLU), while ST6 and CAPN6 only showed a mild change in expression

(Fig. 11A). In group 2 we examined 2 genes, TGF β1 and STC1. In the case of STC1, we could not detect a significant change in expression (in both mutations), contrary to that observed in the gene array, while with TGF β1 we obtained a completely opposite result to that observed in the gene array assay (Fig. 11B), rendering both genes as poor candidates for further studies. Of the 4 genes examined from group 3, ISG15, VCAN and FN1 (VCAN and FN1 are both ECM proteins) we confirmed the results of the gene array showing a significant difference between the two mutations and the wtL1. A significant change was observed also when comparing between the wtL1 and the negative control. With the fourth gene, MUC2 (a key regulator of the gut), we observed the same pattern as in the gene array, namely both mutations expressed higher levels of this gene than the wtL1-transfected cells, but the difference between wtL1 and the negative control was minor (Fig. 11C).

Clusterin expression is downregulated only in L1/H210Q expressing cells In a study from our lab published this year, we found that transcription and protein levels of clusterin are upregulated in L1-expressing cells, and when clusterin expression is silenced by shRNA, the CRC cells do not form metastases when injected into mice [74]. It was interesting to find that clusterin also appeared in this study, as part of group 1 genes that are affected mainly by the H210Q mutation, and is downregulated in cells overexpressing the mutant L1/H210Q. The results obtained by the gene array analysis were also verified by RT-PCR (Fig. 11A). The levels of clusterin in the culture medium of cells (clusterin in mostly a secreted protein) were determined by western blotting (Fig. 12A), and were in line with the results obtained when determining RNA levels namely, a significant downregulation of clusterin was observed only in L1/H210Q cells. In the study by Shapiro et al., [74] clusterin transcription in L1 expressing cells was found to be mediated by the activation of the transcription factor STAT1 and not by NF-κB. This suggests that the homophilic interactions of the ECD domain in L1 (that are mediated by the Ig-2 domain) are responsible for the activation of at least two separate cellular signaling pathways, NF-κB and STAT1. Together with the high number of genes that were altered mostly in L1/H210Q expressing cells, the results strongly indicate a pivotal role for the homophilic ECD interactions in L1-mediated CRC metastasis.

Figure 11. Validation of gene expression patterns from microarrays using quantitative real time PCR . RNA levels of CDH17, KLK6, CD10, CLU, ST6, CAPN6 (A), TGF β1, STC1 (B), VCAN, FN1, MUC2, IGS15 (C) in Ls174T cells expressing wtL1, L1/H210Q and L1/D598N and control cells were determined by qRT-PCR.

Figure 12. Determination of protein levels of genes identified in the gene array using L1/H210Q and L1/D598N expressing cells . A, Expression of clusterin in Ls174T cells was determined by western blotting of the protein extracted from the conditioned medium of cells, 48hr after culturing in serum-free medium. Equal loading was confirmed by normalization of the medium samples to protein levels of the cell lysates. Protein levels for CD10, CDH17 (B), KLK6 (C) and VCAN (D) in Ls174T cells were determined in cell lysates and for KLK6 in both medium and cell lysates.

The role of CD10 in L1-mediated metastasis

The level of CD10 is altered in L1/H210Q expressing CRC cells To investigate further the differences between the two ECD mutations, H210Q and D598N, we chose to focus on four genes: CD10, CDH17, KLK6 and VCAN. The levels of CD10 protein were verified by western blotting and were in accordance with the results obtained for the changes in RNA level (Fig. 12B). Western blots of L1-expressing cells also confirmed the RT-PCR results for CDH17, showing a reduction in this protein when comparing mutant L1 expressing to control cells. However, in L1/H210Q expressing cells, the level of CDH17 was only slightly increased, while in the L1/D598N mutant-expressing cells the changes in the level of this protein were minor (Fig. 12B). We tried to determine

35 the level of KLK6 and VCAN in cell expressing the mutant L1 forms, but we either could not detect any change in protein levels when comparing to wtL1 (KLK6, Fig. 12C), or could not detect the protein at all (VCAN, Fig. 12D).

CD10 (or Neprilisyn, Neutral Endopeptidase) is a cell surface zinc-dependent metalloprotease that functions as a regulator of biological activities of peptide substrates, by reducing the local concentrations available for receptor binding [75]. CD10 expression was reported in many cancers (colon, lung, skin, pancreas, breast, esophagus and liver), both in tumor cells and in the stroma. It is suggested that CD10 has a role in invasion, proliferation, apoptosis and disease progression [76-83], thus making it an interesting target gene of L1- mediated signaling. To examine the functional importance of CD10 in L1-mediated metastasis, we manipulated CD10 expression in Ls174T and compared its effects on cellular properties compared to cells expressing wtL1. To directly examine the role of CD10 expression under the context of L1 expression, we suppressed CD10 expression by stably transfecting wtL1-expressing cells with shCD10 plasmids. In addition, we wished to examine the possible role of CD10 in mediating metastasis in an L1-independent manner, by stably transfecting Ls174T cells with a CD10 plasmid. We expected that a combined examination of these cell lines will shed light on the role that CD10 plays in L1-mediated metastasis and will indicate whether CD10 is a more general metastasis promoting molecule, or whether it is specifically activated only when expressed together with L1. We have successfully isolated CD10 overexpressing CRC cells and validated the expression of the protein by western blot analysis (Fig. 13A). We also generated a shCD10 plasmid (using pSuper kit), transfected it into L1-epxressing Ls174T cells and isolated cell clones that express L1 and exhibit a clear reduction in CD10 expression (Fig. 13B).

