The Role of the Collapsin Response Mediator Protein 1

THE ROLE OF THE COLLAPSIN RESPONSE

MEDIATOR PROTEIN 1 (CRMP1) ON CELL

MIGRATION

AND INVASION IN GLIOMA

Jonah Shiroky

A thesis submitted in partial fulfillment of the

requirements for the degree of

Master’s of Science, Division of Experimental

Medicine

McGill University, Montreal

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TABLE OF CONTENTS

TABLE OF CONTENTS ...... 2

LIST OF FIGURES ...... 4

ACKNOWLEDGEMENTS ...... 5

ABBREVIATIONS ...... 6

ABSTRACT ...... 7

RÉSUMÉ...... 8

CHAPTER 1 : INTRODUCTION ...... 10

PREAMBLE ...... 11

CRMP SEQUENCE HOMOLOGY ...... 13

ALTERNATIVE SPLICING ...... 14

DIFFERENTIAL EXPRESSION PATTERNS OF CRMP ISOFORMS ...... 15

a) Central Nervous System (CNS) ...... 15

b) Outside the CNS ...... 16

STRUCTURE OF CRMP PROTEINS ...... 17

POSTTRANSLATIONAL MODIFICATIONS ...... 18

a) Phosphorylation ...... 18

b) Calpain Cleavage ...... 19

c) Other Modifications ...... 20

BIOLOGICAL FUNCTIONS OF CRMP PROTEINS ...... 20

a) Neurite outgrowth ...... 20

b) The Sema3A (Collapsin-1) Pathway ...... 24

c) CRMP and the Rho Pathway ...... 26

2 d) Neuronal Plasticity ...... 26

e) Cell Migration ...... 28

f) Neuronal Proliferation/Apoptosis...... 30

IMPLICATIONS OF CRMP PROTEINS IN NEURODEGENERATIVE DISEASE ...... 31

THE ASSOCIATION OF CRMP PROTEINS WITH CANCER ...... 31

RATIONALE ...... 44

CHAPTER 2 : MATERIALS AND METHODS ...... 45

CHAPTER 3 : RESULTS...... 54

IN VITRO EFFECTS OF CRMP ON GBM MIGRATION...... 55

INDIRECT IMMUNOFLUORESCENCE OF CRMP PROTEINS ...... 56

IN VIVO TUMOUR IMPLANTATIONS ...... 57

ASSOCIATION BETWEEN CRMP EXPRESSION AND SURVIVAL IN GBM PATIENTS ...... 58

EXPRESSION LEVELS OF CRMP IN GBM CELL LINES...... 59

CHAPTER 4 : DISCUSSION ...... 76

BIBLIOGRAPHY ...... 84

3 LIST OF FIGURES

Figure 1.1: Common properties of CRMP proteins ...... 38

Figure 1.2: Model for Akt/GSK-3β/CRMP2 pathway in neurite outgrowth ...... 40

Figure 1.3: CRMP1 and the Reelin Signaling Pathway ...... 42

Figure 3.1: Verification of CRMP overexpression ...... 60

Figure 3.2: Analysis of cell proliferation ...... 62

Figure 3.3: Effect of CRMP1 on glioma cell migration ...... 64

Figure 3.4: Effects of other CRMP members on glioma cell migration ...... 66

Figure 3.5: Indirect immunofluorescence of CRMP-transfected cell lines ...... 68

Figure 3.6: In vivo tumour implantations ...... 70

Figure 3.7: The effect of CRMP1 and CRMP4 expression on GBM patient survival ...... 72

Figure 3.8: Real-time PCR analysis of CRMP expression ...... 74

Table 1.1: Sequence Homology of CRMP proteins...... 34

Table 1.2: Developmental Expression of CRMP Proteins ...... 36

Table 4.1: Chromosomal Alterations in GBM ...... 82

4 ACKNOWLEDGEMENTS

First and foremost, I would like to thank my supervisor, Josephine Nalbantoglu, who has been a huge source of support throughout my time in her laboratory. Her patience, insight and guidance have helped me become a better person and a better scientist. I am also grateful for her encouragement in my pursuit of a future in medicine. I thank her from the bottom of my heart.

My colleagues have been a huge source of support and over the past two and half years. These individuals include: Cara Lau, Zaf Koty, Zivart Yasruel, David Huang, Patrick Fok, Nadia Houri, Vinit Srivastava, Raj Deol, Mike Sabbagh and Lisa “Smelly” Feldman. I would particularly like to thank Nancy Larochelle, for tolerating my endless questions regarding experimental procedures. Luyu Zheng was the only student to begin at the same time as me, and hold a special place in my heart. Things would not have been nearly as much fun without her. I would also like the thank Dr. Rolando Del Maestro and Dr. Alyson Fournier, and their staff, for their help and kindness. In addition, the workers at the Montreal Neurological Institute (MNI) the animal facility were instrumental in helping me along the way with my in vivo studies. I also would like to express my gratitude to the Montreal Centre for Therapeutics in Cancer (MCETC), for their fellowship support.

Lastly, I would like to thank my family. Their love has shaped who I am today. My parents, Odile and Peter, have always encouraged me to follow my dreams and that anything is possible with hard work. My brother, Ben, has been a huge source of support during my time as a graduate student. I am grateful that these last three years at McGill have brought us closer together than we ever were before. Jenny, Isaac, Jane, Joy, Steven, Erica, Dave, Michelle, Jen, Diana, Greg, Ethan, Andrew, Sapna – I am truly blessed to have you as my grandparents/uncles/aunts/cousins/friends. 5 ABBREVIATIONS

ANOVA Analysis of Variance BDNF brain-derived neurotrophic factor CAR Coxsackie and Adenorvirus Receptor Cdk5 Cyclin-dependent kinase 5 CRMP Collapsin Response Mediator Protein DHPase Dihydropyrimidinase F-actin Filamentous Actin GBM Glioblastoma Multiforme GC Growth Cone GSK-3β glycogen synthase kinase-3β MT Microtubule NB Neuroblastoma NLS Nuclear Localization Sequence NP1 Neuropilin 1 NT Neurotrophin PlexA1 PlexinA1 RMS Rostral Migratory Stream ROCK Rho-associated kinase Sema3A Semaphorin 3A SVZ Subventricular Zone

6 ABSTRACT

GBM is an advanced-stage form of brain cancer with an extremely low survival rate. Tumour reoccurrence is quite common, resulting from micropockets of tumour cells outside the primary mass, which makes surgical resection extremely difficult. Previous research in our laboratory suggested a link between GBM invasiveness and CRMP1 (Collapsin Response Mediator Protein 1), a member of a family of phosphoproteins involved in cytoskeletal dynamics and already considered a tumour invasion suppressor gene in Non-Small Cell Lung Cancer (NSCLC)1. The aim of this study was to examine the role of CRMP1 in GBM cell migration. Several CRMP members and longer alternatively spliced variants (long form - L, normal form - S) were also examined to determine common/distinct properties. CRMP cDNAs were transfected into glioma cell lines (U87, U251N) to generate stable pooled populations which were characterized in vitro and in vivo in terms of cell viability, cell migration and tumour growth. Although there was no significant difference in cell proliferation as compared to control neo-transfected glioma cell lines, significantly fewer cells migrated in a Boyden chemotactic assay in lines overexpressing CRMP1-S, CRMP2-S, or CRMP4-S. In athymic mice, intracerebral implantation of cells overexpressing CRMP1-S yielded tumours that were significantly smaller in volume as compared to those produced by control glioma cells. In addition, the analysis of gene expression profiles in GBM patient samples collected by the National Cancer Institute (REMBRANDT) established a link between patient survival and CRMP1 and CRMP4 levels. In conclusion, our results suggest that CRMPs 1, 2 and 4 may regulate glioma cell migration and CRMP1 and CRMP4, may be involved in GBM patient survival.

7 RÉSUMÉ

Le glioblastome multiforme (GBM) est un stade avancé de cancer du cerveau avec un très faible taux de survie. Sa récurrence est assez fréquente, causée par la présence de cellules tumorales en dehors de la masse primaire, ce qui rend difficile la résection chirurgicale. Des recherches antérieures dans notre laboratoire ont suggéré un lien entre le pouvoir envahissant du GBM et CRMP1 (Collapsin Response Mediator Protein 1), un membre d'une famille de phosphoprotéines impliquées dans la dynamique du cytosquelette et déjà considéré comme un gène suppresseur d’invasion dans le cancer du poumon. Le but de cette étude était d'examiner le rôle du CRMP1 dans la migration cellulaire des GBM. Plusieurs autres membres de la famille CRMP ainsi que leurs isoformes épissées differentiellement (forme longue - L, la forme normale - S) ont également été examinés afin de déterminer les propriétés en commun. L’ADN complémentaire des CRMP a été transfecté dans les lignées de cellules de gliome (U87, U251N) pour générer des populations stables qui ont été caractérisées in vitro et in vivo sur le plan de la viabilité cellulaire, la migration cellulaire et la croissance de la tumeur. Bien qu'il n'y ait pas eu de différence significative dans la prolifération cellulaire par rapport a la lignée contrôle, beaucoup moins de cellules migrèrent dans un essai de type Boyden dans les lignées surexprimant soit CRMP1-S, CRMP2-S, ou CRMP4-S. Dans les souris thimoprivées, l'implantation intracérébrale de cellules surexprimant CRMP1-S a produit des tumeurs qui ont été nettement plus faible en volume par rapport à celles produites par des cellules contrôles. En outre, l'analyse des profils d'expression génique dans les échantillons des patients GBM recueillis par l'Institut national du cancer (Rembrandt) a démontré un lien entre la survie des patients et les niveaux du CRMP1 et CRMP4. En conclusion, nos résultats 8 suggèrent que CRMPs 1, 2 et 4 pourraient contrôler la migration des cellules de gliome et CRMP1 et CRMP4, pourraient être impliqués dans la survie des patients

GBM.

CHAPTER 1 : INTRODUCTION

10 Preamble

The most common primary brain tumour among adults also happens to be the most malignant. Glioblastoma Multiforme (GBM) is classified as a grade IV tumour, and, despite current treatments that include radiotherapy, chemotherapy and surgical excision, the median survival rate remains roughly 1 year2. GBM can occur de novo (primary GBM) or by the transformation of a lower-grade astrocytoma (secondary GBM), each type exhibiting different hallmark genetic alterations.

