GTPase hGBP-1 involvement in glioblastoma malignancy

Stephanie Angel

Undergraduate, Senior

University of Toledo

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

• 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)

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

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

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)

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

hGBP-1 increases glioblastoma invasiveness

• 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)

• 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.

Elevated hGBP-1 in glioblastomas correlates with shorter patient survival

• 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 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.

• Patients with tumors expressing high levels of hGBP-1(red line) had the shortest mean survivals of about 475 days. https://caintegrator.nci.nih.gov/rembrandt/

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

GBP-1 is upregulated in TMZ-resistant U251 glioblastoma cells

• 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.

• 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.

• The expression of hGBP-1 is elevated in U251 cells resistant to TMZ.

(Justinger and Vestal)

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.

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.

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.

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.

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.

0 10 25 50 1000%

20%

40%

60%

80%

100%

120%

U251Control #10Control #5U251-TRhGBP1 #7hGBP1 #2hGBP1 #4

µM/mL TMZ

Colo

nie

s (%

of

con

trol)

• 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.

• 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.

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.

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.

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.

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.

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.

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

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.

Work Cited1. American Brain Tumor Association. (n.d.). Glioblastoma and Malignant Astrocytoma. Retrieved March 2014, from American

Brain Tumor Association: http://www.abta.org/secure/glioblastoma-brochure.pdf

2. Bohman, L. E., Swanson, K. R., Moore, J. L., Rockne, R., Mandigo, C., Hankinson, T., ... & Bruce, J. N. (2010). Magnetic resonance imaging characteristics of glioblastoma multiforme: implications for understanding glioma ontogeny. Neurosurgery, 67(5), 1319.

3. Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, I., Soreq, H., ... & Schlessinger, J. (1985). Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin.

4. Nagane, M., Coufal, F., Lin, H., Bögler, O., Cavenee, W. K., & Huang, H. S. (1996). A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer research, 56(21), 5079-5086.

5. Li, M., Mukasa, A., Inda, M. D. M., Zhang, J., Chin, L., Cavenee, W., & Furnari, F. (2011). Guanylate binding protein 1 is a novel effector of EGFR-driven invasion in glioblastoma. The Journal of experimental medicine, 208(13), 2657-2673.

6. Gene [Internet]. HGNC: HUGO Gene Nomenclature Committee -   . MMP1 matrix metallopeptidase 1 (interstitial collagenase) [ Homo sapiens (human) ]; [updated 2014 March 3; cited 2014 March 2]; [about 5 p.]. Available from: http://www.ncbi.nlm.nih.gov/gene/4312

7. Vetal D, Jeyaratnam J (2011), The Guanylate-Binding Proteins: Emerging insights into the Biochemical Properties and Funcitions of This Family of Large Interferon-Induced Guanosine Triphosphatase, J Interferon Cytokine Res 31(1) 89-97

8. Bourne, H. R., Sanders, D. A., & McCormick, F. (1991). The GTPase superfamily: conserved structure and molecular mechanism. Nature, 349(6305), 117-127.

9. National Institute of Health. (2005). REMBRANDT. Retrieved February 23, 2013, from National Cancer Institute: http://rembrandt.nci.nih.gov

10. Duan, Z., Foster, R., Brakora, K. A., Yusuf, R. Z., & Seiden, M. V. (2006). GBP1 overexpression is associated with a paclitaxel resistance phenotype. Cancer chemotherapy and pharmacology, 57(1), 25-33.

11. Wesolowski, J. R., Rajdev, P., & Mukherji, S. K. (2010). Temozolomide (Temodar). American Journal of Neuroradiology, 31(8), 1383-1384.

12. Shen, W., Hu, J. A., & Zheng, J. S. (2014). Mechanism of temozolomide-induced antitumour effects on glioma cells. Journal of International Medical Research, 42(1), 164-172.

13. Dupont, C. D., & Hunter, C. A. (2012). Guanylate-binding proteins: niche recruiters for antimicrobial effectors. Immunity, 37(2), 191-193.

14. Tanida, I., Ueno, T., & Kominami, E. (2008). LC3 and Autophagy. In Autophagosome and Phagosome (pp. 77-88). Humana Press.

15. Al-Zeer, M. A., Al-Younes, H. M., Lauster, D., Lubad, M. A., & Meyer, T. F. (2013). Autophagy restricts Chlamydia trachomatis growth in human macrophages via IFNG-inducible guanylate binding proteins. Autophagy, 9(1), 50.