Silencing CD10 in L1 expressing cells abolishes L1-mediated cellular properties We set to examine the effects of altering CD10 expression on cellular properties that are promoted by L1. We analyzed possible effects on cell motility and proliferation rates under stressful conditions. In a scratch wound assay, using clones of L1+shCD10 and CD10 overexpressing cells, we observed that L1+shCD10 cells have lost the typical L1-mediated elevated motility (Fig. 13C), and the cells exhibited the same rate of wound closure as the control cells lacking L1. Cells overexpressing CD10, on the other hand, did not exhibit an

36 increase in motility as compared to control cells (Fig. 13C). Examining the cell proliferation rate, we observed a different behavior than in the motility assays. As in the motility assay, cells expressing L1+shCD10 exhibited a loss of the L1-mediated phenomenon, namely a higher growth rate in cells that are starved (in 0.1% serum containing medium). However, in this assay, cells overexpressing CD10 showed increased proliferation, similar to L1 overexpressing cells (Fig. 13D). In addition, we found that L1+shCD10 expressing cells were also less tumorigenic when injected to flanks of nude mice (Fig. 13E, G). Together, these results suggest that CD10 plays a role in L1-mediated cellular properties, since CD10 silencing affected both cell motility and proliferation rates. When L1 is not present, CD10 by itself can only promote an increase in proliferation rate, but not in motility, another indication to the broader effect that L1 expression has in CRC. As with most other downstream “target” molecules of L1 overexpression that were examined by our group (clusterin and SMOC2), CD10 could not confer the full repertoire of effects observed with L1 expression.

Next, we examined the importance of CD10 in L1-mediated metastasis. Cells expressing L1+shCD10, or CD10 were injected into the spleen of nude mice, and after 7.5 weeks, the animals were sacrificed and the livers and spleens were examined for tumors. Consistent with the in vitro results, L1+shCD10 cells were suppressed in their ability to produce macrometastases in the liver. As shown in Figure 13E, metastatic tumors where found only in one mouse out of the 8 mice injected (4 mice for each clone). Cells overexpressing CD10 did not produce any metastases (data not shown). These in vivo results support our in vitro assays suggesting that CD10 has an important role in the L1-mediated effects on cells, but when L1 is not expressed, CD10 overexpression cannot reproduce the these effects by itself.

Figure 13. Silencing CD10 abolishes the L1-mediated properties. A, B, CD10 protein levels in stable clones of CD10 and L1+shCD10 expressing Ls174T cells were determined by western blot. C, wound closure assay done on stable clones of CD10 and L1+shCD10 expressing cells, wild type L1 expressing cells (wtL1) and empty vector transfected Ls174T cells (con) were used as positive and negative controls. D, Growth rate assay using XTT kit, 50,000 cells were cultured for 1, 2, 3 and 6 days, measurement were normalized according to the results of the first day of that same sample. Each line represents the average of two clones. E and G, 1.5x10 6 Ls174T cells expressing wtL1, L1+shCD10 and empty vector were injected to flanks of nude mice, tumors were examined and weighted (F) after 15 days. G, Two clones of L1+shCD1 cells were injected into the spleen of nude mice, mice were sacrificed after 8 weeks and tumors in the spleen and liver were photographed.

CD10 is a target of L1-NF-κB signaling CD10 expression was found to be downregulated in L1/H210Q expressing cells (Fig. 12 B) that were shown to be deficient in activating the NF-κB signaling (Fig.8 A, C). To determine whether CD10 expression depends on NF-κB signaling we examined CD10 levels in stable clones of L1 expressing cell co-transfected with shRNA against p65, the transcriptionally active sub unit of NF-κB. We found that L1+shp65 expressing cells do have lower levels of CD10 when comparing to L1 expressing cells (Fig.14). Put together with the findings that the H210Q mutant form of L1 cannot activate NF-κB signaling, we suggest that CD10 is indeed an L1- NF-κB signaling target gene.

Figure 14. CD10 expression requires NF-κB signaling . CD10 protein levels were determined in stable clones of Ls174T expressing L1+shp65 by western blotting. wtL1 and empty vector (con) expressing cells were used as positive and negative controls.

Discussion

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths, but as of today, the molecular mechanisms underlying this disease are incompletely understood. Gaining metastatic capacities is a pivotal step in disease progression. To successfully metastasize, cells have to detach from the primary tumor and invade into the surrounding tissue, intravasate into the blood or lymph vessels, survive the mechanical stress involved, avoid apoptosis imposed by the immune system, and eventually arrest and proliferate in new microenvironments after extravasation. Metastasis is therefore a complex multistep process that involves changes in many cellular properties that need to be active (or to be shut off) at the right time and place. Recent studies in our laboratory identified L1 as a target gene of the β-catenin-TCF transcriptional complex (the Wnt pathway) in CRC cells where the Wnt signaling pathway is aberrantly activated in ~90% of the cases. L1 was observed in human CRC tissue exclusively in tumor cells of the invasive edge [20]. L1 expression in human CRC cells that lack L1 confers enhanced motility and proliferation, and the capacity to metastasize to the liver. L1 stands as an excellent candidate that has a role as a master orchestrator of metastasis, thanks to its ability to mediate numerous interactions both inside the cell as well as outside of it. Studies from our lab already established the role of the cytosolic tail of L1 that binds to ezrin and activates the NF-κB pathway [60]. As shown in our recent study, NF-κB is not the only target of L1 signaling, since the L1-dependent transcription of clusterin was shown to be mediated by the transcription factor STAT1, independently of NF-κB signaling [74]. While the cytosolic part of L1 is short and does not contain many binding sites for other components, the L1 ECD is very large and consists of many domains that were shown to interact with numerous molecules, therefore making the ECD a prime subject in having a key role in transducing cellular signals beyond its role in cell-cell adhesion.