In situ cell migration is a multi-step dynamic process which involves adhesion to the extracellular matrix (ECM), cell motility through the reorganization of the actin cytoskeleton/microtubule networks, and invasion via degradation of the matrix components (reviewed by Lefranc et al and Giese et al). One of the characteristics that make GBM so deadly is its highly invasive nature within the brain. As a result, tumour micropockets often form outside the primary mass. This makes complete excision near impossible, recurrence often striking around the vicinity of the former surgical site. Curiously, while malignant gliomas are extremely invasive within the brain, they rarely metastasize to other organs.

The idea to study the effects of CRMP on GBM invasiveness originated from previous work on the Coxsackie and Adenovirus Receptor (CAR). CAR is a plasma membrane glycoprotein of the immunoglobulin family (Ig) involved in the

11 attachment and infection of cells by group B coxsackieviruses and adenoviruses3.

While its biological function remains unclear, it appears to be essential for early

Central Nervous System (CNS) development3,4. The expression of CAR has also been linked to tumour progression in several cancer types.

The discovery that CAR might play a role in cancer was rather serendipitous. Cancer researchers first studied CAR expression to determine if adenoviral gene therapy could be a viable therapeutic option. To their surprise,

CAR expression varied greatly among cancer lineages3,5. In glioma, CAR levels inversely correlates with progression towards malignancy5.

Previous research in our lab showed that there was a significant decrease in tumour cell migration upon the reintroduction of CAR into U87 glioma cells6. This effect was mediated by the cytoplasmic domain, suggesting the involvement of a downstream signaling mechanism. Mass spectrometry of neonatal mouse brains revealed that CRMP1, CRMP2 and CRMP4 (among other proteins) could interact with the cytoplasmic tail of CAR (Fok et al., unpublished results). Since CRMP1 had been previously described as an invasion-suppressor gene in non-small cell lung cancer (NSCLC)7, we decided to investigate if it might mediate similar effects in a brain tumour model. The goal of our study was to determine if CRMP1 exerted an effect on GBM migration. Given the high sequence homology between

CRMP family proteins, several other CRMP members (and alternatively spliced variants) were also examined to determine if our results were CRMP1-specific or a shared characteristic among family members.

12 The CRMP family is comprised of five cytosolic phosphoproteins that are highly homologous but possess distinct temporal and spatial expression patterns8-10.

Their high interspecies conservation suggests a prominent role in brain development8,9,11-17. The first CRMP protein was discovered in 1995 (CRMP2) through its involvement in axonal outgrowth and semaphorin 3A

(Sema3A/Collapsin-1) mediated growth cone collapse12,15. Since then, CRMP proteins have been discovered under different biological contexts, and a variety of nomenclature exists (Table 1).

Since the normal functional pathways of CRMP proteins are likely involved in their influence on cancer, they will be discussed in detail. For example, CRMPs can affect microtubule (MT) and F-actin dynamics, a property that may contribute to the cytoskeletal changes required during tumour migration.

Therefore, this introduction will discuss our current understanding of the biological roles of CRMP – in neurite outgrowth/retraction, migration and proliferation/apoptosis – and conclude with their pathological roles, in neurodegenerative diseases and cancer.

CRMP Sequence Homology

The CRMP family shares high sequence homology with several other proteins.

They are likely related to the nematode unc-33 gene, whose mutation causes aberrant outgrowth and uncoordinated movement11,12,15,18-20. Sequence homology

13 is also shared with the bacterial D-hydantoinase (DHP) and Dihydropyrimidinase

(DHPase), the latter being involved in uracil and thymine catabolism, leading to

CRMP proteins being classified as members of the dihydroorotase-like superfamily12,13. In fact, CRMP5 appears to be more closely related to DHPase than to CRMP1-421,22. However, no enzymatic activity has been observed among members12. Although both CRMP1 and CRMP2 possess a cavity similar to the

DHP active site, the DHP catalytic residues are not conserved and amidohydrolytic activity is absent23,24. Thus, it has been suggested that CRMPs may utilize the characteristic dihydroorotase fold to bind molecules for a different biological purpose1,24. However, little has been shown to this effect.

Alternative Splicing

Both CRMP1 and CRMP2 have been characterized at the genomic level and have been demonstrated to be composed of 14 exons25,26. However, there exists an alternatively spliced variant for each protein as a result of an alternate exon located upstream of exon127-29. Alternative splicing leads to a longer (~75kDa) protein product with a unique N-terminus28,30. Splice variants of CRMP1-4 have been discovered in chicks, while variants of CRMP1, CRMP2 and CRMP4 have been identified in humans, mice and rats. CRMP-5, however, appears to exist as a single isoform in all species examined. The common 64kDa form is expressed far more abundantly and appears to be functionally distinct from its longer counterpart29-31.

Of the longer splice variants, CRMP1 and CRMP2 are the most abundant and

14 widely expressed within the CNS28. While the common (short) form is referred to as the B-variant in chick models, it is considered the A-variant in humans, mice and rats. To avoid any misunderstanding, the short common form of CRMP will be referred to as CRMP#-S (short) and the longer splice variant as CRMP#-L (long).

Furthermore, since the vast majority of research has focused on CRMP-S, any reference to CRMP proteins in this work will imply this form unless otherwise noted.

Differential Expression Patterns of CRMP isoforms a) Central Nervous System (CNS)

CRMP proteins are primarily expressed in post-mitotic neurons in the developing

CNS. However, differential expression does exist in certain oligodendrocytes as well10,32-34. Their expression commences during embryogenesis (~E15), peaks soon after birth (~P6), and is dramatically downregulated during adulthood8-12,14-

16,21,22,32,33,35-40. Spatial distribution in the developing brain is quite similar among

CRMP members. Areas of high expression include the developing cerebral cortex, cerebellum, olfactory bulb, hippocampus, retina, dorsal root ganglia (DRG) and spinal cord. CRMP3, however, is mostly expressed in the olfactory bulb, DRG, cerebellum and retina, with weak to undetectable levels in other areas of the CNS.

While CRMP expression is considerably downregulated during adulthood, it still persists in certain areas, including the cerebral cortex, cerebellum, olfactory bulb and hippocampus. Part of the transcriptional regulation of CRMP1 and CRMP4 15 during brain development appears to be under the control of the DNA binding protein, nuclear factor I-A (NFI-A)41. However, it is unclear if other CRMP proteins are regulated similarly.

Recent analyses have discerned several differences in the developmental expression of the longer CRMP-L splice variants as compared to their shorter counterparts.(Table 3). For example, both CRMP1-L and CRMP2-S appear earlier in development than their counterparts30. Also, CRMP2-S persists in adulthood even though CRMP2-L does not. Furthermore, CRMP proteins and their N- terminal splice variants exhibit distinct subcellular localization patterns, which may also differ depending on cell maturation30-32.

b) Outside the CNS

While little work has been done with regard to CRMP proteins outside the CNS, some CRMP expression has also been observed in the Enteric Nervous System

(ENS). In mice, mRNA expression of CRMP1, CRMP2 can be detected in enteric ganglia at E14, while CRMP4 appears at E1242. During adulthood, CRMP2 expression dominates in the small intestine, while CRMP4 is the major isoform present in the large intestine. However, enteric expression of CRMP1 is undetectable in adult mice, paralleling the dramatic downregulation observed within the CNS. It is unclear what role, if any, these three proteins play in the development and maintenance of the ENS.

16 Attempts to detect CRMP expression outside the nervous system have led to varied results. This may reflect the detection methods used and the relatively weak levels present.

Several CRMP proteins are expressed in lung tissue (CRMP1-2, CRMP4), and in post-meiotic germs cells in adult testis (CRMP1, CRMP4-5)7,8,11,13,27,43,44. In addition, both CRMP2 and CRMP4 were faintly detected in the heart, kidney, T- lymphocytes and skeletal muscle. Weak levels of CRMP4 are also found in the liver and placenta.

CRMP4 possesses an alternate mRNA construct (~2.2kb instead of ~5.5kb) that is expressed exclusively in adult testis and encodes a highly distinct N- terminal11,44. Moreover, differential expression of CRMP4 has been linked to myogenic differentiation, in vitro, potentially through an evolutionarily conserved consensus MyoD/myogenin binding site9,13,27. These observations give rise to the possibility that CRMP proteins exert tissue-specific functions that may be quite distinct from their role in the CNS.

Structure of CRMP proteins

CRMP proteins form tetramers in a bi-lobed “lung-shaped” structure, the core comprised of an eight-fold α/β TIM barrel23,24. Oligomerization is promoted by the presence of divalent cations, such as Ca2+ and Mg2 and likely occurs through the binding of two dimers (e.g.: a dimer of CRMP1 binding to a dimer of CRMP2)24,45.

Assembly is not random, as CRMPs possess unique binding affinities towards other

17 members and heterotetrameric interactions are preferred22,46. For example, CRMP3 and CRMP5 prefer to bind with CRMP2 over CRMP121,22,47. While little work has been done to elucidate the functional distinctions among arrangements, it is easy to see how this mechanism could be used to establish functional diversity. Combined with their differential expression, heterotetramerization could allow for specifically localized biological functions. Further investigation is warranted and would likely contribute to a greater understanding of CRMP function and the relevance behind each member’s heterotetrameric binding affinities.

Posttranslational Modifications a) Phosphorylation

CRMPs can be phosphorylated by a number of kinases, most notably Glycogen

Synthase Kinase 3β (GSK-3β), cyclin-dependent kinase 5 (Cdk5) and Rho- associated kinase (ROCK). Phosphorylation by GSK-3β only occurs following a priming phosphorylation at the residue Ser522, which is achieved by Cdk548-51.

With the exception of CRMP3, all CRMP members contain a Cdk5 consensus site[(S/T)PX(K/H/R)] around residue 52251. CRMP1 also possesses an additional potential Cdk5 site at the Thr509 residue50. Alternate priming kinases may exist as

CRMP phosphorylation by GSK-3 is not completely inhibited in Cdk5-/- mice.

