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Wnt Signaling in Human Neural Stem Cells and Brain Tumour Stem Cells
by
Caroline Brandon
A thesis submitted in conformity with the requirements for the degree of Master of Science
Laboratory Medicine and Pathobiology University of Toronto
© Copyright by Caroline Brandon 2010
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Targeting Wnt Signaling in Human Neural Stem Cells and Brain
Tumour Stem Cells
Caroline Brandon
Master of Science
Laboratory Medicine and Pathobiology University of Toronto
2010
Abstract
We sought to determine whether activation of the Wnt signaling pathway altered the function of
hNSCs in vitro. We took three approaches to activate Wnt signaling: Wnt3a, constitutively
stabilized β-catenin (ΔN90), and the GSK3 inhibitor BIO. While Wnt3a and ΔN90 had no effect
on proliferation in both stem cell (+EGF/FGF) and differentiating (-EGF/FGF) conditions, BIO
reduced proliferation in both. All methods of Wnt signaling activation promoted neuronal lineage
commitment during hNSC differentiation. Furthermore, BIO was able to induce mild neuronal
differentiation in stem cell conditions, suggesting that GSK3-inhibition interferes with several
pathways to regulate hNSC fate decisions.
We also probed BTSC function using BIO-mediated GSK3 inhibition. We found that in stem cell
conditions, BIO was able to induce neuronal differentiation, decrease proliferation, and induce
cell cycle arrest. Together this data suggests that GSK3-inhibition, possibly through activation of
Wnt signaling, may offer a novel mechanism for the differentiation treatment of glioblastomas.
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Acknowledgments
Science is obsessed with quantification. How much? How far? How long? What cannot be
quantified is the amount of gratitude I feel for all the support and guidance I have received
throughout my life, especially over the last few years from colleagues, friends, and family. I
thank you all for keeping me sane in trying times, for making me laugh when my first instinct
was to cry, and for stretching my imagination beyond its limited confines. I dedicate this body of
work to my parents. A girl couldn’t ask for bigger and better fans. Without the both of you, goals
would stay goals and dreams would remain dreams. Thank you for helping make them a reality. I
love you both more than science can explain.
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Table of Contents
Acknowledgments.......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Tables ................................................................................................................................ vii
List of Figures .............................................................................................................................. viii
Abbreviations ................................................................................................................................. ix
Chapter 1 ......................................................................................................................................... 1
1 Introduction to the Literature ..................................................................................................... 1
1.1 Introduction to Stem Cells .................................................................................................. 1
1.1.1 Embryonic Stem Cells ............................................................................................ 1
1.1.2 Adult Stem Cells ..................................................................................................... 1
1.2 Neural Stem Cells ............................................................................................................... 2
1.2.1 Regulation of NSC activity ..................................................................................... 3
1.3 Cancer Stem Cells............................................................................................................... 4
1.3.1 Stochastic Model versus the Cancer Stem Cell Hypothesis ................................... 4
1.3.2 Lessons from Leukemia .......................................................................................... 7
1.4 Brain Tumours .................................................................................................................... 7
1.4.1 Identification of Brain Tumour Stem Cells ............................................................ 8
1.4.2 Intrinsic regulators of Brain Tumours and BTSCs ................................................. 9
1.4.3 Extrinsic Regulation of Brain Tumours and BTSCs............................................. 10
1.5 Introduction to Wnt Signaling .......................................................................................... 11
1.5.1 The Canonical Wnt Signaling Pathway ................................................................ 11
1.5.2 Regulatory mechanisms of the Canonical Wnt Pathway...................................... 12
1.5.3 GSK3: the master of multitasking......................................................................... 13
1.6 Wnt Signaling in Development and Stem Cells ............................................................... 18
v
1.6.1 Wnt in Neural Development and NSCs ................................................................ 19
1.7 Wnt Signaling in Cancer and CSCs .................................................................................. 20
1.7.1 Wnt in Colorectal Cancers .................................................................................... 20
1.7.2 Wnt in Skin Cancers ............................................................................................. 20
1.7.3 Wnt in Leukemias ................................................................................................. 21
1.8 Wnt in Brain Tumours ...................................................................................................... 22
1.8.1 Wnt in Medulloblastomas ..................................................................................... 22
1.8.2 Wnt in Glioblastoma Multiforme.......................................................................... 22
1.8.3 Brain tumour stem cells in vitro............................................................................ 23
1.9 Thesis Rationale and Aim................................................................................................. 26
1.10Thesis Hypothesis ............................................................................................................. 26
Chapter 2 ....................................................................................................................................... 27
2 Materials and Methods............................................................................................................. 27
2.1 Cell culture and differentiation protocol........................................................................... 27
2.2 Immunoblotting................................................................................................................. 27
2.3 Immunofluorescence......................................................................................................... 28
2.4 Intracellular Flow Cytometry............................................................................................ 28
2.5 Luciferase Assay............................................................................................................... 29
2.6 Transfection of cells.......................................................................................................... 29
2.7 MTT assays ....................................................................................................................... 29
2.8 BrdU labeling.................................................................................................................... 29
Chapter 3 ....................................................................................................................................... 31
3 Results ...................................................................................................................................... 31
3.1 Wnt signaling promotes a neuronal cell fate choice in human fetal neural stem cells ..... 31
3.1.1 Human fetal neural stem cells express key Wnt pathway components and exhibit low baseline TCF/LEF-mediated transcription......................................... 31
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3.1.2 Wnt3a does not alter human fetal neural stem cell proliferation .......................... 35
3.1.3 Wnt3a promotes a neuronal cell fate choice in human fetal neural stem cells under differentiating conditions ............................................................................ 37
3.1.4 Stabilized β-catenin activates Tcf/Lef—mediated transcription........................... 40
3.1.5 Stabilized β-catenin does not alter human neural stem cell proliferation. ............ 42
3.1.6 Stabilized β-catenin promotes neuronal cell fate choice in human fetal neural stem cells under differentiating conditions ........................................................... 44
3.1.7 BIO—mediated inhibition of GSK3 activates TCF/LEF transcription ................ 47
3.1.8 BIO promotes differentiating human fetal neural stem cells to slow proliferation and exit the precursor state .............................................................. 49
3.1.9 BIO promotes a neuronal cell fate choice in differentiating human fetal neural stem cells............................................................................................................... 51
3.1.10 BIO decreases proliferation and induces neuronal differentiation of human neural stem cells in EGF and FGF........................................................................ 54
3.2 GSK3 inhibition induces neuronal differentiation of brain tumour stem cells ................. 56
3.2.1 Brain tumour stem cells express Wnt pathway components and can activate TCF/LEF-transcriptional activity.......................................................................... 56
3.2.2 BIO induces neuronal differentiation of brain tumour stem cells......................... 59
3.2.3 BIO treatment induces brain tumour stem cells to exit the cell cycle and decrease proliferation............................................................................................ 62
Chapter 4 ....................................................................................................................................... 64
4 Discussions and Future Directions........................................................................................... 64
4.1 Wnt signaling in human neural stem cells ........................................................................ 64
4.2 Targeting the Wnt pathway for differentiation therapy of brain tumour stem cells ......... 69
References..................................................................................................................................... 73
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List of Tables
Table 1 .......................................................................................................................................... 25
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List of Figures
Figure 1 – The Stochastic model and the cancer stem cell model. ................................................. 6
Figure 2 – The canonical Wnt Signaling Pathway........................................................................ 17
Figure 3 - HumanNSCsexpresscanonicalWntsignalingpathwaycomponents................... 32
Figure 4 – hNSC lines activate TCF/LEF transcriptional activity in response to Wnt3a............. 34
Figure 5 – Wnt3a does not alter hNSC proliferation. ................................................................... 36
Figure 6 – Wnt3a promotes a neuronal cell fate in differentiating hNSCs................................... 38
Figure 7 – Stabilized β-catenin activates Tcf/Lef transcriptional activity in hNSCs. .................. 41
Figure 8 – Stabilized β-catenin does not affect proliferation of hNSCs in vitro. ......................... 43
Figure 9 – Stabilized β-catenin promotes a neuronal cell fate choice during differentiation. ...... 45
Figure 10 – BIO-mediated inhibition of GSK3 activates Tcf/Lef transcription ........................... 48
Figure 11 – BIO-mediated GSK3 inhibition promotes hNSCs to exit the precursor state. .......... 50
Figure 12 – BIO promotes a neuronal cell fate choice in differentiating hNSCs. ........................ 52
Figure 13 – BIO decrease proliferation and induces mild neuronal differentiation in EGF and
FGF. .............................................................................................................................................. 55
Figure 14 – GliNS1 expresses Wnt pathway components and is BIO-responsive in vitro. ......... 58
Figure 15 – BIO reduces GliNS1 precursor marker expression in EGF and FGF. ...................... 60
Figure 16 – BIO treatment induces neuronal differentiation of GliNS1 in EGF and FGF........... 61
Figure 17 – BIO treatment promotes cell cycle exit and decreased proliferation......................... 63
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Abbreviations
AML Acute myeloid leukemia APC Adenomatous polyposis coli BCR-ABL Breakpoint cluster region-Ableson murine leukemiaviral oncogene homologue BDNF Brain-derived neurotrophic factor bHLH Basic helix loop helix BIO (2’Z,3’E)-6-Bromoindirubin-3’-oxime (a GSK3 inhibitor) BMP4 Bone morphogenic protein 4 BrdU Bromodeoxyuridine BTSC Brain tumour stem cell CK1 Casein Kinase 1 CML Chronic myeloid leukemia CNS Central nervous system CSC Cancer stem cell DAPI 4', 6-diamidino-2-phenylindole, DNA intercalater DAPT N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester DKK1-4 Dickkopf 1-4 DVL Dishevelled E Embryonic day ECM Extracellular matrix EDTA ethylenediaminetetra-acetic acid EGF Epidermal growth factor EGFR Epidermal growth factor receptor ERBB2 Human epidermal growth factor receptor 2 ES Embryonic Stem cell FAP Familial adenomatous polyposis FBS Fetal Bovine Serum FGF Fibroblast growth factor fgfr1 Fibroblast growth factor receptor 1 Fzd Frizzled receptor GAPDH Glyceraldehyde 3-phosphate dehydrogenase GBM Glioblastoma Multiforme GFAP Glial fibrillary acidic protein Gli Glioma-associated oncogene homologue 1 GSK3α/β Glycogen synthase kinase-3 α isoform/β isoform GTPase guanosine triphosphate hydrolase enzymes Hh Hedgehog HRP Horseradish peroxidase JAK2 Janus kinase 2 JNK c-Jun N-terminal kinase LEF Lymphoid enhancer-binding factor Li+ Lithium LIF Leukemia inhibitory factor LRP5/6 Low density lipoprotein receptor-related protein 5/6 MAP2 Microtubule-associated protein 2 mRNA Messenger ribonucleic acid
x
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NGS Normal goat serum NH2 Amine NICD Notch intracellular domain NOD/SCID Non-obese diabetic/Severe combined immunodeficiency NSC Neural stem cell PBS Phosphate buffered saline PCP Planar Cell Polarity PFA Paraformaldehyde PDGFRA Platelet-derived growth factor receptor A pHH3 Phospho-histone H3 PI3K Phospoinositide 3-kinase PKB Protein Kinase B (also known as AKT) PLO Poly-L-Ornithine PNET Primitive neuroectodermal tumour PSA-NCAM Poly-sialated neural cell adhesion molecule PTCH Patched gene PTEN Phosphatase and tensin homologue PVDF Polyvinylidene fluoride RMS Rostral migratory stream RTK Receptor tyrosine kinase RYK Related to receptor tyrosine kinase SDS sodium dodecyl sulfate SGZ Subgranular zone SL-IC SCID leukemia initiating cell STAT1/3 Signal Transducers and Activators of Transcription-1/3 SVZ Subventricular zone TBST Tris-Buffered Saline Tween-20 TCF T-cell factor TK Thymidine Kinase TS Turcot syndrome WIF Wnt inhibitory factor Wnt Wingless integration-1
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Chapter 1
1 Introduction to the Literature
1.1 Introduction to Stem Cells
The homeostatic replenishment and repair of tissues is one of the most fundamental processes in
cellular biology. These processes are due in large part to the persistence of stem cells throughout
an organism’s lifetime. Stem cells are what allow blood, bone, muscle, epithelia, and gametes to
turn over at astonishing rates throughout an organism’s lifetime. For instance, the mammalian
intestinal lining is turned over every two to seven days1. Stem cells are defined by their unique
functional properties: self-renewal—the ability to regenerate itself, and differentiation—their
ability to mature into multiple cell lineages that comprise the tissue in which they reside.2 These
functions are tightly regulated by intrinsic cues as well as extrinsic cues that are found in the
stem cell microenvironment, known as the niche3. There are two major types of stem cells:
pluripotent embryonic stem (ES) cells and multipotent somatic stem cells (embryonic and adult).
1.1.1 Embryonic Stem Cells
The inner cell mass of the developing embryo contains a population of pluripotent cells that can
be characterized in vitro as Embryonic stem (ES) cells. ES cells in vitro are characterized by
their capacity to self-renew indefinitely as well as their ability to differentiate into all the somatic
cell types comprising all three germ layers of the embryo and adult tissues, as well as the germ
cells4. This differentiation profile of ES cells defines the pluripotent state, a less restricted state
than their adult counterparts, which demonstrate tissue-specific multilineage differentiation
potentiality5.
1.1.2 Adult Stem Cells
Adult stem cells are tissue-specific, self-renewing, and exhibit multipotency. These stem cells
can regenerate the various cell types specific to the tissue from which it resides5. These cells are
responsible for replenishing tissues under homeostatic conditions as well as after injury.
Different tissues have varying rates of turnover. Tissues with a relatively slow turnover rate
include skeletal muscle and the brain, while tissues with high turnover rate include the skin, the
intestine, and the blood, changing over every 4 weeks, 3-5 days, and 1 billion cells per day,
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respectively6. What mediates the proliferative, self-renewing, and differentiating processes of
these cells are highly complex combinations of extrinsic signals such as soluble growth factors
and hormones, molecules from adjacent cells or in the extracellular matrix, constituting a
complex niche5. In many pathologies these signals are altered, leading to the deregulation of
stem cells and their signature processes. Furthermore, intrinsic alterations, such as genetic
mutations or epigenetic changes can alter the hereditable programs of these cells and have
catastrophic consequences for their resident tissues. Loss of self-renewing capacity can result in
premature aging, senescence or tissue degeneration. On the other hand, genetic and epigenetic
changes in stem cells that result in increased proliferative and/or self-renewing capacity, or
blocks in terminal differentiation capacity, can lead to cancer.
1.2 Neural Stem Cells
Until recently, it was thought that neurogenesis, the process by which new mature and
specialized neurons are generated, did not persist through adulthood. However, in the last two
decades, seminal studies by several groups have shown that, in fact, neural stem cells (NSCs) do
persist throughout adulthood and neurogenesis can and does occur in at least two regions within
the brain: the subventricular zone (SVZ) along the fourth lateral ventricle and the subgranular
zone (SGZ) of the hippocampus7,8,9,10. Although adult NSCs from both the SVZ and the SGZ
generate neurons, they occupy very different niches with unique anatomical structures. The
NSCs from the SVZ, called type B cells by their morphologic criteria on electron microscopy,
are relatively slow cycling and express Nestin, Sox2, and GFAP, reminiscent of embryonic radial
glial cells. B cells give rise to transit amplifying cells (called C cells) that are a rapidly dividing
GFAP-negative population that ultimately gives rise to type A cells, neuroblasts that will migrate
through the rostral migratory stream (RMS) to the olfactory bulb and generate mature neurons. C
type cells are thought to be EGF-responsive and can reacquire stem cell characteristic (observed
in B type cells) in the presence of EGF11. Type A cells express early neuronal markers such as
doublecortin and PSA-NCAM12,13. Within the SGZ, there are thought to be two NSC
populations, one of which (type 1) is more quiescent and expresses Nestin, Sox2 and GFAP,
while the second type (type 2) of NSC cycles more frequently and express Nestin and Sox2, but
not GFAP10,14. The relationship between the two populations remains unknown. In both
neurogenic regions, NSCs are closely associated with the vasculature, adjacent to a plethora of
neighboring cells that can contribute to NSC regulation, and are in close contact with basal
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lamina components rich in extracellular matrix (ECM) components such as laminin, enabling
them to respond to local and systemic signals14.
