INFLUENCE OF SPHINGOSINE 1-PHOSPHATE RECEPTOR SUBTYPES OVER GLIOBLASTOMA MULTIFORME MALIGNANT BEHAVIOR Nicholas Young, Int...
Glioblastoma Multiforme <ul><li>Gliomas are brain tumors resembling glial cells </li></ul><ul><li>Astrocytomas are a form ...
Glioblastoma Multiforme (GBM) <ul><li>Most common adult primary brain tumor </li></ul><ul><ul><li>Incidence is 2-3:100,000...
<ul><ul><ul><li>Weil RJ . Glioblastoma multiforme--treating a deadly tumor with both strands of RNA </li></ul></ul></ul><u...
Sphingosine 1-phosphate <ul><li>Formed in cells in 2 ways: </li></ul><ul><ul><li>De novo Synthesis pathway </li></ul></ul>...
Sphingomyelin (SM) Sphingosine 1-phosphate S1P lyase Hexadecenal + Ethanolamine-P
Sphingosine 1-phosphate S1P Rheostat Apoptosis Growth arrest Cell survival Mitogenesis
S1P and S1P receptor targets <ul><li>Bioactive lipid signaling molecule </li></ul><ul><li>Couples to GPCR family of recept...
S1P receptors are GPCRs <ul><li>G-protein coupled receptors </li></ul><ul><li>Signal though unique sets of G proteins to e...
Survival analysis of glioblastoma patients real time PCR analysis  median survival  SphK1 > 0.65  102 days   SphK1  <  0.6...
Preliminary Work <ul><li>Does S1P stimulate malignant behavior of GBM cells? </li></ul><ul><li>Is this response chemotacti...
S1P stimulates motility and invasiveness of U-373 MG glioma cells   Motility Invasion
Chemokenetic effects of S1P on glioma cell motility   U-373 MG
Statement of Hypothesis <ul><li>Hypothesis: </li></ul><ul><ul><li>S1P receptor expression and stimulation with S1P in glio...
Initial Questions <ul><li>How do the individual receptor subtypes influence in vitro GBM cellular behavior? </li></ul><ul>...
My strategy  <ul><li>U-118 MG express low endogenous receptors </li></ul><ul><ul><li>Overexpress S1P 1 , S1P 2 , S1P 3 , a...
U-118 MG * * * * * * * * *
Serum free media Serum free media + 100nm S1P U-118 MG
ERK phosphorylation in U-118 MG and U-373 MG cells
Conclusions <ul><li>S1P 1-3  contribute to GBM cellular growth and DNA synthesis  </li></ul><ul><li>S1P 5  negatively regu...
 
* * * * * #
Conclusions <ul><li>S1P 1-3  contribute to GBM cellular growth and DNA synthesis </li></ul><ul><li>S1P 5  negatively regul...
Additional Questions <ul><li>Why the differential effect of S1P 2  on cell migration and invasion? </li></ul><ul><li>Cellu...
U118-MG Gelatin Matrigel Well Coating Serum free media Serum free media + 100nm S1P Adhesion Assay * * * * * # #
Conclusions <ul><li>S1P 1-3  contribute to GBM cellular growth and DNA synthesis,  and   adhesion </li></ul><ul><li>S1P 5 ...
Relationship between G-protein signaling to cellular motility and invasiveness Gi Rac Lamellapodia formation Rho Stress fi...
Rac and Rho activation in U-118 MG cells
U-118 MG  cntrl U-118 MG  S1P 2  B U-373 MG  Sicntrl S1P Y-27632 PTX -  +  +  + -  -  +  - -  -  -  +
U-118 MG  Migration * * # * *
U-118 MG  Invasion * * * * * *
Conclusions <ul><li>S1P 1-3  contribute to GBM cellular growth, adhesion, and invasion </li></ul><ul><li>S1P 5  negatively...
 
Next Phase <ul><li>Most interesting data came out of experiments on invasiveness </li></ul><ul><li>Histological examinatio...