The importance of the Ig-2 and Ig-6 domains in L1-mediated metastasis As described in the Introduction, more than sixty point mutations in the ECD of L1 were found in brain disease patients spanning the entire ECD and being symptomatic [70, 71], therefore emphasizing the importance of the various L1 ECD regions in the proper function of the molecule. These findings with L1 function in brain cells correlate well with

40 our findings using the human Ls174T CRC cell line that lacks endogenous L1 into which we transfected various wt and mutant forms of L1. Thus, each one of the ECD point mutations that was transfected into CRC cells, failed to confer metastasis (except for the wtL1) when injected into mice. These studies further reiterated the importance that the various L1 ECD domains have in the proper function of L1 in the nervous system and also in its ability to confer metastasis in CRC. We chose to focus our study on two L1 ECD domains: Ig-2 and Ig-6, the first is considered a key component in L1-L1 trans-interactions and the latter contains the integrin-binding RGD sequence. Mutations in both domains were chosen because they disrupt the conformational integrity of these domains that are believed to be most important for L1 activity. Another important guide in choosing the mutants that we studied was the fact that these mutations are “structural” mutations that affect the local structural integrity of the domain, but do not have a bigger effect on the conformation integrity of the ECD, or the protein itself. Another important consideration was that the mutant protein should exhibit proper localization and expression pattern in the cell, to make sure that the effects observed result from the lack of activity of the relevant domain, and not by other more trivial effects such as altered processing and/or subcellular localization.

At the start of the study, we knew (from previous work during my M.Sc thesis studies) that the two mutations we chose for analysis had similar effects on CRC cellular properties (metastasis, proliferation, motility). We hypothesized that, in the end, both mutations will have the same effect on L1 since they operate through the same mechanism of signaling. But, when examining the activation of the NF-κB pathway by the cytoplasmic tail of L1, we found a difference between the two ECD mutations: only the D598N mutant was shown to activate NF-κB signaling. This result brought up the hypothesis that the Ig-2 domain (and thus homophilic interactions) is the mediator of NF-κB activation, while the Ig- 6 domain probably affects another (yet to be discovered) pathway that is also important in L1-mediated metastasis. Supporting this view were the observations that the transcription of 2 out of 3 known L1-NF-κB target genes (that were studied in our lab) were altered only in cells expressing the H210Q L1-mutation.

Homophilic interactions mediated by the Ig-2 domain are crucial for ezrin activation Ezrin was shown in our lab to be a key mediator of L1-dependent NF-κB activity involving the cytoplasmic domain of L1. L1 recruits ezrin from filopodia, binds to it and activates it by phosphorylation that involves ROCK [60]. This complex then binds to I κB and promotes its phosphorylation and degradation by the proteasome. This results in the release of NF- κB that becomes free to translocate into the nucleus where it promotes target gene transcription. In our study, we observed a rather unexpected result that may shed some light on how L1 recruits and activates ezrin. It was expected that L1 promote the phosphorylation (and activation) of ezrin via ROCK activation followed by the binding of activated ezrin to the cytosolic domain of L1. We therefore anticipated a decrease in p-ezrin levels in cells expressing H210Q and an ezrin re-localization to its original localization in filopodia. However, while we found that cells expressing the L1-H210Q had lower levels of p-ezrin, surprisingly the ezrin molecules were still co-localized with the mutant L1 in the membrane at cell-cell contacts. This may imply that L1 and ezrin interact by a mechanism of ezrin activation by ROCK involving homophilic interactions of L1 (promoted by the Ig-2 domain), while the localization and binding of ezrin to L1 is mediated by a different domain of L1, most probably by its cytoplasmic tail.

The role for L1 shedding in promoting metastasis As described in the Introduction, the cleavage of L1 and its shedding into the culture medium can affect certain cellular properties, L1 was found to be cleaved by several proteases, including ADAM10 [67] and BACE1 [84]. We found that L1 and ADAM10 are co-expressed at the invasive front of CRC tumor tissue, and the overexpression of both molecules in CRC cell lines increased the metastatic capacities of these cells in comparison to cells expressing L1 alone [50]. Knowing that L1/H210Q-expressing cells lack metastatic capacity compared to cells expressing wtL1, and since the Ig-2 domain is a key component of homophilic trans interactions, we hypothesized that the shedding of L1, and the binding of soluble/shed L1 molecules back to the cells are important for L1 functions. In a previous study, we saw that inhibition of ADAM10 (and other metalloproteases), resulted in a decrease in cell motility in L1-expressing cells (from previous work during my M.Sc studies). Moreover, when such cells were cultured in conditioned medium from L1- expressing cells (that contains soluble L1 molecules) we observed an increase in cell

