Chemotherapy Resistance in Diffuse-Type Gastric Adenocarcinoma Is Mediated by RhoA Activation in Cancer Stem-Like Cells

Abstract

Purpose The Lauren diffuse type of gastric adenocarcinoma (DGA), as opposed to the intestinal type (IGA), often harbor mutations in RHOA but little is known about the role of RhoA in DGA. Experimental Design We examined RhoA activity and RhoA pathway inhibition in DGA cell lines and in two mouse xenograft models. RhoA activity was also assessed in patient tumor samples. Results RhoA activity was higher in DGA compared to IGA cell lines, and was further increased when grown as spheroids to enrich for cancer stem-like cells (CSC) or when sorted using the gastric CSC marker CD44. RhoA shRNA or the RhoA inhibitor Rhosin decreased expression of the stem cell transcription factor, Sox2, and decreased spheroid formation by 78–81%. DGA spheroid cells had 3–5 fold greater migration and invasion than monolayer cells, and this activity was Rho-dependent. Diffuse GA spheroid cells were resistant in a cytotoxicity assay to 5-fluorouracil and cisplatin chemotherapy, and this resistance could be reversed with RhoA pathway inhibition. In two xenograft models, cisplatin inhibited tumor growth by 40–50%, RhoA inhibition by 32–60%, and the combination by 77–83%. In 288 patient tumors, increased RhoA activity correlated with worse OS in DGA patients (p=0.017) but not in IGA patients (p=0.612). Conclusions RhoA signaling promotes CSC phenotypes in DGA cells. Increased RhoA activity is correlated with worse OS in DGA patients and RhoA inhibition can reverse chemotherapy resistance in DGA CSC and in tumor xenografts. Thus the RhoA pathway is a promising new target in DGA patients.

Integrated Analysis Identifies Molecular Signatures and Specific Prognostic Factors for Different Gastric Cancer Subtypes

“…Cell adhesion– and RhO GTPase–related genes were enriched in diffuse GC, which could be interpreted by the high mutation and methylation level of CDH1 [10] and the high mutation rate of RhoA gene [30], [31]. Cell cycle and p53-DNA repair–related genes were found enriched in intestinal GC, which is in coincidence with previous reports [15], [32].…”

Isolation of spheroid-forming single cells from gastric cancer cell lines: enrichment of cancer stem-like cells

“…Consistent with these results, the protein levels of SOX2 and NANOG, but not OCT4, were increased in SS#2 spheroids (Figure 3B). These data suggest that among those three genes, SOX2 and NANOG are the key factor conferring stemness characteristics in spheroid-forming ability as previous studies reported [19,20]. Furthermore, higher expression levels of those genes in SS#2 than TS#2 demonstrate that our novel method isolated more homogeneous spheroid cells with stem-like characteristics.…”

Role of Rac1 Pathway in Epithelial-to-Mesenchymal Transition and Cancer Stem-like Cell Phenotypes in Gastric Adenocarcinoma

“…5-fluorouracil was purchased from US Biological, and cisplatin was purchased from Enzo Life Sciences. Gastric cancer cell lines were grown as spheroids and spheroids were counted as previously described (14). …”

“…Western blot analysis was performed as previously described (14) using the following antibodies: Akt (sc-8312, Santa Cruz Biotechnology), ERK1 (sc-271270, Santa Cruz Biotechnology), p38 (sc-81621, Santa Cruz Biotechnology), JNK2 (sc-827, Santa Cruz Biotechnology), CD24 (sc-11406, Santa Cruz Biotechnology), c-Myc (sc-40, Santa Cruz Biotechnology), Sox2 (#2748, #3579), Oct-4 (#2750), Nanog (#4893), Slug (#9585), Snail (#3879), phospho-Akt (ser473) (#9271), phospho-p38 (#9211), phospho-ERK1/2 (#9101), phospho-JNK1/2 (#9251), CD44 (#3578), Musashi-1 (#2154),, cleaved caspase-3 (#9661), Rac1 (BD 61065, BD Biosciences), N-cadherin (BD 610920, BD Biosciences), Zeb1 (NBP-1-05987, Novus Biologicals), CD133 (MBS462020, MYBioSource), and β-actin (A5228, Sigma)…”

“…These proteins serve as intracellular molecular switches cycling between a GTP-bound active form and a GDP-bound inactive form. We recently found that RhoA signaling promotes chemotherapy resistance in diffuse type GA cells, and RhoA inhibition could reverse this chemotherapy resistance (14). …”