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Transcript
Running head: SONIC HEDGEHOG PATHWAY IN MEDULLOBLASTOMA
Sonic Hedgehog Pathway in Medulloblastoma
Student 1
University of British Columbia
1
SONIC HEDGEHOG PATHWAY IN MEDULLOBLASTOMA
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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
canonical 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 release of the transcription factor GLI1 from
cytoplasmic sequestration by SUFU and other proteins (Lo et al., 2009). GLI1 is then able to act as the terminal
effector, leading to transcription of various genes, including those involved in 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 this pathway. Using vector
transfection and fluorescence microscopy, they found that GLI alone is found in the nucleus, where it is able to
perform its 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. This tGLI is found in various
cancer cells, but not normal cells (Lo et al., 2009). It is a gain-of-function mutant that retains the regulatory
and functional domains of GLI1 and is still able to activate GLI1 target genes, as well as different genes (Lo et
al., 2009).
Medulloblastoma is a type of malignant brain tumour, found in both children and adults (Kijima &
Kanemura, 2016). It can be divided into four subgroups: WNT, SHH, Group 3, and Group 4 (Kijima & Kanemura,
2016). These subgroups differ both in terms of genotype and phenotype, as well as clinical outcome. 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).
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 underway (Rimkus et al., 2016). SMO inhibitors are likely to be effective for both
SMO mutations themselves, as well as PTCH1 and SHH mutations, due to the fact that the drugs’ effects are
downstream of these mutations (Kool et al., 2014). There is also interest in targeting GLI, due to its role as the
terminal effector of the SHH pathway, as well as its involvement in SHH independent mechanisms (Rimkus et
al., 2016). There is currently one FDA approved GLI inhibitor (Rimkus et al., 2016). Rimkus et al. (2016) suggest
that inhibiting SHH itself or targeting tGLI could also be effective treatments. The advantage of targeting tGLI1
is that this protein is found exclusively in cancerous cells, thereby reducing off-target effects. However, the
treatment would need to be able 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 agonists would be indirect.
SONIC HEDGEHOG PATHWAY IN MEDULLOBLASTOMA
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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 the ability of PTCH1 to repress the SMO receptor. SMO activation then dissociates GLI1 from SUFU. 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.
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
antagonists are a theoretical treatment option.
SONIC HEDGEHOG PATHWAY IN MEDULLOBLASTOMA
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Research Question and Potential Impact
The question this proposal aims to investigate is: is SUFU sufficient to repress truncated GLI1 (tGLI1) in
the sonic hedgehog (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 also important 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 aspect of these similarities or differences between GLI1 and tGLI1, 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. Specifically, I propose that SUFU binds to tGLI1 and is sufficient to repress it: this will be
the aim of my investigation. However, I also posit that other components of this pathway would 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.
Proposed Experiment and Predictions
The first experiment would be a “look” experiment, to ensure that tGLI1 is expressed in
medulloblastoma tumours. While it is known that this protein is expressed in other types of cancer,
medulloblastoma has yet to be tested.
This would be done using immunohistochemistry in tumour cells of a medulloblastoma mouse model.
This model would be a xenograft into an immunocompromised mouse. Xenografts would be developed from
infant, juvenile, and adult medulloblastoma patients. The specific strain of immunocompromised mice used
would be severe combined immunodeficiency (SCID), as they have previously been shown to allow for
medulloblastoma development due to their inability to reject human cells (Chiou et al., 2006).
As for immunohistochemistry, I would use an antibody to visualize localization of tGLI1 throughout the
tumour tissue. Zhu, Carpenter, Han, and Lo (2014) created an antibody which acts specifically at the exon 2-4
junction of tGLI1, which is not present in wildtype GLI1. Thus, I can ensure that I am assaying for tGLI1
specifically.
This experiment would be performed at three different ages: 28 days (infant), 42 days (adolescence),
and 3 months (adulthood). These ages were chosen as mice are weaned at approximately day 28, attain
puberty at day 42, and reach sexual maturity at 8-12 weeks (Dutta & Sengupta, 2016). As mentioned
previously, the SHH variant of medulloblastoma demonstrates an unusual bimodal age distribution, whereby
infants and adults exhibit the greatest risk (Kijima & Kanemura, 2016). Furthermore, it has been theorized that
the differences between age groups is not just an effect of the mutations involved (Kijima & Kanemura, 2016).
Thus, cells from patients of different ages (with different mutations) will be implanted in mice of varying ages
in order to determine how both genetics and age affect the expression of tGLI1.
Due to the relationship between age and mutations, I predict that the largest tumours will be seen in
mice where the age of injection corresponds to the xenograft type. Furthermore, I expect to see tGLI1 present
in all tumours as I predict that it is a common mechanism within SHH medulloblastoma. Even though
adolescent SHH medulloblastoma tumours are uncommon, I predict that they would still contain tGLI1.
SONIC HEDGEHOG PATHWAY IN MEDULLOBLASTOMA
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Assuming the result of this procedure is that tGLI1 is present in at least some of the tumours, I would proceed
with the following experiment.
The second experiment (the focus of this project) would be an “add something” experiment.
Specifically, I would increase SUFU expression using a knocked-in gene in a medulloblastoma xenograft mouse
model. The xenograft model will be constructed as per the first experiment. However, any models not
displaying tGLI1 expression will be omitted. Furthermore, if the prediction that tumour size is greatest when
the tumour type correlates with rodent age, I would use only these models.
