Notch Pathway Blockade in Human Glioblastoma Stem Cells ... · Notch Pathway Blockade in Glioblastoma Stem Cells Defines Heterogeneity and Sensitivity to Neuronal Lineage Commitment
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Notch Pathway Blockade in Human Glioblastoma Stem Cells Defines Heterogeneity and Sensitivity to Neuronal Lineage
Commitment
by
Erick Ka Ming Ling
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Laboratory Medicine and Pathobiology University of Toronto
1.1 Cancer ..................................................................................................................................................1 1.1.1 Brain Cancer ..........................................................................................................................................1 1.1.2 Cancer Biology ......................................................................................................................................1
1.3 Stem Cells and Cancer Stem Cells .....................................................................................................9 1.3.1 Normal neural stem and progenitor cells ...............................................................................................9 1.3.2 Notch Signaling in Stem Cells.............................................................................................................10
1.4 Cancer Stem Cells .............................................................................................................................11 1.4.1 Brain Tumor Stem Cells ......................................................................................................................14 1.4.2 Notch in cancer and cancer stem cells. ................................................................................................15 1.4.3 The Function of Notch Brain Cancer and Brain Cancer Stem Cells....................................................16
1.5 Specific Aims......................................................................................................................................17
2 USING NOTCH PATHWAY BLOCKADE TO DEFINE HUMAN GLIOMA STEM CELL HETEROGENEITY AND SENSITIVITY TO NEURONAL LINEAGE DIFFERENTIATION.......................................................................................................19
2.2 Results ................................................................................................................................................20 2.2.1 Primary Patient Glioma Express Notch Receptors and Ligands..........................................................20 2.2.2 Adherent Cancer Stem Cell Cultures are Highly Homogeneous and Express Primitive Stem Cell
Markers. ..............................................................................................................................................23 2.2.3 Tumor Precursor Lines Express Notch Receptors and Ligands...........................................................26 2.2.4 Notch receptors are activated in Glioma NS lines ...............................................................................28 2.2.5 Notch Pathway antagonism decreases cell proliferation......................................................................30 2.2.6 γ-Secretase inhibitor prevents activated Notch1 nuclear localization and inhibits the propagation of
canonical Notch signal transduction....................................................................................................35 2.2.7 Glioblastoma NS lines downregulate primitive markers in the absence of Notch signals...................42 2.2.8 Notch blockade promotes neuronal lineage differentiation .................................................................47 2.2.9 Neuron like cells are negative for neurotransmitter synthesis genes ...................................................53 2.2.10 Hierarchical clustering of signaling pathway genes reveals differential expression of Notch
components between tumor lines. .......................................................................................................56 2.2.11 Treatment of glioblastoma stem cells with γ-secretase inhibitor increases tumor latency...................60 2.2.12 Gene expression between Responsive and Non-responsive NS tumor lines. ......................................64 2.2.13 Activation of the wingless signaling pathway sensitizes glioblastoma stem cells to Notch blockade
induced differentiation. .......................................................................................................................67 2.2.14 Neuronal precursors treated with γSI and BIO are less proliferative...................................................71 2.2.15 Notch antagonist and Wnt agonists synergistically reduce in-vivo engraftment and tumor growth. ...75
2.3 Discussion...........................................................................................................................................80 2.3.1 The Notch-Hes Axis as a Therapeutic Target......................................................................................80 2.3.2 Modulating Canonical and Non-Canonical Elements of the Notch pathway ......................................82 2.3.3 Functional Synergism in BTSCs..........................................................................................................84 2.3.4 Clinical Implications............................................................................................................................88
Materials and Methods .............................................................................................................................................95 2.3.5 Primary Patient Samples......................................................................................................................95 2.3.6 Tissue Culture......................................................................................................................................95 2.3.7 Vectors and Transfection.....................................................................................................................96 2.3.8 Immunocytochemistry .........................................................................................................................96 2.3.9 Semi-Quantitative and Real Time PCR ...............................................................................................97 2.3.10 Flow Cytometry...................................................................................................................................97 2.3.11 Animals................................................................................................................................................98
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2.3.12 Microarray Data and Analysis .............................................................................................................98
3 SYMMETRIC VERSUS ASYMMETRIC SELF RENEWAL IN CANCER STEM CELLS. ...............................................................................................................99
4.2 Results ..............................................................................................................................................120 4.2.1 Sequence analysis of the Notch1 heterodimerization and PEST domain in CNS tumors..................120
4.4 Materials and Methods ...................................................................................................................125 4.4.1 Genomic DNA extraction ..................................................................................................................125 4.4.2 Nested Polymerase Chain Reaction...................................................................................................125 4.4.3 Sequencing.........................................................................................................................................125
CHAPTER 5 GENERAL DISCUSSION.......................................................................126
5.1. Targeting Notch in Brain Cancers.................................................................................................126 5.1.1. Cancer Stem Cells are Controversial .................................................................................................126
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5.1.2. Cancer Stem Cells as a Therapeutic Target .......................................................................................127 5.2. Notch and the Cancer Niche ..............................................................................................................128 5.2.1. Insight from Neurodegenerative Disease Treatment .........................................................................130 5.2.2. Therapeutic Specificity ......................................................................................................................131 5.2.3. Forcing lineage choice as a treatment for cancer ...............................................................................132 5.2.4. Glioblastoma prevention....................................................................................................................133
5.3. Origins and mechanisms of brain tumors .....................................................................................134 5.3.1. A neural stem cell as the cancer stem cell .........................................................................................134 5.3.2. Symmetrical versus asymmetrical self-renewal in neural stem cells and cancer stem cells ..............135 5.3.3. Cancer as a caricature of development ..............................................................................................136
5.4. Future Direction ..............................................................................................................................139 5.4.1. How can we target non-neurogenic glioblastoma? ............................................................................139 5.4.2. Targeting Notch in CNS tumors ........................................................................................................140
List of Tables Table 2-1 ....................................................................................................................................... 24
List of Figures Figure 1-1........................................................................................................................................ 5
List of top 53 probe sets most significantly elevated in neurogenic NS lines compared to
non-neurogenic NS lines. Principal component gene analysis between neurogenic and non-
neurogenic cell lines reveals 879 genes that are significantly upregulated in neurogenic lines.
