Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells.
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.
Department of Biological Chemistry and Molecular Pharmacology and.
Tingfang
T
Yi
Department of Cell Biology, Harvard Medical School, Boston, MA 02115; and.
Bo
B
Zhai
Department of Cell Biology, Harvard Medical School, Boston, MA 02115; and.
Yonghao
Y
Yu
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
Yoshikawa
Y
Kiyotsugu
Department of Biological Chemistry and Molecular Pharmacology and.
Thomas
T
Raschle
Department of Biological Chemistry and Molecular Pharmacology and.
Manuel
M
Etzkorn
Department of Biological Chemistry and Molecular Pharmacology and.
Hee-Chan
HC
Seo
Department of Cell Biology, Harvard Medical School, Boston, MA 02115; and.
Michal
M
Nagiec
Department of Biological Chemistry and Molecular Pharmacology and.
Rafael E
RE
Luna
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
Ellis L
EL
Reinherz
Department of Cell Biology, Harvard Medical School, Boston, MA 02115; and.
John
J
Blenis
Department of Cell Biology, Harvard Medical School, Boston, MA 02115; and steven_gygi@hms.harvard.edu gerhard_wagner@hms.harvard.edu.
Steven P
SP
Gygi
Department of Biological Chemistry and Molecular Pharmacology and steven_gygi@hms.harvard.edu gerhard_wagner@hms.harvard.edu.
Gerhard
G
Wagner
D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D054377:Chemokine CXCL12 / Q000378:metabolism*; D025461:Feedback, Physiological / Q000502:physiology*; D005260:Female; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009362:Neoplasm Metastasis / Q000503:physiopathology*; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D019718:Receptors, CXCR4 / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*
C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D019718:Receptors, CXCR4
GPCR; MAPK; chemokine receptor