GBM local invasiveness <ul><li>GBM local invasiveness often leads to tumor recurrence and poor patient prognosis </li></ul>
Cellular GBM Invasion <ul><li>GBM cell populations exist far away from original tumor mass </li></ul>
Specific Aims of Next Phase <ul><li>Create suitable spheroid model with more relevance than previous boyden chamber method...
Spheroid invasion Assay
Specific Aims of Next Phase <ul><li>Create suitable spheroid model with more relevance than previous boyden chamber method...
Preliminary Data <ul><li>U-373 MG cells gene chip assay </li></ul><ul><ul><li>CCN1 found to be upregulated with S1P additi...
 
U-118 MG cntrl S1P 2  B
CCN1 Results <ul><li>Both S1P 1and2  stimulate CCN1 upregulation </li></ul><ul><ul><li>- S1P 1  through Gi and S1P 2  at l...
RT-PCR analysis of S1P influence on expression of genes related to GBM invasiveness in U-118 MG cells   cntrl S1P 1 S1P 2 ...
Conclusions <ul><li>S1P 1-2  induce CCN1 expression with S1P treatment </li></ul><ul><li>S1P 1-2  overexpression endogenou...
Regulation of adhesion by   V and   3 integrins
S1P receptor subtype effects on uPAR protein expression in U-118 MG cells S1P Inhib Cntrl  S1P 1   S1P 3   S1P 2 Optical D...
S1P Inhib -  +  +  -  +  +  -  +  +  -  +  +  -  -  P  -  -  P  -  -  P  -  -  P  Cntrl  S1P 1   S1P 3   S1P 2 * * * * * *...
S1P Inhib -  +  +  -  +  +  -  +  +  -  +  +  -  -  P  -  -  P  -  -  P  -  -  P  Cntrl  S1P 1   S1P 3   S1P 2 uPA  activi...
Conclusions <ul><li>S1P 1-2  induce CCN1 expression with S1P treatment </li></ul><ul><li>S1P 1-2  overexpression endogenou...
S1P receptor subtype influence over endogenous SK-1 activity   Cntrl  S1P 1   S1P 3   S1P 2 SK activity  (pmol/min/mg prot...
SK inhibitor -  +  -  +  -  +  -  +  Cntrl  S1P 1   S1P 3   S1P 2 SK inhibitor -  +  -  +  -  +  -  +  Cntrl  S1P 1   S1P ...
Cntrl S1P1 Day Cellular viability (%) sphingosine kinase inhibitor and its effects on cellular viability
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...
Conclusions <ul><li>S1P 1-2  induce CCN1 expression with S1P treatment </li></ul><ul><li>S1P 1-2  overexpression endogenou...
Specific Aims of Next Phase <ul><li>Create suitable spheroid model with more relevance than previous boyden chamber method...
Photographs of Day 6 spheroid invasion assay
4x  10x  20x  40x SF S1P cAb uPA Ab CCN1 Ab SK inh  S1P 1 cellular morphology along  the invasive periphery  during sphero...
Conclusions <ul><li>S1P 1-2  induce CCN1 expression with S1P treatment </li></ul><ul><li>S1P 1-2  overexpression endogenou...
Model of S1P-induced GBM invasiveness through uPA/uPAR and CCN1 mechanisms as regulated by S1P receptors and sphingosine k...
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 ...
Specific Aims of Next Phase <ul><li>Create suitable spheroid model with more relevance than previous boyden chamber method...
X-12   U-87 MG Invasion of human glioma cells into surrounding mouse brain
S1P-induced spheroid invasion in neuronal stem cells and X-12 glioma cells   Neuronal stem cell line SFME and X-12 glioma ...
SK inhibitor effects on X-12 invasion
Conclusions <ul><li>X-12 cells resemble neuronal stem cells in S1P-induced invasiveness  </li></ul><ul><ul><li>Support GBM...
Growth Factor Receptor Growth Factor SK-1 SK-1 sph S1P S1P S1PR Rac MOTILITY INVASION ERK AKT SURVIVAL GROWTH CCN1 uPA  3...
Future work <ul><li>Continue to work with the X-12s in vitro to limit spheroid invasion  </li></ul><ul><ul><li>Targeting S...