Genes are organized in order of significance.
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2.2.13 Activation of the wingless signaling pathway sensitizes glioblastoma stem cells to Notch blockade induced differentiation.
Notch signaling is known to regulate CNS development and neuronal differentiation in concert
with other pathways72. Some of these interactions are postulated to function in a regulatory
manner outside of a developmental context. Therefore, inhibiting Notch signaling combined
with activation of other developmental pathways in glioblastoma stem cells may induce a
synergistic response that may render CSC’s more sensitive to differentiation and inhibit self-
renewal. Notch and Wnt have collectively been implicated in the progression of intestinal
adenomas183, colon cancer184 and may crosstalk directly at several points in the signaling
pathway44,43. Existing reports have suggested LiCl inhibition of GSK3β may inhibit cancer stem
cell self-renewal in sphere assays185. Activation of the wingless pathway by administration of
exogenous Wnt ligands or pharmacologic inhibition of negative regulators (Brandon, Dirks et al,
unpublished data) has been shown to induce a neuronal lineage phenotype with upregulation of
downstream Wnt targets. Therefore, we postulated that blockade of Notch in concert with the
activation of Wnt may synergistically inhibit in-vivo growth of glioblastoma stem cells compared
to modulation of either pathway alone. We constitutively activated canonical Wnt signaling
using 6-bromoindirubin-3'-oxime (BIO)186, a potent inhibitor of GSK-3β. GSK-3β is a kinase
which phosphorylates activated β-catenin in the absence of pathway activation to facilitate
recognition by ubiquitin ligases and targeting by the proteosome51 (Figure 1-3).
Using BIO and γSI, we conducted a two-dimensional dose response analysis and assayed GliNS1
for neuronal (β-IIIT) or glial (GFAP) differentiation (Fig 2-26). After one week in culture, we
observed that γSI induced a dose dependent (0-10μM) bipolar morphology and β-IIIT expression
with a corresponding decrease of GFAP expression. Similar dose dependent (0-1μM)
observations were observed with BIO treatment. Combinations of γSI and BIO induced
significant morphologic changes and increased β-III-tubulin expression beyond levels of
individual treatments. 1μM γSI and 1μM BIO had the largest qualitative induction of neuronal
morphology and marker expression with a concurrent repression of astrocytic marker expression.
Therefore, we utilized these concentrations for further characterization of NS lines.
To examine whether synergistic treatment would promote neurogenesis in non-neurogenic lines,
we treated G166NS with a combination of γSI and BIO. The individual compounds and
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combination treatment were effective in reducing GFAP protein expression but did not increase
expression of neuronal markers upon 1μM BIO and/or 1μM γSI treatment (Figure 2-27). This
demonstrates that despite synergistic activation of Wingless and inactivation of Notch, non-
neurogenic lines remain resistant to neuronal lineage differentiation.
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Figure 2-23
γSI and BIO synergistically induce neuronal lineage differentiation in GliNS1. GliNS1 was cultured for 7 days with doses of γSI, BIO or a combination of the two compounds. Immunocytochemistry was conducted to visualize protein expression of neuronal and glial marker expression. Scale bar 100μm.
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Figure 2-24
Non-neurogenic line remains neuronal marker negative with Wnt activation and Notch
blockade. G166NS cultured for one week with 1μM BIO and/or 1μM GSI downregulate
astrocytic markers but do not upregulate neuronal lineage markers. Scale bar 100μm.
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2.2.14 Neuronal precursors treated with γSI and BIO are less proliferative.