One potential candidate is the Dual Tyrosine-Regulated Kinase 2 (DYRK2), which can phosphorylate CRMP2 and CRMP4 at their Cdk5 site50. In fact, DYRK2 can

18 phosphorylate CRMP4 at a higher rate than Cdk5 in rat primary cortical neurons, suggesting that it may the main priming kinase for CRMP450.

Rho-associated protein kinase (ROK/ROCK/Rho-kinase) is a serine/threonine kinase known to phosphorylate residue Thr555 of CRMP-2 52.

Sequence alignment of CRMP isoforms demonstrates that both CRMP-4 and

CRMP-5 have a serine residue at that location, while CRMP-1 and CRMP-3 have a

Thr554 residue, raising the possibility that all 5 isoforms may be regulated through

ROCK. (Fig.1)

b) Calpain Cleavage

CRMP proteins are susceptible to proteolytic cleavage by calpain, a calcium- dependent protease that is activated by N-methyl-D-aspartate (NMDA) receptors

23,53-59 and possibly downstream of Phospholipase A2 (PLA2) signaling . Calpain cleavage of CRMP yields a single protein of roughly 54-58 kDa, with the exception of CRMP1, which can yield two smaller different cleavage products (55kDa and 58 kDa)53,55,56. This cleavage occurs toward the C-terminus of all CRMP members except for CRMP355. Given that CRMP proteins located in the synaptosome have a higher sensitivity to calpain cleavage than cytosolic CRMP, it has been suggested that cleavage may be a locally restricted phenomenon56.

Truncated CRMPs have been implicated in neuroplasticity and cell death following cerebral ischemia, oxidative stress and glutamate excitotoxicity53,55-58.

However, precise mechanisms remain unclear. Truncated CRMP may exert its

19 effects within the nucleus, as they have been observed to translocate following calpain cleavage56. The CRMP2 sequence possesses a nuclear receptor LxxLL binding motif and two nuclear localization signal (NLS) sequences, one of which has demonstrated functional activity (Arg471+Lys472) following C-terminal cleavage60. It has been suggested that its cleavage may allow CRMP to either dissociate from its binding partners, or to expose its putative NLS, potentially masked by protein folding. Since the NLS sequence found in CRMP2 is highly conserved in all CRMP isoforms (Fig.2), it is conceivable that they may translocate in a similar manner.

c) Other Modifications

Other post-translational modifications include O-glycosylation (O-GlcNAc) and biotinylation61,62. Predicted glycosylation sites exist on the C-terminal tail of

CRMPs and have been proposed to affect either subcellular localization or function by blocking phosphorylation sites1,24.

Biological Functions of CRMP Proteins a) Neurite outgrowth

All five CRMP proteins have demonstrated an influence on neurite outgrowth alone and may mediate this effect in various pathways23,50,55,63-66. For example, various

CRMP proteins have been implicated in the neurite outgrowth effects of

20 neurotrophins, including glial cell-line derived neurotrophic factor (GDNF)67, nerve growth factor (NGF)11, neurotrophin-3 (NT-3)49,68, brain-derived neurotrophic factor (BDNF)49, and fibroblast growth factor (FGF)69. In addition, CRMP1-4 can interact with the extracellular matrix (ECM) protein chondroitin sulfate and, given that CRMPs are released into the ECM following cortical cell death, they may also belong to a group of chondroitin sulfate-bound cues that influence neurite outgrowth70.

Some CRMP functions are likely distinct among members. For example, unlike other CRMPs, the overexpression of CRMP5 can cause a decrease in neurite length, induce supernumerary growth cones and an increase in filopodial length71.

CRMP1 has been implicated in the modulation of the Wnt/β-catenin pathway, which possesses diverse functions that include neurite outgrowth and dendritic arborization72,73. On the other hand, CRMP 3 effects appear to be dendrite-specific, as demonstrated in knockout mice 64. Thus, it appears that while CRMP members share certain mechanistic properties, they may also manifest distinct functional characteristics.

The ability of CRMPs to modulate neurite outgrowth has been primarily attributed to their interactions with cytoskeletal proteins. CRMPs are microtubule associated proteins that promote MT assembly by binding directly to tubulin heterodimers74. The interaction between CRMP and tubulin is phosphorylation- dependent. Once phosphorylation occurs, CRMP proteins are no longer able to interact with tubulin, abolishing their MT-assembly effects and inhibiting neurite

21 outgrowth49-51. This mechanism is likely involved in the neurite extension effects caused by neurotrophins. Stimulation by NGF, NT-3 and BDNF leads to a decrease in CRMP phosphorylation status, the latter two mediating this effect through the inhibition of GSK-3β9,11,49. In the case of NGF, this dephosphorylation occurs in conjunction with an increase in CRMP1, CRMP2 and CRMP4 expression and decrease in CRMP3.

MT assembly function of CRMP proteins is necessary but not sufficient to promote neurite outgrowth74. Their involvement in neurite outgrowth is likely to be further influenced by three additional pathways: dynamic F-actin assembly, cargo transport, and endocytosis. In vitro, CRMP4 can promote F-actin bundling75.

Furthermore, its knockdown leads to the disruption of the F-actin structure in neuronal lamellipodia. While CRMPs colocalize with F-actin regardless of phosphorylation state, it has yet to be determined if their effects on actin dynamics are affected by this modification15,28,31,49,74,76,77 However, since the ability of

CRMP proteins to promote neurite outgrowth is abolished after phosphorylation, it seems unlikely that F-actin assembly would continue. This property may simply be a means of maintaining proper CRMP localization within neuritic processes.

CRMP2, and potentially CRMP1, can act as cargo adaptor proteins by linking essential cytoskeletal proteins and mediating their transport to the distal growth cone. The distal transport of tubulin and the Rac1-associated protein/WASP family verprolin-homologous protein 1 (Sra-1/WAVE) complex, a known regulator of F-actin dynamics, occurs through their binding to CRMP2,

22 which in turn binds to the motor transport protein Kinesin-145,78,79. In addition, observations from crmp1-/- mouse brains suggest that it may be required for the proper distal dendritic transport of Microtubule-Associated Protein 2 (MAP2), a protein thought to be involved in MT assembly80.

Both CRMP2-S and CRMP4-L have been directly implicated in endocytic pathways that promote neurite outgrowth. CRMP2-S is involved in the clathrin- dependent recycling of neuronal cell adhesion molecule L1 through its interaction with Numb81. This interaction may be common among CRMP members, as

CRMP2/Numb binding occurs via a highly conserved region (the PTB domain of

CRMP). Similarly, CRMP4-L can bind the endocytic-exocytic adaptor protein, intersectin, and colocalizes with synaptic vesicle protein 2 (SV2)-positive vesicles30.

As demonstrated previously with CRMP4-L, CRMP splice variants can possess distinct biological roles28,30,31. The functional differences are likely mediated through their highly specific N-terminal domains. Splice variants can mediate their functions through their influence on CRMP-S activity as well as their interactions with other signaling molecules. For example, while CRMP2-L does not affect axon elongation or branching in chick retinal ganglion cells (RGCs), its coexpression with CRMP2-S blocks CRMP2-S-mediated outgrowth28. Moreover, the interaction between the N-terminal domain of CRMP4-L and RhoA is important for the inhibitory effects of outgrowth inhibiting molecules such as chondroitin sulfate proteoglycans31, while CRMP1-L can inhibit the catalytic activity of ROCK

23 by binding to its kinase domain, potentially regulating the phosphorylation status of other CRMP members29.

b) The Sema3A (Collapsin-1) Pathway

The activities of CRMP1-4 (particularly that of CRMP1 and CRMP2) are involved in the signal transduction of Sema3A-induced neuronal growth cone collapse. In contrast, CRMP5 exerts an inhibitory role on Sema3A-induced growth cone collapse, possibly through its heterotetramerization with CRMP271. In addition to growth cone collapse, the Sema3A/CRMP pathway has been linked to effects on the branching morphogenesis in lung tissue82, oligodendrocyte process extension34, chemorepulsion of axons83, neural progenitor cell migration83,84, and dendritic spine morphology85.

CRMP1, CRMP2 and CRMP4 can form a physical complex with the

Sema3A co-receptor, PlexinA1 (PlexA1), acting downstream of PlexA1- activation23. However, CRMP proteins can mediate collapse even in the absence of

PlexA1. An additional co-receptor, neuropilin-1 (NP1), is required for Sema3A signaling and blocks CRMP/PlexA1 interaction under basal conditions.

Additional CRMP protein interactions may be functionally relevant to growth cone collapse. Molecules interacting with CasL (MICAL) is an intracellular protein with enzymatic activity known to influence proper axonal guidance in

Drosophila86,87. Truncation of MICAL can furthermore lead to a dominant negative effect on Sema3A response in vitro. In addition, CRMP1-4 can form a 24 complex with MICAL and PlexA1, and may play a role in activating MICAL enzymatic activity. It has been proposed that this activity is potentiated through

CRMP association with trans-plasma-membrane oxidoreductases (PMO)61 and facilitated by mutual interaction with Plexin71,86. In addition, the Rac-GTPase

Activating Protein (GAP), 2-chimaerin, can interact with PlexA1, CRMP2 and

Cdk5 and its activity is required for Sema3A-induced growth cone collapse48.

These observations have led to the suggestion that CRMP may exist in a large

NP1/PlexA1 regulatory complex. Finally, the activity of neuronal phospholipase

D2 (PLD2), is inhibited by Sema3A signaling and its direct association with

CRMP288. While the downstream effects are unclear, the colocalization of CRMP2 and PLD2 to the distal tips of neurites suggests that this mechanism may be implicated in growth cone collapse as well.

The modulation of CRMP-MT interactions through phosphorylation state is a key mechanism in Sema3A-induced growth cone collapse. Sema3A signaling leads to CRMP phosphorylation by Cdk5 and GSK-3β and is necessary to mediate growth cone collapse48,51. However, additional phosphorylation pathways exist.

Both Fes/Fps tyrosine kinase and the closely-related Fer tyrosine kinase are linked to the Sema3A pathway in part by their ability to phosphorylate CRMP2 and

CRMP5 and their kinase-deficient mutants can significantly diminish the Sema3A response47,89.