Acknowledgements  <ul><li>Dr. Jim Van Brocklyn ,  The Ohio State University Department of Pathology </li></ul><ul><li>Mous...
  “ An expert is a man who has made all the mistakes which can be made, in a narrow field.”  Niels Bohr (1885-1962)  “ Eve...
Extra Slides
cntrl S1P 1 S1P 2 S1P 3 Day Day Day Day Invasive diameter  –   core diameter (  m) Invasive diameter  –   core diameter (...
S1P +  +  +  +  +  +  I P Cntrl  S1P1  S1P2 I D Cntrl  S1P1  S1P2 -   plasminogen S1P -  +  -  +  -  +  -  +  Cntrl  S1P 1...
Sphingosine 1-phosphate De novo synthesis pathway
Sphingomyelin Sphingosine 1-phosphate Degradative pathway (SM)
 
U-373 MG * * * * * * * *
Serum free media Serum free media + 100nm S1P U-373 MG
# # # # #
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Nicholas Young, Sphingosine 1-phosphate Receptor Subtype Influence over Glioblastoma Multiforme Pathology

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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"

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Nicholas Young, Sphingosine 1-phosphate Receptor Subtype Influence over Glioblastoma Multiforme Pathology

  1. 1. INFLUENCE OF SPHINGOSINE 1-PHOSPHATE RECEPTOR SUBTYPES OVER GLIOBLASTOMA MULTIFORME MALIGNANT BEHAVIOR Nicholas Young, Integrated Biomedical Science Graduate Program
  2. 2. Glioblastoma Multiforme <ul><li>Gliomas are brain tumors resembling glial cells </li></ul><ul><li>Astrocytomas are a form of glioma </li></ul><ul><ul><li>WHO Grading </li></ul></ul><ul><ul><ul><li>Pilocytic Astrocytoma – grade I </li></ul></ul></ul><ul><ul><ul><li>Diffuse Astrocytoma – grade II </li></ul></ul></ul><ul><ul><ul><li>Anaplastic Astrocytoma – grade III </li></ul></ul></ul><ul><ul><ul><li>Glioblastoma multiforme – grade IV </li></ul></ul></ul>
  3. 3. Glioblastoma Multiforme (GBM) <ul><li>Most common adult primary brain tumor </li></ul><ul><ul><li>Incidence is 2-3:100,000 </li></ul></ul><ul><li>Current treatment is standardized and ineffective </li></ul><ul><ul><li>tumor resection followed radiation and chemotherapeutic treatment </li></ul></ul><ul><li>Median survival 11.5 months </li></ul><ul><li>5-year survival under 5% </li></ul><ul><li>Molecular based therapy could improve outcome of patients </li></ul>
  4. 4. <ul><ul><ul><li>Weil RJ . Glioblastoma multiforme--treating a deadly tumor with both strands of RNA </li></ul></ul></ul><ul><ul><ul><li>PLoS Med. 2006 Jan;3(1):e31. Epub 2005 Dec 6 </li></ul></ul></ul><ul><ul><li>Brain Tumor Institute, Cleveland Clinic Foundation. </li></ul></ul>Current treatment is standardized and ineffective
  5. 5. Sphingosine 1-phosphate <ul><li>Formed in cells in 2 ways: </li></ul><ul><ul><li>De novo Synthesis pathway </li></ul></ul><ul><ul><li>Degradative pathway </li></ul></ul>
  6. 6. Sphingomyelin (SM) Sphingosine 1-phosphate S1P lyase Hexadecenal + Ethanolamine-P
  7. 7. Sphingosine 1-phosphate S1P Rheostat Apoptosis Growth arrest Cell survival Mitogenesis
  8. 8. S1P and S1P receptor targets <ul><li>Bioactive lipid signaling molecule </li></ul><ul><li>Couples to GPCR family of receptors called S1P receptors </li></ul><ul><ul><li>S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 receptor subtypes </li></ul></ul><ul><li>S1P 1 , S1P 2 , S1P 3 nearly ubiquitous </li></ul><ul><li>S1P 5 found in normal brain tissue </li></ul><ul><ul><li>S1P 1 , S1P 2 , and S1P 3 in GBM tissue </li></ul></ul>
  9. 9. S1P receptors are GPCRs <ul><li>G-protein coupled receptors </li></ul><ul><li>Signal though unique sets of G proteins to elicit cellular responses </li></ul><ul><li>Receptor subtypes all couple to different sets of G-proteins to varying degrees </li></ul><ul><li>Each G-protein signals through distinct signal transduction cascades to elicit cellular responses </li></ul>
