1. GTPase hGBP-1 involvement
in glioblastoma malignancy
Stephanie Angel
Undergraduate, Senior
University of Toledo

2. What is Glioblastoma Multiforme?
• Gliobastoma Multiforme (GBM) consists of stage IV tumors that
arise from supportive cells of the brain called astrocytes.
• Glioblastoma cells reproduce quickly and are supported by an
ample blood supply.
• Glioblastomas are highly invasive, spreading quickly throughout
normal brain tissues.
• Adults with glioblastoma have a median survival of about 14.6
months even with aggressive treatment.
http://www.abta.org/brain-tumor-information/types-of-tumors/glioblastoma.html

3. • Magnetic resonance images of a patient with a glioblastoma
over a 1-month period. Tumor volume expanded by 19-fold
within one month.
(Bohman et al. 2010)

4. Epidermal Growth Factor Receptor (EGFR) in
Glioblastomas
• EGFR is a transmembrane receptor tyrosine kinase which when
bound to epidermal growth factor (EGF) leads to cell
proliferation. In most glioblastomas, EGFR is either amplified or
mutated. The most common mutated EGFR contains an in-frame
deletion producing a truncated receptor that is constitutively
active. 2,3
• EGFR gene amplification occurs in about 40-50% of
glioblastomas with almost half of the tumors exhibiting
amplifications mutant forms of the receptor. 3
• Constitutively active EGF receptor promotes tumorigenicity by
increasing proliferation and inhibiting apoptosis.
3

5. Matrix Metalloproteinase-1 (MMP-1) induction
by EGFR
• Activation of EGFR in glioblastomas induces the expression of
hGBP-1 and MMP-1. 4
• MMP-1 is a secreted collagenase involved in the breakdown of
the extracellular matrix. Activity of this collagenase leads to
increased invasiveness of cancerous cells and is associated
with metastasis, or spread, of cancers. 5,6
• EGFR signaling leads to increased invasiveness by inducing
MMP-1. 4

6. Human Guanylate Binding Protein-1 (hGBP-1)
is required for MMP-1 induction by EGFR
• The GTPase, hGBP-1, is required for the basal and EGFR-
induced MMP-1 transcription in glioma cells. This is unlike
endothelial cells where GBP-1 inhibits MMP-1 expression.
• Overexpression of GBP-1 enhanced glioblastoma invasion.
Reduction of GBP-1 interfered with glioblastoma invasion.
• This indicates hGBP-1 increases the aggressiveness of
glioblastomas.
(Li et al., 2011)

7. What are Guanylate Binding Proteins
(GBPs)?
• Guanylate-Binding Proteins (GBPs)
are immunity-related GTPases
inducible by IFN-γ, a cytokine mainly
produced by T-cells of the immune
system. 7
• GBPs hydrolyze GTP to both GDP and
GMP, a unique characteristic among
GTPases. 8
• MMP-1 induction requires the C-
terminal helical domain of hGBP-1 and
does not require the GTPase activity. 4 Ribbon representation of hGBP1

8. hGBP-1 increases glioblastoma invasiveness
• To study the effects of GBP-1 in tumor
formation, glioblastoma cells were
transducted with either shGFP or shGBP-1
and injected into 4-5 wk old athymic nude
mice. Mice were euthanized after 14-20
days and brain was removed, embedded in
paraffin, and stained with hematoxylin and
eosin.
• Tumor margins are delineated
using dotted lines. Arrowheads
denote invasive extensions from
tumor mass (T). Arrows indicate
invasive tumor cells and
disseminated tumor clusters away
from the tumor mass.
• Control tumors exhibited invasive
extensions into normal brain
tissue similar to highly invasive
glioblastoma. Tumors with
reduced hGBP-1 exhibited
smooth tumor–parenchyma
interfaces and decreased glioma
cell infiltration.
• hGBP-1 is involved in
glioblastoma invasiveness.
(Li et al., 2011)