1.2.1 Regulation of NSC activity
A critical question in the neural stem cell field is what regulates stem cell function, particularly
expansion via self-renewal and directed differentiation of specific target cells, for instance
dopaminergic neurons15. Many extrinsic signals that modulate NSC function have been studied
in depth in an effort to unravel this complex molecular network. Epidermal growth factor (EGF)
has been shown to be a potent mitogen, maintaining multipotentiality and promoting the
expansion of NSCs in vitro and in vivo11,16. Fibroblast growth factor-2 (FGF2) has also been
shown to promote NSC proliferation and suppress neurogenesis in vitro. In fact, NSCs are
typically grown in a serum-free defined medium that includes EGF and FGF, as well as other
survival factors17. However, in vivo, FGF2 has also been shown to increase the number of
newborn neurons in the olfactory bulb, suggesting it may play a role in regulating neurogenesis
in the SVZ18. SGZ neurogenesis was reduced as well as a decrease in BrdU+ cells was observed
in the SGZ when fgfr1 was deleted from the CNS, suggesting it could regulate SGZ neuronal
progenitor proliferation19. Other factors have also been shown to mediate neurogenesis. Brain-
derived neurotrophic growth factor (BDNF), for one, is a major positive regulator of
neurogenesis in both the SVZ as well as the SGZ10. The Notch signaling pathway has been
shown to be necessary for the maintenance of NSC self-renewal in vivo20. Furthermore, it is
thought to be a negative regulator of neurogenesis through the inhibition of a variety of basic
helix-loop-helix (bHLH) transcription factors implicated in neuronal differentiation, including
neurogenin1 and 2, Mash1 and Math121. Alternatively, Leukemia inhibitory factor (LIF), a
positive regulator of pluripotency in ES cells, is a potent inducer of astrocytic differentiation
through activation of signal transducers and activators of transcription 1 and 3 (STAT 1 and 3)
signaling, cooperating with a host of chromatin remodeling complexes to bind the GFAP
promoter, activating astrocyte differentiation22. Interestingly, it seems that the Notch signaling
pathway also contributes to gliogenesis by activating STAT3 through the recruitment of Janus
kinase 2 (JAK2), thereby activating STAT323. The Wnt pathway has also been shown to regulate
NSC proliferation and neurogenesis in vitro and in vivo and will be discussed in greater detail
later. Though this list of extrinsic cues is incomplete, it does hint at the complex network of
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signals that regulate neural precursor function within its in situ niche as well as in the culture
dish.
1.3 Cancer Stem Cells
While human cancers have been recognized as abnormal tissues with morphologically
heterogeneous populations of cells for over 100 years, it has only been in the last decade that
functional heterogeneity has been demonstrated in a number of cancers24, 25, 26, 27, 28. In the 1950s
and 1960s, an unethical study where human subjects were injected with increasing numbers of
their own explanted cancer cells was published that suggested that the growth potential of cancer
might reside within rare cell populations29. Results from this experiment showed that although
over a million cells were injected, only 50% of these injections developed into palpable nodules,
suggesting that not all tumour cells are capable of initiating growth30. There are at least two
models of cancer growth that can help explain these results31.
1.3.1 Stochastic Model versus the Cancer Stem Cell Hypothesis
The stochastic model assumes that each cell within a tumour has equal capacity to proliferate and
regenerate a tumour, although the probability of any given cell doing so is random and very low.
This model would suggest that prospectively isolating subpopulations of cancer cells on the basis
of functional and phenotypic characteristics would not enrich for tumour-initiating capacity. The
cancer stem cell (CSC) model implies there is functional heterogeneity among tumour cells for
the ability to initiate and maintain tumour growth. In contrast to the stochastic model, one would
then predict that an ability to fractionate subsets of tumour cells based on marker expression or
phenotypic characteristics would define different cell populations with differing capacity to
initiate tumour growth. Tumourigenic capacity would then reside within a subpopulation of the
tumour’s cells and not within the bulk of the tumour mass. The CSC hypothesis also predicts
that treatments targeting the bulk tumour population but sparing CSCs would ultimately fail and
lead to recurrence. This hypothesis has been supported by recent studies that demonstrate that
CSCs are less sensitive to radiation and chemotherapeutic treatments32, 33. Therefore, a key to
further understanding cancer biology lies in a more detailed understanding of the mechanisms of
CSC growth and therapeutic resistance.
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CSCs demonstrate the following cardinal functions: tumour initiation, self-renewal, and
differentiation capacity regenerating the phenotypic heterogeneity of the parent tumour34.
Through the development of in vivo functional assays that test tumour-initiating capacity of
discrete cell populations, in conjunction with experimental methods of prospective purification
of tumour cells by surface markers or functional behavior, CSCs have been identified in a
number of human cancers, including blood24, brain25,35, colon27,36, breast26, pancreas37,
melanoma28,38, mesenchymal neoplasms39, neuroblastomas40, and lung41.
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Figure 1 – The Stochastic model and the cancer stem cell model.
The stochastic model and the cancer stem cell model. a) The stochastic model implies that
all cells within a tumour are functionally the same and have equal tumourigenic potential.
b) The cancer stem cell model suggests that cells within a tumour exist in a functional
hierarchy where only a subpopulation of cells have tumourigenic potential, while the other
cells are unable to contribute to the tumour bulk. These tumour-initiating cells are termed
cancer stem cells.
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1.3.2 Lessons from Leukemia
The first evidence of CSCs came from John Dick’s work in blood system cancers, where a rare
population of CD34+ CD38- cells was isolated from acute myeloid leukemia (AML) patients and
infused back into severe combined immunodeficient (SCID) mice42. This discrete and very rare
population of cells resulted in leukemic blast generation while a more differentiated population
of cells (CD34+ CD38+ and CD34-) did not, thereby identifying the SCIC leukemia-initiating cell
(SL-IC). A subsequent study, published by the same group in 1997, was able to increase
engraftment efficiency by infusing SL-ICs into non-obese diabetes (NOD)/SCID mice24. This
superior repopulation assay allowed them to demonstrate that SL-ICs differentiate in vivo to
reacquire the same leukemic phenotype as seen in the patient while retaining self-renewal
capacity. Furthermore, they were able to show that AML is organized in a hierarchical fashion,
much like the normal hematopoietic system, and that the primitive CD34+ CD38- cell population
(also shown to be the SCID-repopulating cells of the normal human hematopoietic system43) is
the target of transformation. This in vivo model of serial transplantation of cells into NOD/SCID
mice has become the gold standard assay for demonstrating in vivo tumour initiation,
multilineage differentiation potential, and self-renewal capacity of CSCs. By following a similar
logic and experimental method, the identification of CSCs in other blood and solid tumours has
been possible.
1.4 Brain Tumours
Brain tumours refer to a heterogeneous collection of neoplasms that occur in the CNS. The two
most common classes of brain tumours are primitive neuroectodermal tumours (PNETs) and
gliomas44. The most common and aggressive type of PNET is medulloblastoma, a cerebellar
tumour that arises primarily in children, accounting for 20-30% of pediatric tumours.
Astrocytomas, the most common gliomas, are graded on a scale of I-IV, I being benign low
grade and IV being glioblastoma multiforme (GBM), a highly invasive and aggressive tumour
normally found within the cerebral hemisphere, with peak onset at 40 to 70 years of age.
Following treatment, the mean survival time is only 10-12 months due to their inherent
therapeutic resistance and diffuse infiltration of the brain tissue45. Current treatment of both
medulloblastomas and the more lethal malignant gliomas consists of a combination of surgical
resection, radiation and chemotherapy. An additional challenge to treatment is that the blood-
8
brain barrier restricts access of therapeutic molecules into the brain milieu in order to preserve
the integrity of brain function. Little advancement has been made in the chemotherapeutic
treatment of glioblastomas (grade IV); temozolomide offering only an additional three months of
median survival as the best advance in the past 40 years. This partially explains why it remains
one of the deadliest cancers and why more efforts are necessary to uncover novel drugs that
effectively target the cancer cells.
1.4.1 Identification of Brain Tumour Stem Cells
In 2003, Al-Hajj et al. isolated cells from fresh human breast cancer cells based on the cell
surface marker expression profile CD44+ CD24−/low Lineage− that exhibited potent tumour-
initiating ability when as few as 1 x 103 cells were injected into the mammary fat pad of
NOD/SCID mice26. These cells could be serially transplanted, demonstrating their self-renewal
and extensive proliferative capacity while also recapitulating phenotypic copies of the patient
tumour, all of which are hallmarks of CSCs. This was the first time CSCs were prospectively
isolated in a solid tumour.
In 2000, a study by Uchida et al. showed that multipotent and self-renewing human neural stem
cells could be prospectively isolated and purified based on CD133+ expression46. Under the
premise that brain-tumour initiating cells could originate from a normal stem or progenitor cell,
the Dirks laboratory identified brain-tumour stem cells (BTSCs) in adult glioblastomas and
childhood medulloblastomas based on the cell surface marker CD133+ 25,35. Having initially
identified CD133+ cells as a clonogenic and multipotent population in vitro, they then showed
that CD133+ were the tumour-initiating population in vivo, generating a tumour with as few as 1
x 102 cells, and could self-renew in vivo, as was demonstrated by serial transplantation of
CD133+ cells into the brains of NOD/SCID mice. Several groups, including our own, have since
reported that CD133+ does not universally identify a tumour-initiating population, but it remains
the most reliable marker available47,48. Recently, a study by Fine’s group demonstrated that up to
60% of freshly isolated glioblastomas do not express CD133, but express SSEA-1 (CD15),
which also identifies a tumourigenic subpopulation when CD133 is not expressed. Thus, an
additional marker of BTSCs is SSEA-1, which enriches for self-renewing and multipotent
tumour-initiating cells that could give rise to SSEA-1- as well as SSEA-1+ cells, establishing a
cellular hierarchy49. Needless to say, more efforts are needed to identify marker signatures that
9
will define BTSC populations. Moreover, it is likely that more than one putative BTSC
population will exist from patient to patient as different brain tumours may have originated from
different cell populations or have undergone different transforming events that will lead to
distinct cell surface profiles.
1.4.2 Intrinsic regulators of Brain Tumours and BTSCs
Cancer has long been considered a disease of genomic alterations: DNA sequence mutations,
copy number changes, chromosomal rearrangements and various other aberrations have all been
shown to contribute to the development and maintenance of many human cancers50. Genome-
wide profiling studies of human glioblastoma confirm decades’ worth of findings that several
core molecular pathways, namely the p53, pRB, p16Ink4/p19Arf and receptor tyrosine kinase
(RTK) pathways, are the main contributors in gliomagenesis and disease progression50,51. The
p53 tumour suppressor—a transcription factor that regulates cell cycle progression and apoptosis
in response to various insults—is mutated or lost in over 60% of sporadic astrocytomas52.
Furthermore, patients with Li-Fraumeni syndrome—a syndrome characterized by the germ-line
mutation of the TP53 gene rendering them susceptible to cancers—are predisposed to various
brain tumours including gliomas53.
PTEN—phosphotase and tensin homologue—is a major inhibitor of the pro-growth PI3K/AKT
pathway and is mutated or lost in over 30% of primary glioblastomas (GBMs)50. Moreover, in
human GBMs harbouring TP53 mutations, 60% of these tumours had concomitant PTEN
alterations including homozygous deletion54. In the same study, it was found that over 60% of
primary GBMs showed loss of heterozygosity (LOH) of chromosome10q, where the PTEN gene
is located. In a genetic study that examined the roles of p53 and Pten in neural and glioma stem
cells, p53-/- pten+/- fetal NSC and tumourspheres showed increased proliferation and resistance to
differentiation cues55. They also showed that p53 and Pten cooperate to regulate Myc—a
transcription factor known to play a role in cell cycle progression and stem cell self-renewal—
showing that elevated levels of Myc impeded stem cell differentiation in NSCs and
tumourspheres, indicating a contribution of p53 and Pten in BTSC regulation. Over 50% of
human GBMs lack a functional Ink4a/Arf locus, thereby altering the p53 and pRb pathways,
both of which have known roles in cell cycle progression and stem cell regulation50,56.
10
Along with the loss of key tumour suppressors, various RTKs, including EGFR, ERBB2, and
PDGFRA are amplified in 45%, 8%, and 13%, respectively, indicating a role for these pro-
proliferative pathways in gliomagenesis. Altogether, the core pathways that harbour the majority
of genetic abnormalities found in human GBMs are pathways shown to regulate cell cycle
progression and stem cell functions in normal NSCs11,57,58. This fact highlights the importance of
studying these pathways within BTSCs in order to gain insight into tumour initiation and
propagation.
1.4.3 Extrinsic Regulation of Brain Tumours and BTSCs
It is well established that the tumour microenvironment can regulate initiation and maintenance
of the malignancy through extrinsic cues, including a variety of developmental signaling
pathways59. Individuals with germ-line alterations in the PTCH (patched) gene, a member of the
hedgehog signaling pathway, are prone to the development of basal cell carcinoma as well as a
medulloblastomas, a cerebellar malignancy60. Furthermore, studies have shown that blocking
hedgehog signaling with an inhibitor known as cyclopamine can halt proliferation and
clonogenicity of stem-like cells from GBMs, as well as prevent tumour growth in vivo61. The
Bone Morphogenic Protein signaling pathway may also be important for GBMs. In a study by
Piccirillo et al., transient exposure to BMP4 was shown to deplete the BTSC population in vitro
by promoting astrocytic differentiation as well as inhibit tumour initiation in vivo62. Notch
signaling may also be important. Pharmacological inhibition of the γ-secretase complex Notch
processing pathway—necessary for proper signal transduction—has been shown to block
proliferation and decrease the number of CD133+ cells in medulloblastomas63. In another study,
Purow et al. showed that primary glioma tissues overexpress the Notch1 intracellular domain
(NICD), indicating pathway activation, and that down-regulation of Notch1 or its ligand Delta-
like-1 leads to increased apoptosis and decreased proliferation in vitro, as well as prolonged
survival in a murine orthotopic brain tumour model64. Wnt signaling, another developmental
pathway implicated in a number of human cancers, has also been shown to play a role in brain
tumour initiation and maintenance but has not been explored in as much detail as the above
pathways65. This will be discussed in further detail later. Therefore, as is the case with normal
NSCs that are regulated by the extrinsic cues within their niche, so too are BTSCs, which receive
signals from their microenvironment that regulate their ability to self-renew and proliferate,
thereby contributing to tumour initiation and maintenance66.
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1.5 Introduction to Wnt Signaling
Multicellular organisms require the precise orchestration of a variety of signaling pathways to
govern the processes necessary for proper development: proliferation, differentiation, cell fate
decisions, and survival67,68. The Wingless-int (Wnt) signaling pathway regulates a large number
of adult and developmental processes, primarily by modulating gene transcription through
several signal transductions69. Wnts, for which mammals have 20, are cysteine-rich secreted
glycoproteins that signal through one of ten Frizzled (Fz) receptors, which are seven-pass
transmembrane proteins with an extracellular N-terminal cysteine-rich domain70. The Wnt-Fz
complex recruits the scaffolding protein Dishevelled (Dvl), bringing together downstream
pathway components for signal transduction71. Downstream of Dvl, the pathway splits into at
least three known branches: the planar cell polarity (PCP) branch, the Wnt/calcium (Wnt/Ca2+)
pathway, and the canonical or Wnt/β-catenin-mediated pathway72. The PCP pathway is mediated
by small GTPases (Rho and Rac1) and the c-jun amino (N)-terminal kinase (JNK), and has been
shown to control cell polarity and orientation within a tissue as well as convergent extension
movements during gastrulation73. In the Wnt/Ca2+, signal transduction through Dvl induces
calcium influx, activating protein kinase C (PKC) and calcium/calmodulin dependent protein
kinase II (CaMKII), and has been shown to play a role in cell migration during gastrulation and
cardiac development72. The most well-characterized or canonical Wnt signaling pathway
involves signaling through the β-catenin and the GSK3-Axin-APC protein complex and
transcriptional activation mediated by Tcf/Lef transcription factors at specific targets.
