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Breast Cancer Research
Published in: Breast Cancer Research 1/2017

Open Access 01-12-2017 | Research Article

Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells

Authors: Asfa Alli-Shaik, Sheena Wee, Lina H. K. Lim, Jayantha Gunaratne

Published in: Breast Cancer Research | Issue 1/2017

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Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.
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Literature
1.
Flower RJ, Rothwell NJ. Lipocortin-1: cellular mechanisms and clinical relevance. Trends Pharmacol Sci. 1994;15(3):71–6.CrossRefPubMed
2.
3.
Perretti M, D'Acquisto F. Annexin A1 and glucocorticoids as effectors of the resolution of inflammation. Nat Rev Immunol. 2009;9(1):62–70.CrossRefPubMed
4.
Scannell M, Flanagan MB, de Stefani A, Wynne KJ, Cagney G, Godson C, et al. Annexin-1 and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages. J Immunol. 2007;178(7):4595–605.CrossRefPubMed
5.
Sugimoto MA, Vago JP, Teixeira MM, Sousa LP. Annexin A1 and the resolution of inflammation: modulation of neutrophil recruitment, apoptosis, and clearance. J Immunol Res. 2016;2016:8239258.CrossRefPubMedPubMedCentral
6.
7.
D'Acquisto F, Paschalidis N, Sampaio AL, Merghani A, Flower RJ, Perretti M. Impaired T cell activation and increased Th2 lineage commitment in Annexin-1-deficient T cells. Eur J Immunol. 2007;37(11):3131–42.CrossRefPubMed
8.
Yang YH, Song W, Deane JA, Kao W, Ooi JD, Ngo D, et al. Deficiency of annexin A1 in CD4+ T cells exacerbates T cell-dependent inflammation. J Immunol. 2013;190(3):997–1007.CrossRefPubMed
9.
Grewal T, Enrich C. Annexins–modulators of EGF receptor signalling and trafficking. Cell Signal. 2009;21(6):847–58.CrossRefPubMed
10.
Alldridge LC, Harris HJ, Plevin R, Hannon R, Bryant CE. The annexin protein lipocortin 1 regulates the MAPK/ERK pathway. J Biol Chem. 1999;274(53):37620–8.CrossRefPubMed
11.
Liu YF, Zhang PF, Li MY, Li QQ, Chen ZC. Identification of annexin A1 as a proinvasive and prognostic factor for lung adenocarcinoma. Clin Exp Metastasis. 2011;28(5):413–25.CrossRefPubMed
12.
Bai XF, Ni XG, Zhao P, Liu SM, Wang HX, Guo B, et al. Overexpression of annexin 1 in pancreatic cancer and its clinical significance. World J Gastroenterol. 2004;10(10):1466–70.CrossRefPubMedPubMedCentral
13.
Lin Y, Lin G, Fang W, Zhu H, Chu K. Increased expression of annexin A1 predicts poor prognosis in human hepatocellular carcinoma and enhances cell malignant phenotype. Med Oncol. 2014;31(12):327.CrossRefPubMed
14.
Geary LA, Nash KA, Adisetiyo H, Liang M, Liao CP, Jeong JH, et al. CAF-secreted annexin A1 induces prostate cancer cells to gain stem cell-like features. Mol Cancer Res. 2014;12(4):607–21.CrossRefPubMedPubMedCentral
15.
Yang Y, Liu Y, Yao X, Ping Y, Jiang T, Liu Q, et al. Annexin 1 released by necrotic human glioblastoma cells stimulates tumor cell growth through the formyl peptide receptor 1. Am J Pathol. 2011;179(3):1504–12.CrossRefPubMedPubMedCentral
16.
Wang LP, Bi J, Yao C, Xu XD, Li XX, Wang SM, et al. Annexin A1 expression and its prognostic significance in human breast cancer. Neoplasma. 2010;57(3):253–9.CrossRefPubMed
17.
Yom CK, Han W, Kim SW, Kim HS, Shin HC, Chang JN, et al. Clinical significance of annexin A1 expression in breast cancer. J Breast Cancer. 2011;14(4):262–8.CrossRefPubMedPubMedCentral
18.
Sobral-Leite M, Wesseling J, Smit VT, Nevanlinna H, van Miltenburg MH, Sanders J, et al. Annexin A1 expression in a pooled breast cancer series: association with tumor subtypes and prognosis. BMC Med. 2015;13:156.CrossRefPubMedPubMedCentral
19.
de Graauw M, van Miltenburg MH, Schmidt MK, Pont C, Lalai R, Kartopawiro J, et al. Annexin A1 regulates TGF-beta signaling and promotes metastasis formation of basal-like breast cancer cells. Proc Natl Acad Sci U S A. 2010;107(14):6340–5.CrossRefPubMedPubMedCentral
20.