9. Elevated hGBP-1 in glioblastomas correlates
with shorter patient survival
•
•
Patients with all forms of gliomas (blue
line) have a median survival of about 500
days.
•
Patients with tumors expressing low levels
of hGBP-1 (yellow line) had the longest
mean survivals of about 1500 days.
•
https://caintegrator.nci.nih.gov/rembrandt/
To determine if GBP-1 expression in
gliomas effects patient survival,
REMBRANDT, The REpository for
Molecular BRAin and Neoplasia DaTa,
was used to create Kaplan-Meier plots
displaying survival data as a step function
(Y-axis) versus survival time (x-axis). This
plot correlates survival time to expression
of hGBP-1 in brain tumors.
Patients with tumors expressing high
levels of hGBP-1(red line) had the
shortest mean survivals of about 475
days.

10. GBP-1 in Drug Resistance
• The expression of hGBP-1 is up-regulated in human cancer cell
lines when they are made resistant to paclitaxel. 10
• Ectopic expression of hGBP-1 in a paclitaxel-sensitive ovarian
cell line resulted in a moderate resistance to paclitaxel.
10
• GBP-1 is responsible for increased invasiveness in
glioblastomas. GBP-1 is also involved in chemotherapeutic drug
resistance. We therefore asked if hGBP-1 is involved in
Temozolomide (TMZ) resistance in glioblastoma?
• TMZ is a the chemotherapeutic drug used to treat glioblastomas,
due to its high penetration of the blood brain barrier. TMZ
functions by methylating cellular DNA, activating the mismatch
repair system which drives the cell to apoptosis. 11

11. GBP-1 is upregulated in TMZ-resistant U251
glioblastoma cells
•
•
U251 cells sensitive to TMZ and resistant
to TMZ (U251 TMZ) were analyzed by
western blot for hGBP-1 expression.
•
Actin was used as a loading control.
•
(Justinger and Vestal)
To explore if hGBP-1 is involved in TMZ
resistance in glioblastomas, expression
levels of hGBP-1 in sensitive and resistant
U251 glioblastoma cells were analyzed.
The expression of hGBP-1 is elevated in
U251 cells resistant to TMZ.

12. hGBP-1 in Glioblastoma
Summary of role of hGBP-1 in Glioblastoma to this point.
• GBP-1 increases glioblastoma invasiveness into normal
brain tissue.
• GBP-1 is directly correlated with shorter survival times in
patients with glioblastoma.
• GBP-1 plays a role in paclitaxel resistance in various cancer
cell lines.
• GBP-1 is upregulated in a glioblastoma cell line resistant to
Temozolomide.
This information led to the hypothesis of my project.

13. Hypothesis: The GTPase, hGBP-1, is involved in
Temozolomide drug resistance in glioblastomas.
To determine if hGBP-1 is involved in TMZ resistance, we can ask two
questions:
1. Is hGBP-1 alone sufficient to cause TMZ resistance?
This can be answered by overexpressing hGBP-1 in TMZ
sensitive glioblastoma cells and asking whether they are now
resistant to TMZ.
2. Is hGBP-1 required for TMZ resistance?
This can be answered by knocking down the expression of
hGBP-1 in TMZ resistant cells that overexpress hGBP-1 and
asking whether these cells are now more sensitive to TMZ.

14. Is GBP-1 Sufficient to Cause TMZ
Resistance?
• To determine if GBP-1 is required for TMZ resistance, U251 glioblastoma
cells were transfected with control plasmid pIRES-hygro2 or plasmid
encoding hGBP1 c-myc-hGBP1-pIRES-hygro2. Transfection solutions
were made at a lipid (µg) to plasmid (µL) ratio of 3:1 using FuGene 6 as
transfection reagent.
• Transfected U251 cells were selected in complete media selected with 50
µg/ml hygromycin.
• After 6 weeks of expansion twelve of the c-myc-hGBP1-pIRES-hygro2
transfected U251 colonies were analyzed by Western Blot.
• The c-myc-hGBP1-pIRES-hygro2
transfected U251 colonies expressed
more hGBP1 than untransfected
U251 cells. Three were chosen for
further studies (hGBP1 #2, #4, and
#7). Actin serves as a loading control
to demonstrate relatively equal
amounts of protein in each sample.