Since neuronal lineage marker expression is prevalent in neurogenic lines treated with
combinations of γSI and BIO, we hypothesized that using dual pathway modulation to force
cancer stem cells into a growth restricted neuronal lineage may be a more viable strategy to treat
glioblastomas in patients. We therefore treated GliNS1 with 1μM γSI and/or 1μM BIO for 1-2
weeks and pulsed with BrdU 24hrs immediately prior to analysis to monitor cells progressing
through S phase. At one week, 46±21% of all cells in BIO/γSI combination treatment expressed
β-III-Tubulin. This was significantly greater than that of individual 1μM GSI (10±5%) and 1μM
BIO (22±10%) treatments alone. A low percentage of vehicle treated cells were β-III-T positive
(5±2%), reflecting the low levels of spontaneous differentiation inherent to the assay (Figure 2-
28). Continued treatment for two weeks increased neuronal marker expression, growth of
bipolar processes and a reduction of numbers of GFAP positive cells (Figure 2-29). 52±25% of
all cells expressed neuronal marker expression in combination treatment versus 1μM GSI
(7±6%), 1μM BIO (14±2%) and vehicle treatment (2±1%). The effect of the combination
treatment is greater than the sum of the individual compounds, suggesting that Notch signaling
inhibition, together with activation of Wnt signaling, synergistically promotes neuronal lineage
differentiation.
The level of BrdU incorporation in cells positive for neuronal markers is much lower than that of
marker negative cells. At one week of γSI/BIO combination treatment, 63±14% of all β-IIIT
positive cells are BrdU negative. At 2 weeks of treatment, 86±4% of β-III-Tubulin positive cells
are BrdU negative and only 7±2% of total cells were double positive. Thus neuronal precursors
expressing neuronal markers have significantly impaired ability to proliferate, which becomes
more pronounced with prolonged Notch/Wnt blockade. This supports the notion that cells with
committed neuronal character are more limited in proliferative potential.
Additionally, we examined expression of glial markers and BrdU incorporation with γSI and/or
BIO. At one week, most of the GFAP positive cells are also BrdU positive, suggesting that cells
at this stage are proliferating glial precursors. This population of cells is unaffected by 1μM γSI
or 1μM BIO but is dramatically reduced to 0±1% when treated with a combination of the two
compounds. At two weeks, individual treatments of γSI and BIO effectively reduce the small
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percentage (5±1%) of doubly positive GFAP/BrdU cells found in vehicle treated controls to
levels similar to that of combination treatment (Figure 2-29). Thus, combining the two drugs
synergistically diminishes the time required to force neuronal lineage differentiation.
Taken together we conclude that neuronal lineage differentiation may contribute to a
proliferative disadvantage found in neurogenic cell lines. An anti-proliferative effect of
Notch/Wnt modulators is observed in neurogenic lines is likely due to a growth restriction
imposed by cell type to and not due to non-specific toxicity, the proliferative disadvantage
conferred to non-neurogenic cell lines is less well understood. While there is a possibility that
the non-neurogenic lines are restricted due to non-specific toxicity, it is more likely that Notch
antagonists inhibit the proliferation of the glioblastoma stem cells but cannot also engage them in
differentiation as they are hypothesized to have a molecular defect in differentiation.
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Figure 2-25
Cells possessing neuronal characteristics are less proliferative. GliNS1 cultured with 1μM
γSI, 1μM BIO or both for 1 or 2 weeks and pulsed with BrdU for 24hrs prior to fixation and
immunofluorescence stain. Quantification was conducted by nuclear localization.
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Figure 2-26
BrdU positive glial precursors are reduced by combined γSI and BIO treatment. GliNS1
glioblastoma line was cultured with 1μM γSI or 1μM BIO and pulsed with BrdU for 24hrs. A)
At one week, most GFAP positive cells are BrdU positive and are eliminated with treated with
γSI and BIO. B) GFAP positive cells are reduced after two weeks of treatment.
A
B
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2.2.15 Notch antagonist and Wnt agonists synergistically reduce in-vivo engraftment and tumor growth.
We demonstrated that Notch and Wnt blockade synergistically induce neuronal lineage
differentiation, associated with lineage specific reduction in proliferation, therefore we tested
whether these cells have impaired engraftment or proliferation in-vivo. GliNS1 was treated ex-
vivo with vehicle, 1μM γSI, 1μM BIO or a combination of the two drugs for 1 week. We then
orthotopically injected viable cells into the brains of NOD/SCID mice. The median survival of
mice injected with vehicle treated cells in this experiment was 74 days, consistent with previous.
Treatment of cells with 1μM GSI had no difference in survival, as animal survived a median 68
days. Since we previously demonstrated an improvement in host survival when the cells were
treated with 10μm γSI, this data illustrates that a threshold concentration is required to abrogate
tumorigenicity. 1μM BIO treated GliNS cells engrafted had a median survival to 116 days. In
contrast, combination treatment with 1μM BIO and 1μM GSI synergistically and substantially
improved median survival to 182 days (P<0.05, Logrank Test), an improvement of over 2-fold
compared to vehicle treated cells (Figure 2-30A).