1.5.1 The Canonical Wnt Signaling Pathway
The canonical pathway—the only Wnt pathway referred to from here in—is mediated by β-
catenin, a cytoplasmic protein whose stability is regulated by the destruction complex72. The
destruction complex comprises several key players: Axin, adenomatous polyposis coli (APC),
glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1). In the absence of Wnt
stimulation, β-catenin is phosphorylated by CK1 at serine 45 (S45), which primes it for further
phosphorylation by GSK3 at threonine 41 (T41), S37 and S33, targeting β-catenin for
ubiquitination and subsequent degradation74. However, when Wnt engages its Fz receptor, Dvl is
recruited to the cytoplasmic tail of Fz and thought to recruit Axin and the other complex
components with it, allowing β-catenin to evade phosphorylation and degradation70.
12
Unphosphorylated stabilized β-catenin is free to translocate to the nucleus where it acts as a
transcriptional co-activator by interacting with a group of transcription factors known as T-cell
Factors/Lymphoid Enhancer Factors (TCF/LEFs) (and various chromatin remodeling proteins)
that together transcribe target genes75.
1.5.2 Regulatory mechanisms of the Canonical Wnt Pathway
Despite the simple picture painted above, the Wnt pathway is extremely complex. For one, along
with the 20 mammalian Wnt ligands, there are 10 mammalian Fz receptors, as well as several
other receptors (e.g. low-density lipoprotein receptor-related protein 5/6—LRP5/6, RYK, Ror2)
that are known to initiate one of three Wnt signaling cascades either in conjunction with Fz
receptors, or on their own72. Not all Wnt-Fz combinations are well understood. Some ligands
have been denoted as “canonical” because they were capable of inducing axis duplication in the
Xenopus following injection of Wnt mRNAs, including Wnt1, Wnt3a, Wnt5a, Wnt7a, Wnt8a,
and Wnt8b76. However, new studies suggest that signal transmission initiated by any of these
ligands may be highly context-dependent and in some cases may act to inhibit TCF/LEF-
mediated transcription77. Wnt inhibitors are very important in regulating signal transmission
throughout development and various cell processes. Inhibitory molecules such as Dickkopf1-4
(Dkk1-4), Wnt inhibitory factors (WIFs), and soluble frizzled-related proteins (sFRPs) act as
Wnt pathway antagonists by either binding directly to the Fz-receptor complex to block Wnt
from binding (i.e. Dkk1), or by binding directly to Wnt ligands (WIFs and sFRPs) through their
cysteine-rich domains, which these inhibitors also possess 78. Thus, at the cell surface level
alone, there is a great deal of complexity in regulating the appropriate signal transduction.
As a means of mediating transcriptional output upon stimulation, the Wnt signaling pathway has
built-in feedback loops that regulate the pathway at several key points. A prime example is the
classic Wnt target genes Axin2 or DKK1. Both proteins are negative regulators of the signaling
pathway and are actively transcribed when TCF/LEF-mediated transcription is on. The negative
feedback loop helps to stabilize β-catenin directly (i.e. Axin2) or indirectly by interfering with
ligand-receptor binding (i.e. Dkk1)70. This complex network of regulatory mechanisms is crucial
for mitigating the risk implicated in a signaling network that has such a broad impact on
fundamental cellular processes throughout an organism’s life. Any deregulation of the pathway
13
will have profound impacts on the feedback loop, and could in turn perpetuate any resulting
phenotype.
Further downstream, there are internal regulators that modulate β-catenin stability through direct
or indirect interaction with various components of the destruction complex. The most notable
interactions involve GSK3, a key negative regulator of β-catenin and central moderator of the
canonical signaling pathway as a whole. However, GSK3 is a downstream switch for many
signaling pathways including Wnt, growth factors, insulin, RTKs, hedgehog, G-protein coupled
receptors (GPCRs) and is involved in almost every cell function from metabolic regulation, cell
development, cell cycle regulation, gene transcription, cell proliferation and apoptosis79.
1.5.3 GSK3: the master of multitasking
Glycogen synthase kinase-3 (GSK3) is a multifaceted kinase that functions in several distinct
pathways. Originally identified for its phosphorylation of glycogen synthase, the rate-limiting
enzyme of glycogen metabolism, it has since been shown to target over 50 substrates in the
RTK—PI3K, Hh, Notch, and Wnt pathways80. In the Drosophila, the loss of GSK3
(Shaggy/Zeste-white3) results in the accumulation of the full-length active form of Cubitus
interruptus (Ci), the fly orthologue of the Gli3 transcription factor, and the subsequent ectopic
expression of Hh-responsive genes81. Thus, it is thought that GSK3 regulates the phosphorylation
and ensuing proteolytic degradation of Ci. Espinosa et al. found that GSK3β also regulated the
Notch signaling pathway in vitro and in vivo through the phosphorylation of the Notch2 receptor
intracellular domain82. Furthermore, they found that GSK3β inhibition by Wnt1 or its
pharmacological inhibition using LiCl abrogated Notch2 phosphorylation and increased Hes1-
reporter activity, Hes1 being a target gene of Notch signaling. Together this suggests that GSK3β
is a negative regulator of the Notch pathway, and may crosstalk with the Wnt pathway.
Two isoforms, GSK3α and GSK3β, are present in mammals83. While GSK3α (52 kDa) is 5 kDa
larger than GSK3β due to an amino-terminal glycine rich extension of unknown function, there
is tremendous functional overlap between the two isoforms in most instances, as was observed in
the GSK3β knockout mouse84,85. Upon analysis of the GSK3β knockout mouse model, it was
observed that one unique function of GSK3β is its necessity for the proper nuclear function of
NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in response to TNFα
(Tumour Necrosis Factor-α)-induced apoptosis in hepatocytes. Interestingly, there was no
14
evidence that Wnt signaling or glucose metabolism was disturbed, suggesting that GSK3α is able
to compensate for the loss of GSK3β in these pathways. However, O’Brien et al. performed a
study comparing Gsk3β+/- mice with lithium-treated mice and found that while Gsk3α protein
levels were not significantly increased in Gsk3β+/- mice brains, there was an increase in
stabilized β-catenin, similar to levels observed in Li+ mice brains86. Therefore, at least in the
brain, the loss of one copy of the Gsk3β allele resulted in the increased stabilization of β-catenin,
suggesting a possible slight preference for GSK3β in the brain. Doble et al. also analyzed the
functional redundancy of GSK3α and GSK3β in Wnt/β-catenin signaling in ES cell lines87. ES
cell lines were generated with 0-4 functional GSK3 alleles and their isoform-specific function in
the canonical Wnt pathway was examined. It was found that GSK3 protein levels did not
increase when either GSK3α or GSK3β was absent, and TCF—mediated transcription was
normal. Only when cells lacked 3 or 4 alleles was gene dosage reflected in TCF activity.
Furthermore, ES cells lacking 3 or 4 of the GSK3 alleles showed impaired differentiation,
particularly down the neuroectoderm lineage, which was restored upon reintroduction of
functional GSK3. While this study validates the redundancy of GSK3α and GSK3β as regulators
of Wnt/β-catenin signaling, as well as their role in ES cell regulation, it does not determine
whether the observed phenotype is Wnt/β-catenin—dependent. This could be addressed by
interfering with β-catenin—mediated signaling downstream of GSK3 to see whether, similar to
with GSK3 re-expression, the neuroectodermal differentiation capacity of GSK3α-/- /β-/- cells can
be rescued.
One peculiar feature of GSK3 is that unlike most kinases, it is highly active in resting cells and is
regulated by inhibition in response to various cellular signals. Such signals include hormone and
growth-factor activation of RTKs leading to the activation of PI3K—mediated activation of PBK
(also known as Akt), and Wnt-induced inactivation of GSK3 via Dishevelled (Dsh), the
mechanism of which is not entirely clear80. Insulin, among other hormone and growth factors,
bind to RTKs at the cell membrane leading to the activation of PI3K, which recruits and
activates the prosurvival factor PKB88. Subsequently, PBK phosphorylates GSK3 on Ser21 (on
the α isoform) or Ser9 (one the β isoform) and inactivates it, resulting in the primary mechanism
of growth-factor inhibition of GSK389. Surprisingly, PKB—mediated inactivation of GSK3 is
not associated with an increase in TCF/LEF transcriptional activity, maintaining a distinction
between the PI3K/PKB pathway and the canonical Wnt signaling pathway90. The inactivation of
15
GSK3α/β in vivo also requires dephosphorylation of tyr 279/216, respectively, in response to
extracellular signaling91. Unlike most protein kinases that rely on sequence recognition, many
target substrates of GSK3 require a priming phosphorylation event by another kinase in order to
generate a recognition site. The primed molecule can then interact with Arg96 (on the GSK3β
isoform), which is proximal to the substrate-binding site, and bind to GSK392. When GSK3 is
inactivated by hormone and growth factor inhibiting phosphorylation on Ser9 at the amino
terminus (or Ser21 on the GSK3α isoform), the NH2-terminal domain bends back and binds to
Arg96, blocking the substrate-binding site93. Therefore, GSK3 would not be capable of binding
phosphoprimed substrates that rely on Arg96 binding but would be able to target proteins that are
independent of this mechanism, such as β-catenin.
GSK3-mediated phosphorylation is a crucial event leading to the subsequent ubiquitination and
degradation of β-catenin85. It has been shown by several groups that Axin, β-catenin and GSK3
exist in a ternary structure and that Axin promotes GSK3-dependent phosphorylation of β-
catenin94,95. Only Axin-associated GSK3 can display activity toward β-catenin while non-
associated GSK3 showed no activity toward β-catenin and is irrelevant in the Wnt pathway96.
Moreover, Axin shows no preference between either GSK3 isoform, and readily binds to either80.
However, it remains unknown how Axin acts to commandeer an entire pool of Wnt-responsive
GSK3 and whether (and if so, how) Axin acts to shield GSK3 from other regulatory interactions
(i.e. PKB phosphorylation). Axin is not the only molecule affecting GSK3 activity toward β-
catenin. Although the mechanism remains unknown, FRAT (frequently rearranged during
advanced T-cell lymphomas), also known as GBP (GSK3-binding protein), binds to GSK3 in a
mutually exclusive manner with Axin and negatively regulates GSK3 activity toward β-catenin97.
Also, DISC1 (Disrupted in Schizophrenia 1) inhibits GSK3β activity specifically toward β-
catenin through direct interaction, resulting in stability of β-catenin and increased target gene
expression in murine neural precursors98. Therefore, direct interaction with GSK3 is one of the
primary means of regulating its kinase activity toward β-catenin.
Lithium (Li+), long used in the treatment of bipolar disorder, has been shown to inhibit GSK3
kinase activity in a non-competitive manner in vitro and in vivo99,100. The effect is reasonably
specific in that no other kinase is affected but it does interfere with other processes and enzymes,
including IMPase (inositol monophosphatase)99. In corroboration with independent genetic
studies, many GSK3 targets have been identified using Li+ and other chemical inhibitors of
16
GSK3, including cyclin D101. BIO ((2’Z,3’E)-6-Bromoindirubin-3’-oxime), the synthetic
derivative of 6-bromoindirubin, was also found to be a potent competitive inhibitor of GSK3
activity and has been shown to mimic the canonical Wnt pathway by maintaining self-renewal
and pluripotency of mouse and human ES cells102. Furthermore, Lluis et al. found that activation
of β-catenin through BIO-mediated GSK3 inhibition increases the efficiency with which somatic
cell reprogramming occurs mediated by cell fusion of several types of hybrids103. However, it
was found that concentrations above 0.005 µM are no longer specific to Gsk3α/β kinase activity
alone and can inhibit the closely related CDK1/CyclinB, CDK2/CyclinA, and CDK5/p35 at
concentrations below 1µM104. Also, while it has yet to be documented in the literature, it is likely
that BIO interferes with other signaling pathways such as Hh, Notch, and RTK-PI3K, given that
GSK3 plays a mediating role in all of them. Therefore, it is important to consider other possible
direct targets and downsteam effects of BIO-mediated GSK3 inhibition other than the canonical
Wnt signaling pathway. In any event, GSK3 remains a central mediator of many critical
pathways, and understanding how this kinase can be modulated genetically and chemically will
likely be crucial for understanding the molecular basis of various developmental processes and
diseases.
17
Figure 2 – The canonical Wnt Signaling Pathway.
A schema of canonical Wnt signaling. a) The signaling pathway is inactive due to the lack
of Wnt ligand. β-catenin is restrained in the Axin/APC/GSK3 destruction complex and
targeted for serial phosphorylation by GSK3 and CK1 on its amino terminal.
Phoosphorylated β-catenin is degraded. TCF/LEF complexes remain in its repressed state.
b) When Wnt ligands bind to the Frizzled-Lrp5/6 receptors on the cell surface, the
destruction complex is recruited to the inner cell membrane and β-catenin can evade
phosphorylation, allowing it to stabilize and accumulate in the cytosol. β-catenin then
translocates to the nucleus and activates TCF/LEF—mediated transcription of target
genes, such as neurogenin1.
18
1.6 Wnt Signaling in Development and Stem Cells
The canonical Wnt pathway has been shown to be crucial throughout development (Table 1).
Embryos with a null mutation for β-catenin show gastrulation defects such as failure to produce
an A-P (anterior-posterior) Axis or to form mesoderm by E7 that result in embryonic lethality78.
Embryos with constitutive activation of β-catenin seems to result in premature epithelial to
mesenchymal transition in the epiblast105. Furthermore, culturing ES cells in vitro while
maintaining pluripotency seems to require GSK3 inhibition, which can be accomplished by
several small molecule inhibitors106. In tissue-specific development, Wnt has also been shown to
be a crucial pathway. Wnt signaling seems to be important in maintaining self-renewal and
expansion of cardiac progenitors and subsequently needs to be repressed in order for
differentiation of cardiomyocytes or smooth muscle cells to take place107. In the skin, Tcf3 is
exclusively expressed in bulge stem cells and is thought to maintain stem cell activity through
transcriptional repression of Wnt target genes108. Cells destined to become transit-amplifying
cells and differentiate to become hair switch from a Tcf3- to a Lef1-mediated transcriptional
program, resulting in the terminal differentiation of bulge stem cells to hair cells109. In the
intestine, Wnt signaling seems to be the predominant force controlling cell fate along the crypt-
villus axis as nuclear β-catenin is observed at the bottom of the crypts where the stem cells are
thought to reside70. In Tcf4-/- mice, the differentiated epithelial compartments of the neonatal
intestine appear normal, however the crypt progenitor pool is ablated110. A similar phenotype is
also exhibited in mice with transgenic expression of the inhibitor Dkk185. Also, the Wnt target
gene, Lrg5, has been shown to mark the crypt stem cell111. TCF reporters have also been shown
to be active in hematopoietic stem cells (HSCs)70. In a recent study by Zhao et al., β-catenin-
deleted HSCs showed decreased long-term self-renewing capacity upon transplantation into
recipient mice but did not affect lineage differentiation112. Interestingly, an earlier study showed
that deleting β-catenin using an interferon-based method showed no decrease in self-renewal or
reconstitution capacity in vivo113. They hypothesized that γ-catenin (plakoglobin) could be
playing a redundant role in HSCs. However, Koch et al. deleted both β- and γ-catenin in HSCs
and found no impairment of self-renewal in a primary reconstitution assay114. This series of
experiments highlights the controversy and the complexity that remains to be unraveled in the
blood field.