42 motility, suggesting that homophilic interactions of L1 to soluble L1 molecules outside the cell are important for achieving the L1-mediated properties. The experiments with conditioned medium could only provide very limited information, since such medium is by definition already partly depleted of important nutrients, and may have a toxic effect on cells, if the cells are cultured in such medium for long periods of time. We observed a dramatic decrease in cell motility when comparing L1 cells that were cultured with conditioned medium, to cells cultured in fresh medium. These observations called for a new experimental model enabling the investigation of the importance of L1 shedding. We chose to adopt the use of Fc-bound particles. Fc-particles conjugated to the ECD of L1 were isolated and purified from cells that secreted them, and then incubated with cells in fresh medium. In this model, cells can be treated for longer periods of time with the particles. The first experiment was to verify that the cells identify the soluble Fc-L1 particles and bind to them. We found that both wtL1 and L1/H210Q expressing cells bind to the Fc-L1 particles and, as expected, L1/H210Q cells did not bind to Fc-L1/H210Q particles. Unfortunately, after numerous attempts we could not detect any effect of the Fc-L1 on other cellular properties. L1 cells treated with metalloprotease inhibitors did not exhibit a “rescue” phenomenon when Fc-L1 particles were added, and cells expressing L1/H210Q did not change their properties when Fc-L1 particles were added. One possible explanation for these observations can be that the L1 ECD conjugated to the Fc particles is not folded in the same manner as the shed L1, and therefore may have a different conformational structure that cannot bind to cell-bound L1 molecules in the same manner, making the Fc particles unable to reproduce the cellular responses obtained with the shed L1 that binds to cells. This may result from the fact that the Fc-L1 particles contain the full length ECD, while the ADAM10 cleaved L1 is smaller (the cleavage site is suspected to be inside the fifth FNIII repeat and not at its end).

The “bigger role” of the Ig-2 domain in L1 functions After establishing that homophilic interactions (mediated through the Ig-2 domain, and abolished when the H210Q mutation is introduced) are important for NF-κB activation, we wished to examine whether there is a more global effect regarding the differences between the two ECD domains (Ig-2 and Ig-6). For this, we examined the gene expression patterns of CRC cells expressing both Ig-2 and Ig-6 mutations and compared them to that of

43 wtL1 expressing cells. While establishing a threshold of 2-fold change, we observed a difference between the two mutations in the number of genes affected. While the H210Q mutation altered the transcription of 142 genes, the D598N altered only 89 genes (in each case 59 genes were common for both). This gave us an initial indication about a possible “wider” role for the homophilic interactions mediated via Ig-2. Adding to the number of genes affected, we observed that the clusterin gene is also affected only by the L1/H210Q mutation. At the time, clusterin was studied in our lab as a L1 downstream effector, and its transcription was found to be controlled by the transcription factor STAT1, while manipulating NF-κB activity (either by overexpressing p65 in control cells, or by silencing it in L1 expressing cells using shRNA) did not affect clusterin expression [74] . The finding that only the H210Q L1 mutant affects the expression of clusterin supported the notion that there is a “wider” role for homophilic L1-L1 interactions that are probably responsible for mediating both NF-κB and STAT1 transcriptional activities. Finally, when comparing the reports on each of the genes in both lists, we have seen that the number of genes that were altered in L1/H210Q expressing cells that are known to have a role in cancer progression (any cancer) is almost 3 times larger than the list of genes altered by D598N (44 against 17). At this point, we suggest that interference with the homophilic interactions of L1 most probably has a bigger effect on L1 functions, implying a more general role in the signals mediated by L1, while some other interactions of L1 probably have a more “pathway- specific” effect.

CD10 transcription is essential for L1-mediated metastasis

Conducting a gene expression array comparison and RT-PCR for wtL1, L1/H210Q and L1/D598N expressing cells, we identified dozens of genes that were either over or under expressed by L1 and that require proper homophilic L1-L1 interactions. One of these genes, CD10, is a peptidase well known to contribute to disease progression in many types of cancer [76, 79-82]. CD10 was found to be involved in the propagation of cancer cell motility and metastasis [75, 78, 85]. We therefore suspected that it has a role in L1-mediated metastasis in CRC. CD10 can affect the response of cells to signals from the outside by shredding the ligands and reducing their concentrations in the close vicinity of the cells [75]. We found that when the expression of CD10 is silenced in L1 expressing cells, the cells are

44 less motile, proliferate at a slower rate and fail to metastasize compared to wtL1 expressing cells. In comparison, cells that express CD10 (but not L1) could not reproduce the full repertoire of effects we see in L1 overexpressing cells. These results imply that while CD10 is an important downstream target of L1, it only functions in metastasis in the context of L1 overexpression and as part of the L1 signaling induced genes. The mechanism/s by which L1 promotes CD10 transcription is yet to be discovered.

Conclusions Metastasis is a series of distinct biological processes that are mediated by many pathways and various regulatory molecules and by the cellular microenvironment. Our lab has identified L1 as a master-orchestrator of metastasis in human colorectal cancer. L1 fulfills this role by its capacity to either propagate cellular signaling directly, via its cytosolic tail, or by the numerous extracellular interactions it can form. In this study we wished to shed more light on the role of the ECD domains of L1, and found that even though many (if not all) of the domains are required for gaining metastatic capacity, the homophilic interactions of L1 (mediated by the Ig-2 domain) are important for the complete activity of the L1 molecule. Interference with homophilic interactions has a bigger effect on the cells, thus making them a good candidate for therapeutic interventions (see figure 15).