While GFAP marker expression is diminished, non-neurogenic lines do not acquire markers of
neuronal lineage commitment with synergistic Notch and Wnt modulation (Figure 2-27).
Nevertheless, we questioned whether synergistic treatment was sufficient to block in-vivo
engraftment and growth a representative glioblastoma cell line from this class of tumors.
G166NS vehicle treated cells injected into NOD/SCID mice had a median survival of 97 days.
1μM γSI and 1μM BIO alone treated cells had a median survival of 66.5 and 89 days
respectively. Treatment of cells with both compounds and orthotopic injection resulted in a
significant (P<0.05, Logrank Test) 1.3-fold increase in median survival to 127 days (Figure 2-
30B).
Comparison of frank tumors in H&E stained paraffin embedded sections shows large intracranial
tumors with brain invasion. Immunostaining of GliNS1-vehicle tumors showed GFAP and β-
III-Tubulin positive cells demonstrating at least some spontaneous differentiation in-situ,
potentially in response to the endogenous neurogenic and gliogenic factors that exist in the brain
milieu. Some GFAP and β-III-tubulin staining was observed in GliNS1-BIO and GliNS1-γSI
cohorts. Interestingly, and importantly, tumors arising from GliNS1-BIO-γSI combination
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treatment possessed large areas of β-III-tubulin staining suggesting persistent neuronal
differentiation after engraftment. We hypothesize that the more synergistic differentiation may
have lead to the increased survival of the dual modified cells. In addition, G166NS was also
stained for markers of differentiation. Consistent with results observed in-vitro, tumors arising
from these cells possessed very little β-III-tubulin immunoreactivity regardless of ex-vivo Notch
or Wnt modulation. Thus, the biases in lineage fates resulting from pathway modulation in-vitro
with dual pathway modification are preserved in-vivo in the absence of continued pharmacologic
modulation. Dual modification may more firmly lock cells into a differentiated sate that is
maintained cell autonomously after engraftment.
We have shown that a Notch antagonist and a Wnt agonist synergistically improve survival by
delaying tumor growth from both neurogenic and non-neurogenic glioblastoma groups. The
synergistic effect of γSI and BIO is greater in neurogenic tumors (2-fold) compared to non-
neurogenic tumors (1.3-fold), we hypothesize that this reflects the more limited proliferative
potential of glioblastoma cells that are directed into the neural lineage.
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Figure 2-27
Notch antagonists and Wnt agonists synergistically improve survival of mice injected with
ex-vivo treated cell lines. A) GliNS1 neurogenic line cultured ex-vivo with 1μM γSI and/or
1μM BIO for 1 week. Combination treatment significantly improves survival. Asymptomatic
animals were sacrificed after 270 days. p<0.05, Logrank Test. B) γSI and BIO synergistically
improve survival with G166NS. P<0.05, Logrank Test.
*
*
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Figure 2-28
GliNS1 glioblastoma NS lines treated ex-vivo with γSI, BIO or combination. Treatment of
tumor lines ex-vivo increases host survival. Examining the expression of lineage markers by
immunofluoresence shows an increase in neuronal marker expression in combined BIO+γSI
treated cells. Star marks the site of the frank tumor.
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Figure 2-29
G166NS glioblastoma NS line treated ex-vivo with γSI, BIO or combination. Treatment of
tumor lines ex-vivo increases host survival but does not affect gross morphology of tumors.
Non-neurogenic lines do not express neuronal lineage markers with γSI and BIO combination
treatment. Star marks the site of the frank tumor.
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2.3 Discussion Glioblastomas are a highly malignant and phenotypically diverse class of tumors demonstrating
remarkable heterogeneity from patient to patient. Despite a growing appreciation for the
complex genomic and molecular variability encapsulating this disease, there have been very few
successful clinical treatments. Insight from cancer breakthroughs, such as the use of Imatinib for
CML187,188 and Trastuzumab in breast cancer189,190, illustrated that inhibition of the key tumor
pathway drivers can significantly improve the outcome of patients. In this study, we antagonized
the Notch signaling pathway, inducing neuronal lineage commitment and reducing
tumorigenicity of a cancer initiating population. Furthermore, based upon expression of Notch
and downstream components, we have identified patterns of gene expression in glioblastoma
stem cells that identify subsets of glioblastoma that are more sensitive to neuronal lineage
commitment. These findings provide potential valuable insight in identifying novel
differentiation strategies for glioblastoma stem cells based on analysis of molecular and pathway
specific predictors of differentiation.