19
1.6.1 Wnt in Neural Development and NSCs
Not surprisingly, Wnt signaling also plays a crucial role in A-P axis formation of the neural tube
through the specification of cell fates78. Various lines of evidence suggest that Wnts have a
posteriorizing role during head formation while inhibiting Wnt, particularly by Dkk1, allows for
anterior neural structure generation115. Various structures within the embryonic cerebral cortex
have been shown to require the activity of specific ligands for proper development. Most
notably, Wnt3a is required at E10.5 to regulate proliferative expansion of a hippocampal
progenitor pool that leads to normal hippocampal development116. In the adult hippocampus, it
has been shown that secreted Wnt3 regulates hippocampal neurogenesis in vitro and in vivo by
promoting the proliferation of neuronal precursors and their subsequent differentiation117. Wnt7a
was also shown to promote neuronal differentiation of E11.5 cortical neural progenitors in vitro,
even in the presence of FGF-2, a potent mitogen118. However, another study by Israsena et al.
suggests that the status of FGF-2 determines whether β-catenin-mediated signaling promotes
proliferation or neuronal differentiation of NSCs119. They propose that while NSCs are in the
SVZ, they receive FGF-2 stimulation, promoting renewal and expansion. However, when these
cells migrate into the cortex and away from the SVZ, they no longer receive FGF-2 signals, and
the β-catenin-mediated pathway promotes a neuronal cell fate. This theory may also help to
explain findings by Chenn and Walsh, who found that when an activated form of β-catenin was
overexpressed in nestin-expressing cells there was an expansion of cells resulting in an
overgrowth of the cerebral cortex, which was thought to be due to increased reentry into the cell
cycle, 120. However, in another study by Ivaniutsin et al. that examined the role of APC in the
developing cerebral cortex, different results were observed with respect to the regulation of NSC
proliferation121. Whereas Chenn and Walsh expressed stabilized β-catenin in neural precursors
prior to commitment to the cerebral cortical fate (~E9.5), APC is deleted under the Emx1
promoter (~E9.5), a promoter that is active in both proliferating and differentiated cortical
neurons122. When APC is deleted under the Emx1 compartment, there is a decrease in the size of
the precursor pool, which seems to be the result of increased apoptosis and a decrease in the
number of cycling cells as indicated by bromodeoxyuridine (BrdU) uptake. Furthermore, loss of
APC resulted in premature neuronal differentiation and caused cerebral cortical cells to adapt
fates typically associated with more dorsal-posterior regions of the CNS. Therefore, while both
studies use models that result in increased β-catenin-mediated signaling, the spatiotemporal
20
regulation of β-catenin-mediated signaling is critical for neural cell fate decisions and may differ
dramatically in a cell-autonomous manner. Along the same lines, it is possible that Wnt signaling
may have differing roles at various levels within the functional hierarchy, for example self-
renewal of NSCs as well as neuronal differentiation of more committed progenitors. Until the
hierarchy can be delineated more effectively, these questions remain unanswered. Further
investigation is required to truly understand the effects of β-catenin-mediated signaling on NSC
function in vitro and in vivo.
1.7 Wnt Signaling in Cancer and CSCs
1.7.1 Wnt in Colorectal Cancers
Given the integral roles that Wnt signaling plays in development as well as homeostasis, it is no
surprise that the canonical pathway has been linked to many human cancers, particularly of the
epithelial variety. For example, around 85% of all sporadic and hereditary colorectal tumours
show loss of APC function123. Of the tumours with wild-type APC, 15% show point mutations
on key serine/threonine residues in the amino-terminus of β-catenin, thought to be putative
targets of GSK3 phosphorylation124. Since Tcf4 is crucial for stem cell maintenance in the crypt,
it is likely that stable activation of the β-catenin/Tcf4 pathway causes the expansion of the crypt
compartment, which is seen in early stages of colorectal tumourigenesis125. Also, the hereditary
disease known as familial adenomatous polyposis (FAP) that eventually leads to colorectal
cancer in inflicted individuals is caused by a loss of function mutation in the APC gene124. This
is similar to another disease known as Turcot’s Syndrome (TS), discussed later, in which a
germline mutation in the APC gene leads to colorectal cancer and, infrequently, to primary brain
tumours126.
1.7.2 Wnt in Skin Cancers
In the context of normal skin homeostasis, TCF3-mediated repression of canonical Wnt signaling
is necessary to maintain bulge stem cell activity, while an active LEF1-mediated pathway seems
to promote terminal differentiation127. In human metastatic melanoma as well as a murine model
(B16), it was observed that activated β-catenin signaling correlated with decreased proliferation
and smaller tumour volume128. Furthermore, the study showed that inducing Wnt signaling up-
regulates markers of melanocyte differentiation. Therefore, it seems the loss of Wnt signaling
21
may be critical for melanoma progression and metastasis. In squamous cell carcinoma (SCC),
however, Malanchi et al. reported the identification of a murine CSC population based on the
expression of CD34 and the exclusion of various lineage markers129. They also report that
cutaneous CSC maintenance is dependent on continuous β-catenin signaling since when β-
catenin was genetically ablated, there was a marked loss of the CD34+ population and CSCs lost
the ability to initiate secondary tumours due to terminal differentiation, indicating a loss of self-
renewal properties. Therefore, in another type of skin cancer, it seems that β-catenin mediated
signaling induces the opposite effect as compared to normal circumstances, signifying the highly
context-dependent nature of Wnt signaling.
1.7.3 Wnt in Leukemias
Wnt has also been shown to be deregulated in various leukemias130. In acute myeloid leukemia
(AML), it has been shown that important Wnt pathway antagonists (sFRPs and DKKs) are
downregulated due to promoter methylation and silencing, potentialliy contributing to the
pathogenesis of AML131. The activation of Wnt/β-catenin signaling has also been implicated in
maintaining chronic myeloid leukemia (CML) stem cell function112. This was demonstrated
using the BCR-ABL leukemia mouse model and crossed into conditionally deleted β-catenin-/-
mice. Results of the study showed that self-renewal of CML stem cells were impaired in BCR-
ABL/β-catenin-/- mice compared to control BCR-ABL mice, as was shown by serial
transplantation assay. It was shown that BCR-ABL protein levels were significantly decreased in
β-catenin-/- mice, but that this observation was specific to BCR-ABL CML and not acute
lymphoid leukemia (ALL). In another study, it was observed that blast crisis (BC) CML stem
cells express a misspliced variant of GSK3β—a negative regulator of β-catenin—that resulted in
the loss of its kinase domain and the activation of canonical Wnt signaling132. Moreover, BC
CML stem cells that expressed this splice variant exhibited enhanced leukemic engraftment that
could be rescued by reintroducing full-length GSK3β. Further investigation is necessary to
uncover novel therapeutic targets specifically within the Wnt pathway.
22
1.8 Wnt in Brain Tumours
1.8.1 Wnt in Medulloblastomas
The dysregulation of Wnt signaling has been well documented in medulloblastomas. Turcot’s
syndrome, a familial cancer syndrome in which patients have a germline mutation in the APC
gene, results in the development of colonic polyps and has also been linked to the development
of primary brain tumours, most of which are medulloblastomas133. Many studies have also
documented mutations in the genes of key components of the Wnt signaling pathway, including
APC, CTNNB1 (the gene for β-catenin), CDH1 (the gene for the adhesion molecule E-cadherin),
and GSK-3β loss of heterozygosity in sporadic medulloblastomas134. Therefore, evidence
suggests that activated Wnt signaling is implicated in the pathogenesis of at least a subset of
medulloblastomas. Interestingly, however, a study conducted in Britain found that children that
presented with medulloblastomas that were immunoreactive for nuclear β-catenin had a
significantly higher overall (OS) and event-free (EFS) survival compared to children who did not
show nuclear immunoreactivity to β-catenin, and thus β-catenin status can actually be used as a
prognostic marker135,136. While it is clear that the Wnt/β-catenin pathway has been directly linked
to medulloblastomas oncogenesis, it is unknown whether it plays a role as a driving
transformation event in tumour initiation, or whether its dysregulation is a secondary event that
is associated with and contributes to disease progression.
1.8.2 Wnt in Glioblastoma Multiforme
In gliomas, specifically glioblastoma multiforme (GBM), less is known about the role of Wnt in
tumourigenesis, however, a few studies using serum-derived lines have attempted to address this
broad question. In one study it was observed that sFRPs, a group of secreted Wnt inhibitors,
slowed the motility of glioma cells while enhancing proliferation and in vivo tumourigenicity137.
Surprisingly, while β-catenin levels were not altered by sFRP-2-mediated inhibition, tyrosine
phosphorylation, which is thought to regulate β-catenin’s association with cadherins, was
significantly decreased and may help to explain the observed phenotype. A separate study by Pu
et al. observed that knockdown of Wnt2 as well as β-catenin induced apoptosis and decreased
cell growth in vitro and in vivo138. They also observed an associated decrease in PI3K/Akt
activity in Wnt2 and β-catenin knockdown lines, which may help to explain the observed
decrease in proliferation. Overexpression of Wnt5a, thought primarily to be a canonical ligand,
23
has also been shown promote proliferation of glioma cells in vitro, while in vivo, Wnt5a-
knockdown results in decreased tumour volume upon orthotopic transplantation in comparison to
the control139. Early studies investigating the role of Wnt1 in the development of the CNS
revealed that Wnt1 regulates proliferation of mid/hindbrain precursor cells, and that
overexpression of Wnt1 causes an expansion of the hindbrain region140,141. Although no reports
implicate Wnt1 in gliomagenesis as of yet, it is conceivable that its mitogenic effects may
contribute to neoplastic progression.
While various secreted proteins have been implicated in gliomas, another study looked at
GSK3β, a negative regulator of β-catenin as well as a point of convergence for several signaling
transduction pathways known to modulate cell proliferation (eg. FGF-1, Insulin, PI3K/Akt)142.
When treated with various GSK3 inhibitors, tumour cells showed decreased expression of neural
precursor markers (Sox2 and Nestin) and increased expression markers associated with glial and
neuronal differentiation (GFAP and β-III-tubulin, respectively). In contrast to the previous
findings, this study suggests that an activation of the canonical Wnt signaling pathway through
inactivation of a key modulator—GSK3β—results in growth arrest and increased terminal
differentiation. It is clear from these confounding results that further investigation of Wnt
signaling in gliomas is warranted to truly appreciate its affect on tumourigenesis and
maintenance.
1.8.3 Brain tumour stem cells in vitro
A caveat of the above studies is that experiments were carried out using serum-derived GBM
lines. It has become increasingly clear, however, that traditional serum-derived GBM lines to do
not recapitulate the genotype and phenotype of the original human GBM, making it a poor in
vitro model for drug discovery and preclinical testing17. GBM lines established in defined serum-
free conditions with bFGF and EGF exhibit consistent growth kinetics, retain similar
morphology to NSCs, express known immature markers (Sox2 and Nestin), retain differentiation
capacity, and retained in vivo tumourigenic and self-renewing properties through serial
transplantation, irrespective of passage number. In contrast, the serum-derived lines varied in
growth kinetics depending on the passage number, altered their morphology to resemble
fibroblast-like cells, lost NSC marker expression and showed varied response to differentiation
cues. Also, serum-derived lines were only tumourigenic at late passages, when their proliferation
24
rate seemed to increase exponentially. When gene expression and cluster analysis were
performed for serum-free, serum-derived, NSCs and parent GBM tissue, results revealed that
gene expression of serum-derived GBM lines did not reflect the gene expression pattern of the
original GBM, nor did it cluster with serum-free GBM lines or NSCs. On the other hand, serum-
free GBM lines retained similar gene expression patterns to the original GBM from which it
originated, regardless of passage, and it clustered with other fresh GBMs, and NSCs143. These
observations reveal that results from studies using serum-derived GBM lines may not report a
biologically relevant phenomenon. What this also suggests is that very little is known about the
role of Wnt on the tumour-initiating population (i.e. BTSCs), which are thought to be the cells
driving the tumour’s progression. Thus, it is important to carry out future investigations in a
culture system that more accurately reflects the in vivo characteristics of human GBMS, such as
serum-free adherent GBM lines144.
25
Table 1
26
1.9 Thesis Rationale and Aim
One view of cancer is that it may represent aberrant organogenesis. In the last ten years, there
has been a large amount of evidence suggesting that tumours are arranged in a functionally
hierarchical fashion, with a subpopulation of tumour stem cells driving tumour initiation and
maintenance, much like adult stem cells within normal tissues. Many developmentally
significant pathways, including the canonical Wnt pathway, have been implicated in cancer
development. Thus it is necessary to gain a firm understanding of the molecular events
governing normal development in order to appreciate what is perturbed in tumour initiation and
progression. There remain many gaps in our understanding of canonical Wnt signaling in human
NSCs. Our goal has been to determine the effect of β-catenin-mediated Wnt signaling on normal
human NSC proliferation, survival, and lineage specification in vitro. Additionally, and in
parallel, we also sought to determine whether Wntsignalingmayplayaroleinregulating
cancer stem cells isolated from human glioblastomas (GBMs). Our ultimate goal is to determine
how targeting the canonical Wnt signaling pathway in human brain tumors may affect the
proliferation and/or differentiation of brain tumour stem cells.
1.10 Thesis Hypothesis
Canonical Wnt signaling plays a role in lineage specification of normal human NSCs. By
extension, we hypothesize that brain tumor stem cells may also retain responsiveness to Wnt
signaling by altering cell fate decisions, and thus may offer a novel target for therapy of GBMs.
27
Chapter 2
2 Materials and Methods
2.1 Cell culture and differentiation protocol
Human fetal brains (5205: Gestational Week (GW) 11; 5281: GW12.5) were mechanically
dissociated in Neurobasal medium (Invitrogen) and made into single cell suspensions with
Accutase (Sigma) treatment. Primary cells were plated onto laminin (10 mg/L, Sigma)-coated
dishes (Primaria) in expansion media made up of NeuroCult Basal Human medium (Stem Cell
Technologies), modified N2 supplement (Ying and Smith, 2003), B27 (20ml/L final, Invitrogen),
penicillin-streptomycin-fungizome (10 ml/L final, Sigma), Heparin (2 µg/ml final, Sigma),
recombinant mouse EGF (10 ng/ml final, Sigma), and recombinant human FGF-2 (Stem Cell
Technologies). Cells were incubated in a 37°C incubator containing 5% CO2, fed every 2-3 days
and passaged 1:2-1:3 when cultures became confluent.
Brain tumour samples were obtained from patients treated in Toronto hospitals following local
ethical board approval. GliNS1 (unknown, M), G377 (19 yr, F), G179 (52 yr, M) were all
diagnosed as glioblastoma multiforme (GBM). GNS cells were cultured as described above with
human NS cells.
In order to differentiate NSCs and BTSCs cells, 0.5-2×106 human NS cells were plated onto
poly-L-ornithine (Sigma) and laminin 10 cm dishes in expansion medium for 24 hours then
switched to expansion medium without EGF. After 7 days, medium was changed to the mix
NeuralCult basal-Neurobasal (1:1), supplemented with N2 (0.5×), Heparin (2 µg/ml final), and
B27 (1×) for 14 days. Recombinant mouse Wnt3a (R&D laboratories) and BIO (sigma) were
used in some cell culturing experiments.
