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Quantitative phospho-proteomic profiling of hepatocyte growth factor (HGF)-MET signaling in colorectal cancer
Shawna L Organ1, Jiefei Tong, Paul Taylor
1Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Canada.
Abstract:
Colorectal cancer (CRC) is the second leading cause of death from cancer. The MET receptor tyrosine kinase and/or its ligand HGF are frequently amplified or overexpressed in CRC. It is known that tyrosine phosphorylated proteins are involved in progression and metastasis of colorectal cancer; however, little is known about the MET phospho-proteome in CRC. High resolution mass spectrometry was used to characterize immunoaffinity-purified, phosphotyrosine (pY)-containing tryptic peptides of the MET-expressing CRC cell model, DLD1. A total of 266 unambiguously identified pY sites spanning 168 proteins were identified. Quantification of mass spectrometry ion currents identified 161 pY sites, including many not previously linked to MET signaling, that were modulated in abundance by HGF stimulation. Overlay of these data with protein-protein interaction data sets suggested that many of the identified HGF-modulated phospho-proteins may be directly or indirectly associated with MET. Analysis of pY sequence motifs indicated a prevalence of Src family kinase consensus sequences, and reciprocal signaling between Src and MET was confirmed by using selective small molecule inhibitors of these kinases. Therefore, using quantitative phospho-proteomics profiling, kinase modulation by ligand and inhibitors, and data integration, an outline of the MET signaling network was generated for the CRC model.
Insights
This study reveals the MET phospho-proteome in colorectal cancer (CRC) using mass spectrometry. It identifies HGF-modulated phosphosites and suggests a reciprocal signaling network between MET and Src family kinases in CRC progression.
Area of Science:
- Oncology
- Molecular Biology
- Proteomics
Background:
- Colorectal cancer (CRC) is a leading cause of cancer-related mortality.
- MET receptor tyrosine kinase and HGF are implicated in CRC progression and metastasis.
- The MET phospho-proteome in CRC remains largely uncharacterized.
Purpose of the Study:
- To characterize the phosphotyrosine (pY) proteome associated with MET signaling in colorectal cancer.
- To identify HGF-modulated phosphosites and their potential association with MET.
- To elucidate the signaling network involving MET and other kinases in CRC.
Main Methods:
- Utilized high-resolution mass spectrometry for phosphoproteomic analysis of MET-expressing DLD1 CRC cells.
- Employed immunoaffinity purification to isolate phosphotyrosine-containing peptides.
- Integrated quantitative mass spectrometry data with protein-protein interaction networks and kinase inhibitor studies.
Main Results:
- Identified 266 unique pY sites across 168 proteins, with 161 sites modulated by HGF stimulation.
- Discovered numerous HGF-modulated phosphosites not previously linked to MET signaling.
- Revealed a prevalence of Src family kinase consensus sequences and confirmed reciprocal signaling between Src and MET.
Conclusions:
- Quantitative phospho-proteomics profiling provides insights into the MET signaling network in CRC.
- HGF stimulation significantly modulates the phosphotyrosine proteome in CRC cells.
- The findings suggest a complex interplay between MET and Src family kinases in colorectal cancer.
