Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells

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

Research output: Contribution to journalArticle

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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 phospho-proteins 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-cat-enin, 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.

Original languageEnglish (US)
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number21
DOIs
StatePublished - May 27 2014

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Chemokine CXCL12
Neoplastic Stem Cells
Breast Neoplasms
Phosphorylation
Phosphoproteins
Neoplasms
CCR Receptors
Neoplasm Metastasis
Proteins
MAP Kinase Signaling System
Mitogen-Activated Protein Kinase Kinases
G-Protein-Coupled Receptors
Isotopes
Cell Movement
Cats
Phosphotransferases
Up-Regulation

Keywords

  • Chemokine receptor
  • GPCR
  • MAPK

ASJC Scopus subject areas

  • General

Cite this

Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells. / Yi, Tingfang; Zhai, Bo; Yu, Yonghao; Kiyotsugu, Yoshikawa; Raschle, Thomas; Etzkorn, Manuel; Seo, Hee Chan; Nagiec, Michal; Luna, Rafael E.; Reinherz, Ellis L.; Blenis, John; Gygi, Steven P.; Wagner, Gerhard.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 111, No. 21, 27.05.2014.

Research output: Contribution to journalArticle

Yi, Tingfang ; Zhai, Bo ; Yu, Yonghao ; Kiyotsugu, Yoshikawa ; Raschle, Thomas ; Etzkorn, Manuel ; Seo, Hee Chan ; Nagiec, Michal ; Luna, Rafael E. ; Reinherz, Ellis L. ; Blenis, John ; Gygi, Steven P. ; Wagner, Gerhard. / Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells. In: Proceedings of the National Academy of Sciences of the United States of America. 2014 ; Vol. 111, No. 21.
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AU - Raschle, Thomas

AU - Etzkorn, Manuel

AU - Seo, Hee Chan

AU - Nagiec, Michal

AU - Luna, Rafael E.

AU - Reinherz, Ellis L.

AU - Blenis, John

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AU - Wagner, Gerhard

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AB - 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 phospho-proteins 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-cat-enin, 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.

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