Label-Free Quantitative Phosphoproteomics Reveals Regulation of Vasodilator-Stimulated Phosphoprotein upon Stathmin-1 Silencing in a Pair of Isogenic Colorectal Cancer Cell Lines.

In this communication, we present the phosphoproteome changes in an isogenic pair of colorectal cancer cell lines, viz., the poorly metastatic HCT-116 and the highly metastatic derivative E1, upon stathmin-1 (STMN1) knockdown. The aim was to better understand how the alterations of the phosphoproteins in these cells are involved in cancer metastasis. After the phosphopeptides were enriched using the TiO2 HAMMOC approach, comparative proteomics analysis was carried out using sequential window acquisition of all theoretical mass spectra-MS. Following bioinformatics analysis using MarkerView and OneOmics platforms, we identified a list of regulated phosphoproteins that may play a potential role in signaling, maintenance of cytoskeletal structure, and focal adhesion. Among these phosphoproteins, was the actin cytoskeleton regulator protein, vasodilator-stimulated phosphoprotein (VASP), where its change in phosphorylation status was found to be concomitant with STMN1-associated roles in metastasis. We further showed that silencing of stathmin-1 altered the expression, subcellular localization and phosphorylation status of VASP, which suggested that it might be associated with remodeling of the cell cytoskeleton in colorectal cancer metastasis.