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The Day and Night of Grb2 in Glioblastoma Multiforme Kettle Moraine SMART Team: Grant Hoppel, Tyler Holman, Harrison Plum, Bailey Rockwell, Kevin Zhang, The Day and Night of Grb2 Sean Murray, Zella Christensen, Alberto Patti, Matt Griesbach, Kimberly Stalbaum Advisor: Stephen Plum Mentor: Shama Mirza, Ph.D., Medical College of Wisconsin Abstract: Growth Factor 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 Bevacizumab (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 The most common and aggressive grade IV glial tumor (a type of brain cancer) • 5 of every 100,000 in the USA • 20% of primary brain tumors • Most invasive and Grows rapidly • Most common age 45 or above • Median survival < 2years Most common symptoms: Headache, Seizure, Focal neurologic deficits, Change in mental status Treatment: Neurosurgery, radiation therapy and chemotherapy One of the treatment options is focused on reducing the growth of tumors by inhibiting angiogenesis. -Avastin (Bevacizumab) therapy Molecular mechanisms underlying the GBM growth Proteomics of Glioblastoma multiforme Rat brain inoculated with U87 MG Growth Factor Receptor Binding Protein – 2 (Grb2)3,4 Inoculate U87 MG human GBM cells Grb2 coordinates pathways within the cell, playing a key role in several intracellular networks. Grb2 is critical for functions such as epithelial morphogenesis, cell motility, and vasculogenesis. No treatment L Angiogenesis Avastin (Bevacizumab) therapy (anti-angiogenic therapy) for recurrent tumors www.avastin.com Avastin limits the production of Vascular Endothelial Growth Factor (VEGF) by the tumor, thereby inhibiting angiogenesis. This will starve the tumor and lead to its shrinkage. -Response rate is not same in all individuals. -40% are non-responders. 1gri.pdb 1gri.pdb Grb2 is a protein involved in the Ras signal transduction pathway. In healthy cells the pathway operates normally, but in many cancer cells the pathway is hyperactive, stimulating uncontrolled cell growth. One of the ways that Grb2 can become hyperactive is by phosphorylating/acetylating the protein. Not enough expression of Grb2 is also bad, especially in embryo development. Grb2 is one possible protein to focus on for cancer treatments. Mass Spectrometer Inlet Sample introduction Ion Source Generates gas phase ions Mass Analyser(s) Separates ions by m/z and fragments ions Detector Computer Counts number of ions for each m/z Instrument control and data acquisition Hence understanding GBM molecular biology is crucial to identify new therapeutic targets. Mass Spectrometery-based Proteomics 3. 4. A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973). Proteolytic Peptides Protein MS L R tumor tumor Proteins differentially expressed in tumor & as a response to therapy none Protein expressed as L a Calmodulin response to therapy 14-3-3 protein epsilon Protein kinase C inhibitor protein 1 Results: Alpha-enolase none Protein Signature Glia maturation factor beta L Growth factor receptor-bound protein 2 Calmodulin Neuron-specific calcium-binding protein hippocalcin 14-3-3 protein epsilon Hippocalcin-like Protein kinase C protein inhibitor4 protein 1 Eukaryotic translation initiation factor 5A-1 Alpha-enolase Malate dehydrogenase Glia maturation factor beta Macrophage inhibitory factor2 Growth factormigration receptor-bound protein Vesicle-fusing ATPase Neuron-specific calcium-binding protein hippocalcin Protein kinase C protein in neurons Hippocalcin-like 4 protein 1 Brain-specific polypeptide PEP-19 Eukaryotic translation initiation factor 5A-1 Brevican core protein Malate dehydrogenase Peroxiredoxin-2 Macrophage migration inhibitory factor Tumor protein D52 Vesicle-fusing ATPase Glycogen phosphorylase, brain form1 Protein kinase C in neurons protein S100 calcium-binding protein B Brain-specific polypeptide PEP-19 SH3-containing GRB2-like protein 3-interacting protein 1 Brevican core protein Beta-soluble NSF attachment protein Peroxiredoxin-2 Alpha-synuclein Tumor protein D52 Ubiquitin L1 brain form Glycogen thioesterase phosphorylase, S100 calcium-binding protein B SH3-containing GRB2-like protein 3-interacting protein 1 Beta-soluble NSF attachment protein Alpha-synuclein Ubiquitin thioesterase L1 R R avastin L avastin L R R none avastin none avastin Grb2 is up-regulated in its expression in sample with no treatment. Grb2 expression is down-regulated after avastin treatment. • Mass spectrometry-based proteomics can identify proteins expressed in disease and healthy states. • Grb2 is up-regulated in its expression in glioblastoma multiforme, and is down-regulated after avastin treatment. • Protein signatures are unique in glioblastoma multiforme with and without avastin therapy – useful to identify new and alternate potential therapeutic targets. Trypsin Protein Identification Data analysis %R A References Eric C. H. Glioblastoma multiforme: The terminator. Proc Natl Acad Sci U S A. 2000; 97(12): 6242–6244. Alessio G, Terrence R. B. Jr., and Donald P. B. Grb2 Signaling in Cell Motility and Cancer. Expert Opin Ther Targets. 2008; 12(8): 1021–1033. 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 Behavior of Glioblastoma Cells Expressing a Truncated Epidermal Growth Factor Receptor Is Mediated through the Ras-ShcGrb2 Pathway. 1996; 271(41), 25639–25645. Nagpal S, Harsh G, Recht L. Bevacizumab improves quality of life in patients with recurrent glioblastoma. Chemother Res Pract. 2011; 2011: 602812. R Overview Vacuum Chamber 1. 2. Avastin (10mg/kg) Proteins differentially expressed during tumor growth Angiogenesis and Avastin Therapy Angiogenesis plays a key role in Tumor growth. Angiogenesis is the growth of new blood vessels. In most scenarios, Angiogenesis is considered a healthy process and increases blood circulation. This however is not always the case. In cases of Angiogenesis when one or more tumors are present , the increased blood circulation leads to increased tumor growth and the spread of cancer. Treat with or without Avastin Grows GBM tumors m/z Theoretical Peptide mass pattern Mass Spectrum
