Quantitative Phosphoproteomic Analysis Reveals System-wide Signaling Pathways Downstream of SDF-1/CXCR4 in Breast Cancer Stem Cells

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2014 May 1

PMID 24782546

Citations 61

Authors

Tingfang Yi

Bo Zhai

Yonghao Yu

Yoshikawa Kiyotsugu

Thomas Raschle

Manuel Etzkorn

Hee-Chan Seo

Michal Nagiec

Rafael E Luna

Ellis L Reinherz

John Blenis

Steven P Gygi

Gerhard Wagner

Affiliations

Soon will be listed here.

Abstract

Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4-mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4-mediated phosphoproteome, including construction of kinase-substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.

Citing Articles

Differential substrate specificity of ERK, JNK, and p38 MAP kinases toward connexin 43.

Latchford L, Perez L, Conage-Pough J, Turk R, Cusimano M, Vargas V J Biol Chem. 2025; 301(3):108178.

PMID: 39798878 PMC: 11870265. DOI: 10.1016/j.jbc.2025.108178.

A1, an innovative fluorinated CXCR4 inhibitor, redefines the therapeutic landscape in colorectal cancer.

Khorramdelazad H, Bagherzadeh K, Rahimi A, Darehkordi A, Najafi A, Karimi M Cancer Cell Int. 2025; 25(1):5.

PMID: 39757159 PMC: 11702187. DOI: 10.1186/s12935-024-03584-y.

Phosphorylation regulates the chromatin remodeler SMARCAD1 in nucleosome binding, ATP hydrolysis, and histone exchange.

Aboulache B, Hoitsma N, Luger K J Biol Chem. 2024; 300(12):107893.

PMID: 39424143 PMC: 11742319. DOI: 10.1016/j.jbc.2024.107893.

SAP deletion promotes malignant insulinoma progression by inducing CXCL12 secretion from CAFs via the CXCR4/p38/ERK signalling pathway.

Jiang G, Xu S, Mai X, Tu J, Wang L, Wang L J Cell Mol Med. 2024; 28(10):e18397.

PMID: 38766687 PMC: 11103456. DOI: 10.1111/jcmm.18397.

CDKN1A/p21 in Breast Cancer: Part of the Problem, or Part of the Solution?.

Manousakis E, Miralles C, Esquerda M, Wright R Int J Mol Sci. 2023; 24(24).

PMID: 38139316 PMC: 10743848. DOI: 10.3390/ijms242417488.

References

Wildenberg G, Dohn M, Carnahan R, Davis M, Lobdell N, Settleman J . p120-catenin and p190RhoGAP regulate cell-cell adhesion by coordinating antagonism between Rac and Rho. Cell. 2006; 127(5):1027-39. DOI: 10.1016/j.cell.2006.09.046. View

Alema S, Salvatore A . p120 catenin and phosphorylation: Mechanisms and traits of an unresolved issue. Biochim Biophys Acta. 2006; 1773(1):47-58. DOI: 10.1016/j.bbamcr.2006.06.001. View

Plotnikov A, Zehorai E, Procaccia S, Seger R . The MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation. Biochim Biophys Acta. 2010; 1813(9):1619-33. DOI: 10.1016/j.bbamcr.2010.12.012. View

Alessi D, Saito Y, Campbell D, Cohen P, Sithanandam G, Rapp U . Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J. 1994; 13(7):1610-9. PMC: 394991. DOI: 10.1002/j.1460-2075.1994.tb06424.x. View

Aoki K, Yamada M, Kunida K, Yasuda S, Matsuda M . Processive phosphorylation of ERK MAP kinase in mammalian cells. Proc Natl Acad Sci U S A. 2011; 108(31):12675-80. PMC: 3150946. DOI: 10.1073/pnas.1104030108. View

Demarchi F, Bertoli C, Sandy P, Schneider C . Glycogen synthase kinase-3 beta regulates NF-kappa B1/p105 stability. J Biol Chem. 2003; 278(41):39583-90. DOI: 10.1074/jbc.M305676200. View

