11.27.12 - lcbg department journal club
DESCRIPTION
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.TRANSCRIPT
Journal ClubJournal Club
Nov 27, 2012
LCBGDr. Hunter’s Lab
Farhoud Faraji Kent Hunter, PhD
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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
Inhibiting microRNA Function
Melanoma Cells
Endothelial Cells
Transwell Assays
Melanoma Cells
FBS gradient
Invasion Endothelial Recruitment
In vivo Selection of Metastatic Clones
AssumptionClones only differ from parental line in molecular and cellular phenotypes related to the metastatic potential
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
Outline1. 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 DNAJA44. ApoE and DNAJA4 Mediate m199 and m1908-
Dependent in vitro and in vivo Metastatic Phenotypes5. ApoE Suppresses Invasion, Endothelial Recruitment, and
Metastasis6. Extracellular ApoE Divergently Targets Melanoma Cell
LRP1 and Endothelial Cell LRP8 receptors7. m1908 and m199a are Robust Prognostic and
Therapeutic Targets in Melanoma Metastasis
MeWo: Melanotic A375: Amelanotic
In vivo Selection of Highly Metastatic Human Melanoma Cell Lines
Identification of Differentially Expressed miRNAs
Array-based miR Global Profiling Taqman Validation
miR-1908miR-214miR-199a are more highly expressed in highly metastatic daughter clones
Functional Validation: Ectopic Expression
Ectopic expression of miR-1908 and miR-199a enhances the metastatic potential of the poorly metastatic MeWo parental line.
Inhibition of miR-1908, miR-199a-5p, and miR-199a-3p diminishes the metastatic potential MeWo and A375 highly metastatic clones.
Functional Validation: miR KnockdownDo not show data for miR expression levels upon knockdown.
Blinded Analysis of Clinical Samples of Human Melanoma
All three miRs are more highly expressed in melanomas that had metastasized
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
Outline1. 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 DNAJA44. ApoE and DNAJA4 Mediate m199 and m1908-
Dependent in vitro and in vivo Metastatic Phenotypes5. ApoE Suppresses Invasion, Endothelial Recruitment, and
Metastasis6. Extracellular ApoE Divergently Targets Melanoma Cell
LRP1 and Endothelial Cell LRP8 receptors7. m1908 and m199a are Robust Prognostic and
Therapeutic Targets in Melanoma Metastasis
miR-199a and miR-1908 impact on primary tumor and in vitro phenotypes
Anoikis Serum Starvation Colony Formation
Endothelial AdhesionProliferation
miR-199a and miR-1908 Promote in vitro Invasion
miR-199a and miR-1908 Promote in vitro Endothelial Cell Recruitment
Each miR required and sufficient for enhanced metastatic nodule endothelial content and perfusion.
miR-199a and miR-1908 Promote in vivo Angiogenesis
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
Outline1. 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 DNAJA44. ApoE and DNAJA4 Mediate m199 and m1908-
Dependent in vitro and in vivo Metastatic Phenotypes5. ApoE Suppresses Invasion, Endothelial Recruitment, and
Metastasis6. Extracellular ApoE Divergently Targets Melanoma Cell
LRP1 and Endothelial Cell LRP8 receptors7. m1908 and m199a are Robust Prognostic and
Therapeutic Targets in Melanoma Metastasis
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
Array Validation
Direct Target Validation
Direct Target Validation
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
Outline1. 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 DNAJA44. ApoE and DNAJA4 Mediate m199 and m1908-
Dependent in vitro and in vivo Metastatic Phenotypes5. ApoE Suppresses Invasion, Endothelial Recruitment, and
Metastasis6. Extracellular ApoE Divergently Targets Melanoma Cell
LRP1 and Endothelial Cell LRP8 receptors7. m1908 and m199a are Robust Prognostic and
Therapeutic Targets in Melanoma Metastasis
ApoE and DNAJA4 Knockdown Promotes Invasion and Endothelial Cell Recruitment
ApoE and DNAJA4 Expression Inhibits Invasion and Endothelial Cell Recruitment
Knockdown of miR targets in the context of miR-1908 and miR-199a knockdown rescues in vitro phenotypes
Knockdown of ApoE and DNAJA4 in the context of miR-1908 & miR-199a knockdown rescues metastasis in vivo
Overexpression of ApoE and DNAJA4 suppresses miR-1908-induced invasion and endothelial cell recruitment
Overexpression of ApoE and DNAJA4 suppresses miR-199a-induced invasion and endothelial cell recruitment
