Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF‑7 Cells Revealed by Targeted, Quantitative Proteomic Analysis

Development
of tamoxifen resistance remains a tremendous challenge
for the treatment of estrogen-receptor (ER)-positive breast cancer.
Small GTPases of the Ras superfamily play crucial roles in intracellular
trafficking and cell signaling, and aberrant small-GTPase signaling
is implicated in many types of cancer. In this study, we employed
a targeted, quantitative proteomic approach that relies on stable-isotope
labeling by amino acids in cell culture (SILAC), gel fractionation,
and scheduled multiple-reaction-monitoring (MRM) analysis, to assess
the differential expression of small GTPases in MCF-7 and the paired tamoxifen-resistant
breast cancer cells. The method displayed superior sensitivity and
reproducibility over the shotgun-proteomic approach, and it facilitated
the quantification of 96 small GTPases. Among them, 13 and 10 proteins
were significantly down- and up-regulated (with >1.5-fold change),
respectively, in the tamoxifen-resistant line relative to in the parental
line. In particular, we observed a significant down-regulation of
RAB31 in tamoxifen-resistant cells, which, in combination with bioinformatic
analysis and downstream validation experiments, supported a role for
RAB31 in tamoxifen resistance in ER-positive breast-cancer cells.
Together, our results demonstrated that the targeted proteomic method
constituted a powerful approach for revealing the role of small GTPases
in therapeutic resistance.