2.3.1 The Notch-Hes Axis as a Therapeutic Target
Our lab recently demonstrated that primary patient glioblastoma samples can be expanded in
serum free conditions on a laminin coated surface as an adherent monolayer157, a technique
which greatly facilitates molecular characterization and functional analysis. We manipulated the
Notch pathway using pharmacologic and genetic strategies with the goal of elucidating the
components required to maintain stemness. Our study of downstream Notch targets showed that
we could relieve repression of downstream proneural transcription factors using pharmacologic
antagonists of γ-secretase. Of the multiple downstream targets, our data show that one of the key
factors in regulating lineage fate in CSCs is Hes5 and not Hes1. Hes5 expression is diminished
in all lines upon treatment with γSI (5/5), demonstrating an exquisite sensitivity to Notch
receptor activity. In contrast, Hes1 is much less responsive with only 60% (3/5) of lines
significantly downregulating Hes1 after γSI treatment. Further, Hes5 is more sensitive to ectopic
Notch activation with NICD1 expression than Hes1. The current understanding of bHLH factors
is not completely understood and remains controversial. Of the multitude of Hes transcription
factors, Hes1 and Hes5 have traditionally been viewed as the major downstream targets of Notch
in murine and human neural stem cells74,191. Owing to partial functional redundancy in the
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context of murine cortical development191, the precise roles for Hes1 and Hes5 has been unclear.
The prevailing paradigm has been that the Notch-Hes1 axis is critical in neurosphere self renewal
whereas Hes5 is a dispensible component82,192. In contrast, a seminal study illustrated that ES
cells and ES sphere colonies derived from RBP-jK-/- mice fail to express Hes5 transcripts but
retain Hes1 expression73 indicating a Notch independent regulatory mechanism. Furthermore,
Notch1-/- and RBP-jK-/- E9.0 murine embryos retain expression of Hes1 and Hes3, but not
Hes5193. Taken together, these studies suggest that Hes5, and not Hes1, is directly regulated by
Notch. In support of this, there evidence that some canonical Notch targets are regulated
independently of the NICD/RBP-jK activation complex. In a study conducted with serum cell
lines, hedgehog signaling can directly regulate Hes1 independent of Notch receptor activation in
C3H/10T1/2 mesodermal and MNS70 neural cells129. In the context of murine postnatal retinal
progenitor cells, activation of hedgehog signaling is a stronger stimulus than activated Notch for
Hes1 expression and occurs independently of RBP-jK194. This suggests that Hes1 may not be an
exclusive Notch target as previously thought. Our study has demonstrated that Hes5 is more
sensitive than Hes1 to Notch blockade and over expression. Therefore, in the cancer stem cell,
Hes5 appears to be regulated by Notch directly whereas Hes1 may be regulated by alternate
pathways and/or Notch. Obviously further deliniation of the relative importance of Hes1 and
Hes5 in glioblastoma stem cells requires further functional analysis of these cells with additional
gain and loss of function experiments.
The persistance of Hes1 expression despite Notch blockade reveals a fascinating possibility into
the potential mechanism of glioblastoma disease recurrence. A seminal study reported that Hes1
maintains the reversibility of cellular quiescence in human fibroblasts and rhabdomyosarcoma by
blocking the effect of Cyclin Dependent Kinase inhibitor p21Cip1 and inducing expression of
TLE1195. In a developmental context, this protective function would intuitively prevent stem and
progenitor cells from undergoing premature quiescence, ensuring that developmental and
homeostatic programs are retained. Likewise, maintenance of Hes1 expression in cancers would
confer a significant survival advantage. Inducing cellular quiescence and terminal differentiation
in cancer is a highly sought after goal for the purposes of treating disease. Our observation that
glioblastoma NS lines retain Notch-independent Hes1 expression illustrates how a normal
protective mechanism may be co-opted by aberrant cancer programming. Indeed, we have
observed that glioblastoma stem cells can be differentiated into less proliferative (potentially
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more quiescent) neuronal cells in-vitro, yet retain the capacity to generate tumors upon in-vivo
orthotopic transplantation when isolated from the effects of γSI. This could reflect a state of
reversible quiescence or reversible differentiation of glioblastoma stem cells, and could have
important implications for guiding anti-Notch directed therapies (or differentiation therapy
strategies in general) and suggests that maximal therapeutic benefit of these types of therapies
may require active maintenance of differentiation signals to the cancer stem cells. Small
molecule inhibitors196 and anti-Notch receptor antibodies197 are just some pharmacologics that
are currently being developed and tested. However, it may be crucial to also target the pathways
which crosstalk with Hes1 in order to minimize the likelihood of disease recurrence. We have
shown that the wingless pathway is a candidate pathway for combinatorial modulation.
Recently, Schreck et al., demonstrated that Hes1 negatively regulates hedgehog by directly
binding the Gli1 gene in a glioblastoma neurosphere model. Interestingly, blockade of Notch
with γSI resulted in a corresponding increase in Gli1 activity as a result of diminished Hes1
expression198. The result was a compensatory upregulation of Shh activity and was postulated to
protect cancer cells from death. From this preliminary study, combining Notch and Shh
inhibitors was more effective at reducing cell proliferation than individual inhibitors. Ultimately,
simultaneous targeting of multiple signaling pathways implicated in tumorigenesis is likely to be
more efficacious in a clinical setting.
