IntroductionPrognostic gene signatures that predict patient risk for metastatic disease are in clinical trials. These gene sets, however, provide little insight into mechanisms of metastasis. Here we exploit the principle that metastatic propensity is modified by the genetic background to link prognostic gene signatures with molecular mechanisms driving metastasis. In doing so we transcend single gene functional analysis and unveil the higher-order transcriptional architecture of heritable predisposition to breast cancer metastasis. Key PointsA.Network analysis of global expression profiles from genetically defined AKXD recombinant inbred panel primary tumors identified a network of co-expressed genes centered on Cnot2 that predicts metastasis free survival in human breast cancer.

Cnot2 over-expression regulates expression of network genes. Modulating Cnot2 expression inversely impacts tumor cell metastatic potential in vivo. Cnot2 binds the metastasis driver genes Brd4-SF, Rrp1b, and Sipa1

B.Small RNA sequencing of the same tumor panel revealed miRNA-3470b as a potential upstream regulator of the Cnot2 network.

miR-3470b down-regulates Cnot2 network hub gene expression. miR-3470b expression down-regulated anti-metastatic genes and upregulated pro-metastatic

genes miR-3470b promotes metastasis.

ConclusionOur systems genetics strategy provides a higher-order view of metastatic susceptibility. We identify and validate a co-expressed module of transcripts that is post-transcriptionally regulated by miR-3470b and whose central node, Cnot2, functionally regulates metastasis. The physical interaction of CNOT2 with previously identified metastasis modifier proteins BRD4-SF, SIPA1, and RRP1B implicates CNOT2 in a larger nuclear complex that regulates metastatic potential, further demonstrating the value of undertaking higher-order analyses to interrogate mechanisms of metastasis.

AKR/J DBA/2J

xP

Metastasis-prone Metastasis-resistant

x

FVB PyMT

AKXDnPyMT+ F1

[ ]

MetastasisSusceptibility Prone Intermediate Resistant

xF1

F2x x x

x x xF3

F>20++

1 Employing meiotic genetics to understand heritable predisposition to metastasis

2 Systems genetics strategy to unveil the higher-order transcriptional structure of metastasis

Microarray based mRNA profiling

miRNA Sequencing

Negatively CorrelatedmiRNA-mRNA

AKXD x PyMT F1 Tumors

Filter for miR-mRNA Target Pairs

NetworkGeneration

Network HubKaplan-Meier

Analysis

BiologicalValidation

Identify miRNAsHighly Represented

In Networks

BiologicalValidation

3 The Cnot2 Network: A module of co-expressed genes regulated by Cnot2

Primary Tumor Burden

Tum

or M

ass

(g)

0.0

0.5

1.0

1.5

6DT1shControl

6DT1sh62

6DT1sh64

Primary Tumor Burden

Primary Tumor Burden

Tum

or M

ass

(g)

0.0

0.5

1.0

1.5

2.0

6DT1Control

6DT1Cnot2

Primary Tumor Burden*

Pulmonary Metastases

Surf

ace

Met

asta

sis

Coun

t

0

20

40

60

80

6DT1Control

6DT1Cnot2

Pulmonary Metastases**

Pulmonary Metastases

Surf

ace

Met

asta

sis

Coun

t

0

20

40

60

80

Pulmonary Metastases*

**

6DT1 shControl 6DT1 sh62 6DT1 sh64

6DT1shControl

6DT1sh62

6DT1sh64

Dis

tant

Met

asta

sis

Free

Sur

viva

l

GSE2034 GSE11121

MonthsMonths

p = 0.004 p = 0.004––– Low risk----- High risk

––– Low risk----- High risk0.0

0.2

0.4

0.6

0.8

1.0

0 50 100 150 0 50 100 150 200

Cnot2 module expression predicts distant metastasis free survival in lymph node negative non-adjuvant-treated breast cancer

In vivo metastasis assays validate Cnot2 as a metastasis suppressor

5 miR-3470b is a candidate post-transcriptional regulator of the Cnot2 network

Cont

rol

miR

-347

0am

iR-3

470b

6DT1Control

6DT1miR-3470a

6DT1miR-3470b

miR-3470a/b RNA

Primary Tumor Burden

Tum

or M

ass (

g)

0.0

0.5

1.0

1.5

2.0

2.5

6DT1Control

6DT1miR-3470a

6DT1miR-3470b

Primary Tumor Burden

*p=0.11

6DT1Control

6DT1miR-3470a

6DT1miR-3470b

Pulmonary Metastases

Surfa

ce M

etas

tasis

Cou

nt

0

20

40

60

80**

Pulmonary Metastases**

miR-3470b down-regulates Cnot2 network hubs

Underlined: Predicted miRNA Recognition Element

*

**

**

*

*

***p=0.05

*

**

**

**

**

****

**

miR-3470b modulates metastasis driver expression to drive a pro-metastatic transcriptional profile

miR-3470a and miR-3470b expression promote metastasis in vivo

Farhoud Faraji1, Ying Hu2, Gang Wu3, Jinghui Zhang3, Kent W. Hunter1

1Laboratory of Cancer Biology and Genetics and 2Laboratory of Population Genetics, National Cancer Institute. Bethesda, MD.3Department of Computational Biology, Saint Jude Children’s Research Hospital. Memphis, TN.

Systems Genetics Analysis Reveals Higher-Order Transcriptional Regulatory Elements of Breast Cancer Metastasis

Contact: farajif@mail.nih.gov

4 CNOT2 binds known metastasis modifier proteins

Cnot2-MYC

Negative

Cnot2-MYC

Rrp1b-H

ACnot2 + Rrp

1bNega

tive

Rrp1b-H

ACnot2 + Rrp

1b

1% Input IP: anti-MYC

anti-HA

WB

anti-MYC

Negative

Cnot2-MYC

Brd4-SF

-FLAG

Cnot2 + Brd4-SF

Negative

Cnot2-MYC

Brd4-SF

-FLAG

Cnot2 + Brd4-SF

1% Input IP: anti-MYC

anti-FLAG

WB

anti-MYC

Protein B = RRP1B

BiFC

MergeDAPI

Transfection Control

Protein B = BRD4-SF

BiFC

MergeDAPI

Transfection Control

Protein B = SIPA1

BiFC

MergeDAPI

Transfection Control

CNOT2 complements YFP fluorescence in conjunction with BRD4 short isoform (BRD4-SF), SIPA1, and RRP1B

CNOT2 Protein B

N-terminal YFPFragment

C-terminal YFPFragment

Absorption

Proteins not in close proximity

No Signal

ReconstitutedYFP

514nm

530nm

Absorption Signal

CNOT2 Protein B

Proteins in close proximity

514nm

CNOT2 co-precipitates BRD4-SF and RRP1B

Bi-molecular fluorescent complementation (BiFC)