11.27.12 - LCBG Department Journal Club

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NIH/NCI/CCR/LCBG Branch Journal Club

Presented Article:
Pencheva N, Tran H, Buss C, Huh D, Drobnjak M, Busam K, Tavazoie SF. Convergent multi-miRNA targeting of ApoE drives LRP1/LRP8-dependent melanoma metastasis and angiogenesis. Cell. 2012 Nov 21;151(5):1068-82.

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11.27.12 - LCBG Department Journal Club

  1. 1. Journal ClubJournal Club Nov 27, 2012 LCBG Dr. Hunter’s Lab Farhoud Faraji Kent Hunter, PhD Your Picture here Your Mentor’s Picture here
  2. 2. microRNA Family of post- transcriptional regulatory small RNAs Concepts: •miR can ‘promiscuously’ bind mRNAs – Each miR has many targets •mRNAs can ‘promiscuously’ bind miRs – Each mRNA is targeted by many miRs He and Hannon. 2009
  3. 3. Inhibiting microRNA Function
  4. 4. Melanoma Cells Endothelial Cells Transwell Assays Melanoma Cells FBS gradient Invasion Endothelial Recruitment
  5. 5. In vivo Selection of Metastatic Clones Assumption Clones only differ from parental line in molecular and cellular phenotypes related to the metastatic potential
  6. 6. Objective • Use in vivo selection to find prometastatic microRNAs in melanoma Strategy • Construct highly metastatic lines from poorly metastatic parental human melanoma lines • Look for microRNAs with: – High expression in highly metastatic clone – Low expression in poorly metastatic parental
  7. 7. Outline 1. Endogenous miR-1908, miR-199a-3p, and miR-199-5p Promote Human Melanoma Metastasis. 2. m1908 and m199a Promote Invasion, Endothelial Recruitment, and Angiogenesis. 3. m1908 and m199a Convergently and Cooperatively Target ApoE and DNAJA4 4. ApoE and DNAJA4 Mediate m199 and m1908- Dependent in vitro and in vivo Metastatic Phenotypes 5. ApoE Suppresses Invasion, Endothelial Recruitment, and Metastasis 6. Extracellular ApoE Divergently Targets Melanoma Cell LRP1 and Endothelial Cell LRP8 receptors 7. m1908 and m199a are Robust Prognostic and Therapeutic Targets in Melanoma Metastasis
  8. 8. MeWo: Melanotic A375: Amelanotic In vivo Selection of Highly Metastatic Human Melanoma Cell Lines
  9. 9. Identification of Differentially Expressed miRNAs Array-based miR Global Profiling Taqman Validation miR-1908 miR-214 miR-199a are more highly expressed in highly metastatic daughter clones
  10. 10. Functional Validation: Ectopic Expression Ectopic expression of miR-1908 and miR-199a enhances the metastatic potential of the poorly metastatic MeWo parental line.
  11. 11. Inhibition of miR-1908, miR-199a-5p, and miR-199a-3p diminishes the metastatic potential MeWo and A375 highly metastatic clones. Functional Validation: miR Knockdown Do not show data for miR expression levels upon knockdown.
  12. 12. Blinded Analysis of Clinical Samples of Human Melanoma All three miRs are more highly expressed in melanomas that had metastasized
  13. 13. Summary 1 • Highly metastatic clones generated from two independent human melanoma cell lines • miR-1908, miR-199a, and miR-214 more highly expressed in highly metastatic clones • Ectopic expression of miR-1908 and miR-199a enhances metastasis. • Knockdown of m1908, m199a-5p, and m199a- 3p inhibits metastasis. • m1908, m199a-5p, and m199a-3p more highly expressed in metastatic clinical samples
  14. 14. Outline 1. Endogenous miR-1908, miR-199a-3p, and miR-199-5p Promote Human Melanoma Metastasis. 2. m1908 and m199a Promote Invasion, Endothelial Recruitment, and Angiogenesis. 3. m1908 and m199a Convergently and Cooperatively Target ApoE and DNAJA4 4. ApoE and DNAJA4 Mediate m199 and m1908- Dependent in vitro and in vivo Metastatic Phenotypes 5. ApoE Suppresses Invasion, Endothelial Recruitment, and Metastasis 6. Extracellular ApoE Divergently Targets Melanoma Cell LRP1 and Endothelial Cell LRP8 receptors 7. m1908 and m199a are Robust Prognostic and Therapeutic Targets in Melanoma Metastasis
  15. 15. miR-199a and miR-1908 impact on primary tumor and in vitro phenotypes Anoikis Serum Starvation Colony Formation Endothelial AdhesionProliferation
  16. 16. miR-199a and miR-1908 Promote in vitro Invasion
  17. 17. miR-199a and miR-1908 Promote in vitro Endothelial Cell Recruitment