2.3.2 Modulating Canonical and Non-Canonical Elements of the Notch pathway
In our study we compared canonical and non-canonical pathway gene expression in glioblastoma
stem cell lines, fetal NS lines and cerebral cortex. We discovered that Notch2, Hes1 and
Jagged1 expression is relatively increased in normal or tumor stem cells compared to human
cortex. Intriguingly, we uncovered patterns of gene expression which may be predictive of the
differentiation potential for glioblastoma stem cells. Glioblastomas that acquire neuronal
phenotypes in response to Notch blockade have patterns of gene expression similar to human
fetal neural stem cell lines. Importantly, this sensitive group of gliomas can be distinguished
from insensitive gliomas by high relative expression of Ascl1 and Dll3. Ascl1, a transcription
factor whose expression is repressed by the Notch target Hes1/Hes5199,115, is crucial for
reprogramming fibroblasts to functional neurons200 and is critical in neuronal differentiation and
patterning200. Paradoxically, we have shown that Ascl1 is expressed in some EGF/FGF cultured
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NS lines, conditions which promote self-renewal and inhibit differentiation. Rectifying this
apparent contradiction, Castro et al., recently reported that Ascl1 has direct functions in all stages
of neurogenesis. In addition to regulating genes associated with Notch signaling, cell fate
specification, neuronal differentiation and neurite morphogenesis, Ascl1 was found to control
genes regulating cell proliferation201. The authors show that Ascl1 can bind the promoters of up
to 603 genes responsible of reactivating cell cycle. Transfection of dominant negative-Ascl1 into
neural stem cells resulted in impaired S-phase progression. Interestingly, the authors discovered
that the Ascl1 promoter binding sequence (GTGGGAC) very closely matched that of the
CBF1/NICD co-activator sequence (GTGGGAA). They proposed a model whereby Ascl1
functions to co-activate cell cycle progression genes when Notch is active, but is displaced by
CBF1/co-repressor complex when Notch is inactive. Therefore, in our neurogenic lines,
CBF1/NICD and Ascl1 may function together to promote cell cycle progression/self-renewal
when Notch is active. Notch blockade therefore promotes the transcription of genes relating to
neuronal lineage commitment and maturation.
We wondered whether other transcription factors downstream of Notch contributed to the
prevalence of neurons and relative paucity of astrocytes upon Notch blockade. In our principal
component analysis of neurogenic versus non-neurogenic NS lines, we identified high expression
of Sox4 in neurogenic lines relative to human cortex and non-neurogenic lines. There is
evidence to support the role of this Sry-related HMG-box gene in neuronal commitment and
CNS development. In mouse models of development, over expression of Sox4 under the GFAP
promoter supports normal differentiation of neurons, but induces massive cerebellar defects due
to the developmental failure of Bergman glia202. In this mouse model, apoptosis occurred
extensively in astrocytes. Expression of Sox4 in neurogenic glioblastoma stem cell lines may
conceivably diminish the population of astrocytes when exposed to the neurogenic environment
of γSI treatments. Thus, considering the importance of this transcription factor in a
developmental context, it may serve an important function in neuronal lineage programming in
cancer stem cells, and would likely benefit from further detailed study.
In this study, we directly modulated the activity of Notch receptors using pharmacologic
inhibitors. While our study was being conducted, other groups have demonstrated that the
activity of Notch signaling can be modulated by affecting genes responsible for receptor
transcription and post-translational processing. Ying and colleagues demonstrated that the
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retinoic acid induced differentiation of glioblastoma neurospheres was dependant on expression
of Krüppel-Like Family 9 (KLF9) transcription factor. KLF9 exerts negative regulatory effect
by direct binding to basic transcription element (BTE) sequences found in the Notch1 promoter.
Underexpressed in glioblastoma intiating cells, ectopic expression induces differentiation and
was sufficient to reduce tumorigenicity in mouse transplant models203. Identifying novel
pharmacologic and genetic mechanisms to perturb Notch signaling will of great importance for
efficiently and terminally antagonizing pathway activity in cancer cells.
2.3.3 Functional Synergism in BTSCs.
To determine whether Notch targeted therapies could be effective in patients we conducted a
proof of concept study using ex-vivo orthotopic models. Mice transplanted with γSI-induced
differentiated neurogenic lines significantly improved survival over transplants with untreated
cells. Non-neurogenic tumor lines treated the same way did not demonstrate an improvement in
host survival, but we acknowledge that numbers of animals tested and numbers of cell lines
tested are small to make the most concrete conclusions. Additionally, we activated the wingless
pathway using pharmacologic inhibitors of GSK-3β in conjunction with Notch blockade and
induced robust neuronal lineage differentiation in neurogenic lines. This combination improved
tumor-free survival of mice transplanted with neurogenic tumor cells. Remarkably, despite the
lack of neuronal differentiation, BIO and γSI combination treatment of non-neurogenic
glioblastoma lines was also effective in significantly prolonging the survival of mice in
orthotopic transplant. It may be possible that BIO and γSI promote the development of a
neuronal marker negative ‘tumor transient amplifying cell’. In the study conducted by Dieter
and colleagues, they demonstrated that colon cancer initiating cells could be subdivided into
three fractions: long term tumor initating cells, transient amplifying cells, and delayed
contributing cells. Of these fractions, transient amplifying tumor cells were only capable of
forming tumors in primary animals. Furthermore, transient amplifying tumor cells were much
less likely to metastasize within the murine host204. In our study, we demonstrated that mice
injected with BIO and γSI treated cells had improved survival compared to injections with
untreated or single treated cells. However, did not test the serial propagation ability of these
tumors. Furthermore, since CNS tumors do not metastasize to distant sites outside the CNS, we
are unable to assess the migratory abilities of putative glioblastoma transient amplifying cells in-
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vivo. Ultimately, combination treatment may diminish tumorigenicity of non-neurogenic lines
by promoting a transient amplifying cell fate possessing limited self-renewal capabilities.