Parsons JT, Horwitz AR, Schwartz MA. Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat Rev Mol Cell Biol. 2010;11(9):633–43.CrossRefPubMedPubMedCentral
21.
Swa HL, Blackstock WP, Lim LH, Gunaratne J. Quantitative proteomics profiling of murine mammary gland cells unravels impact of annexin-1 on DNA damage response, cell adhesion, and migration. Mol Cell Proteomics. 2012;11(8):381–93.CrossRefPubMedPubMedCentral
22.
Swa HL, Shaik AA, Lim LH, Gunaratne J. Mass spectrometry based quantitative proteomics and integrative network analysis accentuates modulating roles of annexin-1 in mammary tumorigenesis. Proteomics. 2015;15(2-3):408–18.CrossRefPubMed
23.
Wiśniewski JR, Nagaraj N, Zougman A, Gnad F, Mann M. Brain phosphoproteome obtained by a FASP-based method reveals plasma membrane protein topology. J Proteome Res. 2010;9(6):3280–9.CrossRefPubMed
24.
Wiśniewski JR, Zougman A, Mann M. Combination of FASP and stagetip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J Proteome Res. 2009;8(12):5674–8.CrossRefPubMed
25.
Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008;26(12):1367–72.CrossRefPubMed
26.
Cox J, Matic I, Hilger M, Nagaraj N, Selbach M, Olsen JV, et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc. 2009;4(5):698–705.CrossRefPubMed
27.
Huang DW, Sherman BT, Tan Q, Kir J, Liu D, Bryant D, et al. DAVID Bioinformatics Resources: expanded annotation database and novel algorithms to better extract biology from large gene lists. Nucleic Acids Res. 2007;35(Web Server issue):W169–175.CrossRefPubMedPubMedCentral
28.
Domanova W, Krycer J, Chaudhuri R, Yang P, Vafaee F, Fazakerley D, et al. Unraveling kinase activation dynamics using kinase-substrate relationships from temporal large-scale phosphoproteomics studies. PLoS One. 2016;11(6):e0157763.CrossRefPubMedPubMedCentral
29.
Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015;43(Database issue):D447–452.CrossRefPubMed
30.
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–504.CrossRefPubMedPubMedCentral
31.
Horn H, Schoof EM, Kim J, Robin X, Miller ML, Diella F, et al. KinomeXplorer: an integrated platform for kinome biology studies. Nat Methods. 2014;11(6):603–4.CrossRefPubMed
32.
Casado P, Rodriguez-Prados JC, Cosulich SC, Guichard S, Vanhaesebroeck B, Joel S, et al. Kinase-substrate enrichment analysis provides insights into the heterogeneity of signaling pathway activation in leukemia cells. Sci Signal. 2013;6(268):rs6.CrossRefPubMed
33.
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–50.CrossRefPubMedPubMedCentral
34.
Isserlin R, Merico D, Voisin V, Bader GD. Enrichment map - a cytoscape app to visualize and explore OMICs pathway enrichment results. F1000Res. 2014;3:141.PubMedPubMedCentral
35.
Matys V, Fricke E, Geffers R, Gossling E, Haubrock M, Hehl R, et al. TRANSFAC: transcriptional regulation, from patterns to profiles. Nucleic Acids Res. 2003;31(1):374–8.CrossRefPubMedPubMedCentral
36.
Li T, Wernersson R, Hansen RB, Horn H, Mercer J, Slodkowicz G, et al. A scored human protein-protein interaction network to catalyze genomic interpretation. Nat Methods. 2017;14(1):61–4.CrossRefPubMed
37.
38.
Kain KH, Klemke RL. Inhibition of cell migration by Abl family tyrosine kinases through uncoupling of Crk-CAS complexes. J Biol Chem. 2001;276(19):16185–92.CrossRefPubMed
39.
Wang Y, Miller AL, Mooseker MS, Koleske AJ. The Abl-related gene (Arg) nonreceptor tyrosine kinase uses two F-actin-binding domains to bundle F-actin. Proc Natl Acad Sci U S A. 2001;98(26):14865–70.CrossRefPubMedPubMedCentral
40.
Miao H, Li DQ, Mukherjee A, Guo H, Petty A, Cutter J, et al. EphA2 mediates ligand-dependent inhibition and ligand-independent promotion of cell migration and invasion via a reciprocal regulatory loop with Akt. Cancer Cell. 2009;16(1):9–20.CrossRefPubMedPubMedCentral
41.
Zeng X, Tamai K, Doble B, Li S, Huang H, Habas R, et al. A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature. 2005;438(7069):873–7.CrossRefPubMedPubMedCentral
42.
Wittmann T, Bokoch GM, Waterman-Storer CM. Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1. J Biol Chem. 2004;279(7):6196–203.CrossRefPubMed
