Quantitative Phosphoproteomics Reveals a Cluster of Tyrosine Kinases That Mediates Src Invasive Activity in Advanced Colon Carcinoma Cells

Abstract

The nonreceptor tyrosine kinase Src is frequently overexpressed and/or activated in human colorectal carcinoma (CRC), and its increased activity has been associated with a poor clinical outcome. Src has been implicated in growth and invasion of these cancer cells by still not well-known mechanisms. Here, we addressed Src oncogenic signaling using quantitative phosphoproteomics. Src overexpression increased growth and invasiveness of metastatic SW620 CRC cells. Stable isotope labeling with amino acids in cell culture in combination with liquid chromatography tandem mass spectrometry allowed the identification of 136 proteins which exhibited a significant increase in and/or association with tyrosine phosphorylation upon Src expression. These mainly include signaling, cytoskeleton, and vesicular-associated proteins. Interestingly, Src also phosphorylated a cluster of tyrosine kinases, i.e., the receptors Met and EphA2, the cytoplasmic tyrosine kinase Fak, and pseudo-tyrosine kinase SgK223, which were required for its invasive activity. Similar results were obtained with metastatic Colo205 CRC cells that exhibit high endogenous Src activity. We concluded that Src uses a tyrosine kinases network to promote its invasive activity in CRC and this implicates a reverse signaling via tyrosine kinase receptors. Targeting these tyrosine kinases may be of significant therapeutic value in this cancer. [Cancer Res 2009;69(6):2279-86]

Src family tyrosine kinases-driven colon cancer cell invasion is induced by Csk membrane delocalization

“…This mechanism may particularly operate in advanced CRC cells, in which tumour cells harbour maximal Csk membrane delocalization together with a high SFK level. In this respect, a moderate expression of wild-type Src in advanced CRC SW620 cells is sufficient to increase oncogenic properties, in particular, the proinvasive activity (Leroy et al, 2009). This may explain why CRC cells can withstand high Csk levels, as observed in tumour biopsies.…”

SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2

“…The identity of this protein was unravelled via a SILAC-based quantitative proteomic analysis, which allowed the identification of SLAP interactors in tumour cells including those phosphorylated on tyrosine residues. A prominent interactor isolated was EPHA2, which we recently described as an important SRC substrate for oncogenic signalling 11 . While SLAP was originally identified by its binding capacity to EPHA2, their interaction has never been addressed in vivo 21 .…”

“…Consequently, upregulation of wild-type SRC in advanced tumour stages may be sufficient to induce oncogenic signalling. A previous phosphoproteomic analysis revealed that SRC phosphorylates a cluster of TK, including the receptors MET and EPHA2 that mediate CRC cell tumorigenicity and invasiveness 11 . How SRC elicits the activation of these RTK was previously unclear but the present study describes a mechanism that favors SRC-EPHA2 interaction through receptor stabilization following SLAP inactivation.…”

“…While CSK is well expressed in CRC and shows strong activity in vitro, CSK mislocalization due to the silencing of its transmembrane interactor Cbp/PAG promotes CSK inactivation and induces SRC pro-invasive activity. The nature of SRC signalling mediating this invasive process is still only partially elucidated, probably because of the capacity of SRC to phosphorylate several hundred substrates, several of them having no clear function in neoplastic transformation [9][10][11][12] . Interestingly, a quantitative phosphoproteomic analysis in metastatic CRC cells revealed that SRC phosphorylates a cluster of downstream TK composed of EPHA2, MET, FAK and SGK223, which were essential for the promotion of tumour cell growth and invasion 11 .…”

“…The nature of SRC signalling mediating this invasive process is still only partially elucidated, probably because of the capacity of SRC to phosphorylate several hundred substrates, several of them having no clear function in neoplastic transformation [9][10][11][12] . Interestingly, a quantitative phosphoproteomic analysis in metastatic CRC cells revealed that SRC phosphorylates a cluster of downstream TK composed of EPHA2, MET, FAK and SGK223, which were essential for the promotion of tumour cell growth and invasion 11 . EPHA2 is a Receptor Tyrosine Kinase (RTK) that binds to membrane-bound ligands called Ephrins and regulates cell-cell contacts through bidirectional signalling in neighboring cells, particularly during developmental and physiological processes 13 .…”