Department of Physiology and NUS Immunology Program, Inflammation and Cancer Lab, National University of Singapore.
Oncogene. 2011 Jul 14;30(28):3174-85. doi: 10.1038/onc.2011.28. Epub 2011 Mar 7.
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.
在实体瘤中，组成性核因子-κB（NF-κB）激活的分子机制尚未阐明。我们发现膜联蛋白-1（ANXA1）参与了这一过程，并且在高转移性乳腺癌细胞中抑制 ANXA1 的表达会阻碍体外和体内的迁移和转移能力。ANXA1 的表达与 NF-κB 活性相关，表明 ANXA1 可能是 IκB 激酶（IKK）和 NF-κB 在高转移性乳腺癌中组成性活性所必需的。凝胶过滤分析表明，ANXA1 与 IKK 复合物和 NF-κB 信号通路的成员共洗脱，免疫沉淀证实 ANXA1 可以与 IKKγ或 NEMO 结合并相互作用，但不能与 IKKα或 IKKβ结合。重要的是，沉默 ANXA1 可阻止 NEMO 和 RIP1 的相互作用，这表明 ANXA1 对于 RIP1 招募到 IKK 复合物是必需的，这对于 NF-κB 的激活可能很重要。NF-κB 的下游靶标包括 uPA 和 CXCR4，它们可以被 ANXA1 沉默所调节。ANXA1 沉默可显著调节乳腺癌细胞系对 CXCL12 的 CXCR4 介导的迁移，表明其在乳腺癌细胞的组织特异性迁移中的重要性。染色质免疫沉淀实验证实，在 ANXA1 过表达的细胞中，NF-κB 在没有外部刺激的情况下被募集到 CXCR4 启动子上，表明 ANXA1 对于 NF-κB 在乳腺癌中的组成性激活以促进转移是至关重要的。最后，我们发现 ANXA1 过表达可增强心内转移模型中的转移并降低存活率，而 ANXA1 缺陷型小鼠与 MMTV-PyMT 小鼠杂交后，其转移率明显低于杂合子同窝仔鼠，表明 ANXA1 是乳腺癌转移的一个重要基因。我们的数据揭示了 ANXA1 可以通过与 IKK 复合物的相互作用，在乳腺癌细胞中组成性地激活 NF-κB，并表明调节 ANXA1 水平具有抑制乳腺癌转移的治疗潜力。