Figure 15. A scheme of the proposed model of L1 interactions and the pathways activated by L1

Supplementary Tables Table 2. Genes that are downregulated mostly in cells expressing the L1/H210Q mutant

D598N Vs. L1 H210Q Vs. L1 Gene symbol Ref Seq p- Fold p- Fold H/D Protein Name value change value change ratio

HBE1 NM_005330 0.836 1.403 0.182 -10.534 7.51 hemoglobin, epsilon 1 BGN NM_001711 0.866 1.197 0.084 -8.195 6.85 biglycan, PG-I DKK4 NM_014420 0.893 1.209 0.171 -8.135 6.73 dickkopf homolog 4 OAS2 NM_016817 0.556 -2.197 0.135 -8.869 4.04 2'-5'-oligoadenylate synthetase 2 adenomatosis polyposis coli down- APCDD1 NM_153000 0.750 -1.417 0.151 -5.612 3.96 regulated 1 IGHM Y14737 0.990 1.011 0.167 -3.710 3.67 immunoglobulin heavy constant Mu MME (CD10) NM_000902 0.988 1.011 0.153 -3.393 3.36 neprilysin/ Neutral Endopeptidase FGF20 NM_019851 0.703 -1.431 0.133 -4.748 3.32 fibroblast growth factor 20 CAPN6 NM_014289 0.296 -2.622 0.044 -8.475 3.23 Calpain 6 SLC16A14 NM_152527 0.015 1.339 0.000 -4.258 3.18 solute carrier family 16, member 14 KLK6 NM_002774 0.803 1.176 0.082 -3.666 3.12 kallikrein 6 KRT75 NM_004693 0.801 1.185 0.101 -3.497 2.95 Keratin 75 MT1G NM_005950 0.350 -6.436 0.160 -18.891 2.94 metallothionein 1G IGFL2 NM_001002915 0.866 -1.179 0.247 -3.320 2.82 IGF-like family member 2 IFI6 NM_022873 0.134 -3.898 0.023 -10.904 2.80 interferon, alpha-inducible protein 6 LIX1L NM_153713 0.753 -1.312 0.168 -3.637 2.77 Lix1 homolog (mouse)-like HIST2H3D NM_001123375 0.907 -1.122 0.282 -3.052 2.72 histone cluster 2, H3d CXCL10 NM_001565 0.910 -1.101 0.257 -2.795 2.54 chemokine (C-X-C motif) ligand 10 RNU12 NR_029422 0.349 -1.633 0.026 -4.123 2.52 RNA, U12 small nuclear cadherin 3, type 1, P-cadherin CDH3 NM_001793 0.562 -1.371 0.055 -3.403 2.48 (placental) LCN15 NM_203347 0.919 1.111 0.346 -2.754 2.48 lipocalin 15 MT1A NM_005946 0.262 -2.104 0.035 -5.137 2.44 metallothionein 1A potassium channel, subfamily T, KCNT1 NM_020822 0.982 1.009 0.058 -2.457 2.44 member 1 GMPR NM_006877 0.501 1.271 0.015 -3.088 2.43 guanosine monophosphate reductase TEKT4P2 NR_038327 0.831 -1.105 0.072 -2.649 2.40 tektin 4 pseudogene 2 ENST00000364 RNA, U5E small nuclear 4, RNU5E-4P 0.946 -1.043 0.181 -2.483 2.38 931 pseudogene IFI27 NM_001130080 0.216 -3.129 0.053 -7.290 2.33 interferon, alpha-inducible protein 27 SLC7A8 NM_012244 0.053 -2.087 0.002 -4.811 2.31 solute carrier family 7 SECTM1 NM_003004 0.009 -1.926 0.000 -4.393 2.28 secreted and transmembrane 1 SNAR-H NR_024342 0.850 -1.063 0.030 -2.409 2.27 small ILF3/NF90-associated RNA H ENST00000362 RNA, Ro-associated Y4 pseudogene RNY4P19 0.304 1.454 0.012 -3.280 2.26 530 19 MT1H NM_005951 0.384 -2.611 0.134 -5.877 2.25 metallothionein 1H

D598N Vs. L1 H210Q Vs. L1 Gene symbol Ref Seq p- Fold p- Fold H/D Protein Name value change value change ratio small nucleolar RNA, H/ACA box SNORA11 NR_002953 0.487 -2.608 0.222 -5.829 2.23 11 interferon-induced protein with IFIT3 NM_001031683 0.771 -1.238 0.203 -2.728 2.20 tetratricopeptide repeats 3 LCN2 NM_005564 0.637 -1.367 0.135 -2.961 2.17 lipocalin 2 MT1F NM_005949 0.294 -2.113 0.059 -4.544 2.15 metallothionein 1F IFI44 NM_006417 0.435 -2.421 0.171 -5.193 2.14 interferon-induced protein 44 DLL4 NM_019074 0.926 -1.050 0.159 -2.235 2.13 delta-like 4 CALCA NM_001741 0.950 1.056 0.391 -2.171 2.06 calcitonin-related polypeptide alpha LGALS9B NM_001042685 0.699 -1.140 0.040 -2.332 2.05 galectin-9 (Gal9) ST6GALNAC4 NM_175039 0.475 -1.173 0.006 -2.394 2.04 ST6 / a2,6-sialyltransferase LRG1 NM_052972 0.893 1.047 0.063 -2.125 2.03 leucine-rich alpha-2-glycoprotein 1 RNU11 NR_004407 0.215 -1.853 0.026 -3.724 2.01 RNA, U11 small nuclear coactosin-like F-actin binding COTL1 NM_021149 0.214 -1.505 0.009 -3.018 2.00 protein 1 CLU NM_001831 0.644 1.094 0.007 -2.122 1.94 Clusterin

Table 3. Genes that are upregulated mostly in cells expressing the L1/H210Q mutant