2.2 Immunoblotting
Cells were lifted off the tissue culture plates with Accutase (Sigma), washed with PBS and lysed
in lysis buffer (50 mM Tris pH=7.4, 1% NP-40, 0.25% NaDeoxycholate, 0.15 M NaCl, 1mM
EDTA, 1 mM Na3VO4, 1mM NaF, 1% SDS) to obtain total protein. Cellular lysate was loaded
with 4X SDS loading buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006%
bromophenol blue, 18 µg/ml β-mercaptoethanol) onto 7.5% and 15% SDS gels and separated by
28
electrophoresis. Proteins were transferred to Imobilon-P PVDF membranes (Millipore) using a
wet transfer apparatus (BioRad) and blocked in 5% non-fat Milk 1×TBST for an hour at room
temperature. The primary antibodies β-catenin (1:1000, Cell Signaling), β-III-tubulin (1:1000,
Chemicon), GFAP (1:1000, Dako), Nestin (1:1000, Chemicon, and β-actin (1:5000, Sigma) were
incubated in 1% non-fat milk 1×TBST either overnight at 4°C or at room temperature for one
hour. Membranes were then probed with the HRP conjugated secondary antibody in 1% non-fat
milk 1×TBST for one hour at room temperature and detected with enhanced chemiluminescence
(ECL) (GE health) and exposed to Amersham hyperfilm ECL film. Blots were stripped with
Restore stripping buffer (Thermo Scientific) for 10-15 minutes at room temperature and reprobed
if necessary.
2.3 Immunofluorescence
Cells were plated at required density (stated in results text and figures) on poly-L-ornithine and
laminin-coated glass coverslips. Cells were fixed in 4% PFA at room temperature for 10-20
minutes, permeabilized with 0.3%-Triton-100X PBS for 5 minutes, and blocked in 1X PBS with
10% FBS for one hour at room temperature. Cells were stained with primary antibody (1:500; β-
III-tubulin, 1:500; GFAP, 1:1000; MAP2, 1:500) in 1X PBS with 10% FBS either for one hour at
room temperature or overnight at 4°C. Cells were then washed in 1XPBS and incubated with
secondary antibodies (Alexa-488; Alexa-568) for one hour at room temperature. Following three
washes, converslips were mounted onto glass slides with DAPI staining. All analysis was
performed on the Zeiss Axiovert 200M microscope.
2.4 Intracellular Flow Cytometry
Cells were dissociated from the tissue culture plate and suspended in 1 ml of 1X PBS. Cells were
fixed with 50 µl of 32% PFA for 10 minutes on ice. Cells were washed with 1X PBS and
permeabilized with 100% ice-cold methanol for 30 minutes on ice, followed by two more washes
with 1X PBS. Cells were blocked in staining buffer (5% NGS, 4mM EDTA, 15mM HEPES,
PBS) for at least one hour at 4°C and then incubated with primary antibody (β-III-tubulin, 1:500;
GFAP, 1:8000) in staining buffer for 30-60 minutes at 4°C. Cells were washed in fresh staining
buffer and incubated in fresh staining buffer with secondary antibodies (Alexa-488; Alexa-405;
Alexa-350; Alexa-633) for 30-60 minutes at 4°C. Cells were washed in fresh staining buffer and
29
left overnight at 4°C to be analyzed the next morning. Cells were analyzed on the BD LSRII-SC
analyzer.
2.5 Luciferase Assay
Luciferase assays were performed as outlined in the Promega Dual-Luciferase Reporter Assay
System (Cat. No. E1910) in 24-well tissue culture plates coated in PLO and laminin. The Renilla
luciferase plasmid was used as a transfection and loading control for every assay. SuperTOPflash
(and FOPflash) luciferase reporter constructs were used to measure TCF/LEF transcriptional
activity and were generous gifts from R.T. Moon.
2.6 Transfection of cells
Transfections were performed according to protocol of the Amaxa mouse Neural Stem Cell
Nucleofector Kit (Lonza). Transfected cells were always replated in full media with EGF and
FGF for a minimum of 24 hours prior to any assay to ensure full recovery post-transfection. Each
plasmid was transfected in the following amounts: TOPflash (or FOPflash): 3µg, Renilla
luciferase: 0.06µg, ΔN90 (or pcDNA empty vector): 5µg. The ΔN90 cDNA expression vector
was a generous gift from B. Alman.
2.7 MTT assays
Cells were seeded in a poly-L-ornithine and laminin-coated 96-well tissue culture plate at
required density (stated in results text and figures). Proliferation was measured on required days
(stated in results text and figures) by adding MTT reagent (1:10, Roche) to the final volume in
each well. Cells and MTT reagent were incubated for 4 hours at 37°C. Equal volume of MTT
solubilizaton buffer to total volume was added to each well and incubated overnight at 37°C to
allow for full lysis of cells. Quantification of viable cells through reading of ultraviolet
absorption spectrum at 575 nm was performed the next day on a Versamax microplate reader
(Molecular Devices, Sunnyvale, CA) equipped with SoftMax Pro software (Molecular Devices,
Sunnyvale, CA).
2.8 BrdU labeling
Cells grown on glass cover slips were pulsed with BrdU (10µM) for 24 hours and then washed
twice with PBS. Cells were then fixed with 4% PFA for 20 minutes at room temperature. Cells
30
were washed with PBS and then treated with 2M HCl for 20 minutes at room temperature. Cells
were then treated with 0.1M sodium borate (NaB4O7) (pH8.5) for 2 minutes at room temperature,
followed by 2 washes with PBS. Cells were stained for BrdU (1:250) according to the previously
described immunocytochemistry protocol.
31
Chapter 3
3 Results
3.1 Wnt signaling promotes a neuronal cell fate choice in human fetal neural stem cells
3.1.1 Human fetal neural stem cells express key Wnt pathway components and exhibit low baseline TCF/LEF-mediated transcription
In order to obtain an understanding of whether Wnt signaling was active in normal human neural
stem cells (hNSCs), we first sought to determine whether key signaling components were
expressed in serum free cultures of human neural stem cells that we had isolated from human
fetal brain. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine the
mRNA expression of various members of the pathway, including Frizzled receptors Fz1, Fz3,
Fz6, Fz7, Fz8, Fz9 and Low density lipoprotein related-receptor protein 5 (Lrp5), the soluble
pathway inhibitor and target gene DKK1, and the central signaling regulators Gsk3βandβ‐
catenin(Figure3a).The mRNA expression patterns of these receptors demonstrated variable
transcript levels and heterogeneity among Wnt pathway components and between hNSC lines
(Figure 3a). We performed western blot analysis on these two hNSC lines to confirm protein
expression of β-catenin, the central mediator of the canonical pathway (Figure 3b). Given that
key components for β-catenin-mediated Wnt signaling are expressed in hNSCs, these cells will
likely respond to experimental manipulation of pathway activity.
32
Figure 3 - HumanNSCsexpresscanonicalWntsignalingpathwaycomponents
hNSCs express Wnt signaling pathway components. a) RT-PCR for various components
reveal that hNSC lines express various Frizzled receptors (1, 3, 6, 7, 8. 9), Lrp5, Dkk1, and
the central components β-catenin and Gsk3β at the mRNA level. Gapdh was used as a
loading control. b) β–catenin was also detected at the protein level by western blotting. β-
actin was used as a loading control.
33
Canonical Wnt signaling is a cascade of molecular interactions converging on the stabilization of
β-catenin and culminating in TCF/LEF—mediated transcription of target genes (Figure 4a).
Wnt3a is one of 19 Wnt ligands identified in the mammalian system. We then tested whether
exogenous stimulation of Wnt signaling resulted in alteration in expression and activity of β-
catenin to assess whether the Wnt signaling cascade is intact in our hNSCs. Within one hour of
exogenous Wnt3a stimulation, β-catenin levels were increased compared to baseline levels in
both hNSC lines, indicating that Wnt3a stimulation resulted in the stabilization and accumulation
of β-catenin (Figure 4b).
The luciferase reporter assay, TOPflash, measures TCF/LEF-mediated transcriptional activity,
which is a measure of canonical Wnt signaling. Eight TCF/LEFconsensus DNA-binding sites lie
upstream of a minimal TK promoter and the firefly luciferase gene (Figure 4c).The negative
control, FOPflash, has eight mutated TCF/LEFbinding sites. When Wnt signaling is activated,
luciferase is transcribed and levels correlate directly to signaling output. As a means of
transfection control, the Renilla luciferase gene is co-transfected into cells to give an estimate of
total transcriptional activity within the bulk transfected cells, serving as a transcriptional
baseline. TOP/FOPflash levels are determined by normalizing it to baseline transcriptional
activity. We used the TOPflash assay to investigate whether the observed stabilization of β-
catenin protein levels correlates with TCF/LEFtranscriptional activity. We found that hNSCs
exhibit very low basal Wnt signaling levels in serum free EGF/FGF conditions in vitro and were
able to respond to Wnt3a stimulation, demonstrating 30-55 fold increase in luciferase levels after
24 hours. Therefore, while hNSCs maintain low basal TCF/LEF transcriptional activity in vitro,
they are capable of responding to Wnt signaling, which may have implications regarding their
functional characteristics.
34
Figure 4 – hNSC lines activate TCF/LEF transcriptional activity in response to Wnt3a.
Human fetal neural stem cells are Wnt-responsive and activate TCF/LEF transcription. a)
A schema depicting the sequence of events that begins with a Wnt ligand binding to a
Frizzled receptor complex, inhibiting the GSK3α/β-APC-Axin destruction complex,
stabilizing β-catenin and activating TCF/LEF transcription of target genes. b) Cells treated
with recombinant mouse Wnt3a (100 ng/ml) for 2 hours respond to Wnt3a stimulation
through the accumulation β-catenin to varying degrees relative to baseline levels. c) The
TOPFlash construct is depicted with 8X TCF/LEFconsensus-binding sites upstream of a
minimal TK promoter and the Firefly luciferase gene. d) hNSCs transfected with TOPflash
(or the negative control FOP) and the Renilla luciferase construct. After 24 hours of
recovery, hNSCs were treated with Wnt3a (100 ng/ml) for 24 hours (or fresh media) and
their TOP/FOP levels were compared in the above bar graph. All TOP and FOP readings
are normalized to the Renilla luciferase output for transfection control.
35
3.1.2 Wnt3a does not alter human fetal neural stem cell proliferation
Within the mammalian CNS, Wnt3a signaling has been shown to be critical for proper
hippocampal development by regulating hippocampal precursor expansion116. Wnt3a is
considered to be a canonical Wnt ligand that activates the β-catenin-mediated pathway. The
current model has Wnt3a forming a receptor-ligand complex with Frizzled and Lrp5/6 that
triggers a series of intracellular events leading to the destabilization of the destruction complex
and the accumulation of β-catenin (Figure 2a).
Exogenous stimulation of hNSCs with Wnt3a is a physiologically relevant approach to activating
β-catenin—mediated signaling and induces a 30 to 55-fold increase in TCF/LEF transcriptional
activity (Figure 4d). We wanted to know whether exogenous Wnt3a stimulation resulted in any
functional consequences in hNSCs. Using the MTT assay to assess proliferation and survival, we
found no significant changes in hNSCs treated with Wnt3a analyzed over 13 days in the presence
of EGF and FGF compared to their untreated counterparts (Figure 5a). Therefore, it seems that
Wnt signaling does not cooperate with EGF and FGF signaling to promote proliferation further.
We were concerned that the potent mitogenic effects of EGF and FGF were masking any
possible negative affects exerted by Wnt3a signaling. Therefore, we performed MTT assays on
Wnt3a-treated hNSCs under differentiating conditions—in the absence of EGF and FGF (-
EGF/FGF)—over 13 days, and no significant change in proliferation of hNSCs treated with
Wnt3a was observed (Figure 5b). Although it is possible that Wnt signaling is not a major
regulator of hNSC function, this is likely not the case. Another explanation is that Wnt signaling
does not directly regulate hNSC proliferation, but may affect other cell fate decisions, such as
lineage fate choice.
36
Figure 5 – Wnt3a does not alter hNSC proliferation.
Wnt3a does not significantly alter hNSCs proliferation. a) hNSCs were seeded at 2000
cells/well (96-well plate), grown for 13 days in stem cell conditions (+EGF/FGF) and in the
presence or absence of recombinant mouse Wnt3a (50 ng/ml). No significant difference in
proliferation, as measured by the MTT assay, was observed between the two groups (n=3).
b) hNSCs were seeded at 2000 cells/well (96-well plate), grown for 13 days under
differentiating conditions (-EGF/FGF) and in the presence or absence of recombinant
mouse Wnt3a (50 ng/ml). No significant difference in proliferation was observed between
the two groups (n=4; unpaired student’s T-test).
37
3.1.3 Wnt3a promotes a neuronal cell fate choice in human fetal neural stem cells under differentiating conditions
Several canonical Wnt species, including Wnt3 and Wnt7a, have been shown to regulate
neuronal differentiation in the murine brain3, 118. Therefore, we hypothesized that Wnt3a may
promote a neuronal fate choice in differentiating hNSCs. In order to test this hypothesis, we
differentiated hNSCs in the absence or presence of exogenous Wnt3a, and analyzed the
expression of lineage-specific markers using two methods of analysis (Figure 6a).
Immunofluorescence revealed that Wnt3a-treated hNSCs differentiated to produce a greater
proportion of βIII-tubulin-positive neurons compared to untreated differentiated hNSCs (Figure
6b). Furthermore, the proportion of GFAP-positive cells was greatly reduced in Wnt3a-treated
hNSCs, suggesting Wnt3a may have an inhibitory effect on astrogenesis (Figure 6b). In order to
quantify this phenomenon, we performed intracellular flow cytometry for the same markers
(Figure 6c). Results indicate a 2.6 fold (p=0.0235; n=4) increase in βIII-tubulin-positive neurons
and a 0.59-fold decrease (p=0.0124; n=4) in GFAP-positive astrocytes in the presence of Wnt3a,
suggesting that Wnt3a activation of the canonical pathway promotes a neuronal fate choice in
cultured hNSCs at the expense of the glial lineage.
38
Figure 6 – Wnt3a promotes a neuronal cell fate in differentiating hNSCs.
Exogenous Wnt3a promotes a neuronal cell fate in differentiating hNSCs. a) Cells were
differentiated for 21 days in the absence or presence of Wnt3a (100 ng/ml) and analyzed for
39
lineages specific markers using immunofluorescence and intracellular flow cytometry. Blue
arrows indicate when media was changed and fresh media and Wnt3a were added to cells.
b) hNSCs differentiated in the presence of Wnt3a significantly increased the number of
βIII-tubulin-expressing cells (red) compared to the untreated differentiated hNSCs.
Furthermore, Wnt3a-treated hNSCs also produced significantly fewer GFAP-expressing
cells (green) compared to untreated hNSCs (DAPI, blue). c) Intracellular flow cytometry
histograms reveal that the number βIII-tubulin-expressing cells increases by 2.598-fold and
a 0.588-fold decrease in the amount of GFAP-positive cells in the presence of Wnt3a
relative to untreated hNSCs. Unstained, red; untreated, green; Wnt3a-treated, blue (n=4;
paired student’s T-test).
40
3.1.4 Stabilized β-catenin activates Tcf/Lef—mediated transcription
The stabilization of β-catenin is critical for its translocation to the nucleus and subsequent
coactivation of Tcf/Lef transcription factors. Cytosolic β-catenin levels are modulated by CK1’s
and GSK3’s constitutive phosphorylation on several conserved amino acid residues (S45, T41,
S37, and S33) located at the amino terminus of the protein. These serial phosphorylation events
prime the protein for ubiquitination and proteasomal degradation. Therefore, stabilization
requires that β-catenin evades phosphorylation and subsequent degradation. In order to
circumvent degradation and the need for extracellular stimulation, a truncated mutant of β-
catenin was produced where the first 90 amino acids are removed from the amino terminus of the
protein, ΔN90 β-catenin (or just ΔN90). This truncation results in the loss of the key
phosphorylation substrates of CK1 and GSK while conserving critical domains integral to its
function, such as the transactivating domain and other protein-protein binding domains also
present in the wild-type (Figure 7a)145.
Previous studies have shown that the expression of ΔN90 within the neural precursor
compartment of the developing murine CNS in vivo results in active Wnt signaling, increased
cell cycle reentry, increased proliferation, and thus an expansion of the precursor pool120.
