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Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

ABSTRACT: Background:Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells. Methods:In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF this has already been defined above cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC-MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells. Results:We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication. Conclusions:Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

SUBMITTER: Wu X

PROVIDER: S-EPMC6003199 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

Wu Xinyan X Zahari Muhammad Saddiq MS Renuse Santosh S Sahasrabuddhe Nandini A NA Chaerkady Raghothama R Kim Min-Sik MS Fackler Mary Jo MJ Stampfer Martha M Gabrielson Edward E Sukumar Saraswati S Pandey Akhilesh A

Clinical proteomics 20180615

<h4>Background</h4>Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells.<h4>Methods</h4>In this stu ...[more]

PMID: 29946230

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Project description:Previously, we showed that Abl kinases (c-Abl, Arg) are activated downstream of PDGF in a manner dependent on Src kinases and PLC-gamma1, and promote PDGF-mediated proliferation and migration of fibroblasts. We additionally demonstrated that Abl kinases bind directly to PDGFR-beta via their SH2 domains.In this study, we extend these findings by demonstrating that Abl kinases also are activated downstream of aPDGF autocrine growth loop in glioblastoma cells, indicating that the PDGFR-Abl signaling pathway also is likely to be important in glioblastoma development and/or progression.We recently showed that Abl kinases are highly active in many breast cancer cell lines, and the Her-2 receptor tyrosine kinase contributes to c-Abl and Arg kinase activation. In this study, we show that Abl kinase SH2 domains bind directly to Her-2, and like PDGFR-beta , Her-2 directly phosphorylates c-Abl. Previously, we demonstrated that PDGFR-beta directly phosphorylates Abl kinases in vitro, and Abl kinases reciprocally phosphorylate PDGFR-beta . Here, we show that PDGFR-beta-phosphorylation of Abl kinases has functional consequences as PDGFR-beta phosphorylates Abl kinases on Y245 and Y412, sites known to be required for activation of Abl kinases. Moreover, PDGFR-beta phosphorylates Arg on two additional unique sites whose function is unknown. Importantly, we also show that Abl-dependent phosphorylation of PDGFR-beta has functional and biological significances. c-Abl phosphorylates three tyrosine residues on PDGFR-beta (Y686, Y934, Y970), while Arg only phosphorylatesY686. Y686 and Y934 reside in PDGFR-beta catalytic domains, while Y970 is in the C-terminal tail. Using site-directed mutagenesis, we show that Abl-dependent phosphorylation of PDGFR-beta activates PDGFR-beta activity, in vitro, but serves to downregulate PDGFR-mediated chemotaxis. These data are exciting as they indicate that Abl kinases not only are activated by PDGFR and promote PDGFR-mediated proliferation and migration,but also act in an intricate negative feedback loop to turn-off PDGFR-mediated chemotaxis.

Dataset Information

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

Publications

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

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