  10. 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)
  11. 11. Preliminary Work <ul><li>Does S1P stimulate malignant behavior of GBM cells? </li></ul><ul><li>Is this response chemotactic ar chemokeneitc in nature? </li></ul>
  12. 12. S1P stimulates motility and invasiveness of U-373 MG glioma cells Motility Invasion
  13. 13. Chemokenetic effects of S1P on glioma cell motility U-373 MG
  14. 14. Statement of Hypothesis <ul><li>Hypothesis: </li></ul><ul><ul><li>S1P receptor expression and stimulation with S1P in glioma cells enhances malignant cellular characteristics </li></ul></ul><ul><ul><ul><li>achieved through external S1P receptor activation </li></ul></ul></ul><ul><ul><ul><ul><li>specific G-protein signal transduction pathway activation to ultimately elicit a cellular response </li></ul></ul></ul></ul><ul><ul><ul><li>this expression and stimulation enhances tumor growth and invasiveness in vivo </li></ul></ul></ul>
  15. 15. Initial Questions <ul><li>How do the individual receptor subtypes influence in vitro GBM cellular behavior? </li></ul><ul><ul><li>What signaling pathways are involved </li></ul></ul><ul><li>Can any of these pathways be manipulated in vitro and in vivo to ultimately decrease malignant behavior of GBM? </li></ul>
  16. 16. My strategy <ul><li>U-118 MG express low endogenous receptors </li></ul><ul><ul><li>Overexpress S1P 1 , S1P 2 , S1P 3 , and S1P 5 </li></ul></ul><ul><li>U-373 MG express high endogenous receptors </li></ul><ul><ul><li>Knock down S1P 1 , S1P 2 , and S1P 3 expression using siRNA </li></ul></ul><ul><li>Use these cell lines to analyze S1P-mediated effects </li></ul><ul><ul><li>Cell growth, migration, invasion, and adhesion </li></ul></ul><ul><ul><ul><li>examine the G-protein signaling pathways involved in these responses </li></ul></ul></ul>
  17. 17. U-118 MG * * * * * * * * *
  18. 18. Serum free media Serum free media + 100nm S1P U-118 MG
  19. 19. ERK phosphorylation in U-118 MG and U-373 MG cells
  20. 20. Conclusions <ul><li>S1P 1-3 contribute to GBM cellular growth and DNA synthesis </li></ul><ul><li>S1P 5 negatively regulates DNA synthesis </li></ul><ul><ul><li>- decreased ERK-phosphorylation </li></ul></ul>
  21. 22. * * * * * #
  22. 23. Conclusions <ul><li>S1P 1-3 contribute to GBM cellular growth and DNA synthesis </li></ul><ul><li>S1P 5 negatively regulates DNA synthesis </li></ul><ul><li>- decreased ERK-phosphorylation </li></ul><ul><li>S1P 1,3 both stimulate S1P-mediated migration and invasion </li></ul><ul><li>While S1P 2 positively stimulates invasion, it decreases S1P-mediated migration </li></ul>
  23. 24. Additional Questions <ul><li>Why the differential effect of S1P 2 on cell migration and invasion? </li></ul><ul><li>Cellular invasion is much more complicated than migration </li></ul><ul><ul><li>Involves ECM degradation, cytoskeletal rearrangement, and adhesion </li></ul></ul><ul><li>Does S1P effect adhesion these cellular activities </li></ul><ul><ul><li>What is the individual receptor subtype influence </li></ul></ul>
  24. 25. U118-MG Gelatin Matrigel Well Coating Serum free media Serum free media + 100nm S1P Adhesion Assay * * * * * # #
  25. 26. Conclusions <ul><li>S1P 1-3 contribute to GBM cellular growth and DNA synthesis, and adhesion </li></ul><ul><li>S1P 5 negatively regulates DNA synthesis and adhesion </li></ul><ul><ul><li>- decreased ERK-phosphorylation </li></ul></ul><ul><li>S1P 1,3 both stimulate S1P-mediated migration and invasion </li></ul><ul><li>While S1P 2 positively stimulates invasion, it decreases S1P-mediated migration </li></ul><ul><li>Does S1P effect cytoskeletal rearrangement? </li></ul><ul><ul><li>What is the individual receptor subtype influence </li></ul></ul>
  26. 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 ?