Therefore, in parallel to our other methods, we set out to understand how ΔN90 might affect
hNSCs in vitro. First, we confirmed that the protein product of the ΔN90 plasmid is detectable
by immunoblotting 48 hours after the transfection. Indeed, when we probe with a carboxy-
terminal recognizing antibody, we do see a second band at around 78 kDa as well as the
endogenous protein located at 92 kDa (Figure 7b). Next, we hoped to confirm that ΔN90
increases Tcf/Lef-mediated transcription, thereby mimicking activated Wnt signaling. Using the
TOPFlash assay we were able to confirm that ΔN90 results in a dramatic increase (167-fold) in
signaling activity relative to endogenous signaling levels (pcDNA transfection control vector)
(Figure 7c).
41
Figure 7 – Stabilized β-catenin activates Tcf/Lef transcriptional activity in hNSCs.
ΔN90-β-catenin activates Tcf/Lef transcription. a) Schema of wildtype (WT) and the
stabilized mutant form of β-catenin (ΔN90) illustrate the 13 armadillo repeats, the Axin,
APC and TCF/LEF binding domains. ΔN90 has an amino terminal truncation of the
protein, which removes key phosphorylation substrates targets by CK1 and GSK3 kinase
activity. b) hNSCs were transfected with either ΔN90 or the control empty vector (pcDNA).
Cell lysates were collected 48 hours post transfection and an immunoblot was performed
for total β-catenin levels. Only ΔN90-transfected cells expressed ΔN90-β-catenin at ~78kDa.
c) hNSCs were transfected with the TOPflash (or FOPflash) and Renilla construct in
conjunction with either the ΔN90 construct (or pcDNA). The TOPFlash assay indicates that
ΔN90 dramatically elevates (~167-fold) Tcf/Lef activity relative to baseline levels (pcDNA)
(n=2).
42
3.1.5 Stabilized β-catenin does not alter human neural stem cell proliferation.
In light of previously reported findings as well as the substantial induction of TCF/LEF activity,
we predicted that expression of ΔN90 in hNSCs would result in increased proliferation. We
performed an MTT assay comparing ΔN90-transfected cells to pcDNA-transfected cells in stem
cell conditions (+EGF/FGF). Surprisingly, there was no significant difference in proliferation
between ΔN90-expressing cells and the control-transfected hNSCs in the presence of EGF and
FGF (Figure 8a). One possibility is that β-catenin does not act synergistically or additively with
the potent mitogenic effects of EGF and FGF in the culturing media, and its effects are masked.
Therefore, we sought to investigate the effects of stabilized β-catenin in conditions where hNSCs
are induced to differentiate (-EGF and FGF). We performed an MTT assay comparing ΔN90-
transfected cells to the control-transfected control cells at days 4 and 8 post-transfection under
differentiating conditions and found no significant difference in proliferation (Figure 8b). These
results indicate that, contrary to findings observed in embryonic mouse neural precursors in vivo,
stabilized β-catenin does not increase the proliferation and/or survival of hNSCs in vitro.
43
Figure 8 – Stabilized β-catenin does not affect proliferation of hNSCs in vitro.
Stabilized β-catenin does not significantly alter proliferation rate of hNSCs. a) hNSCs were
transiently transfected with either pcDNA (transfection control) or ΔN90, seeded at 2000
cells/well (96-well plate) in EGF and FGF and grown for 8 days. Proliferation, as measured
by the MTT assay, was not significantly altered by stabilized β-catenin (n=3). b) hNSCs
were transiently transfected with either pcDNA (transfection control) or ΔN90, seeded at
2000 cells/well (96-well plate) in differentiating conditions (-EGF/FGF) and grown for 8
days. Proliferation, as measured by the MTT assay, was not significantly altered by
stabilized β-catenin (n=3; unpaired student’s T-test).
44
3.1.6 Stabilized β-catenin promotes neuronal cell fate choice in human fetal neural stem cells under differentiating conditions
Several groups have observed that Wnt signaling also plays a role in cell fate determination of
neural precursors117,118. Therefore, we wanted to investigate whether stabilized β-catenin
affected the cell fate choice of hNSCs during differentiation. In order to interrogate this, we
transfected hNSCs with ΔN90 or pcDNA (transfection control) and differentiated them in the
absence of EGF and FGF (Figure 9a)146. We confirmed the presence of the ΔN90 mutant at the
protein level two days post-transfection (Figure 7b). After 21 days, we performed
immunofluorescence for βIII-tubulin and GFAP to identify both a neuronal and astrocyte
population, respectively. We found that constitutively active Wnt signaling in ΔN90-transfected
hNSCs prior to differentiation mildly increased the number of βIII-tubulin-positive neurons but
showed no dramatic change in GFAP-expressing cells (Figure 9b). An immunoblot confirmed
the mild pro-neuronal trend and showed a decrease in GFAP protein levels, indicating a mild
block in astrogenesis in the ΔN90-differentiated population (Figure 9c). Furthermore, nestin
protein levels were decreased in ΔN90-transfected differentiated populations, inferring a more
terminally differentiated population compared to the control differentiated population (Figure
9c). We used intracellular flow cytometry to quantify neuronal and astrocytic populations within
each sample using βIII-tubulin and GFAP, respectively, and found a 1.697-fold increase in the
number of βIII-tubulin expressing cells in differentiated hNSCs transfected with ΔN90 compared
to the pcDNA-transfected control group (Figure 9d). Only a slight decrease was detected in the
number of GFAP-positive cells present in the ΔN90-transfected sample (0.987-fold) (figure 9d).
While these results are preliminary and must be repeated several times to confirm the observed
pro-neuronal trend, they do suggest that activation of the canonical Wnt signaling pathway helps
to promote a neuronal cell fate choice in differentiating hNSCs. Furthermore, while previous
reports focus on the pro-proliferative affects of ΔN90 in the precursor pool of the murine nervous
system, no such affects were observed in hNSCs in vitro (Figure 8).
45
Figure 9 – Stabilized β-catenin promotes a neuronal cell fate choice during differentiation.
Stabilized β-catenin promotes a neuronal cell fate under differentiating conditions. a)
hNSCs were transiently transfected with either pcDNA or ΔN90 and differentiated by
sequential removal of EGF and FGF for a period of 21 days. b) Differentiated hNSCs were
46
stained for neuronal (βIII-tubulin, red) and glial (GFAP, green) markers. Cells transfected
with ΔN90 produced a greater proportion of βIII-tubulin—expressing cells and no change
in GFAP—positive cells numbers compared to the transfection control (pcDNA). c)
Western blots for βIII-tubulin and GFAP also confirmed that stabilized β-catenin (ΔN90)
promoted a neuronal cell fate choice in differentiating hNSCs as well as a decrease in
GFAP. Furthermore, immunoblotting for the precursor marker nestin was drastically
reduced in ΔN90-transfected hNSCs. d) Intracellular flow cytometry histograms for βIII-
tubulin and GFAP reveal a 1.697-fold increase in the proportion of βIII-tubulin-positive
cells and a slight decrease (0.987-fold) in the number of GFAP-positive cells detected in
ΔN90-transfected cells compared to the control. Unstained, red; pcDNA control, green; and
ΔN90, blue. (n=1).
47
3.1.7 BIO—mediated inhibition of GSK3 activates TCF/LEF transcription
Concurrently, a third strategy to perturb canonical Wnt signaling is by treating with GSK3 small
molecule inhibitors since chemical inhibitors represent a clinically relevant method of altering
Wnt signaling. These are thought to have potent effects on Wnt signaling, but may also interfere
with other pathways. We decided to use the small molecule GSK3 inhibitor, BIO, to further
interrogate cell fate choices of hNSCs because it is easily accessible and other independent
groups have published functional studies using BIO as a method of Wnt stimulation104. BIO
((2’Z,3’E)-6-Bromoindirubin-3’-oxime), is a reversible ATP competitive inhibitor of GSK3α/β
that can serve as a Wnt signaling agonist (Figure 10a)102. While BIO is specific to GSK3α/β,
concentrations above 0.005 µM have also been shown to inhibit the closely related cyclin-
dependent kinases (CDKs) 1, 2, and 5, which may influence proliferation rates104.
GSK3 was initially identified as a kinase involved in glucose metabolism, but was later
discovered to play an integral role as a negative regulator of the canonical Wnt pathway147.
GSK3 participates as a key member of the destruction complex by phosphorylating β-catenin,
priming it for ubiquitination and subsequent proteasomal degradation. When GSK3 kinase
activity is disrupted, β-catenin can evade phosphorylation and translocate to the nucleus to
interact with the TCF/LEF transcription factor complex, mediating target gene transcription
(Figure 10a). BIO has been shown to stabilize β-catenin and mimic Wnt signaling104. To
validate that BIO acts as a Wnt signaling agonist in our system, we performed a TOPFlash assay
on hNSCs treated with BIO for 24 hours (Figure 10b). We used 1µM of BIO because
concentrations below 0.5µM showed almost no TCF/LEF activation after 24 hours (data not
shown) and its only off-target substrates were the CDKs mentioned above. Results indicate that
1µM of BIO significantly activates TCF/LEF transcription by approximately 26.4-fold relative to
baseline transcriptional activity, thereby mimicking canonical Wnt activation. Although higher
concentrations (2 µM) illicited a higher TCF/LEF-mediated signal as measured by TOPFlash
activity, this concentration was accompanied by increased cell sloughing, suggesting cytotoxic
effects (data not shown). Therefore, chemical activation of Wnt signaling by GSK3 inhibition
offers another strategy for assessing the function of hNSCs in vitro.
48
Figure 10 – BIO-mediated inhibition of GSK3 activates TCF/LEF transcription
a) BIO ((2’Z,3’E)-6-Bromoindirubin-3’-oxime) inhibits both GSK3α and GSK3β kinase
activity, resulting in the stabilization and accumulation of β-catenin and transcriptional
activation of the TCF/LEF complex. b) hNSCs were transfected with the TOPflash
construct (or FOP) and the Renilla luciferase construct. After 24 hours of recovery, hNSCs
were treated with BIO (1µM) for 24 hours (or fresh media for endogenous levels) and their
TOP/FOP levels were compared in the above bar graph. All TOP and FOP readings are
normalized to the Renilla luciferase output for transfection control. BIO was able to induce
a 26.4-fold increase in TCF/LEF activity relative to endogenous TCF/LEF activity (*:
p<0.01; n=3; unpaired student’s T-test).
49
3.1.8 BIO promotes differentiating human fetal neural stem cells to slow proliferation and exit the precursor state
Activation of Wnt signaling by ΔN90 and Wnt3a did not show any affect on proliferation of
hNSCs under differentiating conditions. However, when we performed an MTT assay for hNSCs
treated with BIO under differentiating conditions, we found a significant reduction in
proliferation of hNSCs treated with BIO (Day 4: p≤0.001; Day 10: p≤0.0001, Figure 11a). This
observation may be due to a GSK3-mediated pathway that does not converge solely on Wnt
signaling. Also described earlier, BIO may also inhibit key CDKs at the concentration used.
Therefore, the affect on proliferation may be attributed to CDK inhibition, rather than GSK3
inhibition. Further investigation is required to determine which target molecule is responsible for
the observed decrease in proliferation.
HNSCs were differentiated in the absence of EGF and FGF for 21 days and then analyzed for
precursor marker expression by intracellular flow cytometry. BIO-treated hNSCs showed a
significant decrease in the number of Nestin-positive (0.334-fold) and Sox2-positive (0.41-fold)
cells compared to untreated differentiated hNSCs (Figure 11b). Together, the decrease in
proliferation paired with the loss of immature marker expression suggests that BIO-mediated
inhibition of GSK3 may promote hNSCs to exit the precursor state toward a more differentiated
cell type.
50
Figure 11 – BIO-mediated GSK3 inhibition promotes hNSCs to exit the precursor state.
a) hNSCs were seeded at a density of 2000 cells/well (96-well plate) and grown under
differentiating conditions (-EGF/FGF) for 10 days either in the presence or absence of BIO
(1µM). Treatment with BIO resulted in a significant decrease in hNSC proliferation (*:
p≤0.001, **: p≤0.0001) (n=3; unpaired student’s T-test). b) Intracellular flow cytometry
histograms reveal that BIO-treated differentiated hNSCs exhibit a significant decrease in
then proportion of Nestin-positive and Sox2-positive cells compared to their untreated
counterparts. Unstained, red; untreated, green; BIO-treated, blue (n=3; paired student’s
T-test).
51
3.1.9 BIO promotes a neuronal cell fate choice in differentiating human fetal neural stem cells
Since activation of canonical Wnt signaling by other approaches results in a neuronal fate choice
during hNSC differentiation, and that BIO causes a loss in precursor marker expression, we
predicted that BIO treatment in the absence of growth factors would result in an increased
proportion of neurons within the differentiated population. In order to validate our predictions,
we differentiated our hNSCs for 3 weeks in the absence or presence of BIO and then analyzed
our cells for expression of lineage-specific markers (Figure 12a). hNSCs differentiated in the
presence of BIO showed a substantial increase in βIII-tubulin-positive cells compared to
untreated differentiated hNSCs when analyzed by immunofluorescence (Figure 12b).
Simultaneously, there was a drastic reduction in the number of GFAP-positive cells when
differentiated in the presence of BIO (Figure 12b). Intracellular flow cytometry for βIII-tubulin
and GFAP was performed in order to quantify these changes. hNSCs showed a significant
increase in neuronal differentiation (1.9-fold) at the expense of glial differentiation (0.4-fold)
when GSK3 is inhibited by BIO in differentiating hNSCs (Figure 12c). Together with earlier
data, our results seem to suggest that BIO-mediated GSK3 inhibition enhances differentiation
and specifically promotes a commitment to the neuronal lineage at the expense of an astrocytic
lineage in the absence of growth factors.
52
Figure 12 – BIO promotes a neuronal cell fate choice in differentiating hNSCs.
BIO promotes a neuronal cell fate choice in differentiating hNSCs. a) Cells were
differentiated for 21 days in the absence or presence of BIO (1µM) and analyzed for
53
lineages specific markers using immunofluorescence and intracellular flow cytometry. Blue
arrows indicate when media was changed and fresh media and BIO were added to cells. b)
hNSCs differentiated in the presence of BIO (1µM) (-EGF/FGF + BIO) showed a drastic
increase in βIII-tubulin—expressing (green) cells and a substantial decrease in GFAP—
expressing (red) cells compared to untreated differentiated (-EGF/FGF) hNSCs, as
determined by immunofluorescence. c) Intracellular flow cytometry histograms show that
the proportion of βIII-tubulin-positive cells increases by 1.9-fold and the number of GFAP-
positive cells decreases to 0.4-fold of the untreated proportion. Unstained, red; untreated,
green; BIO-treated, blue (n=3; paired student’s T-test).
54
3.1.10 BIO decreases proliferation and induces neuronal differentiation of human neural stem cells in EGF and FGF
In vitro, NSCs are bathed in a pool of mitogens, including EGF and FGF, which are known to
promote progenitor expansion148. Since BIO significantly reduced proliferation in the absence of
growth factors, we wanted to know whether BIO-mediated GSK3 inhibition could overcome the
mitogenic effects of EGF and FGF to decrease proliferation of hNSCs under stem cell
conditions. We performed MTT assays on hNSCs treated with BIO (and untreated control
hNSCs) for 10 days in EGF and FGF, and found that BIO treatment resulted in a significant
decrease in proliferation of hNSCs (Day 4, p=0.0082; Day 10, p=0.0003, Figure 13a). One
explanation for the observed decrease in proliferation is that cells are undergoing differentiation
to produce a more quiescent population. Given how important EGF and/or FGF are in
maintaining self-renewal and proliferation of stem cells in vitro7, we wanted to know whether
BIO was potent enough to overcome these mitogenic signals and induce neuronal differentiation.