25 c) CRMP and the Rho Pathway

Unlike Sema3A, lysophosphatidic acid (LPA) and ephrin-A5 both induce growth cone collapse through the RhoA signaling pathway, which leads to ROCK- mediated phosphorylation of CRMP229,52. Similarly, the consequence of this phosphorylation is an inability for CRMP to bind either tubulin or Numb, while F- actin association remains intact76.

N-terminal splice variants have demonstrated distinct roles in Rho signaling. As previously mentioned, RhoA has a higher binding affinity for

CRMP4-L than CRMP4-S, and their association is required for neurite outgrowth inhibition during exposures to myelin or aggrecan31. Furthermore, stimulation by the myelin associated inhibitor, Nogo-A, increases RhoA/CRMP4-L interaction

(but not that of CRMP4-S) and also appears to mediate neurite outgrowth inhibition. CRMP1-L, unlike CRMP4-L, induces RhoA-mediated neurite retraction by binding directly to the kinase domain of ROCK, which leads to the inhibition of its kinase activity29.

d) Neuronal Plasticity

While CRMPs are vastly downregulated during adulthood, their expression can still be detected in areas that retain postnatal neurogenic ability, including the SVZ,

RMS, olfactory bulb, and dentate granule layer of the hippocampus37,39,90-92. They have been shown to exert effects on Long Term Potentiation (LTP), neuronal polarity, and their upregulation is associated with neurogenic differentiation in 26 several cell types18,21,27,64,65,80,93. The effects of CRMP proteins on neuronal polarity, likely through neurite/axonal induction, are mediated through the upstream

Ras/PI3-K/Akt pathway, whose activation leads to phosphorylation49,63,77.

The calpain cleavage products of CRMP proteins have been linked to neuroplastic effects induced through hyperalgesia, neuropathic pain, ischemia and excitotoxicity94-96. During ischemic injury and glutamate cytotoxicity, the influx of

Ca2+ through the NMDA receptor leads to calpain activation and the cleavage of all five CRMP proteins53,55-57. Both CRMP2 and CRMP3 cleavage have been implicated in the modulation of NMDA/glutamate excitotoxicity effects53,55,95. In the case of truncated CRMP2, this effect has been attributed its ability to downregulate the NMDA subunit, NR2B.

The regulation of the interaction between CRMP5 and the glycine transporter 2 (GlyT2), may be also be involved in neuroplasticity. GlyT2 is important in mediating glycine vesicular reuptake and its inhibition leads to a switch from a glycine to GABA phenotype97. In its phosphorylated form, CRMP5 can bind GlyT2 and may contribute to GlyT2 trafficking and/or function98. The presence of putative calpain cleavage sites in the CRMP5 binding region may also provide a regulatory mechanism for this interaction.

The upregulation of CRMP proteins has has been observed in several different models of axonal regeneration, including olfactory and sciatic axotomy, as well as following hypoglossal nerve crushing15,39,66. In addition, their phosphorylation as well as splice variants have been linked to the inhibition of

27 axonal regeneration. For example, the phosphorylation of CRMP2 by ROCK is necessary to mediate the inhibition of axonal regeneration caused by Nogo and myelin-associated glycoprotein (MAG), both myelin-derived inhibitory molecules99. CRMP4-L has also been linked to Nogo-dependent neurite inhibition31. In addition, the expression of CRMP2 and CRMP5 in certain oligodendrocytes, coupled with the high level of Sema3A present in scar tissue fibroblasts suggests that CRMP proteins may also affect the regenerative functions of oligodendrocytes10,100,101.

e) Cell Migration

CRMP proteins have been implicated in neuronal (CRMP1, CRMP2 and CRMP4) and tumour (CRMP1 and CRMP4) cell migration7,14,39,75,80,91,102,103, as well as the infiltrative properties of T-lymphocytes that occur during certain diseases (CRMP2 and CRMP4)1,104. Of these associations, the effect of CRMP1 on neuronal migration through Reelin (Rln) signaling is the best understood. Reelin (Rln) is a secreted protein that binds to ApoER2/VLDR receptors and whose knockout leads to an inversion of cortical layers, referred to as the Reeler phenotype105. Both

CRMP1 and cytoplasmic adaptor protein disabled-1 (Dab1) are phosphorylated by

Fyn downstream of Rln signaling and upregulated in rln-/- mice103. Furthermore, knockout of crmp1 in dab1+/- mice lead to a reeler phenotype, which is not observed in dab1+/- mice alone. Other studies on crmp1 knockouts have shown that

CRMP1 effects on neuronal migration and positioning are not restricted to the

28 cerebral cortex and can affect the cerebellum and hippocampus80,91. In addition, he upregulation of CRMP proteins occurs in neuronal precursor cells, originating from the subventricular zone (SVZ), that are migrating to the olfactory bulb, or to a site of hippocampal damage14,39,102. Thus, it appears that CRMP1 may help in the proper migration and positioning of neuronal precursors during development and following injury.

While the precise mechanisms remain unclear, there is some evidence to suggest that the effects of CRMP proteins on migration are due to their regulation of cytoskeletal dynamics. For example, in B35 neuroblastoma cells, the knockdown of CRMP4 enhanced cell migration while disrupting F-actin in lamellipodia75. In addition, CRMP proteins have been implicated in the effects of neurofibromin on migration and neurite outgrowth. Neurofibromin has been shown to directly associate directly with CRMP2, CRMP4, and potentially with

CRMP1106,107. Moreover, neurofibromin can regulate CRMP2 activity by reducing its phosphorylation, potentially through direct interactions as well as the suppression of the CRMP2-phosphorylating kinases ROCK, GSK-3β and Cdk5107.

Recent experiments have demonstrated that the knockdown of neurofibromatosis type 1 (NF1), the gene for neurofibromin, results in an increase in cell migration and a reduction in neurite outgrowth, likely mediated through its regulation of F- actin dynamics107,108. It has been proposed these results are due to the increased activity of the ROCK/LIMK and Ras/PI3-K/Akt pathways, respectively, both

29 normally suppressed by neurofibromin, which would consequently result in an increase in CRMP phosphorylation.

f) Neuronal Proliferation/Apoptosis

The link between CRMP proteins and apoptotic/replicative pathways is unclear.

Studies of CRMP2 as a potential modulator of apoptosis have led to contrasting results109-112. In crmp1-/- mice, granule cells residing in the external granular layers of the cerebellum demonstrated a decrease in cell proliferation and apoptosis91.

However, this effect is likely transient, as it was only observed in the developing brain.

Truncated CRMP proteins colocalize with TUNEL-positive nuclei in ischemic mouse and human brain tissues55,56. In addition, the overexpression of cleaved CRMP2 or CRMP3 has been shown to induce cell death, while CRMP4 cleavage is observed following oxidative stress55,57,111. These observations suggest that calpain-mediated cleavage of CRMP proteins may have an important role in apoptosis, likely mediated through nuclear localization. CRMP2, along with

CRMP4-5, are hypophosphorylated in the mouse model for perinatal hypoxia and ischemia (HI), suggesting that phosphorylation may also be involved113.

30 Implications of CRMP Proteins in Neurodegenerative Disease

Abnormal CRMP expression, phosphorylation and cleavage have been linked to several neurodegenerative diseases, including schizophrenia, Down’s Syndrome,

Mucopolysaccharidosis type IIIB (MPSIIIB), and most notably, Alzheimer’s

Disease (AD)40,45,50,55,59,65,114-119. In AD, CRMP2 is highly phosphorylated and localized around dystrophic neurites and amyloid plaques. This suggests that

CRMP hyperphosphorylation may contribute to AD disease pathology, likely through its cytoskeletal effects.

The Association of CRMP Proteins with Cancer

Several studies that have analyzed the gene expression and secretion patterns of cancer tissues have identified certain CRMP abnormalities.

Upregulation of CRMP1, CRMP3 or CRMP4 has been detected in the gene expression profile studies of malignant prolactin (PRL) pituitary tumours,120 astrocytoma, osteosarcoma and squamous cell carcinoma as well as human medulloblastoma (MB) and glioma tissue samples121,122. In addition, both CRMP2 and CRMP5 secretions have been observed, in colorectal cancer (CRC) and in certain cancer patients suffering paraneoplastic neurological disease (PNDs), respectively10,35,123. In the case of CRMP5, auto-antibodies have been detected in the serum of neuroendocrine small cell carcinoma (SCLC) and thymoma patients exhibiting multifocal signs121,122,124. However, the presence of CRMP5 auto- antibodies did not correlate with survival rate122. Furthermore, none of the 31 aforementioned studies have established whether these altered expression levels impact prognosis or what role, if any, they possess. They also neglected to test for phosphorylation, a key regulator of CRMP activity, or to discriminate between splice variants, which have demonstrated functional diversity. Another study that examined Ataxia telangiectasia mutated (ATM) mouse tumours observed CRMP2 hyperphosphorylation on its ROCK sites111. Thus, the impact and accuracy of the previously observed upregulations are unclear.

The role of CRMP1 as an invasion suppressor gene in non-small cell lung carcinoma (NSCLC) is most the well-understood association between CRMP members and cancer pathology1. The expression of CRMP1 inversely correlates with lung tumour invasiveness, in vitro, along with advanced disease, lymph node metastasis, early postoperative recurrence, and survival, in vivo7. While the chromosomal location of CRMP1 (chr. 4p16) is a region whose deletion is associated with the transition from carcinoma in situ to invasive lung cancer, the decrease in CRMP1 expression in invasive lung cancer has been more closely linked to transcriptional inactivation125.

The transcriptional factor NF-κB is implicated in the regulation of several tumour invasion and metastasis genes126. Under normal conditions, active NF-κB exists as a p65/p50 heterocomplex. However, several highly invasive cancer models have demonstrated a shift towards the p50 subunit. Similarly, as NSCLC cells become more invasive, p50 expression increases126. The p50 subunit can bind crmp1 directly through a putative NF-κB site that has been shown to inhibit

32 CRMP1-S expression. Conversely, the transfection of antisense p50 in invasive

NSCLC cell lines results in an upregulation of CRMP1-S, a decrease in invasive ability, and a return to less invasive cell morphology. It must be noted, however that NF-κB controls numerous genes, and therefore other proteins are likely implicated. In addition, the low expression of CTGF correlates with metastasis, advanced staging, higher postoperative recurrence and lower survival rate in

NSCLC127. This too has been linked to downstream CRMP1 modulation, as the overexpression of CTGF in these cells led to a dose-dependent increase in CRMP1-

S and decrease in invasiveness.