Before xenotransplantation, I would increase the level of SUFU in the tumour cells using gene knock-in.
Targeted gene knock-in would 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 would be chosen
that targets a region of the genome with no known function. The construct would 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
would need to include its own promoter. Furthermore, the construct would have a positive (neo r) and
negative (HSV-tk) drug selection marker, to allow for selection of properly recombined cells prior to
xenotransplantation (Hall, Limaye, & Kulkarni, 2009). Other essential components include cDNA of the SUFU
sequence, as well as a poly A tail sequence. This construct can be seen in Figure 3.
The control would be a knock-in construct where the SUFU gene has been replaced by the lacZ gene, 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. I would also use a wildtype medulloblastoma
model mouse for comparison in the assay.
This system would be assayed with western blot, staining for tGLI1, SUFU, and lacZ. The tGLI1 antibody
from the first experiment would be used. Additionally, antibodies for SUFU and the control knocked-in gene,
lacZ, would be utilized. This experiment will also be performed at day 28, day 42, and 3 months, in accordance
with the experiment above.
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 wildtype medulloblastoma lane will have bands
for isolated tGLI1 and SUFU, but no or minimal tGLI1-SUFU complex. This is due to the fact that
medulloblastoma tumours have increased GLI1 mediated transcription, indicating that it is not in a repressive
complex with SUFU. The lacZ lane will have bands corresponding to the lacZ protein, ensuring that the
protocol was performed properly.
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 gene knock-in constructs.
Possible Results and Discussion
SONIC HEDGEHOG PATHWAY IN MEDULLOBLASTOMA
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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. This would mean that
SUFU is not sufficient to repress tGLI1. Thus, the tGLI1 protein would function differently than the GLI1
protein, in terms of interaction with SUFU. If there are bands for tGLI1, SUFU, and tGLI1-SUFU, this would
indicate that SUFU can suppress tGLI1, but it is not sufficient, as some tGLI1 remains unbound. If only tGLI1SUFU and tGLI1 bands are seen, this would indicate that SUFU was not increased enough in the experimental
mice or there was another factor acting on SUFU to degrade it before it could interact with tGLI1.
If my prediction is accurate and bands are seen for tGLI1-SUFU with or without SUFU (but not tGLI1),
this would indicate that SUFU is sufficient to repress tGLI1 in SHH medulloblastoma patients. This is an
important result as it could dictate future research. Specifically, it could further investigation into tGLI1
antagonists as potential therapeutics for medulloblastoma. The more evidence accumulates indicating that
GLI1 and tGLI1 function via the same pathway, the more interest there will be in targeting the cancer cell
specific tGLI1.
Additionally, future research should look into the differences between GLI1 and tGLI1. Specifically,
tGLI1 targets both the same genes as GLI1 and novel genes. One such novel gene is CD24. Thus, future
research should aim to elucidate how the various pathway components affect CD24 transcription, as opposed
to genes induced by the GLI1 pathway. In this way, researchers can determine if there will be any side effects
when targeting tGLI1. 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. 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.
Lay Person Summary
See next page.
SONIC HEDGEHOG PATHWAY IN MEDULLOBLASTOMA
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SONIC HEDGEHOG PATHWAY IN MEDULLOBLASTOMA
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References
Carpenter, R. L, & Lo, H. (2012). Hedgehog pathway and GLI1 isoforms in human cancers. Discovery Medicine,
13(69), 105-113.
Chiou, S., Kao, C., Lin, H., Tseng, W., Liu, R., Chung, C., Ku, H., Lin, C., & Wong, T. (2006). Monitoring the growth
effect of xenotransplanted human medulloblastoma in an immunocompromised mouse model using in vitro
and ex vivo green fluorescent protein imaging. Child’s Nervous System, 22, 475-480. doi: 10.1007/s00381-0050026-y
Dutta, S., & Sengupta, P. (2016). Men and mice: Relating their ages. Life Sciences, 152, 244-248.
http://dx.doi.org/10.1016/j.lfs.2015.10.025
Kijima, N., & Kanemura, Y. (2016). Molecular classification of medulloblastoma. Neurologia medico-chirurgica,
E-publication ahead of print, 1-11. doi: 10.2176/nmc.ra.2016-0016
Kool, M., Jones, D. T. W., Jäger, N., Northcott, P. A., Pugh, T. J., Hovestadt, V., … Pfister, S. M. (2014). Genome
sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition. Cancer
Cell, 25(3), 393–405. http://dx.doi.org/10.1016/j.ccr.2014.02.004
Lo, H., Zhu, H., Cao, X., Aldrich, A., & Ali-Osman, F. (2009). A novel splice variant of GLI1 that promotes
glioblastoma cell migration and invasion. Cancer Research, 69(17), 6709-6798. doi: 10.1158/0008-5472.CAN09-0886
Rimkus, T. K., Carpenter, R. L., Qasem, S., Chan, M., & Lo, H. (2016). Targeting the sonic hedgehog signaling
pathway: Review of smoothened and GLI inhibitors. Cancers, 8(22), 1-23. doi: 10.3390/cancers8020022
Taylor, M. D., Liu, L., Raffel, C., Hui, C., Mainprize, T. G., Zhang, X., … Hogg, D. (2002). Mutations in SUFU
predispose to medulloblastoma. Nature Genetics, 31(3), 306-310. doi: 10.1038/ng916
Zhu, H., Carpenter, R. L., Han, W., & Lo, H. (2014). The GLI1 splice variant TGLI1 promotes glioblastoma
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