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.

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. 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. Inhibiting microRNA Function

4. Melanoma Cells
Endothelial
Cells
Transwell Assays
Melanoma Cells
FBS gradient
Invasion Endothelial Recruitment

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. 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. 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. MeWo: Melanotic A375: Amelanotic
In vivo Selection of Highly Metastatic
Human Melanoma Cell Lines

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. Functional Validation: Ectopic Expression
Ectopic expression of miR-1908 and miR-199a enhances the
metastatic potential of the poorly metastatic MeWo parental line.

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. Blinded Analysis of Clinical Samples of
Human Melanoma
All three miRs are more highly expressed in melanomas
that had metastasized

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. 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. miR-199a and miR-1908 impact on primary
tumor and in vitro phenotypes
Anoikis Serum Starvation Colony Formation
Endothelial AdhesionProliferation

16. miR-199a and miR-1908
Promote in vitro Invasion

17. miR-199a and miR-1908 Promote in vitro
Endothelial Cell Recruitment

18. Each miR required and sufficient for enhanced metastatic nodule
endothelial content and perfusion.
miR-199a and miR-1908 Promote in vivo
Angiogenesis

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. 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. 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. Array Validation

23. Direct Target Validation

24. Direct Target Validation

26. 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

27. 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

28. ApoE and DNAJA4 Knockdown Promotes
Invasion and Endothelial Cell Recruitment

29. ApoE and DNAJA4 Expression Inhibits
Invasion and Endothelial Cell Recruitment

30. Knockdown of miR targets in the context of miR-1908 and
miR-199a knockdown rescues in vitro phenotypes

31. Knockdown of ApoE and DNAJA4 in the context of miR-
1908 & miR-199a knockdown rescues metastasis in vivo

32. Overexpression of ApoE and DNAJA4 suppresses miR-
1908-induced invasion and endothelial cell recruitment

33. Overexpression of ApoE and DNAJA4 suppresses miR-
199a-induced invasion and endothelial cell recruitment

34. Concurrent Knockdown of ApoE and DNAJA4 in the
Context of Triple miR Knockdown Rescues in vitro
Invasion and Endothelial Cell Recruitment

35. Concurrent Knockdown of ApoE and DNAJA4 in the
Context of Triple miR Knockdown Rescues Angiogenesis
and Metastatic Nodule Perfusion in vivo

36. ApoE and DNAJA4 Inhibit Metastatic Effect of
miR-1908 Expression in vivo

37. 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

38. 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

39. ApoE Secretion is Inversely Correlated with
miR-199a and miR-1908 Expression

40. ApoE Neutralizing Antibody Promotes in vitro
Invasion and Endothelial Cell Recruitment

41. Heat Shock Factor DNAJA4
Regulates ApoE Expression

42. 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

43. ApoE Has No Effect on
Cell Growth or Survival

44. Pretreatment with Recombinant ApoE
Suppresses Metastatic Potential
Suggests a role for ApoE
downstream signaling in
metastasis suppression

45. ApoE Expression is Reduced in Nodal and
Distal Metastases Relative to Primary Tumor

46. Genetic Inactivation of ApoE
Promotes Metastasis
Pretreatment with ApoE inhibits metastatic colonization.
• Implicates stromal ApoE in metastasis suppression

47. 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

48. 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

49. LRP1 mediates ApoE-mediated suppression
of in vitro invasion in melanoma cells

50. LRP1 mediates ApoE-mediated
suppression of metastasis in vivo

51. LRP8 mediates ApoE-mediated suppression
of in vitro migration of endothelial cells

52. LRP8 mediates ApoE-mediated suppression
of in vitro migration of endothelial cells

53. LRP8 mediates ApoE-mediated suppression
of in vitro migration of endothelial cells

54. ApoE inhibits endothelial recruitment into
subcutaneously implanted matrigel plugs

55. ApoE null mice have increased endothelial
cell density in metastatic nodules
ApoE suppresses of in vivo angiogenesis of metastatic nodules

56. 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

57. 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

58. miR-199a-3p, miR-199a-5p, and miR-1908
Predict Metastasis Free Survival

59. Knockdown of miR-199a-3p, miR-199a-5p,
and miR-1908 by Transfection with anti-miR
LNAs Suppresses Metastasis

61. Transfection with anti-miR LNAs Suppresses
Systemic Metastases of Intra-cardiac
Injected Tumor Cells

62. Intravenous treatment with anti-miR LNAs
Inhibits Expression of miR-199a and miR-1908

63. Intravenous treatment with anti-miR LNAs
Suppresses Metastasis

64. 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

65. 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

66. Thank you for your attention
Questions?