The Day and Night of Grb2 in Glioblastoma Multiforme Kettle Moraine SMART Team: Grant HoppelThe, Tyler Day Holman, and Night Harrison of Grb2 Plum, Bailey Rockwell, Kevin Zhang, Sean Murray, Zella Christensen, Alberto Patti, Matt Griesbach, Kimberly Stalbaum Advisor: Stephen Plum Mentor: Shama Mirza, Ph.D., Medical College of Wisconsin

Abstract: Receptor Binding Protein – 2 (Grb2) is an essential protein in cell motility, signaling, and most importantly, cell division. In a healthy cell, Grb2 interacts with various growth factors, stimulating the Ras signal transduction pathway, which facilitates cell growth and division. One such growth factor is VEGF (vascular endothelial growth factor), which promotes capillary branching, or angiogenesis. In a cancerous cell, this process goes horribly wrong. Overproduction of VEGF causes overexpression of GRB2 and overstimulation of the Ras pathway, leading to tumor growth. The growing tumor requires greater amounts of oxygen, supplied through angiogenesis, to sustain itself, causing increased production of VEGF and ultimately more tumor growth. Through this process, Grb2’s ability to link angiogenesis and tumor growth can result in deadly cancers, including one of the most severe forms of brain cancer, Glioblastoma Multiforme (GBM). One method of treating this form of cancer targets VEGF, inhibiting both angiogenesis and tumor growth through the Grb2-Ras pathway. One medicine developed for this purpose is (commonly called Avastin), which has been shown to lower the function of VEGF. By inhibiting the production of VEGF, the overstimulation of Grb2 is negated and, under ideal circumstances, the tumor is deprived of oxygen and nutrients, resulting in atrophy.

Glioblastoma Multiforme1 Molecular mechanisms Proteomics of Glioblastoma multiforme The most common and aggressive grade underlying the GBM growth IV glial tumor (a type of brain cancer) Rat brain inoculated with U87 MG • 5 of every 100,000 in the USA Binding Protein – 2 (Grb2)3,4 Inoculate U87 MG • 20% of primary brain tumors Grb2 coordinates pathways within the human GBM cells • Most invasive and Grows rapidly cell, playing a key role in several Treat with or • Most common age 45 or above intracellular networks. Grows GBM Grb2 is critical for functions such as tumors without • Median survival < 2years Avastin epithelial morphogenesis, cell motility, Most common symptoms: and vasculogenesis. Headache, Seizure, Focal neurologic deficits, No treatment Avastin

Change in mental status (10mg/kg) Treatment : Neurosurgery, radiation therapy and chemotherapy L R L R tumor tumor One of the treatment options is focused on reducing the growth of tumors by inhibiting angiogenesis. -Avastin (Bevacizumab) therapy Proteins differentially Proteins differentially expressed during expressed in tumor & as a Angiogenesis and Avastin Therapy tumor growth response to therapy

Angiogenesis plays a key role in Tumor growth. none avastin none avastin Angiogenesis is the growth of new blood Protein expressed asL a R L R vessels. In most scenarios, Angiogenesis is Calmodulin response to therapy      1gri.pdb 14-3-3 protein epsilon       considered a healthy process and increases 1gri.pdb ProteinResults: kinase C inhibitor protein 1       Alpha-enolase none  avastin  none avastin blood circulation. This however is not always Grb2 is a protein involved in the Ras signal transduction pathway. In healthy cells GliaProtein maturation Signature factor beta L R L R the case. In cases of Angiogenesis when one CalmodulinGrowth factor receptor-bound protein 2       the pathway operates normally, but in many cancer cells the pathway is 14-3-3Neuron-specific protein epsilon calcium-binding protein hippocalcin       or more tumors are present , the increased hyperactive, stimulating uncontrolled cell growth. One of the ways that Grb2 can ProteinHippocalcin-like kinase C proteininhibitor 4 protein 1       blood circulation leads to increased tumor Alpha-enolaseEukaryotic translation initiation factor 5A-1       become hyperactive is by phosphorylating/acetylating the protein. Not enough GliaMalate maturation dehydrogenase factor beta     Angiogenesis growth and the spread of cancer. GrowthMacrophage factor migration receptor-bound inhibitory protein factor 2       expression of Grb2 is also bad, especially in embryo development. Grb2 is one Neuron-specificVesicle-fusing ATPase calcium-binding protein hippocalcin     possible protein to focus on for cancer treatments. Hippocalcin-likeProtein kinase C proteinin neurons 4 protein 1       Avastin (Bevacizumab) therapy (anti-angiogenic therapy) for recurrent tumors EukaryoticBrain-specific translation polypeptide initiation PEP-19 factor 5A-1     MalateBrevican dehydrogenase core protein       Avastin limits the production of Vascular Endothelial www.avastin.com Mass Spectrometer MacrophagePeroxiredoxin-2 migration inhibitory factor       Vesicle-fusingTumorGrb2 protein is D52ATPase up- regulated in its expression  in sample   Growth Factor (VEGF) by the tumor, thereby inhibiting ProteinGlycogen kinase phosphorylase, C in neurons brain protein form 1   angiogenesis. This will starve the tumor and lead to its Brain-specificS100with calcium-binding no polypeptide treatment. protein PEP-19 B       BrevicanSH3-containingGrb2 core expression protein GRB2-like protein 3-interactingis down protein-regulated 1   after avastin   shrinkage. Inlet Ion Source Mass Analyser(s) Detector Computer Peroxiredoxin-2Beta-soluble NSF attachment protein       Generates gas Sample Separates ions by m/z and fragments Counts number of Instrument TumorAlpha-synuclein protein D52     -Response rate is not same in all individuals. introduction treatment. phase ions ions ions for each m/z control and data GlycogenUbiquitin thioesterasephosphorylase, L1 brain form       -40% are non-responders. acquisition S100 calcium-binding protein B       SH3-containing GRB2-like protein 3-interacting protein 1       Hence understanding GBM molecular biology is crucial to identify new therapeutic targets. Beta-soluble NSF attachment protein   Alpha-synuclein Overview   Vacuum Chamber Ubiquitin thioesterase L1       • Mass spectrometry-based proteomics can identify References Mass Spectrometery-based Proteomics proteins expressed in disease and healthy states. 1. Eric C. H. Glioblastoma multiforme: The terminator. Proc Natl Acad Sci U S A. 2000; 97(12): 6242–6244. Proteolytic 2. Alessio G, Terrence R. B. Jr., and Donald P. B. Grb2 Signaling in Cell Motility and Cancer. Expert Opin Ther Targets. 2008; Protein MS 12(8): 1021–1033. Peptides • Grb2 is up-regulated in its expression in 3. Sally A. P, Motoo N, Hong L, Ivana H, Gerry R. B, James R. F, Webster K. C, and H.-J. Su Huang. Enhanced Tumorigenic glioblastoma multiforme, and is down-regulated after Behavior of Glioblastoma Cells Expressing a Truncated Receptor Is Mediated through the Ras-Shc- Grb2 Pathway. 1996; 271(41), 25639–25645. avastin treatment. 4. Nagpal S, Harsh G, Recht L. Bevacizumab improves quality of life in patients with recurrent glioblastoma. Chemother Res Trypsin Pract. 2011; 2011: 602812. Data • Protein signatures are unique in glioblastoma

analysis multiforme with and without avastin therapy – useful A Protein %R to identify new and alternate potential therapeutic A SMART Team project supported by the National Institutes of Health Science Education Partnership Award Identification m/z (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973). Theoretical Peptide mass pattern Mass Spectrum targets.