The modulation of CRMP1 transcription may also help to explain the tumor-suppressive effects of the cyclo-oxygenase 2 (COX-2) inhibitor, celecoxib, on lung cancer. While an increase in COX-2 expression has been observed in several cancer types, recent evidence suggests that the celecoxib effects are COX-2- independent128,129. Celecoxib can increase mRNA and protein expression of

CRMP1-S in a dose-dependent manner, which is likely established through the inhibition of Sp1 binding and the enhancement of C/EBP binding130. COX-2 inhibitors may also act by decreasing CRMP degradation, as it has been shown to prevent NMDA-induced CRMP4 cleavage in rat cortical neurons58. Recent clinical trials have been established to test celecoxib’s therapeutic impact on NSCLC, however results thus far have not been encouraging124,131,132.

33 Table 1.1

A Alternate Names for CRMP

CRMP1 DRP-1, Ulip3, C-22, DPYSL1

CRMP2 DRP-2, Ulip2, TOAD-64, CRMP-62, CRM, mUNC, DPSYL2

CRMP3 DRP-4, Ulip4, POP66, DPYSL4

CRMP4 DRP-3, Ulip1, hUlip, TUC-4, nsp1, DPYSL3

CRMP5 Ulip6, CRAM, DPSYL5

B Human CRMP Sequence Homology

CRMP1 CRMP2 CRMP3 CRMP4 CRMP5

CRMP1 - 76% 69% 74% 50%

CRMP2 76% - 75% 76% 50%

CRMP3 69% 75% - 70% 48%

CRMP4 74% 76% 70% - 50%

CRMP5 50% 50% 48% 50% -

Table 1.1: Sequence Homology of CRMP proteins. A, The discovery of CRMP proteins under various biological contexts has resulted in multiple names for each isoform. Among these are: DRP/DPYSL:

Dihydropyrimidinase Related Protein 13; Ulip:Unc-33 LIke Protein ;

TOAD-64: Turned Off After Division 64 kDa 15; POP66: paraneoplastic oligodendrocyte protein of 66 kDa 38; TUC:

TOAD/Ulip/CRMP133. B, Human CRMP sequences were aligned using the ClustalW 2.0.8 Multiple Sequence alignment software, demonstrating the (Accession Numbers: CRMP1 - Q14194, CRMP2 -

Q16555, CRMP3 - O14531, CRMP4 - Q14195, CRMP5 - Q9BPU6)

Developmental Expression of CRMP CRMP of Expression Developmental Rats in Protein

2 . 1 Table Table

Table 1.2: Developmental Expression of CRMP Proteins. The level of CRMP expression found at several time points during embryonic

(Ex), postnatal (Px), and adult stages in rats. Detection levels were categorized as: - (very weak/absent), + (weak), ++ (moderate), or +++ (strong). The table was adapted from Quinn et al. 2003, and

Inatome et al. 2000.

37 Figure 1.1

A GSK-3β Cdk5 ROCK

p p p p p CRMP1 505 EVPATPKY--ATPAPSAKSSPSKHQPPPIRNLHQSNFSLSGAQIDDNNPRRTGHRI 558

CRMP2 505 EVSVTPKT--VTPASSAKTSPAKQQAPPVRNLHQSGFSLSGAQIDDNIPRRTTQRI 558 CRMP3 505 EVMVPAKP--GSGAPARASCPGKISVPPVRNLHQSGFSLSGSQADDHIARR TAQKI 558 CRMP4 503 DLTTTPKG--GTPAGSARGSPTRPNPP-VRNLHQSGFSLSGTQVDEG-VRSASKRI 556

CRMP5 497 VVVHPGKKEMGTPLADTPTRPVTRHGG-MRDLHESSFSLSGSQIDDHVPKRASARI 552

B NLS CRMP1 471 RKAFPEHLYQRVKIRNK 487

CRMP2 471 RKPFPDFVYKRIKARSR 487

CRMP3 471 RKTFPDFVYKRIKARNR 487

CRMP4 471 CSPFSDYVYKRIKARRK 487

CRMP5 464 LRSFPDTVYKKLVQREK 480

Figure 1.1: Common properties of CRMP proteins. CRMP protein sequences were aligned using ClustalW 2.0.8 Multiple Sequence

Alignment software and analyzed for common phosphorylation sites and NLS sequences. A, Phosphorylation is a major regulatory post- translational modification among CRMP proteins. The consensus sites for the major ser/thr kinases responsible are highlighted: GSK-3β (red),

Cdk5 (blue), and ROCK (green). The consensus site for Cdk5

[(S/T)PX(K/H/R)] is shown in bold. B, A functional NLS sequence

(Arg471 and Lys472) was identified on CRMP260. However, based on

CRMP sequence alignment, CRMP1 and CRMP3 possess the same sequence, while CRMP4 and CRMP5 share another potential bipartite

NLS. The basic residues required for nuclear internalization are labeled in bold.

2 . 1 Figure Figure

Figure 1.2: Model for Akt/GSK-3β/CRMP2 pathway for neurite outgrowth and axon formation. Three main CRMP functions have been suggested to mediate the effects on neurite outgrowth and axon formation. 1) CRMPs bind to tubulin dimers, promoting MT assembly.

This effect is abolished by phosphorylation. The main kinases implicated are GSK-3β, Cdk5 and ROCK. 2) CRMP2 (and possibly

CRMP1) can act as a cargo adaptor protein and assist in the transport of the Sra-1/WAVE complex, a known effector of F-actin dynamics. 3)

CRMP2 (and possibly CRMP4) is known to mediate endocytotic effects that may be necessary for neurite extension. Specifically,

CRMP2 can bind Numb and contribute to endocytosis of L1.

3 . 1 Figure Figure

Figure 1.3: CRMP1 and the Reelin Signaling Pathway. While the role of CRMP-1 in Reelin signaling is unknown, CRMP1, Dab1 and

Reln knockouts suggest a potential link. Similar to Dab1, CRMP-1 regulation may be the result of tyrosine phosphorylation by Fyn. For review of Reelin pathway see: Bielas S. et al, 2004.

43 RATIONALE

The aim of this study was to determine the role of CRMP1 in GBM migration and invasion. Since CRMP1 had been previously described as an invasion-suppressor gene in non-small cell lung cancer (NSCLC)7, it was hypothesized that it would exert a similar effect. During the course of this investigation, we also examined other CRMP isoforms and splice variants in an effort to determine if our results were unique to CRMP1.

CHAPTER 2 : MATERIALS AND METHODS

45 Cell Lines and Cell Culture – The human U87-MG glioma cell line was obtained from the American Type Culture Collection (Rockland, MD). The human U251N cell line was a kind gift of Dr. Peter Forsythe, University of Calgary. Cell lines were maintained (unless otherwise stated) at 37° Celsius and 5% CO2 in Dulbecco’s

Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), and an antibiotic cocktail (final concentration: 30 µg/ml gentamicin, 100 units of penicillin/ml, 100 µg of streptomycin/ml) (Invitrogen).

Transfections – The CRMP plasmids were graciously donated by the laboratory of

Alyson Fournier (MNI). Each plasmid was under the control of the cytomegalovirus (CMV) promoter. Along with the CRMP cDNA, each plasmid expressed a neomycin phosphotransferase gene to allow the selection of stably transduced cells with geneticin (G418 sulfate). The CRMP proteins were tagged with the V5 epitope. A third plasmid containing only the neomycin resistance gene was used as the control (neo). Cell lines were transfected using FuGENE 6 transfection reagent (Roche) then selected for 10 days with G418 (600 µg/ml). To ensure that non-transduced cells were eliminated, control plates of parental U87 and

U251N cells were treated with G418. Clones were pooled together to generate bulk populations stably expressing CRMP1-S, CRMP1-L, CRMP2-S, CRMP2-L,

CRMP3, CRMP4-S, CRMP4-L and neo. In the case of CRMP1-S, two separate pooled populations were initially generated.

Antibodies and fluorochromes – Monoclonal mouse anti-V5 antibodies were purchased from the Invitrogen and used at dilutions of 1:5000 and 1:1000 for

Western Blot and immunofluorescence, respectively. Hoechst 33258 pentahydrate was used for nuclear staining (1:10,000) for immunofluorescence and chemotaxis assays. Alexa Fluor-conjugated phalloidin (1:40) was used to detect F-actin for immunofluorescence (Molecular Probes, Inc.). Polyclonal rabbit anti-tubulin (1:80) and polyclonal rabbit anti-β-actin (1:1000) antibodies were purchased from Sigma.

Alexa Fluor-conjugated secondary antibodies (1:250) were purchased from

Molecular Probes.

SDS-PAGE and Western Blot – Cells grown in 10 cm plates were washed twice in

PBS and lysed in SDS-loading buffer (2% SDS, 10% glycerol, 0.125 M Tris-HCL pH 6.8, 1 tablet of mini, EDTA-free protease inhibitor cocktail tablet per 10 mL loading buffer). Lysates were then heated for 5 minutes at 100°C. Total protein concentration was determined by bicinchoninic (BCA) protein assay. Samples were supplemented with 5% β-mercaptoethanol and were loaded (10 µg/lane) on a

12% polyacrylamide-SDS gel. Following electrophoresis, proteins were transferred to a nitrocellulose membrane at 100V for 1hr in a mini-transblot II apparatus (Bio- rad) (Transfer buffer: 25mM Tris, 192 mM glycine, 20% methanol, pH 8.3).

