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Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NF-κB: implication in breast cancer metastasis

Oncogene volume 30, pages 3174–3185 (2011)Cite this article

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Abstract

The molecular mechanisms underlying constitutive nuclear factor-κB (NF-κB) activation in solid tumors has not been elucidated. We show that Annexin-1 (ANXA1) is involved in this process, and suppression of ANXA1 in highly metastatic breast cancer cells impedes migration and metastasis capabilities in vitro and in vivo. ANXA1 expression correlates with NF-κB activity, suggesting that ANXA1 may be required for the constitutive activity of IκB kinase (IKK) and NF-κB in highly metatstatic breast cancer. Gel-filtration analysis demonstrated that ANXA1 co-elutes with the members of the IKK complex and NF-κB signaling pathway, and immunoprecipitation confirmed that ANXA1 can bind to and interact with IKKγ or NEMO, but not IKKα or IKKβ. Importantly, silencing of ANXA1 prevents the interaction of NEMO and RIP1, which indicates that ANXA1 is required for the recruitment of RIP1 to the IKK complex, which may be important for the activation of NF-κB. Downstream targets of NF-κB include uPA and CXCR4, which can be modulated by ANXA1 silencing. CXCR4-mediated migration of breast cancer cell lines in response to CXCL12 was significantly modulated by ANXA1, indicating its importance in the tissue-specific migration of breast cancer cells. Chromatin immunoprecipitation experiments confirmed that in ANXA1 overexpressed cells, NF-κB was recruited to CXCR4 promoter without external stimulation, indicating that ANXA1 is critical for the constitutive activation of NF-κB in breast cancer to promote metastasis. Finally, we show that ANXA1 overexpression enhances metastasis and reduces survival in an intracardiac metastasis model, while ANXA1-deficient mice crossed with MMTV-PyMT mice display significantly less metastasis than their heterozygous littermates, indicating that ANXA1 is an important gene in breast cancer metastasis. Our data reveal that ANXA1 can constitutively activate NF-κB in breast cancer cells through the interaction with the IKK complex, and suggests that modulating ANXA1 levels has therapeutic potential to suppress breast cancer metastasis.

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Acknowledgements

This work was supported by grants from the Biomedical Research Council of Singapore (07/1/21/19/507) and A*Star (SIgN 07-011) to LL and Biomedical Research Council of Singapore (04/1/21/19/319) to SCH. We thank Professor Rod Flower (William Harvey Research Institute, Queen Mary's School of Medicine and Dentistry, London) for generously providing us with the pCDNA3.1-V5 ANXA1 construct and Professor Michael Karin (University of California San Diego, CA, USA) for the HA-IKKα, HA-IKKβ and HA-IKKγ plasmid constructs.

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Authors and Affiliations

  1. Department of Physiology and NUS Immunology Program, Inflammation and Cancer Lab, National University of Singapore, Singapore

    P Bist, S C Leow, Q H Phua, S Shu, Q Zhuang, W T Loh, T H Nguyen & L H K Lim

  2. Oncology Research Institute, National University of Singapore, Singapore

    J B Zhou

  3. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    S C Hooi

  4. NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore

    L H K Lim

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  1. P Bist
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Bist, P., Leow, S., Phua, Q. et al. Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NF-κB: implication in breast cancer metastasis. Oncogene 30, 3174–3185 (2011). https://doi.org/10.1038/onc.2011.28

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