To address this possibility, we treated hNSCs with BIO for 14 days in the presence of EGF and
FGF and then performed intracellular flow cytometry for the known neural progenitor marker
nestin as well as neuronal- and astrocyte-specific markers βIII-tubulin and GFAP, respectively
(Figure 13b). Given that cells slowed down when treated with BIO in the presence of EGF and
FGF, we suspected that hNSCs were exiting the progenitor state, thereby losing nestin
expression. However, over four independent experiments, we found no significant change in
nestin expression in our hNSCs after treating with BIO. Likewise, we found no consistent trend
in the fold-change of GFAP-expressing cells when hNSCs are treated with BIO in the presence
of EGF and FGF. However, we do see a 1.8-fold increase in βIII-tubulin expression, suggesting a
trend toward induction of neurogenesis when hNSCs are treated with BIO in the presence of
EGF and FGF. Therefore, it seems that a GSK3-mediated decrease in proliferation may reflect
some change in metabolic and/or cell cycle rate, hNSC neuronal differentiation in stem cell
conditions, or both effects. It is possible that GSK3 plays a role in both proliferation and
neuronal commitment, as is suggested from earlier data in differentiating hNSCs, but that the
pathways responsible for regulating each function are not necessarily one and the same and may
exert different effects depending on the larger molecular context. In sum, a trend is seen where
GSK3-inhibition in EGF and FGF is able to overcome EGF and FGF to induce differentiation.
55
Figure 13 – BIO decrease proliferation and induces mild neuronal differentiation in EGF
and FGF.
BIO decreases proliferation and promotes a proportion of hNSCs to exit from the
progenitor state. a) hNSCs were seeded at 2000 cells/well (96-well plate) in EGF and FGF,
and grown in the absence or presence of BIO (1µM) for 10 days. BIO-treated hNSCs
showed a significant decrease in proliferation as observed by MTT assay (*: p≤0.01, **:
p≤0.001) (n=3; unpaired student’s T-test). b) The bar graph represents the average fold
changes in marker expression when hNSCs are treated with BIO (1µM) for 14 days in stem
cell conditions (+EGF/FGF) (n=4; paired student’s T-test).
56
3.2 GSK3 inhibition induces neuronal differentiation of brain tumour stem cells
3.2.1 Brain tumour stem cells express Wnt pathway components and can activate TCF/LEF-transcriptional activity.
Glioblastoma multiforme (GBMs) is an aggressive type of glial brain tumour that is
characterized by its cellular heterogeneity, a high mitotic index, invasiveness, and necrotic foci45.
GBM tumours demonstrate cell populations that stain for both precursor and differentiated
markers. These precursor phenotype cells are thought to contain the tumour-initiating population
(BTSCs) responsible for the growth and maintenance of the bulk tumour25. Given that BIO-
mediated inhibition of GSK3 activity was able to attenuate proliferation and promote neuronal
lineage commitment under specific signaling contexts in normal hNSCs, we wanted to determine
whether BIO could have similar functional affects on BTSCs. Since hNSCs and BTSCs are
functionally similar in several respects (i.e. proliferation, multilineage differentiation, and self-
renewal) we predict that GSK3 inhibition will lead to increased BTSC neuronal differentiation.
First, we wanted to confirm that our BTSC lines (G377, G179, and GliNS1) express Wnt
pathway components, similar to our hNSC lines. RT-PCR revealed that BTSC lines express key
components at the mRNA level, including the receptors frizzled Fz1, 3,6,7,8,9, and Lrp5, the
central mediators β-catenin and Gsk3β, and the canonical secreted inhibitor Dkk1 (Figure 14a).
As with our normal hNSC lines, BTSC lines also show unique heterogeneous expression patterns
of Wnt pathway components, suggesting that our in vitro conditions preserve in vivo expression
signatures. We then confirmed protein-level expression of β-catenin in our BTSC lines by
western blot and found that while all BTSCs expressed β-catenin to varying degrees, GliNS1
possessed the greatest amount of β-catenin compared to two other BTSC lines (Figure 14b).
Interestingly, however, GliNS1 also showed the highest level of active GSK3 isoforms (GSK3β
Y216 more so than GSK3α Y279) of all BTSC lines, making GliNS1 a sensible cell line for
BIO-mediated GSK3-inactivation. SW480 is an adenocarcinoma cell lines with an APC mutation
that results in the stabilization of β-catenin, and therefore makes for a suitable positive control
for β-catenin. It too possesses high levels of active GSK3β, but this has little bearing on β-
catenin levels due to the functional loss of APC. From this point on we chose to carry out our
preliminary investigations using GliNS1 for this and several reasons. First, GliNS1 is a cell line
57
derived from a glioblastoma that has been confirmed by clinical pathology. Moreover, with the
foresight to eventually move to in vivo studies, GliNS1 has been shown to be highly
tumourigenic in orthotopic tumour engraftment models by other members of the Dirks
laboratory. And furthermore, it has also been included in our microarray studies and therefore
makes gene expression data readily available to gain further insight into the function of this cell
line.
In order to determine whether GliNS1 was BIO-responsive through the β-catenin TCF/LEF
pathway, we performed the TOPFlash assay and found that 1µM of BIO for 24 hours was
sufficient to induce a 44-fold increase in signaling activity (145.5 versus 3.32 Relative
Luciferase Units—RLU) over baseline TCF/LEF transcriptional levels (Figure 14c). Taken
together, this suggests that GliNS1 expresses active forms of GSK3 and is also BIO-responsive.
Therefore, BIO-mediated GSK3 inhibition may represent one strategy for perturbing various
signaling pathways to gain functional insight into BTSC regulation and function.
58
Figure 14 – GliNS1 expresses Wnt pathway components and is BIO-responsive in vitro.
Brain tumour stem cells express Wnt pathway components and activate TCF/LEF—
mediated transcription in response to GSK3 inhibition. a) RT-PCR for various components
reveal that BTSC lines express various Frizzled receptors (1, 3, 6, 7, 8. 9), Low density
lipoprotein related-receptor protein 5 (Lrp5), Dickkopf 1 (Dkk1), and the central
components β-catenin and glycogen synthase kinase 3β (GSK3β) at the mRNA level.
GAPDH was used as a loading control. b) An immunoblot of three BTSC lines for activated
forms of GSK3α and/or GSK3β (Y279 and Y216, respectively) and total β-catenin. The
adenocarcinoma line SW480 was used as a control for β-catenin. β-actin was used as a
loading control. c) TOPFlash assay for GliNS1 BIO-responsiveness. GliNS1 cells were
treated with BIO (1µM) for 24 hours (or fresh media for endogenous levels) and TOP/FOP
levels were compared in the above bar graph. All TOP and FOP readings are normalized
to the Renilla luciferase output for transfection control.
59
3.2.2 BIO induces neuronal differentiation of brain tumour stem cells
Having confirmed that GliNS1 responds to BIO through the activation of TCF/LEF transcription
similarly to hNSCs, we wanted to determine whether BIO also induces neuronal differentiation
in a highly mitogenic environment. We cultured GliNS1 in EGF and FGF in the presence or
absence of BIO for 14 days and analyzed each population for progenitor and lineage-specific
markers using both immunofluorescence and intracellular flow cytometry. Immunofluorescence
for precursor markers revealed that there was a noticeable decrease in nestin and sox2 expression
in BIO-treated GliNS1 relative to untreated cultures (Figure 15a). These changes were quantified
using intracellular flow cytometry, confirming that BIO-treatment led to a 0.637-fold and 0.627-
fold decrease in the number of nestin and sox2 expressing cells compared to untreated GliNS1
grown under identical conditions (Figure 15b). What this may imply is that BIO is inducing
GliNS1 cells to exit the precursor state and differentiate, which can be characterized by the
expression of more mature lineage-specific markers. Given this possibility, we also analyzed
BIO-treated and untreated cells for neuronal and astrocytic markers using both
immunofluorescence and intracellular flow cytometry. Immunofluorescence for βIII-tubulin
revealed that in the presence of EGF and FGF, BIO-treated GliNS1 showed a marked increase in
neuronal differentiation relative to the untreated GliNS1 population, as evidenced by the increase
in βIII-tubulin-positive cells (Figure 16a). Furthermore, intracellular flow cytometry was used to
quantify the change in neuronal marker expression and found that the number of GliNS1 cells
expressing βIII-tubulin significantly increased by 4.797-fold with BIO treatment (Figure 16b).
Furthermore, while only a few GliNS1 cells express GFAP in the presence of EGF and FGF,
BIO treatment reduced the number of GFAP-positive cells present in culture to 0.236-fold of the
untreated levels (Figure 16a,b). Together, this data supports our prediction that BIO-mediated
GSK3 inhibition leads to the depletion of the Sox2+/Nestin+ precursor population and the
simultaneous induction of neuronal differentiation of GliNS1 BTSCs, even in highly mitogenic
stem cell conditions.
60
Figure 15 – BIO reduces GliNS1 precursor marker expression in EGF and FGF.
GliNS1 exhibit decreased precursor marker expression with BIO treatment. a)
Immunofluoescence for nestin (Red) and sox2 (green). GliNS1 treated with BIO (1µM) for
14 days in the presence of EGF and FGF produced fewer nestin- and sox2-expressing cells
relative to untreated GliNS1, as revealed by immunofluorescence. b) Intracellular flow
histograms confirmed the change in nestin and sox2 expression with BIO treatment,
revealing a 0.637- and 0.627-fold decrease in the number of nestin-positive and sox2-
positive cells, respectively, relative to untreated cells. Unstained, red; untreated, green;
BIO-treated, blue (n=3; paired student’s T-test).
61
Figure 16 – BIO treatment induces neuronal differentiation of GliNS1 in EGF and FGF.
BIO induces neuronal differentiation of GliNS1. a) GliNS1 treated with BIO (1µM) for 14
days in the presence of EGF and FGF produced a greater proportion of βIII-tubulin+ (red)
cells and fewer GFAP+ (green) cells compared to their untreated control, as assessed by
immunofluorescence. b) Intracellular flow cytometry histograms confirmed that BIO-
treated GliNS1 produced 4.797-fold more βIII-tubulin+ cells and a 0.236-fold decrease in
GFAP-expressing cell. Unstained, red; untreated, green; BIO-treated, blue (n=3; paired
student’s T-test).
62
3.2.3 BIO treatment induces brain tumour stem cells to exit the cell cycle and decrease proliferation
The ultimate goal of any chemotherapy is to kill or arrest the growth of the tumour cells.
Therefore, we wanted to know whether BIO-induced lineage commitment was potentially
associated with terminal differentiation, which would be marked by cell cycle exit. Forcing
neuronal lineage commitment could be a viable treatment strategy if these cells terminally exited
the cell cycle and could therefore no longer contribute to clonal expansion and tumor bulk. In
order to address the affect of BIO on bulk proliferation rate of GliNS1, we performed an MTT
assay over a period of 14 days in EFG and FGF and in the presence or absence of BIO. While no
significant difference was detected at earlier time points, proliferation differed significantly by
Day 14, suggesting a greater proportion of cells are exiting the cell cycle when exposed to BIO-
mediated GSK3-inactivation long-term (Figure 17a). To confirm that cells were exiting the cell
cycle by day 14, we treated GliNS1 with BIO and performed a BrdU pulse for 24-hours 6 days
and 13 days after treatment began to mark cells going through S-phase of the cell cycle in that
time period. We then stained for BrdU and found that after 7 days of BIO treatment, there was no
difference in the total number of BrdU+ cells between the BIO-treated and untreated BTSCs
(BIO: 77.39% versus untreated: 78.56%), confirming what we observe in our MTT assay (Figure
17b). After 14 days of BIO treatment, we observed a 22% reduction in the number of BrdU+
cells compared to our untreated GliNS1 cultures (BIO: 32.53% versus Untreated: 54.11%)
(Figure 17b), revealing that prolonged treatment of BIO results in increased proportion of cells
undergoing cell cycle arrest, contributing to an observed decrease in proliferation.
Together with earlier findings that BIO treatment for a period of 14 days results in a reduction in
precursor marker expression and an increase in the neuronal lineage marker, βIII-tubulin, results
suggest that a proportion of GliNS1 exits the cell cycle and terminally differentiate down the
neuronal lineage to produce a more quiescent population of cells. Therefore, the inhibition of
GSK3, possibly through the promotion of canonical Wnt signaling, may offer a novel
mechanism for differentiation therapy of GBMs.
63
Figure 17 – BIO treatment promotes cell cycle exit and decreased proliferation.
Long-term BIO treatment promotes BTSCs to exit the cell cycle. a) MTT assay performed
over a period of 14 days for GliNS1 grown in EGF and FGF with/without BIO (1µM) (***:
p≤0.001) (unpaired student’s T-test). b) GliNS1 grown in EGF and FGF with/without BIO
(1µM) for 7 and 14 days, then pulsed with BrdU (10µg/ml) for 24 hours prior to analysis.
There was a 22% reduction in the number of BrdU+ cells at day 14, indicating a trend
toward cell cycle exit when GliNS1 is treated with BIO (unpaired student’s T-test).
64
Chapter 4
4 Discussions and Future Directions
4.1 Wnt signaling in human neural stem cells
Several groups have reported an in vivo expansion of the murine neural precursor pool at the
expense of neuronal differentiation in response to constitutive or targeted activation of β-
catenin—mediated signaling120,149,150. However, other studies also in mice have shown that Wnt
signaling directs neuronal differentiation of neural progenitors, both in vitro and in vivo117,118.
The lack of consensus likely stems in part from the fact that Wnt is highly context-specific,
exerting varied effects depending on the receiving cell, developmental timing, and the other
confounding signals present within the microenvironment. Several questions remain unresolved
that could help reconcile the inconsistencies observed within the field. For one, it remains
unclear where Wnt is acting within the cellular hierarchy. Likely, Wnt regulates most cellular
populations along the hierarchy, but in context-specific ways. For example, it is possible that
Wnt promotes self-renewal of the true stem cell under the appropriate circumstances. However,
committed progenitors within the same environment may interpret this signal as an instruction to
differentiate into a neuron. Moreover, if the microenvironment changes, Wnt may cooperate with
other signals to alter the cell fate decisions of the receiving cell. For example, Israsena et al.
found that the presence of FGF2 determined whether β-catenin effects proliferation or whether it
promotes neuronal differentiation119.
A second reason for the lack of consensus is that the parameters of the experiment dictate the
results we observe. For example, the seminal study by Chenn and Walsh (2002) involves the
expression of constitutively active β-catenin (ΔN90) under the control of the nestin second intron
enhancer, which has been shown to be active by E10 in a diverse group of cells that exhibit
heterogeneity in their transcription factor repertoire120,151. However, nestin expression is
detected as early as E7.75 in many proliferative regions of the developing mouse CNS152.
Therefore, ΔN90 is likely introduced into a stem cell compartment that is already
developmentally locked in a proliferative expansion program that precedes the neurogenic burst
beginning around E10.5153. A similar explanation can be attributed to the findings reported in the
study by Kim et al. (2009), GSK3 deleted under the same nestin promoter resulted in
65
hyperproliferation of neural progenitors and suppressed neuronal differentiation149. One possible
explanation for both these findings is that stabilized β-catenin is introduced into a compartment
of cells already predisposed to proliferation or symmetrical expansion. A study by Hirabayashi et
al. show this by dissecting NPCs from the cortices of E10.5 and E13.5 mice and infecting them
with stabilized β-catenin (S33Y β-catenin)118. After 2 days in the presence of FGF2, E10.5 NPCs
showed a decrease in Tuj1+ (βIII tubulin) cells relative to the control-infected NPCs, while E13.5
NPCs infected with S33Y β-catenin resulted in an almost 4-fold increase in Tuj1+ cells.
Therefore, the developmental age of the NPCs exposed to stabilized β-catenin greatly affects
how they respond154. In other words, the parameters of the experiments dictate the observed
phenotypes. A complete in vivo time course is necessary to understand the role of β-catenin—
mediated signaling at each developmental stage. One way to address this is to create an inducible
system where Wnt signaling can be perturbed in a controlled and specified manner in vivo. Both
the Estrogen Receptor (ER)-tamoxifen and tetracycline (tet)-inducible systems are commonly
used to control spatial and temporal targeting of specific perturbations155,156. This would provide
greater insight into how Wnt signaling regulates CNS development in a spatial-temporal manner.