15. Does elevated GBP-1 cause TMZ
resistance?
• To determine if elevated GBP-1 in glioblastomas causes TMZ
resistance, the three hGBP1 colonies (hGBP1 #2, #4, and #7), two
control pIRES-hygro2 transfected U251 colonies (control #5 and #10), a
regular U251 cell line, and a TMZ resistant U251-TR cell line were used
to perform a colony-forming assay.
• Each of the seven cell lines were plated into 10 cm dishes at 1,000 cells
per dish. Beginning on the day after plating cells were exposed to the
drug TMZ at either 0 µM/ml , 10 µM/mL, 25 µM/mL, 50 µM/mL, or 100
µM/mL in complete media for 48 hours, replenishing the drug after 1
day. Cells were then fed fresh media with no drug every 2 to 3 days for
a period of 7 days.
• Afterwards dishes were washed with PBS, cells were fixed for 10
minutes in ice-cold 100% methanol and stained with 1% crystal violet in
35% methanol. Colonies containing greater than 50 cells were counted
and the numbers recorded.
• Each of the seven cell lines used in the colony-forming assay were also
analyzed for hGBP1 expression by Western Blot.

16. Upregulation of hGBP1 does not result in
Temozolomide resistance in U251 cells
• Figure shows
representative examples of
colony assays.
• The TMZ resistant U251
cells show little cell death
with the highest level of
TMZ. The sensitive U251
cells are killed by this
dosage. The control and
hGBP-1 over-expressing
U251 are no more resistant
to TMZ than sensitive U251
cells.

17. •
The number of colonies in each dish were counted. For each cell line and each drug
concentration a mean from three plates was calculated. The means for cells with no
exposure to TMZ were set at 100% (control). The legend indicates which color corresponds
to which cell line.
•
Results reveal U251-TR growth remains static as exposure to TMZ increases, indicating
resistance. While there are some minor differences in the colony counts for the other cells
lines, these differences do not reach statistical significance.
•
Colonies (% of control)
120%
100%
U251
80%
Control #10
Control #5
60%
U251-TR
40%
hGBP1 #7
hGBP1 #2
20%
hGBP1 #4
0%
0
10
25
50
µM/mL TMZ
100
Western blot analysis of hGBP1 concentrations in each
cell line after the colony assay indicated overexpressing
hGBP1 in the c-myc-hGBP1-pIRES-hygro2 transfected
cells.

18. Interpretation of Colony-Forming Assay
Results
• Overexpressing hGBP1 in glioblastoma cells did not result in resistance to TMZ,
therefore GBP-1 alone is not sufficient to cause TMZ resistance in
glioblastomas.
• The second part to determining if GBP-1 is involved in TMZ resistance asks if
GBP-1 is required for TMZ resistance in glioblastomas. Although GBP-1 alone
cannot cause resistance, it may act in concert with another protein. To determine
if GBP-1 is required for TMZ resistance, shRNAs would be used to knockdown
hGBP-1 expression in TMZ-resistant glioblastomas. A colony-forming assay
would then determine if sensitivity was restored in transduced TMZ-resistant
cells.
• The previous western blot analysis revealed TMZ resistant U251 cells were no
longer up-regulating hGBP1. These results are similar to the consequences of
shRNA knockdown. Despite normal expressing levels of hGBP-1, results from
the colony-forming assay demonstrated these U251-TR cells continued to
remain resistant. Therefore GBP-1 is not required for TMZ resistance in
glioblastomas
• Experiments demonstrated GBP-1 is neither sufficient nor required for
Temozolomide resistance in glioblastomas. We therefore conclude the
GTPase, hGBP-1, is not involved in Temozolomide drug resistance in
glioblastomas.