D598N Vs. L1 H210Q Vs. L1 Gene symbol Ref Seq p- Fold p- Fold H/D Protein Name value change value change ratio olfactory receptor, family 51, OR51E1 NM_152430 0.907 1.093 0.085 4.485 4.10 subfamily E, member 1 carcinoembryonic antigen-related CEACAM5 NM_004363 0.907 -1.190 0.330 4.512 3.79 cell adhesion molecule 5 cystic fibrosis transmembrane CFTR NM_000492 0.842 -1.130 0.053 4.108 3.63 conductance regulator carcinoembryonic antigen-related CEACAM6 NM_002483 0.932 -1.124 0.409 3.221 2.86 cell adhesion molecule 6 dehydrogenase/reductase (SDR DHRS2 NM_005794 0.921 -1.088 0.218 3.059 2.81 family) member 2 KRTAP9-4 NM_033191 0.796 1.300 0.231 3.641 2.80 keratin associated protein 9-4 CENPE NM_001813 0.547 1.431 0.048 4.002 2.80 centromere protein E KRTAP9-2 NM_031961 0.633 1.444 0.106 3.999 2.77 keratin associated protein 9-2 RIMBP2 NM_015347 0.988 1.012 0.258 2.589 2.56 RIMS binding protein 2 NPSR1 NM_207172 0.871 1.115 0.157 2.820 2.53 neuropeptide S receptor 1 defensin, alpha 6, Paneth cell- DEFA6 NM_001926 0.960 1.037 0.233 2.548 2.46 specific SLC7A11 NM_014331 0.777 1.191 0.119 2.924 2.45 solute carrier family 7 polymeric immunoglobulin PIGR NM_002644 0.609 1.927 0.250 4.695 2.44 receptor

D598N Vs. L1 H210Q Vs. L1 Gene symbol Ref Seq p- Fold p- Fold H/D Protein Name value change value change ratio MIR4525 NR_039751 0.742 1.133 0.033 2.719 2.40 microRNA 4525 TC2N NM_152332 0.984 1.007 0.046 2.403 2.39 tandem C2 domains, nuclear GPSM2 NM_013296 0.866 -1.048 0.014 2.497 2.38 G-protein signaling modulator 2 PCDHB14 NM_018934 0.771 -1.114 0.040 2.514 2.26 protocadherin beta 14 regenerating islet-derived family, REG4 NM_001159352 0.480 2.099 0.166 4.713 2.25 member 4 RPL37 NR_037665 0.938 1.039 0.127 2.308 2.22 ribosomal protein L37 cadherin 17, LI cadherin (liver- CDH17 NM_004063 0.198 1.748 0.013 3.851 2.20 intestine) ODZ1 NM_001163278 0.938 1.049 0.203 2.310 2.20 odz, odd Oz/ten-m homolog 1 MIR4295 NR_036177 0.941 -1.052 0.245 2.315 2.20 microRNA 4295 MIR548C NR_030347 0.883 1.080 0.137 2.359 2.18 microRNA 548c THEG NM_016585 0.760 1.264 0.223 2.709 2.14 theg spermatid protein KRTAP9-8 NM_031963 0.598 1.466 0.148 3.133 2.14 keratin associated protein 9-8 alpha thalassemia/mental ATRX NM_000489 0.907 1.067 0.176 2.259 2.12 retardation syndrome X-linked KRTAP4-2 NM_033062 0.829 1.183 0.272 2.468 2.09 keratin associated protein 4-2 AT rich interactive domain 4A ARID4A NM_023000 0.903 1.061 0.142 2.211 2.08 (RBP1-like) CCDC68 NM_001143829 0.788 1.093 0.040 2.273 2.08 coiled-coil domain containing 68 CD44 molecule (Indian blood CD44 NM_001001389 0.883 -1.108 0.257 2.301 2.08 group) KRTAP17-1 NM_031964 0.455 2.138 0.168 4.441 2.08 keratin associated protein 17-1 pellino E3 ubiquitin protein ligase PELI1 NM_020651 0.732 1.097 0.019 2.270 2.07 1 ribosomal protein L23a RPL23AP4 ENST00000434638 0.897 1.030 0.015 2.104 2.04 pseudogene 4 integrin, alpha 2 (CD49B, alpha 2 ITGA2 NM_002203 0.825 1.156 0.221 2.361 2.04 subunit of VLA-2 receptor) trimethyllysine hydroxylase, TMLHE NM_018196 0.945 1.029 0.116 2.098 2.04 epsilon RNA, U4 small nuclear 9, RNU4-9P ENST00000364951 0.868 1.069 0.094 2.154 2.01 pseudogene ITSN2 NM_006277 0.992 -1.004 0.106 2.022 2.01 intersectin 2 LARS NM_020117 0.803 1.114 0.100 2.238 2.01 leucyl-tRNA synthetase

Table 4. Genes that are downregulated mostly in cells expressing the L1/D598N mutant

