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?