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: [email protected]
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)
Recommended