D598N Vs. L1 H210Q Vs. L1 Gene symbol Ref Seq p- Fold p- Fold H/D Protein Name value change value change ratio ubiquitin interaction motif UIMC1 NM_001199297 0.031 -7.851 0.378 -2.014 3.9 containing 1 TGFB1 NM_000660 0.143 -4.097 0.821 1.218 3.36 transforming growth factor, beta 1 DDIT4 NM_019058 0.044 -3.695 0.848 -1.108 3.33 DNA-damage-inducible transcript 4 small nucleolar RNA, H/ACA box SNORA38B NR_003706 0.029 -3.483 0.660 -1.223 2.85 38B GRAMD1A NM_020895 0.211 -2.949 0.962 -1.039 2.84 GRAM domain containing 1A CA11 NM_001217 0.213 -3.450 0.785 -1.289 2.68 carbonic anhydrase XI CTSF NM_003793 0.209 -2.704 0.987 1.012 2.67 cathepsin F MIR5047 NR_039969 0.079 -2.840 0.867 -1.090 2.61 microRNA 5047 MSN NM_002444 0.060 -3.762 0.497 -1.513 2.49 moesin guanine nucleotide binding protein GNG7 NM_052847 0.310 -2.830 0.855 -1.197 2.36 (G protein), gamma 7 small nucleolar RNA, C/D box SNORD83A NR_002571 0.021 -11.132 0.090 -4.829 2.31 58A CTGF NR_000027 0.145 -3.034 0.676 -1.338 2.27 small nucleolar RNA, C/D box 83A SNORD58A NM_001901 0.000 -2.691 0.023 -1.232 2.18 connective tissue growth factor NYN domain and retroviral NYNRIN NM_025081 0.271 -2.388 0.895 -1.104 2.16 integrase containing aspartate beta-hydroxylase domain ASPHD1 NM_181718 0.248 -2.173 0.980 1.016 2.14 containing 1 SNORD9 NR_003029 0.045 -3.528 0.354 -1.650 2.14 small nucleolar RNA, C/D box 9 transforming growth factor beta 1 TGFB1I1 NM_001042454 0.177 -2.938 0.662 -1.383 2.12 induced transcript 1 small nucleolar RNA, H/ACA box SNORA60 NR_002986 0.018 -3.973 0.192 -1.871 2.12 60 NT5E NM_002526 0.209 -2.471 0.799 1.186 2.08 5'-nucleotidase, ecto (CD73) small nucleolar RNA, H/ACA box SNORA14A NR_002955 0.226 -2.107 0.932 -1.050 2.01 14A

Table 5. Genes that are upregulated mostly in cells expressing the L1/D598N mutant

D598N Vs. L1 H210Q Vs. L1 Gene Ref Seq p- Fold p- Fold H/D Protein Name symbol value change value change ratio

STC1 NM_003155 0.045 4.659 0.761 1.214 3.84 stanniocalcin 1 ACTL8 NM_030812 0.058 3.491 0.995 -1.004 3.48 actin-like 8 EPB41L3 NM_012307 0.120 2.871 0.802 -1.164 2.47 erythrocyte membrane protein band 4.1-like 3 IL33 NM_033439 0.108 5.183 0.417 2.139 2.42 interleukin 33 MUC5B NM_002458 0.152 2.505 0.807 1.154 2.17 mucin 5B

D598N Vs. L1 H210Q Vs. L1 Gene Ref Seq p- Fold p- Fold H/D Protein Name symbol value change value change ratio PGAM1 NM_002629 0.083 2.795 0.605 -1.311 2.13 phosphoglycerate mutase 1 ALDH1A1 NM_000689 0.457 2.185 0.979 1.027 2.13 aldehyde dehydrogenase 1 family, member A1 SLC9A2 NM_003048 0.062 2.638 0.595 1.268 2.08 solute carrier family 9, subfamily A GINS2 NM_016095 0.148 2.498 0.744 1.208 2.07 GINS complex subunit 2 (Psf2 homolog) PTGDR NM_000953 0.349 2.022 0.994 -1.006 2.01 prostaglandin D2 receptor (DP)

Table 6. Genes that are downregulated in cells expressing both L1 mutations

D598N Vs. L1 H210Q Vs. L1 Gene symbol Ref Seq p- Fold p- Fold Average Protein Name value change value change fold change MYL9 NM_006097 0.141 -4.342 0.089 -5.798 -5.070 myosin, light chain 9, regulatory DKK1 NM_012242 0.068 -4.988 0.148 -3.332 -4.160 dickkopf 1 homolog IRF9 NM_006084 0.176 -3.200 0.091 -4.588 -3.894 interferon regulatory factor 9 ISG15 NM_005101 0.128 -2.883 0.039 -4.867 -3.875 ISG15 ubiquitin-like modifier MIR200C NR_029779 0.002 -3.916 0.005 -2.997 -3.456 microRNA 200C protein tyrosine phosphatase, PTPRU NM_133178 0.223 -3.921 0.342 -2.822 -3.372 receptor type U FN1 NM_212478 0.264 -3.110 0.253 -3.199 -3.154 Fibronectin 1 CADPS NM_003716 0.325 -2.495 0.180 -3.646 -3.071 Ca++-dependent secretion activator guanidinoacetate N- GAMT NM_000156 0.196 -2.381 0.077 -3.562 -2.972 methyltransferase IFI44L NM_006820 0.389 -2.080 0.139 -3.831 -2.956 interferon-induced protein 44-like HIST1H2BG NM_003518 0.291 -2.036 0.093 -3.397 -2.717 histone cluster 1, H2bg MIR548T NR_036093 0.054 -2.619 0.054 -2.617 -2.618 MicroRNA 548T HOXA13 NM_000522 0.172 -2.854 0.266 -2.291 -2.572 homeobox A13 transient receptor potential cation TRPV6 NM_018646 0.176 -2.640 0.229 -2.335 -2.487 channel, subfamily V, member KLHDC7B NM_138433 0.270 -2.557 0.302 -2.391 -2.474 kelch domain containing 7B TIMP2 NM_003255 0.211 -2.700 0.299 -2.240 -2.470 TIMP metallopeptidase inhibitor 2 VCAN NM_004385 0.208 -2.371 0.181 -2.526 -2.448 versican INTS5 NM_030628 0.156 -2.011 0.052 -2.839 -2.425 integrator complex subunit 5 family with sequence similarity 173, FAM173A NM_023933 0.110 -2.319 0.105 -2.355 -2.337 member A EHD2 NM_014601 0.258 -2.532 0.379 -2.024 -2.278 EH-domain containing 2 sterile alpha motif domain SAMD9L NM_152703 0.307 -2.122 0.242 -2.399 -2.261 containing 9-like KRT31 NM_002277 0.274 -2.191 0.264 -2.232 -2.211 keratin 31 TERC NR_001566 0.255 -2.206 0.263 -2.173 -2.190 telomerase RNA component KRT80 NM_182507 0.182 -2.093 0.143 -2.283 -2.188 keratin 80