Concurrent Knockdown of ApoE and DNAJA4 in the Context of Triple miR Knockdown Rescues in vitro
Invasion and Endothelial Cell Recruitment
Concurrent Knockdown of ApoE and DNAJA4 in the Context of Triple miR Knockdown Rescues Angiogenesis
and Metastatic Nodule Perfusion in vivo
ApoE and DNAJA4 Inhibit Metastatic Effect of miR-1908 Expression in vivo
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
Outline1. 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 DNAJA44. ApoE and DNAJA4 Mediate m199 and m1908-
Dependent in vitro and in vivo Metastatic Phenotypes5. ApoE Suppresses Invasion, Endothelial Recruitment, and
Metastasis6. Extracellular ApoE Divergently Targets Melanoma Cell
LRP1 and Endothelial Cell LRP8 receptors7. m1908 and m199a are Robust Prognostic and
Therapeutic Targets in Melanoma Metastasis
ApoE Secretion is Inversely Correlated with miR-199a and miR-1908 Expression
ApoE Neutralizing Antibody Promotes in vitro Invasion and Endothelial Cell Recruitment
Heat Shock Factor DNAJA4 Regulates ApoE Expression
Recombinant ApoE rescuesinhibition of in vitro invasion and endothelial cell recruitment in thecontext of DNAJA4 knockdown
Neutralizing ApoE antibody rescuesin vitro invasion and endothelial cell recruitment in the context of DNAJA4 expression
ApoE Has No Effect on Cell Growth or Survival
Pretreatment with Recombinant ApoE Suppresses Metastatic Potential
Suggests a role for ApoEdownstream signaling in metastasis suppression
ApoE Expression is Reduced in Nodal and Distal Metastases Relative to Primary Tumor
Genetic Inactivation of ApoE Promotes Metastasis
Pretreatment with ApoE inhibits metastatic colonization.• Implicates stromal ApoE in metastasis suppression
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
Outline1. 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 DNAJA44. ApoE and DNAJA4 Mediate m199 and m1908-
Dependent in vitro and in vivo Metastatic Phenotypes5. ApoE Suppresses Invasion, Endothelial Recruitment, and
Metastasis6. Extracellular ApoE Divergently Targets Melanoma Cell
LRP1 and Endothelial Cell LRP8 receptors7. m1908 and m199a are Robust Prognostic and
Therapeutic Targets in Melanoma Metastasis
LRP1 mediates ApoE-mediated suppression of in vitro invasion in melanoma cells
LRP1 mediates ApoE-mediated suppression of metastasis in vivo
LRP8 mediates ApoE-mediated suppression of in vitro migration of endothelial cells
LRP8 mediates ApoE-mediated suppression of in vitro migration of endothelial cells
LRP8 mediates ApoE-mediated suppression of in vitro migration of endothelial cells
ApoE inhibits endothelial recruitment into subcutaneously implanted matrigel plugs
ApoE null mice have increased endothelial cell density in metastatic nodules
ApoE suppresses of in vivo angiogenesis of metastatic nodules
Summary 61. LRP1 mediates ApoE-mediated suppression
of in vitro invasion in melanoma cells2. LRP1 mediates ApoE-mediated suppression
of metastasis in vivo 3. LRP8 mediates ApoE-mediated suppression
of in vitro migration of endothelial cells4. ApoE suppresses angiogenesis in metastatic
nodules in vivo
Outline1. 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 DNAJA44. ApoE and DNAJA4 Mediate m199 and m1908-
Dependent in vitro and in vivo Metastatic Phenotypes5. ApoE Suppresses Invasion, Endothelial Recruitment, and
Metastasis6. Extracellular ApoE Divergently Targets Melanoma Cell
LRP1 and Endothelial Cell LRP8 receptors7. miR-1908 and miR-199a are Robust Prognostic and
Therapeutic Targets in Melanoma Metastasis
miR-199a-3p, miR-199a-5p, and miR-1908 Predict Metastasis Free Survival
Knockdown of miR-199a-3p, miR-199a-5p, and miR-1908 by Transfection with anti-miR
LNAs Suppresses Metastasis
Transfection with anti-miR LNAs Suppresses Systemic Metastases of Intra-cardiac
Injected Tumor Cells
Intravenous treatment with anti-miR LNAs Inhibits Expression of miR-199a and miR-1908
Intravenous treatment with anti-miR LNAs Suppresses Metastasis
Summary 71. miR-199a and miR-1908 predict distant
metastasis free survival in patient cohorts2. Transfection with anti-miR LNAs suppresses
metastasis of intravenous and intra-cardiac injected melanoma cells
3. Systemic treatment with anti-miR LNAs suppresses metastasis
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
Thank you for your attention
Questions?