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Running head: SONIC HEDGEHOG PATHWAY IN MEDULLOBLASTOMA
Sonic Hedgehog Pathway in Medulloblastoma
Jelaina Holroyd
University of British Columbia
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Background
The sonic hedgehog (SHH) pathway is an important component of embryonic development and
disruption of this pathway has been associated with many different types of cancer (Rimkus et al., 2016). The
SHH pathway proceeds as follows: the glycoprotein ligand SHH binds to the transmembrane receptor
Patched1 (PTCH1), thereby inhibiting its repression on another transmembrane protein, smoothened (SMO)
(Kool et al., 2014; Rimkus et al., 2016). This leads to the transcription factor GLI1 being released from
confinement to the cytoplasm by SUFU and other proteins (Lo et al., 2009). GLI1 is then able to act as the
terminal effector, as it executes the final step of this pathway: gene transcription (Lo et al., 2009). These genes
are involved in such functions as pathway feedback and cell proliferation (Rimkus et al., 2016). See Figure 1 for
a full outline of this pathway.
Taylor et al. (2002) elucidated the importance of the protein SUFU in the SHH pathway. Using vector
transfection and fluorescence microscopy, they found that GLI is located in the nucleus, where it can function
as a transcription factor. However, when wildtype SUFU is present, GLI is instead found in the cytoplasm.
Mutant, non-functional SUFU leads to GLI again being found within the nucleus. Thus, SUFU is necessary for
preventing GLI mediated transcription.
Lo et al. (2009) identified a truncated GLI1, which they termed tGLI1. It is a gain-of-function mutant
that retains the regulatory and functional domains of GLI1, allowing it to activate both GLI1 target genes and
different genes, such as CD24 (Lo et al., 2009). Furthermore, tGLI1 is known to be induced by SHH, but it is
unclear if it interacts with other components of the pathway (Carpenter & Lo, 2012). tGLI1 is found in various
cancer cells, but not normal cells (Lo et al., 2009). However, many cancer types remain to be studied, such as
medulloblastoma.
Medulloblastoma is a malignant brain tumour that can be divided into four subgroups: WNT, SHH,
Group 3, and Group 4 (Kijima & Kanemura, 2016). These subgroups differ both in genotype and phenotype, as
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well as in clinical outcome (Kijima & Kanemura, 2016). An interesting feature of the SHH variant is its bimodal
age distribution: infants and adults have the greatest risk, with a relatively low number of adolescent patients
(Kijima & Kanemura, 2016). Accordingly, mutations leading to SHH medulloblastoma can be segregated
according to age: PTCH1 mutations are seen across all age groups, SUFU mutations in infants, and SMO
mutations in adults (Kool et al., 2014). However, these mutations are not exhaustive and other genes have
been implicated in medulloblastoma, such as increased CD24 expression (Sandén et al., 2015).
Not surprisingly, drugs have been developed to treat SHH medulloblastoma. SMO targeting has
received the most attention, leading to FDA approval of two SMO inhibitors for basal cell carcinoma, with
trials for other cancer types – including medulloblastoma – underway (Rimkus et al., 2016). SMO inhibitors are
likely to be effective for both SMO mutations themselves, as well as PTCH1 and SHH mutations, since the
drugs’ effects are downstream of these mutations (Kool et al., 2014). There is also interest in targeting GLI1,
due to its role as the terminal effector of the SHH pathway and its involvement in SHH independent
mechanisms (Rimkus et al., 2016). There is currently one FDA approved GLI1 inhibitor (Rimkus et al., 2016).
Rimkus et al. (2016) suggest that inhibiting SHH itself or targeting tGLI1 could also be effective treatments. It is
advantageous to target tGLI1 due to its exclusive location in cancerous cells, thereby reducing off-target
effects (Rimkus et al., 2016). However, the treatment would need to target tGLI1 specifically, despite this
protein’s similarity to GLI1. Alternatively, SUFU agonists could be used. However, tGLI1 antagonists are
preferable since tGLI1 is the final effector, while SUFU lies upstream and thus treatments targeting this protein
would be indirect.
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Figure 1. The sonic hedgehog (SHH) pathway in medulloblastoma patients. The SHH ligand binds to the PTCH1 receptor. This binding
prevents PTCH1 from repressing the SMO receptor. SMO activation then dissociates GLI1 from SUFU, as illustrated by the lightning
bolt. GLI1 acts as the terminal effector of the pathway, inducing transcription of target genes. SMO antagonists and GLI inhibitors are
currently under investigation, with some receiving FDA approval.
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Research Question and Potential Impact
The question this proposal aims to investigate is: does SUFU interact with tGLI1 in the SHH pathway in
medulloblastoma patients?
This question is novel because, while tGLI1 has been known for quite some time, its role still remains
highly unknown. The SHH pathway is well described in the literature, but it has yet to be determined if tGLI1
plays a similar role to GLI1 in this pathway.