Membranes were then blocked with 5% (w/v) skim milk in TBS-T (20mM Tris-

HCL pH 7.6, 0.14 NaCl, 0.1% Tween-20) under gentle shaking. Primary

47 antibodies diluted in 5% skim milk TBS-T were applied with gentle shaking overnight at 4°C. After extensive washing with TBS-T, diluted HRP-conjugated secondary antibodies (anti-rabbit at 1:3000, anti-mouse at 1:1000 in 5% skim milk and TBS-T) were applied with gentle shaking for 1hr at room temperature. Protein detection was achieved using SuperSignal Substrate (Pierce Biotechnology, Inc,

Rockford, IL, USA) as per standard manufacturer’s protocol. Chemiluminescence was detected using a cooled charge-coupled device (CCD) camera attached to an image-capturing system (GeneGnome Syngene). As a loading control, membranes were extensively washed in TBS-T under gentle shaking, and then reblotted with polyclonal anti-β-actin.

Cell Proliferation Assay – Cells were seeded in 10cm2 dishes at a density of 5x105 cells and allowed to grow for 24, 48, or 72 hrs. Following their allotted time, dishes were trypsinized and resuspend in standard supplemented DMEM. Subsequently, 5

µL of the cell suspension was diluted with 5 µL of Trypan Blue and the entire volume was loaded into a hemocytometer. Cells were counted using the hemocytometer and this value was used to calculate total number of cells per plate.

Dead cells were excluded from these calculations based on Trypan Blue infiltration.

The count was run in duplicate and average doubling time was analyzed using

Prism software (Graph Pad).

48 Cell Migration Assay – Glioma migration was assessed a Boyden transwell chemotaxis assay as described in 6, Transwell chambers were comprised of an upper chamber with a 8μm polycarbonate filter insert (Corning, Acton, MA, USA), which were placed inside the wells of 24-well tissue plates (Nunc). Cells were first serum starved for 24 hrs in DMEM containing 0.1 % Bovine Serum Albumin

(BSA). Following serum starvation, cells were suspended in DMEM (1% FBS, 1 mg/ml BSA) and seeded in the top chamber at a density of 5000 cells/chamber.

The bottom chamber was seeded with conditioned medium from control cell lines

(U87 or U251N) to act as the directional chemoattractant. Cells were allowed to migrate for 16hrs and were subsequently fixed with 4% PFA in PBS for 30 min at room temperature. Cells were then washed twice with PBS, then stained with

Hoechst dye (1:10,000 in PBS) for 30 min in the dark, then washed twice more with PBS. Cells that remained on the upper surface of the filter insert were removed gently using a cotton-tipped applicator. The remaining cells that had trans-migrated to the bottom of the filter insert were quantified using a Leica wide- field fluorescence microscope and a 4x objective. Four random fields were quantified and each assay was run in at least triplicate. Statistical analysis was performed using Prism software (Graph Pad). Student’s t-test was used for comparison of two groups (unpaired, two-tailed). For multiple groups, one-way

Analysis of Variance (ANOVA) was used along with the Tukey-Kramer post-test for pair-wise comparisons.

49 Indirect Immunofluorescence – Cells were grown on 12mm coverslips inserted into

24-well plates or 8-well chamber slides (Nunc, Rochester NY) for two days under standard culture conditions described previously. Cells were fixed with 4% paraformaldehyde (PFA) in PBS, washed in PBS then blocked for 30 mins with 5%

BSA and 0.1% Tween in PBS. Primary antibodies, diluted with blocking buffer at the appropriate dilution (mentioned previously), were then applied and cells were incubated overnight at 4°C. Cells were then washed three times with PBS, blocked for another 15 minutes, and secondary antibodies were applied for 30 minutes at room temperature. After three washes of PBS, coverslips were applied. Controls consisted of chambers that followed the same protocol but were treated with blocking buffer instead of primary antibodies. For double staining, antibodies were applied at the same time with the exception of Alexa Fluor Phalloidin and Hoechst, which were applied at the same time as the secondary antibodies. Slides were visualized under an oil immersion objective (63x) on a Leica DMIRE2 (Richmond

Hill, ON, Canada) wide-field fluorescence microscope and processed using

Openlab (Lexington, MA) software.

In vivo tumour implantations – Stereotactic injections were performed as previously described134. CD1 nu/nu athymic nude mice (6 wks old, female, Charles River

Canada, St-Constant, Quebec) were anaesthetized with 0.04 ml of anaesthetic cocktail (2 ml of ketamine, 1 ml xylene, 0.6 ml saline, 0.4 ml acepromazine for 4 ml of cocktail) by i.p injection and placed in a stereotactic apparatus (Kopf). A

50 small hole was drilled 1 mm anterior and 2 mm lateral to the bregma. Cells were suspended (1 x 105 cells) in 3 µl of Hank's balanced salt solution (HBSS) and injected stereotactically using a Hamilton syringe at a depth of 3.5 mm. To ensure that the suspension did not flow back through the needle track, the 3 µL was injected at a rate of 0.5 µL every 2 minutes, and the needle was left in position for an extra 15 minutes following injection. Mice were euthanized (31 days and 22 days later for U87 and U251N cell lines, respectively) and their brains were quickly frozen using isopentane chilled with liquid nitrogen. Coronal sections (10 µm) were prepared and stained with hematoxylin and eosin. Tumor volumes were then calculated using the formula a × b2 × 0.4 (a = longest axis, b = width perpendicular to axis). All animal experimentation was carried out according to the guidelines of the Canadian Council on Animal Care.

Gene Expression Analysis of Glioma Patients – Kaplan-Meier curves were generated on the National Cancer Institute (NCI) REpository for Molecular BRAin

Neoplasia DaTa (REMBRANDT) home page (http://rembrandt.nci.nih.gov). Gene expression data was analyzed for 193 glioma patients. Upregulation was defined as

≥2 fold expression, while downregulation was defined as ≤ 2 fold expression, using

Affymetrix reporter probes (Reporters - crmp1: 202517_at; crmp2: 200762_at; crmp3: 214301_s_at, 205492_s_at, 205493_s_at; crmp4: 201431_at, 201430_at; crmp5: 222797_at, 224100_s_at). Data was last accessed January 29, 2009.

51 Real-Time Quantitative PCR – Confluent plates of U87 and U251N cells were trypsinized, then pelleted to remove all culture media. Cells were subsequently lysed using the RNeasy Mini Kit (Qiagen) and homogenized using the

QIAShredder kit (Qiagen). RNA was extracted following the RNeasy Mini Kit protocol. The extracted RNA was used to synthesize cDNA using the Moloney

Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen). Primers for Real-Time PCR were generated using PrimerExpress software (ABI Prism) and purchased from Alpha DNA (Montreal, Canada): CRMP1-L (forward:

CGGCCGGGACAATGG, reverse: GGGATTGGTCATCGTTGATGA), CRMP1-S and

CRMP1-L (forward: CACGAAGCAGCTGACACCAA, reverse:

TCCCGAACGCCATCGTA), CRMP2-S (forward: GGTTGCACCCTTTTCAATCTTG, reverse: TAAGACATCTCTCTCCTGGGAAAAAAT), CRMP2-S and CRMP2-L

(forward: GGATTTGAGCGTTTGCCATT, reverse: CCTCATGATGGCAGGTGGTTA),

CRMP3 (forward: GCATTGAGGAGCGCATGTC, reverse:

ACCGCGACGAACTCATTCTC), CRMP4- S (forward: CGGCCCTCTTCGAATTCAC, reverse: ACAGCTTTGGAATCAGATAGACGAT), CRMP4-L (forward:

AAATACGGCGGCATGTTCTG, reverse: GCATCGAAATCCAGCGTCTT), CRMP5

(forward: CAACCCCAAGACGACACATG, reverse: ACGTGTCGGGCTGTTTAGTTTT).

Real-time quantitative PCR was performed on samples using Power SYBR Green

PCR Master Mix (Applied Biosystems), combined with the cDNA samples and appropriate primers. The reaction was carried out on the ABI PRISM 7000

Sequence Detection System (Applied Biosystems). GAP-DH was used as a

52 reference gene. Observed CRMP levels were divided by those found for GAP-DH from the appropriate cell line. Real-time PCR samples were run in triplicate and on cDNA generated from 3 different plates for each cell line.

CHAPTER 3 : RESULTS

54 In Vitro Effects of CRMP on GBM Migration

To determine whether CRMP proteins had an effect on glioma migration and invasion, U87 and U251N cell lines were transfected with plasmids containing a V5 epitope tagged-CRMP member/variant and the neomycin gene . Pooled populations were collected following G418 selection and CRMP expression was subsequently verified by Western blot analysis (Fig.3.1) using anti-V5 antibodies.

The expected bands for the short (~62-64kDa) and long (~72kDa) forms of CRMP were observed.

In vitro cell migration was determined by the Boyden Chamber Chemotaxis

Assay. Briefly, cell lines were serum starved for 24hrs, then seeded in Boyden chambers. Culture media was collected from the plates of parental cell lines that did not undergo serum starvation. This media was placed under the Boyden chamber to provide the necessary chemoattractants to induce migration. Following

16 hours of incubation time, the number of cells that migrated through the pores of the Boyden chamber was quantified. Both parental cell lines (U87, U251N) and neomycin-resistant transfection controls (U87-neo, U251N-neo) were compared to

CRMP-transfectants. Overexpression of CRMP1-S significantly decreased migration in U87 and U251N cells as compared to controls (Fig. 3.3B, C). This was also observed in cells that overexpressed CRMP2-S or CRMP4-S (Fig 3.4B, C).

However, CRMP1-L (Fig. 3.4A), CRMP3 and CRMP4-L (Fig. 3.4B, C) were unable to significantly alter migration rates.

55 In order to ensure that these findings were not affected by alterations in proliferation rate, the average doubling time of U87-CRMP1-S and U251N-

CRMP1-S cells was calculated (Fig. 3.2). Cell counts were determined over several days using a hemocytometer. Dead cells were excluded from these counts on the basis of Trypan Blue staining. In these observations there was no significant difference between CRMP1-transfected cells and controls. Furthermore, the doubling times were all longer than the duration allotted for Boyden chamber migration. Therefore, it can be concluded that the altered migration rates observed were unlikely to be affected by changes in proliferation rates.