  27. 28. Rac and Rho activation in U-118 MG cells
  28. 29. U-118 MG cntrl U-118 MG S1P 2 B U-373 MG Sicntrl S1P Y-27632 PTX - + + + - - + - - - - +
  29. 30. U-118 MG Migration * * # * *
  30. 31. U-118 MG Invasion * * * * * *
  31. 32. Conclusions <ul><li>S1P 1-3 contribute to GBM cellular growth, adhesion, and invasion </li></ul><ul><li>S1P 5 negatively regulates all the cellular activities examined </li></ul><ul><ul><li>- decreased ERK-phosphorylation </li></ul></ul><ul><li>S1P 1,3 both stimulate S1P-mediated migration and invasion </li></ul><ul><li>While S1P 2 positively stimulates invasion, it decreases S1P-mediated migration </li></ul><ul><ul><li>- Rac not decreased with S1P 2 </li></ul></ul><ul><ul><li>- Rho is activated with S1P 2 </li></ul></ul><ul><ul><li>- S1P-induced Rho activation inhibits migration and contributes stress fiber formation through a G12/13 mediated pathway </li></ul></ul>
  32. 34. Next Phase <ul><li>Most interesting data came out of experiments on invasiveness </li></ul><ul><li>Histological examination of GBM tissue sections shows localized invasiveness </li></ul><ul><li>Why is limiting invasiveness important to GBM patients? </li></ul>
  33. 35. GBM local invasiveness <ul><li>GBM local invasiveness often leads to tumor recurrence and poor patient prognosis </li></ul>
  34. 36. Cellular GBM Invasion <ul><li>GBM cell populations exist far away from original tumor mass </li></ul>
  35. 37. Specific Aims of Next Phase <ul><li>Create suitable spheroid model with more relevance than previous boyden chamber method </li></ul><ul><li>Examine S1P-mediated effects of the proteins involved in invasion </li></ul><ul><li>Establish the S1P receptor subtype influence over GBM invasiveness </li></ul><ul><li>Decrease in vitro GBM invasiveness by targeting S1P-mediated pathobiology </li></ul><ul><li>Establish and characterize a relevant in vivo GBM model to examine invasiveness </li></ul>
  36. 38. Spheroid invasion Assay
  37. 39. Specific Aims of Next Phase <ul><li>Create suitable spheroid model with more relevance than previous boyden chamber method </li></ul><ul><li>Examine S1P-mediated effects of the proteins involved in invasion </li></ul><ul><li>Establish the S1P receptor subtype influence over GBM invasiveness </li></ul><ul><li>Decrease in vitro GBM invasiveness by targeting S1P-mediated pathobiology </li></ul><ul><li>Establish and characterize a relevant in vivo GBM model to examine invasiveness </li></ul>
  38. 40. Preliminary Data <ul><li>U-373 MG cells gene chip assay </li></ul><ul><ul><li>CCN1 found to be upregulated with S1P addition in </li></ul></ul><ul><ul><ul><li>CCN1 is a secreted matricellular protein involved in increasing cellular adhesion and invasiveness </li></ul></ul></ul><ul><ul><li>uPA also found to be upregulated </li></ul></ul><ul><ul><ul><li>plasminogen plasmin (fibrin degradation) </li></ul></ul></ul><ul><li>Areas to investigate </li></ul><ul><ul><li>CCN1 ( see if it shows promise and continue if yes) </li></ul></ul><ul><ul><li>-uPAR-uPA system </li></ul></ul><ul><ul><li>-  V-  3 integrin comlex </li></ul></ul><ul><ul><ul><ul><li>-upregulated in GBM along invasive periphery </li></ul></ul></ul></ul>
  39. 42. U-118 MG cntrl S1P 2 B
  40. 43. CCN1 Results <ul><li>Both S1P 1and2 stimulate CCN1 upregulation </li></ul><ul><ul><li>- S1P 1 through Gi and S1P 2 at least partially through G 12/13 Rho kinase </li></ul></ul><ul><li>S1P 2 mediated invasiveness is neutralized with CCN1 antibody </li></ul><ul><ul><li>- this could therefore serve as a potential therapeutic target in minimizing local GBM invasiveness </li></ul></ul>