While a consensus remains to be reached in the murine system, less is known about how these
trends hold up in the human CNS, and what role canonical Wnt signaling plays in hNSCs. The
data from mice may not hold up in humans because of marked differences in development, such
as cell cycle control and sheer differences in generation of numbers of mature cell types. For
example, the murine neocortex is formed over a 6-day period and requires about 11 cell cycles
whereas neurogenesis in the human neocortex requires about 34 cell cycles and occurs over a
period of approximately 120 days, representing the exponential expansion of the human cerebral
cortex157. Several limitations have hindered our ability to interrogate the functional regulation of
hNSCs. For one, access to human CNS tissue can be challenging, particularly obtaining growth
of precursors from postnatal brain. Second, in vivo experiments are virtually impossible for
obvious reasons. Third, neural development in the human CNS does not occur in a linear fashion,
nor is it as well characterized as the murine system (although progress is being made)158. Much
of what is known is inferred from studies in the mouse, or from some studies of nonhuman
primates159. Therefore, very few studies focus on Wnt in human neural precursors.
In this particular study, we hoped to gain insight into the role of β-catenin—mediated signaling
in hNSCs, particularly how it regulates cell fate choices. Using cell biology, molecular genetics,
66
and chemical biology, we activated canonical Wnt signaling and interrogated the functional
implications of this activation on hNSCs. By all three methods, our data suggests that activating
the canonical Wnt signaling pathway results in a neuronal cell fate choice during differentiation.
Interestingly, however, ΔN90 and Wnt3a were only able to promote a neuronal lineage in
differentiating conditions, but were unable to induce neuronal differentiation under stem cell
conditions (data not shown). Wnt signaling did affect lineage choice in the absence of EGF and
FGF, confirming that Wnt signaling does play a role in neuronal cell fate choice. The context
may be reminiscent of how Wnt signaling might regulate neural precursors in vivo, promoting a
pro-neuronal program in progenitors that migrate away from the stem cell niche and the grips of
potent mitogenic signals. BIO-mediated GSK3 inhibition was able to induce mild neuronal
differentiation in stem cell conditions. However, nestin showed no dramatic change, indicating
that these neurons likely have not terminally differentiated. This suggests that Wnt signaling may
not be sufficient to override EGF and FGF, but GSK3-inhibition may interfere with these and
several other pathways in cooperation with Wnt activation, enabling a switch in cell fate, even in
stem cell conditions. Therefore, the effects of β-catenin-mediated signaling probably depend
heavily on other factors simultaneously present within the microenvironment.
This can also be said about the role of Wnt signaling in proliferation in our study. Unlike what
was reported in the literature for murine NSCs120,160, neither stabilized β-catenin nor Wnt3a was
able to alter proliferation in stem cell and differentiating conditions. Perhaps Wnt must cooperate
with other signals from the niche in vivo to promote proliferation, and the loss of a niche in vitro
removes a context that favours proliferation. A second possibility may be that hNSC
proliferation is not governed primarily by canonical Wnt signaling. The pro-proliferative
transcription factor c-Myc, a known target of Wnt signaling, is also a target of FGF/PI3K
signaling149. It is conceivable that the FGF activation of c-Myc transcription saturates c-Myc
levels, and TCF/LEF—transcriptional activation of c-Myc results in minimal contribution to
overall c-Myc transcription in the cells, resulting in little change in proliferation. Or finally,
perhaps the bulk proliferation rate for the entire population did not change, but rather a shift
occurred where committed neuronal progenitors (instead of multipotent stem cells) expanded in
response to Wnt activation, giving rise to the post-mitotic neurons seen at day 21. This may
explain why we see no change in overall proliferation for the first two weeks (when MTTs were
67
performed), but after 21 days we have a more quiescent terminally differentiated neuronal
population relative to the control population.
BIO-mediated inhibition of GSK3 resulted in a significant decrease in proliferation of hNSCs
both in differentiating or stem cell conditions. While BIO does mimic Wnt signaling by
inactivating GSK3, it also affects other pathways that converge on this multi-tasking kinase,
including Notch, Shh, and PI3K/Akt pathways, all of which have been shown to influence NSC
function79,161,162. It is quite likely that a concerted effort among these pathways is required to
regulate hNSC proliferation. To address the extent to which other pathways contribute to control
proliferation, rescue experiments targeting each independent pathway should be performed to
identify cooperating signaling mechanisms involved in the process. Another reason may be some
off target effects of BIO on various CDK/cyclin pairs that may down-regulate cell cycle
reentry104, however this has yet to be confirmed as well.
What is interesting is that while GSK3-inhibition was able to significantly decrease proliferation
in stem cells conditions, it was only able to induce a mild neuronal differentiation in hNSCs.
Therefore, it is probable that Wnt plays a role in specifying a neuronal fate, but may require the
cooperation of several other signals to exert its effects. Similarly, while Wnt may promote
neuronal fate choice, it may not directly regulate proliferation, again pointing to the possibility
that although these processes are often considered to be coupled functionally, various and
differing signals may govern each process somewhat exclusively. Nevertheless, results indicate
that GSK3 plays a key role in regulating the balance between hNSC proliferation and neuronal
differentiation.
Though our study suggests that GSK3 is a negative regulator of neuronal differentiation, elegant
work by Kim et al. showed that elimination of GSK3α/β in the murine CNS results in the
expansion of the neural precursor pool and a decrease in neurogenesis in vivo149. One
explanation for the observed discrepancy is that perhaps the role for GSK3 in hNSCs differs
from its role in mNSCs at various stages in development. Also, while we did not extend our
investigation beyond canonical Wnt signaling, Kim et al. show that Notch, Shh, and FGF/PI3K
signaling also contribute to the observed neural precursor expansion, suggesting that other major
players converge upon GSK3 to regulate neural precursor activity in vivo. When we treat hNSCs
with a gamma secretase inhibitor (DAPT) that inhibits Notch signaling in combination with BIO,
68
we observe synergistic cooperation resulting in increased neuronal differentiation and a decrease
in BrdU incorporation (data not shown). Therefore, while our findings concerning Notch
signaling agree with Kim et al.’s findings, we find that chemical inactivation of GSK3 induces
the opposite effect, promoting neuronal differentiation. Discrepancies may also lie in the fact that
we are employing a small molecule inhibitor to target GSK3 while Kim et al. have deleted GSK3
genetically under the second enhancer of the human nestin gene149,152,163. This particular
regulatory element targets neuroepithelial (NE) cells in a developmental stage when they are
predisposed to proliferative expansion to develop the SVZ. In humans, neurogenesis begins
around E33 and unlike rodents, continues throughout fetal development only to subside
perinatally153. Our in vitro hNSC lines are established from GW 8-11 (E56-E77) fetal CNS, a
gestational period characterized by the formation of a distinct SVZ, widespread cortical
neurogenesis and histological organization of the CNS structures164. While in culture our hNSC
lines are multipotent, it is possible that these cells were not derived from the quiescent stem cell
population in vivo, but rather were neurogenic precursors that reacquired multipotency in vitro
due to EGF exposure, a phenomenon previously observed with with murine transit-amplifying
cells in culture11. These EGF-responsive transit-amplifying neurogenic precursors have been
localized to the SVZ in mice, and are likely derived from the SVZ in humans as well165.
Therefore, one can presume that our hNSC lines are comprised of a heterogeneous population of
quiescent NSCs and converted multipotent neurogenic transit-amplifying neurogenic progenitors
derived from the expanded SVZ found in the human fetal CNS. Therefore, unlike previous
studies, our populations are likely developmentally more mature and are no longer locked in the
expansion phase when they are perturbed for Wnt signaling, possibly explaining why other
observed expansion of the VZ/SVZ zones, while we observe neurogenesis in response to GSK3-
inhibition149.
Results suggest that Wnt acts in concert with other signaling pathways to tightly regulate hNSCs
function, likely in a spatial-temporal specific manner. Harnessing our knowledge of these
intricate regulatory mechanisms enables us to develop novel pharmacologic strategies, such as in
vivo neuronal replacement therapy through the forced differentiation of hNSCs to treat various
neurodegenerative diseases. Chemical inhibition of GSK3/β-catenin stabilization in ventral
midbrain murine neural precursors results in the production of TH+ dopaminergic neurons,
indicating that Wnt signaling can induce neuronal differentiation of specific classes of
69
neurons166. Further investigation is required to determine whether Wnt stimulates the production
of specific neuronal subtypes or whether it selects for the survival of specific subtypes, not to
mention how it plays a role in specifying human neuronal subclasses. Neuronal specificity is of
great importance as different neuronal subtypes are implicated in different psychiatric
diseases167. For example, serotonergic neurons have been implicated in depression while
dopaminergic neurons have been widely implicated in the pathobiology of schizophrenia.
Furthermore, the canonical Wnt signaling pathway has been implicated in Alzheimer’s Disease
(AD). Several in vivo studies in mice showed that activation of Wnt by GSK3 inhibition showed
a reduction in β-amyloid (Aβ) peptide production, the leading neuropathological characteristic of
AD168,169. Therefore, addressing these outstanding questions in future studies can lead to novel
therapeutic potential in the form of cell-specific replacement therapy. It also offers new tools for
in vitro drug screens to uncover new drugs that target specific neuronal subtypes for the
treatment of various neurological disorders and diseases.
4.2 Targeting the Wnt pathway for differentiation therapy of brain tumour stem cells
Following pioneering work by the Dick and Clarke laboratories identifying leukemic24 and breast
cancer stem cells26, respectively, Singh et al. prospectively identified and isolated brain tumour
stem cells (BTSCs) by the cell surface antigen CD13325. It was shown that cells within the bulk
of the brain tumour were functionally heterogeneous, and that a subpopulation of the cells, the
CD133+ population, was enriched for tumourigenic potential while the remaining CD133- cells
were unable to initiate tumour formation in vivo. BTSCs are characterized by their ability to self-
renew, exhibit a differentiation capacity phenotypically identical to the original patient tumour,
and in vivo tumour initiating potential. While CD133 may mark the CSC population of a subset
of brain tumours, other groups have also identified SSEA-1/CD15 as tumour-initiation enriching
marker in brain tumours, hinting that each brain tumour may possess phenotypically and
functionally unique tumour-initiating populations49. Tied into BTSC heterogeneity is the
molecular heterogeneity of GBM subclasses. Using integrated genomic analysis has unraveled
four distinct molecular subclasses of GBMs that harbour distinct genomic alterations and
respond differently to clinical treatment170,171. Given the heterogeneity observed within tumours
as well as among them, in vitro drug screens of patient-specific BTSC lines and personalized
70
medicines seem like the only possible avenue for true therapeutic advancement in the treatment
of gliomas.
BTSC lines have been established in serum-free conditions that mimic the culturing conditions
of hNSC lines established within the Dirks laboratory172,144. Along with the ease of manipulation,
these lines retain patient-specific phenotypes, making them a valuable and reliable model of the
human disease for genetic and chemical screens. Moreover, BTSCs and hNSCs show remarkable
similarities. Both populations can be propagated in vitro as monolayers (or neurospheres) in the
presence of growth factors, both possess the ability to self-renew, and both exhibit a range of
multipotent differentiation. Furthermore, microarray and principle component analysis reveals
that hNSCs and BTSCs cluster tightly together with a stem cell signature, but differ significantly
from human normal cortex. Given the similarities, understanding the basic biology of hNSCs can
provide insight into the biology of BTSCs and targetable mechanisms that represent therapeutic
avenues for treating brain tumours. Along this line of logic, we attempted to translate the
knowledge gained in interrogating Wnt signaling in hNSCs and investigate the potential for
GSK3 as a novel target for differentiation therapy for a subset of gliomas.
While this study is preliminary and restricted to the GliNS1 BTSC line, data suggest that GSK3-
inhibition results in the depletion of Sox2+/Nestin+ cells and the production of βIII-tubulin+
neurons. This is encouraging as neurons are thought to be a more quiescent cell type compared to
mature astrocytes, a cell type commonly found in gliomas157,45. Furthermore, over a 2-week
period of BIO-treatment, BTSCs showed decreased proliferation and 22% less BrdU
incorporation relative to their untreated counterparts, suggesting that BIO treatment resulted in
fewer cells undergoing mitosis. Importantly, BrdU has also been shown to incorporate into cells
undergoing DNA synthesis during DNA damage repair processes173. Therefore, another
interpretation is that BrdU is marking cells undergoing DNA damage repair and possibly cell
death, implying that BIO treatment may have function as a pro-survival treatment given the
decrease in cells undergoing DNA damage and apoptosis. In order to rule out this possibility, it
will be important to perform co-staining with BrdU and other markers of cell division, such as
Ki67 and pHH3. Inducing cell cycle arrest or terminal differentiation may be an important
feature of chemotherapy since intracranial pressure due expansion of tumour bulk is one the most
detrimental features of brain cancer.
71
While the Wnt pathway has been implicated in the pathobiology of medulloblastomas related to
Turcot’s syndrome174, other studies show that activated Wnt signaling predicts a favourable
outcome in sporadic childhood medulloblastomas175. However, little has been reported about the
Wnt signaling pathway in gliomas. GSK3, a central mediator of the Wnt pathway, has received
increasing attention in the brain tumour field. A recent study, investigates Bmi and GSK3 in
serum-derived glioma lines and found that inhibiting GSK3β by siRNA or chemical inhibition
using LiCl and SB216763 resulted in a reduction in clonogenicity as well as the depletion of
stem cell markers and an increase GFAP+, CNPase+ and βIII-tubulin expressing cells176. It was
also found that down-regulation of Bmi1, a member of the polycomb group of proteins involved
in stem cell maintenance, seemed to coincide with a decrease in GSK3β expression, suggesting a
link between these two important molecules. These findings suggest that Bmi1, which has
already been shown to maintain BTSC self-renewal, may operate in a GSK3β-dependent
manner177. Furthermore, GSK3-inhibition has been shown to induce cell death of serum-derived
tumour lines, implicating GSK3 in the regulation of glioma cell survival178.
PTEN regulates many cellular processes, including proliferation and survival, primarily by
inhibiting the PI3K pathway, and is commonly deleted or down-regulated in glioblastomas179.
Recently it has been shown that GSK3—mediated phosphorylation on threonine 366 of the
PTEN protein results in its destabilization and functional inhibition, and that inhibition of GSK3
can stabilize PTEN levels in vitro161. This provides another link between GSK3 and another
major culprit often implicated in the pathobiology of glioblastomas.
Just as it is possible that Wnt signaling must act in concert with other pathways to exert cell
cycle arrest and subsequent neuronal cell fate choice, one can surmise that BIO-mediated GSK3
inhibition may exert its differentiation effects simultaneously through various pathways in
BTSCs. Understanding the molecular mechanisms governing this phenomenon will be
paramount in identifying possible novel treatments intended to induce terminal differentiation
and cell cycle exit of BTSCs. Future studies should focus on the possible interaction between
GSK3 and classic pathways altered in glioblastomas (i.e. PTEN, pRb, EGFRvIII, etc).
Furthermore, GSK3-inhibiting agents should be included in chemical and genetic screens
performed on patient-specific BTSC lines in order to assess the therapeutic potential of targeting
this kinase. Preliminary screens are currently underway in the Dirks laboratory. And finally,
GSK3 inhibitors such as BIO should be tested in vivo in mouse models that present with gliomas
72
to determine whether GSK3 is a realistic therapeutic target for treatment. Interestingly, lithium
use—a known GSK3 inhibitor often used to treat mood disorders—correlates with decreased
brain tumour incidence, suggesting that preemptive targeting of GSK3 may protect against brain
tumour initiation180. Together, our findings and other studies suggest that GSK3 is implicated in
the regulation of glioma stem cell biology, and may reveal a novel target for chemotherapy for a
subset of glioblastomas.
73
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