Translational Biomedical Proteomics, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 61 Biopolis Drive, Singapore, 138673, Singapore.
Department of Physiology, Immunology Programme, Centre for Life Sciences, Yong Loo Lin School of Medicine, National University of Singapore, 28 Medical Drive, Singapore, 117456, Singapore.
Breast Cancer Res. 2017 Dec 12;19(1):132. doi: 10.1186/s13058-017-0924-4.
Annexin-1 (ANXA1) plays pivotal roles in regulating various physiological processes including inflammation, proliferation and apoptosis, and deregulation of ANXA1 functions has been associated with tumorigenesis and metastasis events in several types of cancer. Though ANXA1 levels correlate with breast cancer disease status and outcome, its distinct functional involvement in breast cancer initiation and progression remains unclear. We hypothesized that ANXA1-responsive kinase signaling alteration and associated phosphorylation signaling underlie early events in breast cancer initiation events and hence profiled ANXA1-dependent phosphorylation changes in mammary gland epithelial cells.
Quantitative phosphoproteomics analysis of mammary gland epithelial cells derived from ANXA1-heterozygous and ANXA1-deficient mice was carried out using stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry. Kinase and signaling changes underlying ANXA1 perturbations were derived by upstream kinase prediction and integrated network analysis of altered proteins and phosphoproteins.
We identified a total of 8110 unique phosphorylation sites, of which 582 phosphorylation sites on 372 proteins had ANXA1-responsive changes. A majority of these phosphorylation changes occurred on proteins associated with cytoskeletal reorganization spanning the focal adhesion, stress fibers, and also the microtubule network proposing new roles for ANXA1 in regulating microtubule dynamics. Comparative analysis of regulated global proteome and phosphoproteome highlighted key differences in translational and post-translational effects of ANXA1, and suggested closely coordinated rewiring of the cell adhesion network. Kinase prediction analysis suggested activity modulation of calmodulin-dependent protein kinase II (CAMK2), P21-activated kinase (PAK), extracellular signal-regulated kinase (ERK), and IκB kinase (IKK) upon loss of ANXA1. Integrative analysis revealed regulation of the WNT and Hippo signaling pathways in ANXA1-deficient mammary epithelial cells, wherein there is downregulation of transcriptional effects of TEA domain family (TEAD) suggestive of ANXA1-responsive transcriptional rewiring.
The phosphoproteome landscape uncovered several novel perspectives for ANXA1 in mammary gland biology and highlighted its involvement in key signaling pathways modulating cell adhesion and migration that could contribute to breast cancer initiation.
膜联蛋白-1（ANXA1）在调节多种生理过程中发挥着关键作用，包括炎症、增殖和凋亡，而 ANXA1 功能的失调与多种类型癌症的肿瘤发生和转移事件有关。尽管 ANXA1 水平与乳腺癌疾病状况和结局相关，但它在乳腺癌发生和进展中的具体功能作用尚不清楚。我们假设 ANXA1 反应性激酶信号改变及其相关磷酸化信号是乳腺癌起始事件中的早期事件的基础，并因此对乳腺上皮细胞中依赖于 ANXA1 的磷酸化变化进行了分析。
使用基于稳定同位素标记的氨基酸细胞培养（SILAC）的质谱法对来自 ANXA1 杂合和 ANXA1 缺失小鼠的乳腺上皮细胞进行定量磷酸蛋白质组学分析。通过上游激酶预测和改变的蛋白质和磷酸蛋白质的综合网络分析，得出 ANXA1 扰动下的激酶和信号变化。
我们总共鉴定了 8110 个独特的磷酸化位点，其中 372 个蛋白质上的 582 个磷酸化位点有 ANXA1 反应性变化。这些磷酸化变化大多数发生在与细胞骨架重排相关的蛋白质上，包括焦点黏附、应激纤维，以及微管网络，这为 ANXA1 在调节微管动力学方面提供了新的作用。调节的全局蛋白质组和磷酸蛋白质组的比较分析突出了 ANXA1 的翻译和翻译后效应的关键差异，并表明细胞黏附网络的紧密协调重布线。激酶预测分析表明，钙调蛋白依赖性蛋白激酶 II（CAMK2）、P21 激活激酶（PAK）、细胞外信号调节激酶（ERK）和 IκB 激酶（IKK）的活性在 ANXA1 缺失时被调节。综合分析显示，WNT 和 Hippo 信号通路在 ANXA1 缺失的乳腺上皮细胞中受到调节，其中 TEA 结构域家族（TEAD）的转录效应下调，提示 ANXA1 反应性转录重布线。
磷酸蛋白质组学图谱为 ANXA1 在乳腺生物学中的作用提供了一些新的视角，并强调了其在调节细胞黏附和迁移的关键信号通路中的作用，这可能有助于乳腺癌的发生。
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