We demonstrated Wnt pathway activation synergizes with Notch inhibition to activate neuronal
genes and upregulate markers of lineage commitment. How do our results fit into the currently
established mechanisms of crosstalk? Evidence from the literature illustrates both direct and
indirect interactions between the two pathways. Indirectly, some elements of the Notch pathway
are targets of downstream activated Wnt signaling. For instance, the Jagged1 promoter is a
known target of the canonical β-catenin activation complex205,184. Katoh and Katoh
demonstrated that β-catenin can activate Notch signaling by upregulating transcription of
Jagged1. Direct protein-protein interactions can occur between Wnt-Notch and may modulate
activity of both pathways. Activated β-catenin can bind to the intracellular domain of activated
Notch and functions to prolong the half-life of NICD in-vitro206,43. In murine e14.5 neural stem
cell models, β-catenin together with NICD1, participates in the activation of Hes genes to
suppress neuronal differentiation206. Confusingly, studies also demonstrate that negative
regulators of Wnt can potentiate Notch signaling. Paradoxically, the kinase responsible for
marking β-catenin for degradation, GSK-3β, also has the ability to directly phosphorylate
Notch1207 and Notch2208, promoting nuclear localization and activation of downstream Hes
targets. Our data show that blocking GSK-3β in BTSCs can be a potent differentiating signal
and may support a model that GSK-3β is a phosphorylase that potentiates nuclear localization of
activated Notch (Figure 2-30). This context suggests that β-catenin is not required to regulate
proliferation. Also, induction of differentiation via BIO/γSI synergy may be independent of
activated β-catenin protein. To test this hypothesis, experiments confirming the physical
interaction between NICD and GSK-3β, and experiments demonstrating the effect of β-catenin
over expression in G-NS cells are required. The recipricol activity of these pathways is not
without precedent, as this putative model may have implications in memory consolidation in
Wistar rats. Conboy and colleagues demonstrated that Notch signaling in the adult rat
hippocampus was downregulated 12 hours following a passive avoidance training stimulus
consisting of a delinated area of a cage which administered an electric shock209. If the Notch
receptor was activated ectopically in-vivo after the animals completed the training period, the rats
failed to condition to the shock stimulus. Interestingly, diminished Notch activity was associated
with increased Wnt activity measured by GSK-3β phosphorylation and accumulation of
86
activated-β-catenin. The authors hypothesized that in order for proper learned conditioning to
occur, downregulation of Notch was required to allow for neuronal differentiation and neurite
outgrowth. Thus, while learning and memory are poorly understood processes, the findings in
this thesis indicate that physiological mechanisms governing memory may be conserved in
glioblastoma stem cells. Ultimately, our data support the hypothesis that the functional synergy
observed with BIO and γSI is due to recipricol Notch and Wnt pathway activation.
Herein, we have implicated a role for Wnt pathway by identifying upregulation of APC in
neurogenic tumors. We showed that activation of Wnt signaling and simultaneous blockade of
Notch promotes glioblastoma stem cell differentiation. The Wnt pathway is known to contribute
to a number of congenital cancer syndromes, most notably Familial Adenomatous Polyposis
(FAP). Inheritance of germline inactivating mutations within the APC gene induces growth of
numerous (hundreds to thousands) colorectal tumors manifesting as polyps in the second or third
decade of life210. This condition requires aggressive surgical treatment. FAP can also contribute
to a spectrum of CNS tumors. Referred to as Turcot’s Syndrome, these patients were found to
commonly develop medulloblastoma and, less frequently, glioblastoma. While this clinical
condition appears to contradict our findings that Wnt activation may diminish tumor growth, a
study that characterized glioblastoma of Turcot’s Syndrome revealed atypical molecular features
including more defects in mismatch repair and markedly improved outcome compared to
classical glioblastoma211. We did not conduct a full genotype analysis on all the glioblastoma
stem cell lines in this study and therefore FAP carrier status is unknown. While tumor response
to Wnt activation was variable, all of the lines studied showed diminished proliferation
suggesting that Wnt activation was not a proliferative simulus. Whether BIO treatment has the
same effect on CNS tumors of FAP origin is yet to be studied. Thus, synergistic inhibition of
Notch and activation of Wnt will be a useful strategy in some, but maybe not all brain tumors.