  41. 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
  42. 45. Conclusions <ul><li>S1P 1-2 induce CCN1 expression with S1P treatment </li></ul><ul><li>S1P 1-2 overexpression endogenously upregulate uPAR transcription </li></ul><ul><li>S1P 1-3 overexpression endogenously induce uPA transcription </li></ul><ul><li>Integrins  V and  3 are expressed in all U-118 MG cells </li></ul>
  43. 46. Regulation of adhesion by  V and  3 integrins
  44. 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
  45. 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
  46. 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
  47. 50. Conclusions <ul><li>S1P 1-2 induce CCN1 expression with S1P treatment </li></ul><ul><li>S1P 1-2 overexpression endogenously upregulate uPAR transcription and protein expression </li></ul><ul><li>S1P 1-3 overexpression endogenously induce uPA transcription and protein expression </li></ul><ul><li>Integrins  3 and  3 are expressed in all U-118 cells and contribute to cellular adhesion in these cells </li></ul><ul><li>S1P 1 overexpression endogenously enhances uPA activity </li></ul>
  48. 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
  49. 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
  50. 53. Cntrl S1P1 Day Cellular viability (%) sphingosine kinase inhibitor and its effects on cellular viability
  51. 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
  52. 55. Conclusions <ul><li>S1P 1-2 induce CCN1 expression with S1P treatment </li></ul><ul><li>S1P 1-2 overexpression endogenously upregulate uPAR transcription and protein expression </li></ul><ul><ul><li>Inhibition of SK prevents uPAR expression </li></ul></ul><ul><li>S1P 1-3 overexpression endogenously induce uPA transcription and protein expression </li></ul><ul><li>Integrins  3 and  3 are expressed in all U-118 cells and contribute to cellular adhesion in these cells </li></ul><ul><li>S1P 1 overexpression endogenously enhances uPA activity and SK activity </li></ul><ul><ul><li>Inhibition of SK prevents uPA activity </li></ul></ul>
  53. 56. Specific Aims of Next Phase <ul><li>Create suitable spheroid model with more relevance than previous boyden chamber method </li></ul><ul><li>Examine S1P-mediated effects of the proteins involved in invasion </li></ul><ul><li>Establish the S1P receptor subtype influence over GBM invasiveness </li></ul><ul><li>Decrease in vitro GBM invasiveness by targeting S1P-mediated pathobiology </li></ul><ul><li>Establish and characterize a relevant in vivo GBM model to examine invasiveness </li></ul>
  54. 57. Photographs of Day 6 spheroid invasion assay
  55. 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
  56. 59. Conclusions <ul><li>S1P 1-2 induce CCN1 expression with S1P treatment </li></ul><ul><li>S1P 1-2 overexpression endogenously upregulate uPAR transcription and protein expression </li></ul><ul><ul><li>Inhibition of SK prevents uPAR expression </li></ul></ul><ul><li>S1P 1-3 overexpression endogenously induce uPA transcription and protein expression </li></ul><ul><li>Integrins  3 and  3 are expressed in all U-118 MG cells and contribute to cellular adhesion in these cells </li></ul><ul><li>S1P 1 overexpression endogenously enhances uPA activity and SK activity </li></ul><ul><ul><li>Inhibition of SK prevents uPA activity </li></ul></ul><ul><li>Antibody treatment with uPA or CCN1 neutralize U-118 MG invasiveness </li></ul><ul><li>SK inhibitor treatment inhibits U-118 MG invasiveness </li></ul>
