Supplemental Figure S1. A. Western blot for putative CSC markers in AGS, MKN-45, and NCI-N87 cells grown as monolayers or as spheroids. Supplemental Figure S2. A. Western blot of CD44(-) and CD44(+) AGS, MKN-45 and NCI-N87 spheroid cells for CD44, Sox2, Oct-4, Nanog, and c-Myc. Supplemental Figure S3. A. Western blot of AGS and NCI-N87 monolayer and spheroid cells for total and phosphorylated MAPK proteins. Supplemental Figure S4. A. Migration and invasion assays of GA monolayer and spheroid cells. Supplemental Figure S5. A. Western blot of AGS and NCI-N87 spheroid cells for total and phosphorylated JNK, active Rac1, N-cadherin, Slug, Snail, and Zeb1. Supplemental Figure S6. Proliferation assays for GA monolayer cells (A) and spheroid cells (B) following treatment with the Rac1 inhibitor NSC23766, 5-fluorouracil (5-FU), cisplatin, and/or carrier (DMSO). Supplemental Figure S7. A. Western blot of MKN-45 cells for total Rac1 following transduction with Rac1 shRNA (sh.Rac1) or scrambled control shRNA (sh.Scr).
ARTICLE ABSTRACT
Rac1, a Rho GTPase family member, is dysregulated in a variety of tumor types including gastric adenocarcinoma, but little is known about its role in cancer stem-like cells (CSCs). Therefore, Rac1 activity and inhibition were examined in gastric adenocarcinoma cells and mouse xenograft models for epithelial-to-mesenchymal transition (EMT) and CSC phenotypes. Rac1 activity was significantly higher in spheroid-forming or CD44+ gastric adenocarcinoma CSCs compared with unselected cells. Rac1 inhibition using Rac1 shRNA or a Rac1 inhibitor (NSC23766) decreased expression of the self-renewal transcription factor, Sox-2, decreased spheroid formation by 78%–81%, and prevented tumor initiation in immunodeficient mice. Gastric adenocarcinoma CSCs had increased expression of the EMT transcription factor Slug, 4.4- to 8.3-fold greater migration, and 4.2- to 12.6-fold greater invasion than unselected cells, and these increases could be blocked completely with Rac1 inhibition. Gastric adenocarcinoma spheroid cells were resistant to 5-fluorouracil and cisplatin chemotherapy, and this chemotherapy resistance could be reversed with Rac1 shRNA or NSC23766. The PI3K/Akt pathway may be upstream of Rac1, and JNK may be downstream of Rac1. In the MKN-45 xenograft model, cisplatin inhibited tumor growth by 50%, Rac1 inhibition by 35%, and the combination by 77%. Higher Rac1 activity, in clinical specimens from gastric adenocarcinoma patients who underwent potentially curative surgery, correlated with significantly worse survival (P = 0.017). In conclusion, Rac1 promotes the EMT program in gastric adenocarcinoma and the acquisition of a CSC state. Rac1 inhibition in gastric adenocarcinoma cells blocks EMT and CSC phenotypes, and thus may prevent metastasis and augment chemotherapy.Implications: In gastric adenocarcinoma, therapeutic targeting of the Rac1 pathway may prevent or reverse EMT and CSC phenotypes that drive tumor progression, metastasis, and chemotherapy resistance. Mol Cancer Res; 15(8); 1106–16. ©2017 AACR.
