Abstract Introduction: The diffuse type of gastric adenocarcinoma (GA) was recently found to frequently harbor activating mutations in RHOA. RhoA is a member of the small GTPase-Ras-like proteins which are involved in cell signaling for cell migration and cell cycle. Methods: RhoA activity and inhibition was examined in diffuse GA cell lines grown as spheroids (i.e. cancer stem cell conditions) and as monolayers in various in vitro assays. RhoA knockdown combined with chemotherapy was examined in a mouse xenograft model. Results: RhoA activity was much higher in diffuse GA cell lines MKN-45 and SNU-668 compared to intestinal GA cell lines NCI-N87 and AGS. RhoA activity was further increased in diffuse GA cells when grown as spheroids rather than as monolayers. RhoA inhibition with shRNA or the RhoA inhibitor Rhosin decreased spheroid formation and deceased expression of the stem cell transcription factor, Sox2. Diffuse GA cells when grown as spheroids had significantly greater migration, invasion, and anchorage-independent growth, and these properties could all be blocked with RhoA shRNA or Rhosin. Diffuse GA spheroid cells (compared to monolayer cells) were resistant to 5-fluorouracil and cisplatin chemotherapy, and this chemotherapy resistance could be reversed with RhoA inhibition. In the MKN-45 xenograft model, cisplatin inhibited tumor growth by 50%, RhoA shRNA by 60%, and the combination by 83%. Clinical tumor samples of diffuse GA are currently being analyzed for RhoA expression and correlated with extent of disease and response to chemotherapy. Conclusions: RhoA signaling is upregulated in diffuse GA cells grown as spheroids and promotes malignant transformation phenotypes such as migration and invasion. RhoA inhibition can reverse chemotherapy resistance in GC spheroid cells and in tumor xenografts, and thus the RhoA pathway is a promising new target of therapy for diffuse GA. Citation Format: Changhwan Yoon, Soo-Jeong Cho, Bulent A. Aksoy, Do Joong Park, Sam S. Yoon. RhoA activation in diffuse type gastric adenocarcinoma promotes cancer stem cell phenotypes including chemotherapy resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4414. doi:10.1158/1538-7445.AM2015-4414