19. Temozolomide in Autophagy
• Glioblastoma cells, when exposed to 100 µmol/l Temozolomide,
exhibit cell arrest, prohibition of cellular migration, and autophagy.
Temozolomide induces autophagy instead of apoptosis in
glioblastoma cells. 12
• hGBP1 can associate with membranes by localizing to vacuoles
containing bacterial cells. 13
• This information led us to the second hypothesis of my project:
The GTPase, hGBP-1, associates with autophagosomal
membranes in glioblastoma cells during periods of stress,
such as serum starvation.

20. Does GBP-1 associate with autophagosomal
membranes during times of stress?
• To determine if hGBP1 associates with autophagosomal membranes during times of
stress, SNB75 glioblastoma cells were plated onto coverslips and transfected with
either pEGFP or pEGFP-LC3 encoding a green fluorescent LC3. LC3 is a
microtubule-associated protein recruited to autophagosomal membranes. Detecting
for LC3 is a reliable marker for autophagosomes.
14
• After 48 hours serum deprivation, hGBP-1 distribution was determined with
immuno-affinity purified rabbit anti-hGBP-1 and Alexa 596 conjugated goat anti-rabbit
secondary giving hGBP1 a red fluorescence. Dapi stained nuclei fluorescent blue.
Images were taken using confocal microscopy. Confocal microscopes capture sliced
images of samples.
• The yellow vesicles indicate co-localization of hGBP1 and LC3, therefore hGBP-1
does associate with autophagosomal membranes.

21. Significance of GBP-1 and LC3
co-localization
• hGBP1 is capable of recruitment to bacterial inclusion bodies, triggering
a re-routing of the inclusion bodie for lysosomal degradation. Therefore
hGBP1 is capable of association with phospholipid membranes. 15
• hGBP1 plays a role in immunity, commonly induced by the release of
cytokine IFN-γ in macrophages.
• hGBP1 association with autophagosomal membranes could reveal a
new mechanism in which these immunity-based GTPases fight off
diseases.
• Future directions would reveal the role hGBP1 recruitment to
autophagosomes play in autophagy in glioblastoma cells. It is possible
hGBP1 induces formation of autophagosomes or triggers a re-routing of
the autophagosomes. Much research is left to determine the reason for
autophagosomal recruitment of hGBP1 in glioblastomas.

22. Conclusions
• GBP-1 increases invasiveness of glioblastoma cells and shortens patient
survival.
• GBP-1 is involved in Paclitaxel resistance in a variety of cancerous cell lines.
• GBP-1 is not sufficient to cause Temozolomide resistance in sensitive
glioblastoma cells.
• GBP-1 is not required for Temozolomide resistance in resistant glioblastoma
cells.
• GBP-1 is not involved in Temozolomide resistance.
• During periods of stress, GBP-1 is capable of associating with autophagosomal
membranes.

23. Future Studies
• To discover proteins that could be involved in Temozolomide
resistance a ChIP analysis of proteins elevated in TMZ-resistant
cells compared to sensitive cells would lead us to a new
direction.
• To further understand hGBP-1 association with autophagosomal
membranes an immunofluorescence time-course analysis of
serum deprivation could reveal when the association occurs in
glioblastomas and for how long.
• To explore how hGBP-1 associates with autophagosomal
membranes hGBP-1 can be immunoprecipitated from
glioblastoma cells after the appropriate time of serum
deprivation and associated proteins or lipids determined by
mass spec analysis

24. Acknowledgements
I want to thank Dr. Vestal for her continuous mentorship,
advice, and words of encouragement throughout my
research project.
I also want to thank my parents and fiancé for their
constant support in all my ventures.

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