My talk presenting my thesis work per invitation of Research and Development Division of Genzyme Corporation to the Lipid Storage Disorders Department. October 5, 2007. Boston, MA: "Sphingosine 1-phosphate Receptor Subtype Influence over Glioblastoma Multiforme Pathology". My PhD was earned through the Integrated Biomedical Sciences, September 2007, The Ohio State University, College of Medicine Columbus, OH. Area of focus for my PhD: Biochemical and Molecular Disease Mechanisms. Dissertation title: Sphingosine 1-phosphate Receptor Subtype Influence Over Glioblastoma Multiforme Malignant Behavior"

1. INFLUENCE OF SPHINGOSINE 1-PHOSPHATE RECEPTOR SUBTYPES OVER GLIOBLASTOMA MULTIFORME MALIGNANT BEHAVIOR Nicholas Young, Integrated Biomedical Science Graduate Program

6. Sphingomyelin (SM) Sphingosine 1-phosphate S1P lyase Hexadecenal + Ethanolamine-P

7. Sphingosine 1-phosphate S1P Rheostat Apoptosis Growth arrest Cell survival Mitogenesis

10. Survival analysis of glioblastoma patients real time PCR analysis median survival SphK1 > 0.65 102 days SphK1 < 0.65 357 days Relationship between S1P and GBM 0 800 200 600 400 1000 1200 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion Surviving SphK-1 > 0.65 SphK-1 < 0.65 Survival (days)

12. S1P stimulates motility and invasiveness of U-373 MG glioma cells Motility Invasion

13. Chemokenetic effects of S1P on glioma cell motility U-373 MG

17. U-118 MG * * * * * * * * *

18. Serum free media Serum free media + 100nm S1P U-118 MG

19. ERK phosphorylation in U-118 MG and U-373 MG cells

22. * * * * * #

25. U118-MG Gelatin Matrigel Well Coating Serum free media Serum free media + 100nm S1P Adhesion Assay * * * * * # #

27. Relationship between G-protein signaling to cellular motility and invasiveness Gi Rac Lamellapodia formation Rho Stress fiber formation G 12/13 PTX Y-27632 ROCK Gi and G12/13 couple to the small GTPases Rho and Rac - results in actin cytoskeleton rearrangements 1. How do the S1P receptor subtypes influence Rho and Rac activation? 2. How does this influence actin cytoskeletal rearrangement? 3. Does Rho/Rac activation influence migratory and invasive response? S1P 2 ?

28. Rac and Rho activation in U-118 MG cells

29. U-118 MG cntrl U-118 MG S1P 2 B U-373 MG Sicntrl S1P Y-27632 PTX - + + + - - + - - - - +

30. U-118 MG Migration * * # * *

31. U-118 MG Invasion * * * * * *

38. Spheroid invasion Assay

42. U-118 MG cntrl S1P 2 B

44. RT-PCR analysis of S1P influence on expression of genes related to GBM invasiveness in U-118 MG cells cntrl S1P 1 S1P 2 S1P 3 S1P - + + - + + - + + - + + inhib - - P - - P - - P - - P CCN1 uPA uPAR ITG  v ITG  3 GAPDH

46. Regulation of adhesion by  V and  3 integrins

47. S1P receptor subtype effects on uPAR protein expression in U-118 MG cells S1P Inhib Cntrl S1P 1 S1P 3 S1P 2 Optical Density uPAR GAPDH * * * * * * - + + - + + - + + - + + - - P - - P - - P - - P

48. S1P Inhib - + + - + + - + + - + + - - P - - P - - P - - P Cntrl S1P 1 S1P 3 S1P 2 * * * * * * * * uPA GAPDH Optical Density S1P receptor subtype effects on uPA protein expression in U-118 MG cells

49. S1P Inhib - + + - + + - + + - + + - - P - - P - - P - - P Cntrl S1P 1 S1P 3 S1P 2 uPA activity * * * * * Optical Density S1P receptor subtype effects on uPA activity in U-118 MG cells