This question is important to both the scientific community and general public since tGLI1 has been
found exclusively in cancerous cells, thereby raising the possibility that it could be an effective treatment
target. However, before such a treatment could be implemented, an understanding of the various interactions
of tGLI1 is needed. Scientists need to know how GLI1 and tGLI1 differ in order to create effective treatments
without undesirable side effects. This study aims to determine one such similarity or difference, through
elucidating the relationship of tGLI1 and SUFU.
Hypothesis
I posit that tGLI1 functions in much the same way as GLI1 itself in medulloblastoma. Although tGLI1 is
found exclusively in cancer cells and is able to activate novel genes, I predict that it is still mediated by the
same factors as GLI1, due to preserved functional domains. Specifically, I propose that SUFU binds to tGLI1,
which will be the aim of my investigation. However, I also posit that other components of this pathway treat
tGLI1 similarly to GLI1, with upstream SHH, PTCH1, and SMO ultimately releasing tGLI1 from SUFU repression.
This proposed pathway is visualized in Figure 2.
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Figure 2. The proposed tGLI1 sonic hedgehog (SHH) pathway in medulloblastoma patients. It is theorized to be identical to the GLI1
pathway, with the exception that novel genes are additionally expressed and this pathway is only present in cancer cells. tGLI1
inhibitors are a theoretical treatment option.
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Proposed Experiment and Predictions
Experiment 1
Protocol
The first experiment will be a “look” experiment, to ensure that tGLI1 is expressed in medulloblastoma
tumours.
This will be done using immunohistochemistry in tumour cells of a medulloblastoma mouse model.
This model is a xenograft into immunocompromised mice. Xenografts are made by transplanting tissues or
organs of one species into a different species. These xenograft models will be developed from infant, juvenile,
and adult medulloblastoma patients since this disorder changes across the lifespan in terms of mutations,
phenotype, and prevalence (Kijima & Kanemura, 2016). The specific strain of immunocompromised mice used
will be severe combined immunodeficiency (SCID), as they have previously been shown to allow
medulloblastoma development due to their inability to reject human cells (Chiou et al., 2006).
Immunohistochemistry in tumour tissue will be done using a tGLI1 antibody developed by Zhu,
Carpenter, Han, and Lo (2014) that acts specifically at the exon 2-4 junction, a region not present in wildtype
GLI1.
Controls
To ensure antibody specificity, I will include a non-tumour tissue control.
Predictions
I expect to see tGLI1 present in some or all of the tumours, as I theorize that the increased CD24
expression seen in some medulloblastoma tumours is due to the presence of tGLI1 (Sandén et al., 2015). The
control mice will have no staining, as tGLI1 is not present in non-cancer cells (Lo et al., 2009). Assuming the
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result of this procedure is that tGLI1 is present in at least some of the tumours, I will proceed with the
following experiment.
Experiment 2
Protocol
The second experiment (the focus of this project) will be an “add something” experiment. Specifically, I
will increase SUFU expression using a knocked-in gene in a medulloblastoma xenograft mouse model. The
model will be constructed as per the first experiment. However, any models not displaying tGLI1 expression
will be omitted.
Before xenotransplantation, I will increase the level of SUFU in the tumour cells using gene knock-in.
Targeted gene knock-in will be used as opposed to random integration with a transgenic construct in order to
prevent side effects due to disruption of essential genomic sequences. Thus, a construct will be chosen that
targets a region of the genome with no known function. The construct will recombine with this region, leading
to a third copy of the SUFU gene. Since it is being inserted into a non-functional region, the construct will need
to include its own promoter. Furthermore, the construct will have a positive (neor) and negative (HSV-tk) drug
selection marker, to allow for selection of properly recombined cells prior to xenotransplantation (Hall,
Limaye, & Kulkarni, 2009). Since SUFU has many introns (21 with 11 alternate splice variants), the construct
will use SUFU cDNA, along with a poly A tail sequence (NCBI, n.d.). This construct can be seen in Figure 3.
This system will be assayed with western blot, blotting for tGLI1, SUFU, tGLI1-SUFU complex, and lacZ.
The tGLI1 antibody from the first experiment will be used, along with lacZ and SUFU antibodies.
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Figure 3. Construct for gene knock-in. Homologous regions are found at either end to allow for recombination into a non-functional
region of the genome. The promoter sequence is needed due to the construct’s location within non-functional DNA. The SUFU cDNA
will allow for increased SUFU expression. The poly A tail sequence (pA) and drug selection marker sequences (NeoR and HSV-tk) are
essential components of cDNA gene knock-in constructs (Hall, Limaye, & Kulkarni, 2009).
Controls
The first control will be the knock-in construct with the lacZ gene instead of SUFU, to ensure that the
results are not due to the technique used. I chose lacZ as it will create a functional protein for assay in the
western blot, but it will not affect cell functioning.