  18. 18. Each miR required and sufficient for enhanced metastatic nodule endothelial content and perfusion. miR-199a and miR-1908 Promote in vivo Angiogenesis
  19. 19. Summary 2 • miR-199a suppresses tumor growth • miR-1908, miR-199a reduce in vitro tumor cell proliferation • miR-1908, miR-199a promote in vitro invasion and endothelial cell recruitment • miR-1908, miR-199a promote angiogenesis and perfusion of metastatic nodules in vivo
  20. 20. Outline 1. Endogenous miR-1908, miR-199a-3p, and miR-199-5p Promote Human Melanoma Metastasis. 2. m1908 and m199a Promote Invasion, Endothelial Recruitment, and Angiogenesis. 3. m1908 and m199a Convergently and Cooperatively Target ApoE and DNAJA4 4. ApoE and DNAJA4 Mediate m199 and m1908- Dependent in vitro and in vivo Metastatic Phenotypes 5. ApoE Suppresses Invasion, Endothelial Recruitment, and Metastasis 6. Extracellular ApoE Divergently Targets Melanoma Cell LRP1 and Endothelial Cell LRP8 receptors 7. m1908 and m199a are Robust Prognostic and Therapeutic Targets in Melanoma Metastasis
  21. 21. Identification of ApoE and DNAJA4 as Targets of miR-199a and miR-1908 Strategy: find transcripts inversely correlated to miR expression in all cases. • miR OE • miR KD • parental vs metastatic clone
  22. 22. Array Validation
  23. 23. Direct Target Validation
  24. 24. Direct Target Validation
  25. 25. Summary 3 • ApoE and DNAJA4 expression inversely correlates with miR-1908 and miR-199a expression • miR-1908 and miR-199a directly target ApoE and DNAJA4
  26. 26. Outline 1. Endogenous miR-1908, miR-199a-3p, and miR-199-5p Promote Human Melanoma Metastasis. 2. m1908 and m199a Promote Invasion, Endothelial Recruitment, and Angiogenesis. 3. m1908 and m199a Convergently and Cooperatively Target ApoE and DNAJA4 4. ApoE and DNAJA4 Mediate m199 and m1908- Dependent in vitro and in vivo Metastatic Phenotypes 5. ApoE Suppresses Invasion, Endothelial Recruitment, and Metastasis 6. Extracellular ApoE Divergently Targets Melanoma Cell LRP1 and Endothelial Cell LRP8 receptors 7. m1908 and m199a are Robust Prognostic and Therapeutic Targets in Melanoma Metastasis
  27. 27. ApoE and DNAJA4 Knockdown Promotes Invasion and Endothelial Cell Recruitment
  28. 28. ApoE and DNAJA4 Expression Inhibits Invasion and Endothelial Cell Recruitment
  29. 29. Knockdown of miR targets in the context of miR-1908 and miR-199a knockdown rescues in vitro phenotypes
  30. 30. Knockdown of ApoE and DNAJA4 in the context of miR- 1908 & miR-199a knockdown rescues metastasis in vivo
  31. 31. Overexpression of ApoE and DNAJA4 suppresses miR- 1908-induced invasion and endothelial cell recruitment
  32. 32. Overexpression of ApoE and DNAJA4 suppresses miR- 199a-induced invasion and endothelial cell recruitment
  33. 33. Concurrent Knockdown of ApoE and DNAJA4 in the Context of Triple miR Knockdown Rescues in vitro Invasion and Endothelial Cell Recruitment
  34. 34. Concurrent Knockdown of ApoE and DNAJA4 in the Context of Triple miR Knockdown Rescues Angiogenesis and Metastatic Nodule Perfusion in vivo
  35. 35. ApoE and DNAJA4 Inhibit Metastatic Effect of miR-1908 Expression in vivo
  36. 36. Summary 4 • ApoE and DNAJA4 expression inversely impacts invasion and endothelial cell recruitment in vitro • ApoE and DNAJA4 epistatically interact with miR-199a and miR-1908 to regulate: – in vitro invasion and endothelial recruitment – in vivo angiogenesis and metastatic coloinzation
  37. 37. Outline 1. Endogenous miR-1908, miR-199a-3p, and miR-199-5p Promote Human Melanoma Metastasis. 2. m1908 and m199a Promote Invasion, Endothelial Recruitment, and Angiogenesis. 3. m1908 and m199a Convergently and Cooperatively Target ApoE and DNAJA4 4. ApoE and DNAJA4 Mediate m199 and m1908- Dependent in vitro and in vivo Metastatic Phenotypes 5. ApoE Suppresses Invasion, Endothelial Recruitment, and Metastasis 6. Extracellular ApoE Divergently Targets Melanoma Cell LRP1 and Endothelial Cell LRP8 receptors 7. m1908 and m199a are Robust Prognostic and Therapeutic Targets in Melanoma Metastasis
  38. 38. ApoE Secretion is Inversely Correlated with miR-199a and miR-1908 Expression
  39. 39. ApoE Neutralizing Antibody Promotes in vitro Invasion and Endothelial Cell Recruitment
  40. 40. Heat Shock Factor DNAJA4 Regulates ApoE Expression