  57. 60. Model of S1P-induced GBM invasiveness through uPA/uPAR and CCN1 mechanisms as regulated by S1P receptors and sphingosine kinase <ul><li>SK appears critical to elicit most invasive response </li></ul><ul><ul><li>S1P 1 expression enhances SK activity </li></ul></ul><ul><ul><li>S1PRs are all in some way involved in </li></ul></ul><ul><ul><ul><li>uPA expression </li></ul></ul></ul><ul><ul><ul><li>uPAR expression </li></ul></ul></ul><ul><ul><ul><li>CCN1 expression </li></ul></ul></ul><ul><ul><ul><li>uPA activity </li></ul></ul></ul><ul><li>S1P 1 having the key role of all receptor subtypes </li></ul>
  58. 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
  59. 62. Specific Aims of Next Phase <ul><li>Create suitable spheroid model with more relevance than previous boyden chamber method </li></ul><ul><li>Examine S1P-mediated effects of the proteins involved in invasion </li></ul><ul><li>Establish the S1P receptor subtype influence over GBM invasiveness </li></ul><ul><li>Decrease in vitro GBM invasiveness by targeting S1P-mediated pathobiology </li></ul><ul><li>Establish and characterize a relevant in vivo GBM model to examine invasiveness </li></ul>
  60. 63. X-12 U-87 MG Invasion of human glioma cells into surrounding mouse brain
  61. 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
  62. 65. SK inhibitor effects on X-12 invasion
  63. 66. Conclusions <ul><li>X-12 cells resemble neuronal stem cells in S1P-induced invasiveness </li></ul><ul><ul><li>Support GBM tumor stem cell theory </li></ul></ul><ul><li>SK inhibitor treatment also reduces invasiveness of these cells </li></ul><ul><li>Endogenous invasive responses could be a result of SK activity enhancement </li></ul><ul><ul><li>Matrigel contains many growth factors </li></ul></ul><ul><ul><li>S1P 1 can induce SK activity </li></ul></ul><ul><li>New Model of invasion should include growth factors and MMP contributions in future work </li></ul>
  64. 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
  65. 68. Future work <ul><li>Continue to work with the X-12s in vitro to limit spheroid invasion </li></ul><ul><ul><li>Targeting S1P-induced invasive mechanisms </li></ul></ul><ul><li>Use the effective in vitro targets to ultimately limit in vivo GBM tumor progression </li></ul>
  66. 69. Acknowledgements <ul><li>Dr. Jim Van Brocklyn , The Ohio State University Department of Pathology </li></ul><ul><li>Mouse brain tumor work: </li></ul><ul><li>Dr. Sean Lawler, The Ohio State University Department of Neurosurgery </li></ul><ul><li>Dr. Oskar Nowicki, The Ohio State University Department of Neurosurgery </li></ul><ul><li>Committee members: </li></ul><ul><li>Dr. Jim Van Brocklyn , The Ohio State University, Department of Pathology </li></ul><ul><li>Dr. Allan Yates , The Ohio State University, IBGP </li></ul><ul><li>Dr. Ken Jones , The Ohio State University, College of Medicine </li></ul><ul><li>Dr. Gary Stoner , The Ohio State University, College of Medicine </li></ul>
  67. 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) 
  68. 71. Extra Slides
  69. 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
  70. 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
  71. 74. Sphingosine 1-phosphate De novo synthesis pathway
  72. 75. Sphingomyelin Sphingosine 1-phosphate Degradative pathway (SM)
  73. 77. U-373 MG * * * * * * * *
  74. 78. Serum free media Serum free media + 100nm S1P U-373 MG
  75. 79. # # # # #
  76. 80. ROCK

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