87
Figure 2-30
Putative Mechanism of Notch-Wnt Synergy. GSK-3β antagonists combined with γSI induces
neuronal lineage differentiation in neurogenic BTSCs. This interaction could putatively occur
through a positive feedback mechanism where GSK-3β interacts with the activated domain of
Notch.
88
2.3.4 Clinical Implications
The identification of the factors which distinguish neurogenic and non-neurogenic tumors in this
study may be clinically useful for identifying patients which may respond to therapies directed
against the Notch signaling pathway. Philips et al212, demonstrated that bulk tumors could be
grouped into three subclasses: proneural, proliferative and mesenchymal with markers from the
Akt and Notch pathway distinguishing Mesenchymal and Proneural groups respectively. The
proneural class included gliomas ranging from Grade II to Grade IV and tended to consist of
patients younger in age compared to the proliferative and mesenchymal groups (~40 y/o vs ~50
y/o). Verhaak and The Cancer Genome Atlas Research Network213 uncovered similar findings
with respect to glioblastoma subtypes. In their study, molecular genetic classification identified
four distinct glasses of glioma: classical, proneural, neural and mesenchymal. Of the proneural
class, they identified Notch, Ascl1 and Sox genes being highly expressed compared to other bulk
glioma. Our work has demonstrated that cancer stem cells can be prospectively organized based
upon a genetic signature and that the two groups possess functional differences in signaling
pathway dependency that can be exploited with γSI.
Interestingly, Verhaak et al. showed that there were significant differences between clinical
subtypes and response to aggressive chemotherapy regimens. Patients with Grade IV glioma can
be treated with ‘standard’ chemotherapy or ‘aggressive’ chemotherapy. Aggressive strategies
comprise of multiple rounds of high dose chemotherapy plus concurrent radiotherapy and have
been shown to improve gross patient survival. However, when grouped according to the genetic
profile of their glioma, patients with proneural GBMs do not possess any improvement in
mortality when compared to traditional chemo/radiotherapy regimens. Therefore, our work may
suggest a treatment modality in this subclass of GBMs. γSI’s may be a strategy to improve the
survival of patients with the proneural class of glioblastoma.
Therapeutically, functional synergy could minimize adverse drug reactions. Indeed, γSI at high
doses has been documented to induce severe gastric toxicity, the effects of which may be
mitigated by some combinations of adjuvant therapy with the corticosteroid dexamethasone214.
Similarly, while BIO has not yet been approved for the use in human patients, Wnt activation
through Lithium Chloride (LiCl) is an approved clinical treatment for neurological/psychiatric
disorders and thus effectively passes the blood brain barrier and functions in the CNS215. Lower
89
doses of combined drugs which have the same effect as high doses would be effective at
minimizing cost, reducing toxic burden, maximizing therapeutic value and ultimately preserving
patient compliance.
Historically, this differentiation therapy has been successfully applied in the treatment acute
promyelocytic leukemia (APL). All-trans retinoic acid (ATRA) administered to patients with
APL induced differentiation to mature granulocytes and effected a complete remission in a high
percentage of patients216. There is evidence in-vitro to demonstrate that this approach may be
viable in human glioma217. Furthermore, novel methods of targeting the Notch axis have been or
are currently under development. Perhaps like herceptin218, a developing strategy is the use of
antagonistic monoclonal antibodies specific for individual Notch receptors197,197.
Understandably, there is concern that targeting a signaling pathway integral to normal stem cell
homeostasis may cause undesirable side effects. Studies in rats have shown that administration
of γSI via intracranial osmotic pumps improves the performance of test subjects in water maze
challenges219, perhaps reflecting increases in functional neurogenesis as a result of Notch
blockade. However, extrapolation to human subjects should be done with caution as long term
memory or cognitive deficit in these laboratory animals have not been explored. Further, NSCs
are known to mobilize to sites of brain injury and ischemic damage. It is unknown how
plasticity and CNS repair would cope in an environment a depleted of a neural stem cell pool220.
Therefore, uncertainty regarding effects on memory and impaired response to brain ischemia
must be considered in a clinical setting.
Our findings have clear clinical implications. We have prospectively identified characteristics
which predict responsiveness to Notch antagonists. Further development and characterization of
the hits revealed in this study may lead to clinical applications where patient glioma samples
may be screened for Notch1, Ascl1 or Sox4 expression in advance of prescribing a
chemotherapeutic regimen. Information gleaned from patient screens may thus lead to the use of
γ-secretase inhibitors and Wnt agonists to induce neuronal lineage differentiation in susceptible
glioma.
90
Supplemental Figure 1
Growth factor withdrawal of NS lines induces multipotent differentiation..
A) HF240NS human fetal neural stem cells, B) GliNS1 glioblastoma line, C) G144NS
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