43.
44.
Yu CF, Liu ZX, Cantley LG. ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. J Biol Chem. 2002;277(22):19382–8.CrossRefPubMed
45.
Aksamitiene E, Kiyatkin A, Kholodenko BN. Cross-talk between mitogenic Ras/MAPK and survival PI3K/Akt pathways: a fine balance. Biochem Soc Trans. 2012;40(1):139–46.CrossRefPubMed
46.
Bist P, Leow SC, Phua QH, Shu S, Zhuang Q, Loh WT, et al. Annexin-1 interacts with NEMO and RIP1 to constitutively activate IKK complex and NF-kappaB: implication in breast cancer metastasis. Oncogene. 2011;30(28):3174–85.CrossRefPubMed
47.
48.
Zaidi SK, Sullivan AJ, Medina R, Ito Y, van Wijnen AJ, Stein JL, et al. Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription. Embo J. 2004;23(4):790–9.CrossRefPubMedPubMedCentral
49.
Miano JM, Long X, Fujiwara K. Serum response factor: master regulator of the actin cytoskeleton and contractile apparatus. Am J Physiol Cell Physiol. 2007;292(1):C70–81.CrossRefPubMed
50.
Wozniak MA, Modzelewska K, Kwong L, Keely PJ. Focal adhesion regulation of cell behavior. Biochim Biophys Acta. 2004;1692(2-3):103–19.CrossRefPubMed
51.
Miller AL, Wang Y, Mooseker MS, Koleske AJ. The Abl-related gene (Arg) requires its F-actin-microtubule cross-linking activity to regulate lamellipodial dynamics during fibroblast adhesion. J Cell Biol. 2004;165(3):407–19.CrossRefPubMedPubMedCentral
52.
53.
Ezratty EJ, Partridge MA, Gundersen GG. Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase. Nat Cell Biol. 2005;7(6):581–90.CrossRefPubMed
54.
Goncharova EA, James ML, Kudryashova TV, Goncharov DA, Krymskaya VP. Tumor suppressors TSC1 and TSC2 differentially modulate actin cytoskeleton and motility of mouse embryonic fibroblasts. PLoS One. 2014;9(10):e111476.CrossRefPubMedPubMedCentral
55.
56.
Robertson J, Jacquemet G, Byron A, Jones MC, Warwood S, Selley JN, et al. Defining the phospho-adhesome through the phosphoproteomic analysis of integrin signalling. Nat Commun. 2015;6:6265.CrossRefPubMedPubMedCentral
57.
Rusan NM, Fagerstrom CJ, Yvon AM, Wadsworth P. Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin. Mol Biol Cell. 2001;12(4):971–80.CrossRefPubMedPubMedCentral
58.
59.
Rubin CI, Atweh GF. The role of stathmin in the regulation of the cell cycle. J Cell Biochem. 2004;93(2):242–50.CrossRefPubMed
60.
Di Paolo G, Antonsson B, Kassel D, Riederer BM, Grenningloh G. Phosphorylation regulates the microtubule-destabilizing activity of stathmin and its interaction with tubulin. FEBS Lett. 1997;416(2):149–52.CrossRefPubMed
61.
Li N, Jiang P, Du W, Wu Z, Li C, Qiao M, et al. Siva1 suppresses epithelial-mesenchymal transition and metastasis of tumor cells by inhibiting stathmin and stabilizing microtubules. Proc Natl Acad Sci U S A. 2011;108(31):12851–6.CrossRefPubMedPubMedCentral
62.
Liu F, Sun YL, Xu Y, Wang LS, Zhao XH. Expression and phosphorylation of stathmin correlate with cell migration in esophageal squamous cell carcinoma. Oncol Rep. 2013;29(2):419–24.CrossRefPubMed
63.
Bhardwaj A, Ganesan N, Tachibana K, Rajapakshe K, Albarracin CT, Gunaratne PH, et al. Annexin A1 preferentially predicts poor prognosis of basal-like breast cancer patients by activating mTOR-S6 signaling. PLoS One. 2015;10(5):e0127678.CrossRefPubMedPubMedCentral
64.
Yang CC, Graves HK, Moya IM, Tao C, Hamaratoglu F, Gladden AB, et al. Differential regulation of the Hippo pathway by adherens junctions and apical-basal cell polarity modules. Proc Natl Acad Sci U S A. 2015;112(6):1785–90.CrossRefPubMedPubMedCentral
Metadata
Title
Phosphoproteomics reveals network rewiring to a pro-adhesion state in annexin-1-deficient mammary epithelial cells
Authors
Asfa Alli-Shaik
Sheena Wee
Lina H. K. Lim
Jayantha Gunaratne
Publication date
01-12-2017
Publisher
BioMed Central
Published in
Breast Cancer Research / Issue 1/2017
Electronic ISSN: 1465-542X
DOI
https://doi.org/10.1186/s13058-017-0924-4

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