OHayre M, Salanga C, Kipps T, Messmer D, Dorrestein P, Handel T . Elucidating the CXCL12/CXCR4 signaling network in chronic lymphocytic leukemia through phosphoproteomics analysis. PLoS One. 2010; 5(7):e11716. PMC: 2908618. DOI: 10.1371/journal.pone.0011716. View

Seternes O, Mikalsen T, Johansen B, Michaelsen E, Armstrong C, Morrice N . Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway. EMBO J. 2004; 23(24):4780-91. PMC: 535098. DOI: 10.1038/sj.emboj.7600489. View

Grassie M, Moffat L, Walsh M, MacDonald J . The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ. Arch Biochem Biophys. 2011; 510(2):147-59. DOI: 10.1016/j.abb.2011.01.018. View

10.

Wong W, Scott J . AKAP signalling complexes: focal points in space and time. Nat Rev Mol Cell Biol. 2004; 5(12):959-70. DOI: 10.1038/nrm1527. View

11.

She Y, Rosu-Myles M, Walrond L, Cyr T . Quantification of protein isoforms in mesenchymal stem cells by reductive dimethylation of lysines in intact proteins. Proteomics. 2011; 12(3):369-79. PMC: 3440571. DOI: 10.1002/pmic.201100308. View

12.

Tsujimura K, Ogawara M, Takeuchi Y, Imajoh-Ohmi S, Ha M, Inagaki M . Visualization and function of vimentin phosphorylation by cdc2 kinase during mitosis. J Biol Chem. 1994; 269(49):31097-106. View

13.

Li M, Satinover D, Brautigan D . Phosphorylation and functions of inhibitor-2 family of proteins. Biochemistry. 2007; 46(9):2380-9. DOI: 10.1021/bi602369m. View

14.

Zheng Y, Xia Y, Hawke D, Halle M, Tremblay M, Gao X . FAK phosphorylation by ERK primes ras-induced tyrosine dephosphorylation of FAK mediated by PIN1 and PTP-PEST. Mol Cell. 2009; 35(1):11-25. PMC: 2715139. DOI: 10.1016/j.molcel.2009.06.013. View

15.

Hoover A, Strand G, Nowicki P, Anderson M, Vermeer P, Klingelhutz A . Impaired PTPN13 phosphatase activity in spontaneous or HPV-induced squamous cell carcinomas potentiates oncogene signaling through the MAP kinase pathway. Oncogene. 2009; 28(45):3960-70. PMC: 2785129. DOI: 10.1038/onc.2009.251. View

16.

Gupta P, Onder T, Jiang G, Tao K, Kuperwasser C, Weinberg R . Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell. 2009; 138(4):645-659. PMC: 4892125. DOI: 10.1016/j.cell.2009.06.034. View

17.

Tan C, Bodenmiller B, Pasculescu A, Jovanovic M, Hengartner M, Jorgensen C . Comparative analysis reveals conserved protein phosphorylation networks implicated in multiple diseases. Sci Signal. 2009; 2(81):ra39. DOI: 10.1126/scisignal.2000316. View

18.

Al-Hajj M, Wicha M, Benito-Hernandez A, Morrison S, Clarke M . Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003; 100(7):3983-8. PMC: 153034. DOI: 10.1073/pnas.0530291100. View

19.

Liu H, Grundstrom T . Calcium regulation of GM-CSF by calmodulin-dependent kinase II phosphorylation of Ets1. Mol Biol Cell. 2002; 13(12):4497-507. PMC: 138649. DOI: 10.1091/mbc.e02-03-0149. View

20.

Zhao Y, Bjorbaek C, Moller D . Regulation and interaction of pp90(rsk) isoforms with mitogen-activated protein kinases. J Biol Chem. 1996; 271(47):29773-9. DOI: 10.1074/jbc.271.47.29773. View