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Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors
Published on: July 17, 2020
SHP2 negatively regulates TGF-β signaling by destabilizing the TGF-β type I receptor
Cen Zhao1, Ihor Yakymovych2, Mariya Yakymovych2
1Department of Medical Biochemistry and Microbiology, SciLifeLab, Uppsala University, Box 582, Uppsala, 751 23, Sweden. cen.zhao@imbim.uu.se.
Background:
SRC homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), acting as a central node of many signaling pathways, has an emerging role in many diseases, including cancer. Transforming growth factor β (TGF-β) affects a wide spectrum of biological processes, including cell proliferation, apoptosis, differentiation and migration, during embryonic development and oncogenesis. TGF-β regulates cell proliferation in a cell-context-dependent manner; loss of TGF-β-mediated growth inhibition is a major characteristic of cancer cells. SHP2 regulates canonical and non-canonical TGF-β signaling in cancer cells and fibroblasts, however, the mechanism by which TGF-β activates SHP2 and the role of SHP2 in TGF-β-mediated growth inhibition has remained unclear.
Methods:
The phosphorylation and activation of SHP2 was assessed after TGF-β stimulation of normal and breast cancer cells. The interaction between SHP2 and SRC was determined by co-immunoprecipitation (co-IP) assay. Pharmacological inhibition and gRNA-mediated knockout were applied to assess the role of SHP2 in TGF-β signaling, and RNA-sequencing to identify gene expression patterns in SHP2 knockout cells treated with TGF-β. Functional studies were performed using breast cancer cells to validate the role of SHP2 in TGF-β -mediated growth inhibition.
Results:
We report that TGF-β activated SHP2 by promoting its tyrosine phosphorylation by SRC. SHP2 depletion in breast cancer cells reduced ubiquitination and degradation of TβRI by impeding the interaction between TβRI and SMAD7, which is a negative regulator of TβRI. Pharmacological or genetic inhibition of SHP2 facilitated TGF-β-induced SMAD2 phosphorylation and transcriptional responses. Consequently, inhibition of SHP2 profoundly enhanced TGF-β-induced cell growth arrest and senescence, including promotion of TGF-β-induced expression of the cell cycle inhibitor p15 at both gene and protein level.
Conclusions:
Our findings uncover a mechanism by which SHP2 is activated by TGF-β in a SRC-dependent manner, and functions in a negative feedback mechanism to regulate TGF-β signaling.
Insights
Transforming growth factor β (TGF-β) activates SRC homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) via SRC phosphorylation. Inhibiting SHP2 enhances TGF-β-induced growth arrest, revealing a negative feedback loop in cancer cells.
Area of Science:
- Cellular Biology
- Molecular Oncology
- Signal Transduction
Background:
- SRC homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) is a key regulator in various signaling pathways and diseases, including cancer.
- Transforming growth factor β (TGF-β) influences critical cellular processes like proliferation and apoptosis, with its growth-inhibitory function often lost in cancer cells.
- The precise mechanism of TGF-β-induced SHP2 activation and SHP2's role in TGF-β-mediated growth inhibition remain incompletely understood.
Purpose of the Study:
- To elucidate the mechanism by which TGF-β activates SHP2.
- To investigate the role of SHP2 in TGF-β-mediated growth inhibition in cancer cells.
- To identify potential therapeutic targets by understanding the SHP2-TGF-β signaling axis.
Main Methods:
- Assessed SHP2 phosphorylation and activation upon TGF-β stimulation in normal and breast cancer cells.
- Utilized co-immunoprecipitation (co-IP) to determine the interaction between SHP2 and SRC.
- Employed pharmacological inhibition and gRNA-mediated knockout of SHP2, alongside RNA-sequencing, to analyze TGF-β signaling pathways and gene expression.
- Conducted functional studies in breast cancer cells to validate SHP2's role in TGF-β-mediated growth inhibition.
Main Results:
- TGF-β activates SHP2 through SRC-mediated tyrosine phosphorylation.
- SHP2 depletion hinders TβRI ubiquitination and degradation by disrupting the TβRI/SMAD7 interaction.
- SHP2 inhibition potentiates TGF-β-induced SMAD2 phosphorylation and transcriptional activity.
- Inhibition of SHP2 significantly enhances TGF-β-induced cell growth arrest, senescence, and p15 expression.
Conclusions:
- Uncovered a novel mechanism of SHP2 activation by TGF-β dependent on SRC.
- Demonstrated that SHP2 acts as a negative regulator in the TGF-β signaling pathway.
- Highlighted the potential of targeting the SHP2-TGF-β axis for cancer therapy by enhancing growth inhibition.
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