51. S1P receptor subtype influence over endogenous SK-1 activity Cntrl S1P 1 S1P 3 S1P 2 SK activity (pmol/min/mg protein) # # U-118 MG

52. SK inhibitor - + - + - + - + Cntrl S1P 1 S1P 3 S1P 2 SK inhibitor - + - + - + - + Cntrl S1P 1 S1P 3 S1P 2 uPAR GAPDH uPA activity Cntrl * SK activity (pmol/min/mg protein) sphingosine kinase inhibitor and its effects on uPAR expression and uPA activity U-118 MG

53. Cntrl S1P1 Day Cellular viability (%) sphingosine kinase inhibitor and its effects on cellular viability

54. cntrl S1P 1 U-118 MG DMSO SK sicntrl siSK siSK inh A B uPA activity DMSO SK sicntrl siSK siSK inh A B siRNA to SK mimics sphingosine kinase inhibitor in reducing uPA activation

57. Photographs of Day 6 spheroid invasion assay

58. 4x 10x 20x 40x SF S1P cAb uPA Ab CCN1 Ab SK inh S1P 1 cellular morphology along the invasive periphery during spheroid invasion

61. SK-1 SK-1 sph S1P S1P S1PR Rac MOTILITY INVASION ERK AKT SURVIVAL GROWTH CCN1 uPA  3  v pro- uPA CCN1 CCN1 CCN1  v  v  3  3 (ECM) ADHESION uPAR (Endothelial cell Recruitement) ANGIOGENESIS plasminogen plasmin INVASION uPAR uPAR uPA pro- uPA pro- uPA matriptase CCN1 Ab uPA Ab SK inh

63. X-12 U-87 MG Invasion of human glioma cells into surrounding mouse brain

64. S1P-induced spheroid invasion in neuronal stem cells and X-12 glioma cells Neuronal stem cell line SFME and X-12 glioma cell line were grown in defined media to enrich stem-like cancer cells as neurospheres

65. SK inhibitor effects on X-12 invasion

67. Growth Factor Receptor Growth Factor SK-1 SK-1 sph S1P S1P S1PR Rac MOTILITY INVASION ERK AKT SURVIVAL GROWTH CCN1 uPA  3  v pro- uPA CCN1 CCN1 CCN1  v  v  3  3 (ECM) ADHESION uPAR FAS FASL APOPTOSIS (Endothelial cell Recruitement) ANGIOGENESIS Pro-MMP plasminogen plasmin MMP INVASION uPAR uPAR uPA pro- uPA pro- uPA matriptase

70. “ An expert is a man who has made all the mistakes which can be made, in a narrow field.” Niels Bohr (1885-1962) “ Everything should be as simple as it is, but not simpler.” Albert Einstein (1879-1955) “ When you steal from one author, it's plagiarism; if you steal from many, it's research.” Wilson Mizner (1876-1933)

71. Extra Slides

72. cntrl S1P 1 S1P 2 S1P 3 Day Day Day Day Invasive diameter – core diameter (  m) Invasive diameter – core diameter (  m) Invasive diameter – core diameter (  m) Invasive diameter – core diameter (  m) inhibition of spheroid invasiveness with SK inhibitor and uPA/CCN1 ab U-118 MG cells

73. S1P + + + + + + I P Cntrl S1P1 S1P2 I D Cntrl S1P1 S1P2 - plasminogen S1P - + - + - + - + Cntrl S1P 1 S1P 3 S1P 2 uPA activity uPA activity uPA activity is not nonspecific and can be immunoprecipitated

74. Sphingosine 1-phosphate De novo synthesis pathway

75. Sphingomyelin Sphingosine 1-phosphate Degradative pathway (SM)

77. U-373 MG * * * * * * * *

78. Serum free media Serum free media + 100nm S1P U-373 MG

79. # # # # #

80. ROCK