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11.06.13 - 2013 NIH Research Festival Poster
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11.06.13 - 2013 NIH Research Festival Poster

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  • 1. Systems Genetics Analysis Reveals Higher-Order Transcriptional Regulatory Elements of Breast Cancer Metastasis Farhoud Faraji , Ying Hu , Gang Wu , Jinghui Zhang , Kent W. Hunter 1 2 3 3 1 Laboratory of Cancer Biology and Genetics and 2Laboratory of Population Genetics, National Cancer Institute. Bethesda, MD. 3 Department of Computational Biology, Saint Jude Children’s Research Hospital. Memphis, TN. 1 Introduction Prognostic 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 Points A.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 A.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. Conclusion Our 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. The Cnot2 3 A module of co-expressed Network: by Cnot2 genes regulated 1 2 Employing meiotic genetics to understand heritable predisposition to metastasis AKR/J Systems genetics strategy to unveil the higher-order transcriptional structure of metastasis AKXD x PyMT F1 Tumors DBA/2J Microarray based mRNA profiling x P Metastasis-prone F1 x F2 x x F3 x + FVB PyMT ] + 4 Prone x Network Hub Kaplan-Meier Analysis Intermediate Resistant 5 51 4 Signal Absorption 51 4 nm * p=0.05 nm C-terminal YFP Fragment CNOT2 miR-3470b down-regulates Cnot2 network hubs Proteins in close proximity No Signal N-terminal YFP Fragment Biological Validation miR-3470b is a candidate post-transcriptional regulator of the Cnot2 network Bi-molecular fluorescent complementation (BiFC) Proteins not in close proximity Identify miRNAs Highly Represented In Networks Biological Validation CNOT2 binds known metastasis modifier proteins Absorption Negatively Correlated miRNA-mRNA Filter for miR-mRNA Target Pairs AKXDnPyMT+ F1 Metastasis Susceptibility miRNA Sequencing Network Generation x x [ F>20 Metastasis-resistant x Contact: farajif@mail.nih.gov *** * ** m 0n 53 Reconstituted YFP * * ** Protein B CNOT2 Protein B ** ** ** Cnot2 module expression predicts distant metastasis free survival in lymph node negative non-adjuvant-treated breast cancer CNOT2 complements YFP fluorescence in conjunction with BRD4 short isoform (BRD4-SF), SIPA1, and RRP1B GSE11121 1.0 Protein B = BRD4-SF 0.8 Protein B = SIPA1 Protein B = RRP1B miR-3470b modulates metastasis driver expression to drive a pro-metastatic transcriptional profile 0.6 ** ** 0.2 ** ––– Low risk ----- High risk ––– Low risk ----- High risk p = 0.004 50 100 150 0 50 Months BiFC p = 0.004 100 150 Transfection Control BiFC Transfection Control BiFC Transfection Control 200 Months In vivo metastasis assays validate Cnot2 as a metastasis suppressor Primary Tumor Burden Primary Tumor Burden ** 0.5 DAPI Merge 6DT1 Control 6DT1 Control 80 Surface Metastasis Count 0.5 6DT1 Cnot2 Pulmonary Metastases Pulmonary Metastases * ** WB 6DT1 shControl 6DT1 sh62 6DT1 sh64 Control WB 40 anti-MYC anti-MYC 6DT1 shControl 6DT1 sh62 6DT1 sh64 1% Input IP: anti-MYC 1.5 1.0 0.5 0.0 1% Input IP: anti-MYC 6DT1 miR-3470a Primary Tumor Burden Primary Tumor Burden p=0.11 * 2.0 anti-HA anti-FLAG 60 20 6DT1 Control 2.5 0 0.0 miR-3470a/b RNA Merge 20 PrimaryTumor Burden Primary Tumor Burden 1.0 DAPI CNOT2 co-precipitates BRD4-SF and RRP1B 40 6DT1 Cnot2 1.5 Merge 60 0 0.0 DAPI 80 Surface Metastasis Count 1.0 6DT1 sh64 Tumor Mass (g) 1.5 6DT1 sh62 Ne ga ti v e Cn ot 2M YC Rr p1 bHA Cn ot 2 + Rr Ne p1 ga b ti v e Cn ot 2M YC Rr p1 bHA Cn ot 2 + Rr p1 b Surface Metastasis Count 80 6DT1 shControl Ne ga tiv e Cn ot 2M YC Br d4 -S FFL Cn AG ot 2 + Br Cn d4 ot -S 2F M YC Ne ga tiv e Br d4 -S FFL Cn AG ot 2 + Br d4 -S F * 2.0 Tumor Mass (g) Pulmonary Metastases Pulmonary Metastases miR-3470a and miR-3470b expression promote metastasis in vivo 6DT1 miR-3470b Pulmonary Metastases Pulmonary Metastases ** ** 60 40 20 0 6DT1 Control 6DT1 miR-3470a 6DT1 miR-3470b miR-3470a 0.0 0 Tumor Mass (g) ** 0.4 miR-3470b Distant Metastasis Free Survival GSE2034 Underlined: Predicted miRNA Recognition Element 6DT1 Control 6DT1 miR-3470a 6DT1 miR-3470b

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