  41. 41. Recombinant ApoE rescues inhibition of in vitro invasion and endothelial cell recruitment in the context of DNAJA4 knockdown Neutralizing ApoE antibody rescues in vitro invasion and endothelial cell recruitment in the context of DNAJA4 expression
  42. 42. ApoE Has No Effect on Cell Growth or Survival
  43. 43. Pretreatment with Recombinant ApoE Suppresses Metastatic Potential Suggests a role for ApoE downstream signaling in metastasis suppression
  44. 44. ApoE Expression is Reduced in Nodal and Distal Metastases Relative to Primary Tumor
  45. 45. Genetic Inactivation of ApoE Promotes Metastasis Pretreatment with ApoE inhibits metastatic colonization. • Implicates stromal ApoE in metastasis suppression
  46. 46. Summary 5 • Extracellular ApoE Inhibits in vitro invasion and endothelial cell recruitment • DNAJA4 regulates ApoE expression • Pretreatment with recombinant ApoE suppresses metastatic potential • ApoE expression is reduced in nodal and distal metastases relative to primary tumor • Absence of stromal ApoE promotes metastatic colonization
  47. 47. Outline 1. Endogenous miR-1908, miR-199a-3p, and miR-199-5p Promote Human Melanoma Metastasis. 2. m1908 and m199a Promote Invasion, Endothelial Recruitment, and Angiogenesis. 3. m1908 and m199a Convergently and Cooperatively Target ApoE and DNAJA4 4. ApoE and DNAJA4 Mediate m199 and m1908- Dependent in vitro and in vivo Metastatic Phenotypes 5. ApoE Suppresses Invasion, Endothelial Recruitment, and Metastasis 6. Extracellular ApoE Divergently Targets Melanoma Cell LRP1 and Endothelial Cell LRP8 receptors 7. m1908 and m199a are Robust Prognostic and Therapeutic Targets in Melanoma Metastasis
  48. 48. LRP1 mediates ApoE-mediated suppression of in vitro invasion in melanoma cells
  49. 49. LRP1 mediates ApoE-mediated suppression of metastasis in vivo
  50. 50. LRP8 mediates ApoE-mediated suppression of in vitro migration of endothelial cells
  51. 51. LRP8 mediates ApoE-mediated suppression of in vitro migration of endothelial cells
  52. 52. LRP8 mediates ApoE-mediated suppression of in vitro migration of endothelial cells
  53. 53. ApoE inhibits endothelial recruitment into subcutaneously implanted matrigel plugs
  54. 54. ApoE null mice have increased endothelial cell density in metastatic nodules ApoE suppresses of in vivo angiogenesis of metastatic nodules
  55. 55. Summary 6 1. LRP1 mediates ApoE-mediated suppression of in vitro invasion in melanoma cells 2. LRP1 mediates ApoE-mediated suppression of metastasis in vivo 3. LRP8 mediates ApoE-mediated suppression of in vitro migration of endothelial cells 4. ApoE suppresses angiogenesis in metastatic nodules in vivo
  56. 56. Outline 1. Endogenous miR-1908, miR-199a-3p, and miR-199-5p Promote Human Melanoma Metastasis. 2. m1908 and m199a Promote Invasion, Endothelial Recruitment, and Angiogenesis. 3. m1908 and m199a Convergently and Cooperatively Target ApoE and DNAJA4 4. ApoE and DNAJA4 Mediate m199 and m1908- Dependent in vitro and in vivo Metastatic Phenotypes 5. ApoE Suppresses Invasion, Endothelial Recruitment, and Metastasis 6. Extracellular ApoE Divergently Targets Melanoma Cell LRP1 and Endothelial Cell LRP8 receptors 7. miR-1908 and miR-199a are Robust Prognostic and Therapeutic Targets in Melanoma Metastasis
  57. 57. miR-199a-3p, miR-199a-5p, and miR-1908 Predict Metastasis Free Survival
  58. 58. Knockdown of miR-199a-3p, miR-199a-5p, and miR-1908 by Transfection with anti-miR LNAs Suppresses Metastasis
  59. 59. Transfection with anti-miR LNAs Suppresses Systemic Metastases of Intra-cardiac Injected Tumor Cells
  60. 60. Intravenous treatment with anti-miR LNAs Inhibits Expression of miR-199a and miR-1908
  61. 61. Intravenous treatment with anti-miR LNAs Suppresses Metastasis
  62. 62. Summary 7 1. miR-199a and miR-1908 predict distant metastasis free survival in patient cohorts 2. Transfection with anti-miR LNAs suppresses metastasis of intravenous and intra-cardiac injected melanoma cells 3. Systemic treatment with anti-miR LNAs suppresses metastasis
  63. 63. Model miR-199a and miR-1908 are robust prognostic and therapeutic targets. ApoE is a potent suppressor of melanoma metastasis via its effects on tumor cell invasion and metastatic endothelial cell recruitment
  64. 64. Thank you for your attention Questions?

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