D598N Vs. L1 H210Q Vs. L1 Gene symbol Ref Seq p- Fold p- Fold Average Protein Name value change value change fold change PGAP3 NM_033419 0.063 -2.002 0.030 -2.363 -2.182 post-GPI attachment to proteins 3 SLC46A3 NM_181785 0.325 -2.230 0.355 -2.117 -2.173 solute carrier family 46, member 3 XAF1 NM_017523 0.269 -2.199 0.292 -2.113 -2.156 XIAP associated factor 1 HIST1H2AM NM_003514 0.340 -2.047 0.293 -2.218 -2.133 histone cluster 1, H2am ARSA NM_000487 0.043 -2.020 0.029 -2.184 -2.102 arylsulfatase A SMPD1 NM_000543 0.052 -2.088 0.050 -2.105 -2.097 sphingomyelin phosphodiesterase 1 ZNF581 NM_016535 0.040 -2.155 0.052 -2.032 -2.094 zinc finger protein 581 MT1E NM_175617 0.034 -2.117 0.038 -2.068 -2.092 metallothionein 1E SHISA4 NM_198149 0.047 -2.031 0.039 -2.117 -2.074 shisa homolog 4

Table 7. Genes that are upregulated in cells expressing both L1 mutations

D598N Vs. L1 H210Q Vs. L1 Gene symbol Ref Seq p- Fold p- Fold Average Protein Name value change value change fold change MUC2 NM_002457 0.049 4.693 0.046 4.814 4.754 mucin 2 HOXB9 NM_024017 0.149 5.224 0.373 2.616 3.920 homeobox B9 ATOH1 NM_005172 0.201 3.085 0.203 3.073 3.079 atonal homolog 1 NEUROG3 NM_020999 0.455 2.062 0.194 3.768 2.915 neurogenin 3 3-hydroxy-3-methylglutaryl-CoA HMGCS2 NM_005518 0.565 2.460 0.455 3.251 2.855 synthase 2 (mitochondrial) Rho GTPase activating protein ARHGAP11B NM_001039841 0.059 2.297 0.020 3.069 2.683 11B CCDC99 NM_017785 0.056 2.287 0.022 2.935 2.611 coiled-coil domain containing 99 myosin, heavy chain 13, skeletal MYH13 NM_003802 0.047 2.411 0.033 2.649 2.530 muscle family with sequence similarity FAM111B NM_001142704 0.063 2.406 0.045 2.653 2.530 111, member B peptidase M20 domain containing PM20D2 NM_001010853 0.055 2.558 0.062 2.466 2.512 2 NOX1 NM_007052 0.082 2.372 0.060 2.600 2.486 NADPH oxidase 1 coenzyme Q3 homolog, COQ3 NM_017421 0.035 2.737 0.085 2.141 2.439 methyltransferase CASC5 NM_144508 0.087 2.221 0.048 2.636 2.428 cancer susceptibility candidate 5 cytoskeleton associated protein 2- CKAP2L NM_152515 0.092 2.032 0.027 2.799 2.416 like EXO1 NM_130398 0.059 2.414 0.065 2.350 2.382 exonuclease 1 LIAS NM_006859 0.011 2.405 0.012 2.347 2.376 lipoic acid synthetase KIF15 NM_020242 0.068 2.277 0.059 2.369 2.323 kinesin family member 15 ZNF100 NM_173531 0.168 2.253 0.162 2.282 2.268 zinc finger protein 100

D598N Vs. L1 H210Q Vs. L1 Gene symbol Ref Seq p- Fold p- Fold Average Protein Name value change value change fold change SPAG5 NM_006461 0.059 2.403 0.114 2.007 2.205 sperm associated antigen 5 SGOL1 NM_001199252 0.065 2.189 0.062 2.217 2.203 shugoshin-like 1 LIN7C NM_018362 0.090 2.145 0.080 2.212 2.179 lin-7 homolog C IQ motif containing GTPase IQGAP2 NM_006633 0.119 2.010 0.069 2.345 2.177 activating protein 2 PLK4 NM_014264 0.043 2.040 0.024 2.307 2.174 polo-like kinase 4 BRCA1 NM_007294 0.121 2.026 0.097 2.156 2.091 breast cancer 1, early onset SHC SH2-domain binding protein SHCBP1 NM_024745 0.028 2.148 0.039 2.014 2.081 1 TSPAN8 NM_004616 0.023 2.012 0.019 2.098 2.055 tetraspanin 8

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Publications during the Ph.D. studies:

1. Shvab A, Haase G, Ben-Shmuel A, Gavert N, Brabletz T, Dedhar S, Ben-Ze'ev A. (2015) Induction of the Intestinal Stem Cell Signature Gene SMOC-2 is Required for L1- Mediated Colon Cancer Progression. Oncogene, doi: 10.1038/onc.2015.127.

2. Shapiro B, Tocci P, Haase G, Gavert N, Ben-Ze'ev A. (2015) Clusterin, A Gene Enriched in Intestinal Stem Cells, is Required for L1-mediated Colon Cancer Metastasis. Oncotarget doi: 10.18632/oncotarget.5360.

3. Gavert N, Shvab A, Sheffer M, Ben-Shmuel A, Haase G, Bakos E, Domany E, Ben- Ze'ev A. c-Kit is suppressed in human colon cancer tissue and contributes to L1-mediated metastasis. Cancer Research 2013, 73(18):5754-5763.