RETRACTED: 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 harbors 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 with IGA cell lines and was further increased when grown as spheroids to enrich for cancer stem-like cells (CSCs) 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% to 81%. DGA spheroid cells had 3- to 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% to 50%, RhoA inhibition by 32% to 60%, and the combination by 77% to 83%. In 288 patient tumors, increased RhoA activity correlated with worse overall survival 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 overall survival 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.

Figure 1. RhoA GTPases in gastric cancer cell lines

A. Western blot demonstrating levels of active and total Rho GTPases RhoA, Rac1, and Cdc42 in diffuse and intestinal gastric cancer cell lines (A), in diffuse gastric cancer cell lines grown as monolayers or as spheroids (B), and in CD44(+) and CD44(−) fraction of diffuse gastric cancer spheroid cells (C). D. CD44 (Green) and RhoA (red) immunofluorescence photos after diffuse gastric cancer cells were separated into CD44(+) and CD44 (−) fractions and then grown as spheroids. Scale bar 50 μm. E. Spheroid formation assay following transduction of diffuse gastric cancer cell lines with RhoA shRNA (sh.RhoA) or scrambled shRNA (sh.Scr) or after treatment with Rhosin (a RhoA inhibitor) or carrier (DMSO). Bars represent standard deviation. *p<0.05.

Figure 2. RhoA activity and EMT, invasion, and migration

Western blot of CD44, the EMT-related protein N-cadherin, and the EMT transcription factors Snail, Slug, and Zeb1 in MKN-45 and SNU-668 cells grown as monolayers or as spheroids (A) and after separation of spheroid cells into CD44(+) and CD44(−) fractions (B). C. Western blot and zymography for MMP-2 and MMP-9 for diffuse gastric cancer spheroid cells separated into CD44(+) and CD44(−) fractions. D. Migration and invasion assay for MKN-45 and SNU-668 cells grown as monolayers or spheroids. E. 3-D invasion assay with CD44(+) and CD44(−) spheroid cells. F. Migration and invasion assay for diffuse gastric cancer spheroid cells separated into CD44(+) and CD44(−) fractions and treated with the RhoA inhibitor Rhosin or carrier (DMSO). Bars represent standard deviation. *p<0.05.

Figure 3. PI3K/Akt pathway activates RhoA

A. Western blot of MKN-45 and SNU-668 spheroid cells for Akt, RhoA, JNK, and Slug following treatment with the PI3K inhibitor LY294003 or carrier (DMSO). B. Immunofluorescence images of MKN-45 and SNU-668 spheroids treated with the LY294002 or carrier (DMSO). C. Photos and graphs of migration and invasion assays for MKN-45 and SNU-668 cells grown as spheroids and treated with the Akt inhibitor LY294002 or carrier (DMSO). D. Western blot for JNK, ERK, p38, and Slug for MKN-45 and SNU-668 cells grown as spheroids and transduced with RhoA shRNA (sh.RhoA) or scrambled shRNA (sh.Scr). E. Western blot for RhoA, JNK, and Slug after MKN-45 and SNU-668 cells were grown as spheroids and treated with the JNK inhibitor SP60015 or carrier (DMSO). Bars represent standard deviation. Bars represent standard deviation. *p<0.05.

Figure 4. Diffuse gastric CSCs and chemoresistance

A–D. Proliferation assays for diffuse gastric cancer spheroid cells and monolayer cells following treatment with RhoA shRNA (sh.RhoA) or scrambled control shRNA (sh.Scr), 5-fluorouracil (5-FU) or cisplatin chemotherapy, the RhoA inhibitor Rhosin, the PI3K inhibitor LY294002, or the JNK inhibitor SP600125. E. Photos and graph of spheroid formation assay for CD44(+) MKN-45 or SNU-668 cells with the PI3K inhibitor LY294002, RhoA inhibitor Rhosin, JNK inhibitor SP600125, or carrier (DMSO). Bars represent standard deviation. *p<0.05.

Figure 5. Cisplatin chemotherapy combined with RhoA inhibition in MKN-45 diffuse gastric cancer xenografts

MKN-45 cells were transduced with RhoA shRNA (Smo.shRNA) or control (Scr.shRNA) and grown and xenografts. A. Tumor growth curves for MKN-45 xenografts treatment with Scr.shRNA or RhoA.shRNA and PBS or cisplatin. B. Photos of representative tumor from each treatment group. C. Photos and graphs following immunohistochemical analysis of tumors for proliferation using Ki-67 (green), apoptosis using cleaved caspase 3 (red), CD44 (green), Slug (red) and Sox2 (white). Scale bar 20 μm. D) Western blot of tumor lysates for CD44, cleaved caspase 3, Slug, and Sox2. Bars represent standard deviation. *p<0.05 compared to control. **p<0.05 compared to all other groups.

Figure 6. ROCK inhibition in SNU-668 xenografts; RhoA activity and survival in diffuse gastric cancer patients

A. Tumor growth curves for SNU-668 xenografts treatment with cisplatin and/or fasudil. B. Photos of representative tumor from each treatment group. C. Graphs following immunohistochemical analysis of tumors for proliferation using Ki-67 and apoptosis using cleaved caspase 3 and immunofluorescence analysis for CD44 + Slug and CD44 + Sox-2. Cisplatin (Cis), Fasudil (Fas). D. Kaplan-Meier overall survival curves for patients undergoing surgical resection for diffuse gastric cancer stratified by low versus high RhoA activity in the primary tumor. E. Diagram of RhoA pathway in diffuse gastric cancer CSCs. Bars represent standard deviation. *p<0.05.