Secondly, I will use a construct with random nucleotides the length of SUFU as a control. In this way, I
can ensure that the region I am targeting truly is non-functional, provided these controls are phenotypically
identical to wildtype mice.
Incidentally, I will also use a wildtype medulloblastoma control, to allow for comparison of SUFU, tGLI1,
and SUFU-tGLI1 levels to baseline.
Predictions
I predict that the western blot will have bands of a high molecular weight corresponding to the bound
tGLI1-SUFU complex and potentially bands for SUFU, if there is an excess of SUFU relative to tGLI1. This will
occur in all experimental groups, as they were previously shown to express tGLI1. There will be no bands for
isolated tGLI1, as it will only be found within the complex.
The lacZ control will have both bands corresponding to the LacZ protein (ensuring that the protocol
was performed properly) and identical bands to the wildtype control. Furthermore, the LacZ protein will be
functional, indicating proper integration.
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The random nucleotide control will be phenotypically identical to wildtype and cannot be assayed via
the western blot.
Since medulloblastoma is known to have increased CD24 expression, I predict that the wildtype control
will have bands for isolated tGLI1 (which can activate CD24) and SUFU, but no or minimal tGLI1-SUFU complex
(which theoretically cannot activate CD24).
Other Potential Results
Although my hypothesis predicts that the experimental group will have bands for the tGLI1-SUFU
complex and potentially isolated SUFU, other results are possible. Specifically, experimental mice may have
bands for isolated SUFU and tGLI1, indicating that the two proteins are not interacting and thereby suggesting
that tGLI1 interacts with SUFU differently than GLI1. If there are bands for tGLI1, SUFU, and tGLI1-SUFU, this
will indicate that SUFU interacts with tGLI1, but it is not sufficient to bind all of the tGLI1 present.
Alternatively, if all bands are present but SUFU and tGLI1-SUFU bands are weak, this could indicate that SUFU
is degraded before it can interact with tGLI1. The inference if only tGLI1-SUFU and tGLI1 bands are seen is that
SUFU was not amply increased to interact with all tGLI1. The predicted and alternative results are shown in
Figure 4.
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Figure 4. Possible results of the western blot. The leftmost lane indicates my predicted result. Note that SUFU is not required to be
present. The first alternative result indicates SUFU does not bind to tGLI1, implying that tGLI1 functions differently than GLI1, in
terms of interaction with SUFU. The second alternative also indicates that SUFU is not sufficient to prevent tGLI1 from being found
alone, although some binding has occurred. The presence of less SUFU and tGLI1-SUFU complex in the third alternative (as
demonstrated by the faint and thin bands) could indicate that SUFU is being degraded. If the fourth alternative is seen, the inference
is that not enough SUFU was added to react with all tGLI1 present. The two right lanes indicate the results of controls. Note that
these controls may or may not have a weak tGLI1-SUFU band. Molecular weight of SUFU is 53.9 kDa (Gene Cards, n.d.); LacZ is 116
kDa (Ow et al., 2010); tGLI1 is 146 kDa (Zhu et al., 2014).
Discussion
If my prediction is accurate and bands are seen for tGLI1-SUFU with or without SUFU (but not tGLI1),
this will indicate that SUFU interacts with tGLI1 in SHH medulloblastoma patients. While the inference is that
SUFU is able to repress tGLI1, this cannot be concluded until downregulation of tGLI1 mediated gene
expression is seen upon SUFU upregulation. Thus, this would be my next experiment.
Altogether, this research could further investigation into tGLI1 antagonists as potential therapeutics for
medulloblastoma. The more evidence that GLI1 and tGLI1 function via the same pathway, the more interest
there will be in targeting the cancer cell specific tGLI1. tGLI1 targets both the same genes as GLI1 and novel
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genes, such as CD24 (Lo et al., 2009). Thus, future research should aim to elucidate how the various pathway
components affect CD24 transcription, as opposed to genes induced by the GLI1 pathway. While tGLI1
appears to be an ideal target, due to its similarity to GLI1 but localization specific to cancer cells, the novel
genes activated by tGLI1 may lead to complications and side effects. Thus, details about these genes need to
be elucidated before tGLI1 treatment can make its way into clinical trials.
This research proposal aims to investigate the role tGLI1 plays, specifically in relation to SUFU, in the
SHH pathway of medulloblastoma patients. This is a novel and important research topic with potential
implications for future cancer treatment.
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Lay Person Summary
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References
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vitro and ex vivo green fluorescent protein imaging. Child’s Nervous System, 22, 475-480. doi:
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Gene Cards. (n.d.). SUFU gene: SUFU negative regulator of hedgehog signaling [Data file]. Retrieved from
http://www.genecards.org/cgi-bin/carddisp.pl?gene=SUFU
Hall, B., Limaye, A., & Kulkarni, A. B. (2009). Overview: Generation of gene knockout mice. Current Protocols in
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