Indirect Immunofluorescence of CRMP Proteins

In order to observe subcellular localization patterns, CRMP-transfected cell lines were labeled with anti-V5 antibodies or co-labeled with anti-V5 and either phalloidin (for F-actin), tubulin or Hoechst. All CRMP-transfected U87 cell lines manifested process extension, with the exception of CRMP1-L (Fig. 3.5A). While

CRMP was detected fairly uniformly within the cell, the strongest signal appeared in the cell body around the nucleus. In both U87 and U251N cell lines, CRMP1-L was weakly detectable at the cell periphery and almost entirely centrally located

(Fig. 3.5A). In the case of U251N cells, CRMP proteins (with the exception of

CRMP1-L) were also primarily localized at the cell periphery in intense patches.

These patches colocalized strongly with phalloidin and tubulin, suggesting that these may represent sites of membrane ruffling (Fig. 3.5B). Some of the CRMP1-

56 S, CRMP2-S and CRMP4-S-transfected U87 cells exhibited elongated processes.

A few U87-CRMP4-L cells did so as well but to a lesser extent. It is also interesting to note that, similar to observations in NSCLC cells, CRMP1-S was able to colocalize with the mitotic spindle in U87 cells (Fig. 3.5A)83,84. Since CRMP proteins have previously been observed to translocate to the nucleus, cells were treated with Hoechst for nuclear labeling. No nuclear localization was detected

(Fig. 3.5B). However, it has been suggested that translocation into the nucleus likely requires C-terminal cleavage for entry83. Since the V5-epitope is C- terminally located and may therefore be cleaved during this process, the possibility that nuclear CRMP exists in these cells cannot be completely overlooked.

In Vivo Tumour Implantations

In order to determine if CRMP1-S-mediated migrational effects could be observed in vivo, athymic (CD1 nu/nu) mice were implanted intracerebrally with one of two pooled populations of U251N-CRMP1-S (.1, .2) or U251N-neo (control). Mice were all euthanized ~3 weeks later and their brains were collected and sectioned.

Following hematoxylin and eosin staining (to visualize the tumour and brain cytoarchitecture), tumour volumes were calculated (Fig. 3.6). Tumours from

U251N-CRMP1-S implantations were either extremely small or undetectable (n =

7, mean volume = 0.01 mm3 for both populations). In contrast, U251N-neo (n = 3) grew quite large (mean volume = 152.64 mm3). These results indicate that the

57 overexpression of CRMP1-S in U251N affects in vivo cell survival and/or cell proliferation in mice.

Association between CRMP Expression and Survival in GBM Patients

Since January 29, 2009, the gene expression profiles of 193 tissue samples from glioma patients has been accumulated and made available online through the

National Cancer Institute’s REMBRANDT database. Through this website,

Kaplan-Meier survival curves were generated based on the levels of CRMP gene expression detected in the tumours. The downregulation of crmp1 expression in tumour samples correlated with a significant decrease in the probability of long- term survival compared to upregulated and intermediate expression (p = 0.0001)

(Fig. 3.7A). The majority of the patient samples analyzed demonstrated a two-fold or higher upregulation in crmp4 gene expression. In addition, the upregulation of crmp4 correlated with higher survival rates (p=0.0039 vs. intermediate) (Fig. 3.7B).

The analysis of crmp3 expression varied greatly depending on which reporter was used. Two reporters (205492_s_at, 205493_s_at) detected that a decrease in crmp3 expression correlated with a decrease in survival. With the latter reporter, results were significant (p = 0.048 vs. other samples). However, reporter 214301_s_at displayed a reverse trend – downregulation correlating with increased survival (p =

0.0010) and upregulation correlating with decreased survival (p = 0.0001). The analysis of crmp2, and crmp5 did not yield any correlations between gene expression and survival. In addition, the fact that only 5 out of 193 glioma samples

58 exhibited altered crmp2 levels suggested that it is unlikely to participate in glioma pathogenesis (Fig. 3.7C). Therefore, the result of these analyses suggests that both crmp1 and crmp4 expression levels are linked to a difference in GBM patient survival, while the association between GBM and crmp3 remains inconclusive.

Expression Levels of CRMP in GBM Cell Lines

Real-time PCR was performed in order to ascertain the endogenous expression of

CRMP transcripts in U87 and U251N cells (Fig. 3.8). In order to do so, cDNA was generated out of the RNA extracts originating from a confluent plate of each cell line. Real-time PCR using the SYBR detection reagent was run in triplicate using cDNA samples generated from three different plates. The analysis showed that

CRMP1-L is not expressed in either U87 or U251N cell lines. In addition, U87 cells expressed very little CRMP3, CRMP4-S, CRMP4-L or CRMP5, while they had high expression levels of CRMP1-S and CRMP2-S and CRMP2-L. In U251N cells, CRMP isoforms and splice variants were expressed at roughly the same level.

However, the highest level of expression in U251N belonged to CRMP1-S. These results indicate that endogenous levels of CRMP are either not sufficient to influence cell migration or that the endogenous CRMP proteins are inactive.

59 Figure 3.1

Figure 3.1: Verification of CRMP overexpression. U87 and U251N cells were transfected with a plasmid containing a CRMP construct tagged with the V5 epitope and the neo gene for selection purposes.

Following G418 selection, stable populations were pooled and expression was verified by Western Blot. A. Western Blot of U87 and

U251N cells for expression of CRMP1-S or CRMP1-L. B and C,

Verification of U87 and U251N (respectively) cell lines overexpressing various CRMP family constructs. Actin was used as a loading control.

61 Figure 3.2

A 3 U87-neo U87-CRMP1-S.1

2 U87-CRMP1-S.2

1 Cell count (10^6 cells)

0 24 48 72 Time (hrs)

4 U251N-neo U251N-CRMP1-S 3

1 Cell count (10^6 cells)

0 24 48 72 Time (hrs)

Figure 3.2: Analysis of cell proliferation. A, Two pooled populations of CRMP1-S (.1 and .2) were plated at a density of 5 x 105 cells and allowed to grow for 24, 48 or 72 hours. Cells were subsequently trypsinized, stained with Trypan Blue (to exclude dead cells), and then counted by hemocytometer. The graph displays the total cell count, which varied between populations because of slight differences in seeding density and thus average doubling time was calculated (U87- neo = 24.64 hrs, U87-CRMP1-S.2= 31.56 hrs, CRMP1-S.2 = 31.58 hrs; n= 2). B. The same experiment was performed on U251N-CRMP1-S cells. The average doubling time was: U251N-neo = 18.51 hrs,

U251N-CRMP1-S = 21.30 hrs.

63 Figure 3.3

2000 A U87 U87-neo U251N U251N-neo

1000 Migration (# of cells)

B **

1500 * U87-neo U87-CRMP1-S.1

1000 U87-CRMP1-S.2

500

Migration (# cells) * p < 0.05 ** p < 0.01

C * 1000 * U251N-neo U251N-CRMP1-S.1 750 U251N-CRMP1-S.2

500

Migration (# cells) 250 * p < 0.05

0 64

Figure 3.3: Effect of CRMP1 on glioma cell migration. A,

Transfection-control (U87-neo and U251N-neo) cell lines were compared to parental U87 and U251N. No significant change in migration was observed. B and C, Boyden migration assays were performed on two pooled U87-CRMP1S (A) and U251N-CRMP1S (B) cell lines. Migration of these cell lines was significantly diminished compared to control.

65 Figure 3.4

A U87 U251N 1500 * 2000 **

1500 1000

1000

500

Migration (# of cells) Migration (# of cells)

0 0 CRMP1-S CRMP1-L CRMP1-S CRMP1-L

B U87 ** ** 1500 * + ** **

1000

500 Migration (# of cells)

0 neo CRMP2-S CRMP3 CRMP4-S CRMP4-L

C U251N

** 1500 *** * ***

1000 * * *

500 Migration (# of cells) 66 0 neo CRMP2-S CRMP3 CRMP4-S CRMP4-L

Figure 3.4 Effects of CRMP family proteins and N-terminal splice variants on glioma cell migration. Boyden chamber migration assays were performed on U87 and U251N cells that overexpressed one of:

CRMP1-S, CRMP1-L, CRMP2-S, CRMP3, CRMP4-S, or CRMP4-L.

A, The N-terminal CRMP1 splice variant (CRMP1-L) had a significantly higher cell migration rate than CRMP1-S (*: p < 0.05, **: p <0.01). B and C, U87 and U251N cells lines (respectively) demonstrated a significant decrease in migration when transfected with

CRMP2-S or CRMP4-S, but not with CRMP3 or CRMP4-L. One-way

ANOVA was statistically significant (A and B, p < 0.0001; Tukey-

Kramer post-test - *: p < 0.05, **: p < 0.01, ***: p < 0.001; Student’s t- test - +: p> 0.05).

67 Figure 3.5

A CRMP1-S CRMP1-L CRMP2-S

U87 CRMP3 CRMP4-S CRMP4-L U251

N-

CRM

P2 B Phalloidin V5 Merge

CRMP1-S CRMP1-L CRMP2-S

Tubulin V5 Merge

U251-CRMP2

CRMP3 CRMP4-S CRMP4-L

Hoechst V5 Merge

Figure 3.5: Indirect immunofluorescence of CRMP-transfected cell lines. A, CRMP-transfected U87 and U251N (not pictured) cell lines were stained with V5 antibodies. Cell lines that were transfected with

CRMP1-S, CRMP2-S, CRMP4-S and, to a lesser extent, CRMP4-L exhibited long neurite-like extensions. In addition, CRMP1-S was observed to co-localize with the mitotic spindle (inset). The most intense staining was generally localized to the cell body. CRMP-1L was observed exclusively in the cell body and not at the cell periphery.

B, U251N-CRMP2 cells were co-stained with V5 antibodies along with phalloidin, tubulin or Hoechst. In U251N cells, all CRMP proteins

(with the exception of CRMP1-L) colocalized with phalloidin and tubulin at the cell periphery in areas that appear to be sites of membrane ruffling. U87 cells exhibited partial colocalization around the cell periphery (not pictured), but membrane ruffling was rarely observed. No colocalization was observed between Hoechst and V5- tagged CRMP proteins.

Figure 3.6

Figure 3.6: In vivo tumour implantations. CD1 nu/nu nude mice were implanted intracerebrally with one of two pooled populations (U251N-

CRMP1.1, U251N-CRMP1.2) (n = 7, each) or with control cells

(U251N-neo) (n = 3). Mice were sacrificed 3 weeks and their brains were harvested, sectioned, and then stained with hematoxylin and eosin. Tumour volumes were measured as described in Materials and

Methods. CRMP1 overexpression led to a significant decrease in tumour volume.

