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

Abstract

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.

Knockdown of SAR1B suppresses proliferation and induces apoptosis of RKO colorectal cancer cells

“…A recent study from Huang and Wang (40) revealed that SAR1B knockdown promoted the migration and invasion of CRC cells. While this previous study focused on the effects of SAR1B on CRC migration, invasion and EMT, the present study focused on the effects of SAR1B on CRC proliferation, colony formation and apoptosis.…”

SAR1B GTPase is necessary to protect intestinal cells from disorders of lipid homeostasis, oxidative stress, and inflammation

“…Therefore, these earlier observations led us to the hypothesis that lipogenesis would be restricted while β-oxidation would be stimulated, and this assumption turned out to be true. Many factors may explain cellular homeostasis infringement in response to the SAR1B deletion, including the accumulation of lipids, abnormalities of protein transport, defects in nascent vesicle formation and membrane bending, poor recruitment of coat components, irregularities in the epithelial-mesenchymal transition, and alterations of cholesterol biogenesis (20, 21). Evidently, reasonable efforts must continue to understand how SAR1B, which regulates the formation and assembly of the ER-derived COPII vesicles and hence the trafficking of most cellular proteins, is involved in the control of lipid metabolic pathways.…”

Global and Targeted Profiling of Gtp‐binding Proteins in Biological Samples by Mass Spectrometry

“…In this regard, we developed and utilized MS‐based approaches for targeted quantitative proteomic analysis of small GTPases of the Ras superfamily in cancer cells and tissues. By employing MRM, in conjunction with the use of SILAC or crude synthetic SIL peptides, we achieved high‐throughput targeted quantitative profiling of the altered expressions of small GTPases accompanied with cancer metastasis and drug resistance in cultured human cancer cells, adipocyte differentiation in murine cells, and development of neurodegenerative diseases in patient‐derived tissue samples (Huang & Wang, 2018, 2019; Huang et al, 2018, 2019; Yang, Huang, & Wang, 2020). By virtue of its superior sensitivity, reproducibility, and specificity, MRM has been widely used for targeted proteomics during the past two decades; however, owing to the use of low‐resolution mass analyzer in MS/MS analysis, the method is more susceptible to background interference from sample matrix.…”

“…Furthermore, by comparing the same samples analyzed by DDA and MRM methods, we found that the LC–MRM method outperformed the shotgun proteomic method in reproducibility and sensitivity. By harnessing the same targeted proteomic workflow, we also explored the roles of small GTPases associated with acquired resistance to tamoxifen in estrogen‐receptor (ER)‐positive MCF‐7 breast cancer cells (Huang & Wang, 2018) and metastatic transformation of colorectal cancer cells (Huang & Wang, 2019).…”