Targeted Quantitative Proteomic Approach for Probing Altered Protein Expression of Small GTPases Associated with Colorectal Cancer Metastasis

Genes encoding the
small GTPases of the Ras superfamily are among
the most frequently mutated or dysregulated in human cancer. No systematic
studies, however, have yet been conducted for assessing the implications
of small GTPases in the metastatic transformation of colorectal cancer
(CRC). By utilizing a recently established high-throughput multiple-reaction
monitoring (MRM)-based workflow together with stable isotope labeling
by amino acids in cell culture (SILAC), we investigated comprehensively
the relative expression of the small GTPase proteome in a pair of
matched primary/metastatic CRC cell lines (SW480/SW620). Among the
83 quantified small GTPases, 25 exhibited at least a 1.5-fold difference
in protein expression in SW480 and SW620 cells. In particular, SAR1B
protein was found to be substantially down-regulated in SW620 relative
to SW480 cells. In addition, bioinformatic analyses revealed that
diminished SAR1B mRNA expression is significantly
associated with higher CRC stages and unfavorable patient prognosis,
in support of a potential role of SAR1B in suppressing CRC metastasis.
In addition, diminished SAR1B expression could stimulate epithelial–mesenchymal
transition (EMT), thereby promoting motility and in vitro metastasis
of SW480 cells. In summary, we profiled systematically, by employing
an MRM-based targeted proteomic method, the differential expression
of small GTPase proteins in a matched pair of primary/metastatic CRC
cell lines. Our results revealed the potential roles of SAR1B in suppressing
CRC metastasis and in the prognosis of CRC patients.