71 Figure 3.7

ALL GLIOMA CRMP1 Down-Reg. >= 2.0X CRMP1 Intermediate

ALL GLIOMA CRMP4 Up-Reg. >= 2.0X CRMP4 Intermediate

C CRMP1 CRMP2 CRMP3 CRMP4 CRMP5 Upregulated 6 0 77 171 15

Downregulated 29 5 19 0 17

Intermediate 158 188 97 30 161

Figure 3.7: The effect of CRMP1 and CRMP4 expression on GBM patient survival. Kaplan-Meier survival curves generated based on

CRMP expression. A, The two-fold downregulation of CRMP1 was associated with a significantly decreased survival rate compared to upregulated and intermediate CRMP1 levels (log p value vs. other samples = 0.0004). B, Glioma patients with tumours that exhibited a two-fold or more increase in CRMP4 expression had a significantly higher probability of survival (log-rank p value up-regulated vs. all other samples = 0.0039). C, Table of expression levels for each CRMP family member in tumour samples analyzed. Data for crmp3, crmp4, and crmp5 was based on Affymetrix reporters 314301_s_at,

201430_s_at, 222797_at, respectively.

Figure 3.8

Figure 3.8: Real-time PCR analysis of CRMP expression. Real-time

PCR was run on cDNA synthesized from U87 and U251N cells, in order to ascertain their level of CRMP expression. The cDNA was generated using the RNA extracted from 3 different plates of each cell line. PCR samples were run in triplicate and normalized to GAPDH levels.

CHAPTER 4 : DISCUSSION

76 Despite multimodal treatment that includes surgical resection, radiation and chemotherapy, the median survival rate for GBM continues to be 12-15 months2.

This is due in part to its highly invasive nature within the brain, which makes complete surgical excision nearly impossible. In over 95% of cases, tumours recur within 2 to 3 centimeters of the resection cavity2,135. Furthermore, migrating tumour cells are less susceptible to apoptosis135,136. Thus, the development of anti- invasive therapies has the potential to be extremely useful in increasing GBM treatment efficacy. Our research has demonstrated that several CRMP family members can inhibit glioma cell migration in vitro (Figs. 3.3, 3.4). In addition, the results obtained from in vivo tumour implantation experiments demonstrated that the overexpression of CRMP1-S could lead to a significant decrease in tumour volume in mice. Importantly, we have found evidence that altered gene expression of either crmp1 or crmp4 can significantly affect the survival rates of brain tumour patients (Fig. 3.7). These results suggest that, similar to its to role in NSCLC,1

CRMP1 (and possibly CRMP4) may act as an invasion suppressor gene in GBM.

The overexpression of CRMP1-S, CRMP2-S and CRMP4-S in U87 and

U251N cell lines inhibited glioma cell migration in vitro. In addition, U87 cell lines exhibited long processes, which is in agreement with previous observations50,63,66,68,74. GBM motility requires the dynamic remodeling of F-actin at the leading edge and the MT network at the cell rear136,137. Therefore, the likelihood that CRMP proteins are inhibiting migration through cytoskeletal regulation seems quite plausible. CRMP proteins have demonstrated an ability to

77 modulate both F-actin and MT dynamics28,74,75,78. Other than the increase in cell process outgrowth observed in some cells, U87 and U251N transfectants appeared otherwise normal. The transfection of mutant CRMP constructs with disabled MT or F-actin activity may help to determine if either of these interactions is necessary in mediating migration suppression.

The ability to bind and modulate MT assembly is a common property of all

CRMP isoforms and appears crucial for neurite/axonal outgrowth as well Sema3A- induced growth cone collapse43. While most glioma cell lines express Sema3A,

NP-1 and PlexA1, they are unable to undergo Sema3A-mediated collapse83.

Furthermore, the knockdown of GSK-3β, in glioma cells can reduce cell migration138, while migrating glioma cells exhibit an increase in phosphorylated

Akt and GSK-3β at their leading edge139. In U87 and U373 glioma cell lines, the inhibition of ROCK prevented directional migration137. Not only did these cells become immobile, but they developed neurite-like processes at random.

Considering the fact that both kinases are implicated in glioma migration, CRMP phosphorylation levels in glioma merits future investigation. Indeed, abnormal

CRMP phosphorylation has been previously linked to AD pathology140 and in another cancer type111. Furthermore, several molecules, known to influence a modulatory effect on CRMP phosphorylation, are frequently abnormally altered

(Table 4.1B). Alterations in the Ras/PI3K/Akt pathway occur roughly in 90% of

GBM tumours141. Most notably, the mutation or homozygous deletion of NF1, whose gene product neurofibromin is thought to interact with CRMP1, CRMP2 and

78 CRMP4 to suppress phosphorylation106,107, occurs 18% of the time. The inactivation of NF1 may therefore lead to an increase in CRMP inactivation due to the upregulation of phosphorylation pathways.

Several laboratories have been successful in constructing CRMP mutants that mimic either the phosphorylated or dephosphorylated state at Cdk5, GSK-3β and ROCK sites49,62,76,85,142. Transfection of these mutants into glioma cell lines may provide some insight on the involvement of CRMP phosphorylation in GBM invasiveness. Similarly, the recent development of commercial phospho-CRMP2 antibodies allow for the possibility of analyzing brain tumour samples for abnormal phosphorylation levels.

Alternatively, CRMP activation may be disrupted through the lower expression of other CRMP family members. While the functional relevance is as of yet unknown, CRMP proteins prefer to form heterotetramers46. For example, the decrease in expression of CRMP1 and/or CRMP4 may be sufficient to abolish

CRMP2 functional activity or promote an as of yet unknown molecular pathway.

This may explain our findings that CRMP2 overexpression led to inhibition of migration in vitro, presumably by affecting the balance of CRMP isomers and thus heterotetramerization.

Both CRMP1 and CRMP4 expression levels were linked to GBM patient mortality. It must be noted, however, that our REMBRANDT analyses does not indicate whether these alterations are the cause or rather the consequence of glioma progression. The former is suggested by our additional experimental data. Based

79 on REMBRANDT data, patients with CRMP4-upregulated tumours had a significantly higher probability of survival (Fig. 3.7B). This finding is corroborated by the discovery that CRMP4 expression inversely correlates with increasing glioma tumour grade121. The Sema3A pathway is involved not only in chemorepulsion of axons but in neural progenitor cell migration83,84. However, its chemorepulsive effect is absent in glioma cells. Since CRMP1 is implicated in the

Sema3A pathway, its downregulation may be required in order to permit tumour migration. While crmp3 is situated at a frequently mutated (~80%) chromosomal location (chr10q25.2-26)35, neither crmp1 or crmp4 (Table 4.1) are situated at common areas of chromosomal deletion or amplification in GBM tumours143. This would suggest that the altered CRMP expression patterns are the result of transcriptional regulation or epigenetic modifications.

The transcription factor, nuclear factor-κB (NF- κB), is a potential candidate in explaining the significant decrease in CRMP1 expression. The NF- κB1/p50 subunit, known to regulate several genes implicated in brain tumour invasion144,145, has recently been demonstrated to be capable of binding the CRMP1 promoter region and inhibiting crmp1 activity in lung adenocarcinoma126. In addition, the increase in NF-κB1/p50 in astrocytic tumour patient samples correlated significantly with increasing tumour grade and a decrease in patient survival146.

The inhibition of CRMP1 by NF-κB1/p50 may therefore be one of several molecular switches required in the glioma invasion pathway.

80 Migrating glioma cells possess a decreased proliferation rate and a stronger resistance to apoptosis compared to stationary glioma cells135,136. While we did not observe a change in the proliferation rate of CRMP1-transfected cells, a decrease in proliferation and apoptosis was indeed observed with granule cell precursors in mice that were CRMP-/-91. Since the in vitro environment can be drastically different from in vivo, it can not be ruled out that the downregulation of CRMP1 in glioma may be implicated in these effects. Furthermore, our in vivo model demonstrated a significant decrease in tumour volume with CRMP1-S overexpression, suggesting either a decrease in proliferation and/or increase in apoptosis. Hypoxic conditions, which are common to glioma tumours, can lead to calpain-mediated proteolysis of CRMP1 proteins, the products of which have been implicated in apoptosis56. Therefore, the downregulation of CRMP1 may be necessary to evade ischemia-induced apoptosis resulting from the typical GBM environment.

In conclusion, our results indicate the involvement of CRMP1 and CRMP4 in GBM pathologenesis. The increased mortality associated with low CRMP1 expression along with the ability of CRMP1 to inhibit in vitro tumour migration and in vivo proliferation and/or apoptosis suggests a role as a potential invasion- suppressor gene in GBM. While the precise molecular signaling is as of yet unknown, this effect is likely due to a role in cell motility, specifically through the dynamic regulation of MT and actin cytoarchitecture, and its dynamic modulation through phosphorylation pathways.

81 Table 4.1

A Human Chromosome Location

CRMP1 4p15-p16.1

CRMP2 8p21-p22

CRMP3 10q26

CRMP4 5q32

CRMP5 2p23.3

Frequency of Alteration

PI3K Mut 15%

PTEN Mut, homo del. 36%

Akt Amp 2%

Ras Mut 2%

NF1 Mut, Homo del. 18%

Table 4.1: Chromosomal Alterations in GBM. A, The chromosomal location of all CRMP members. Gene amplification/deletion events that correlate with GBM pathology have been identified on chromosomes 6, 7, 9, 10, 12, 13, 17, 19, 22143.

Based on these observations, only crmp3 appears to be succeptible to such mutation. Several members of the Ras/PI3K pathway have been implicated in

GBM pathologenesis and act upstream of CRMP phosphorylation (B). While

PTEN and NF1 mutations/deletions are inactivating, Akt, Ras, and PI3K mutations lead to overactivation. All five genetically altered proteins potentially lead to an increase in CRMP phosphorylation. Overall, abnormalities in the Ras/PI3K signaling pathway occur 88% of the time141.

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