Cancer Therapy: Preclinical CD44ExpressionDenotes aSubpopulation ofGastricCancer Cells inWhich Hedgehog Signaling Promotes Chemotherapy Resistance

Purpose: Gastric cancers may harbor a subset of cells with cancer stem cell (CSC) properties, including chemotherapy resistance, and CD44 is a gastric CSC marker. The Hedgehog (HH) pathway is a key developmental pathway that can be subverted by CSCs during tumorigenesis. Here, we examine the role of HH signaling in CD44(þ) gastric cancer cells. Experimental Design: Gastric cancer cell lines, tumor xenografts, and patient tumors were examined. Results:Gastric cancer cell lines AGS,MKN-45, andNCI-N87 grown as spheroids or sorted for CD44(þ) were found to have upregulation of HH pathway proteins. HH inhibition using Smoothened (Smo) shRNAor vismodegib (VIS) decreased spheroid formation and colony formation. CD44(þ) cells, compared with unselected cells, were also resistant to 5-fluorouracil and cisplatin chemotherapy, and this resistance was reversed in vitro and in xenografts with Smo shRNA or VIS. CD44(þ) cells also had significantly more migration, invasion, and anchorage-independent growth, and these properties could all be blocked with HH inhibition. Clinical tumor samples from a phase II trial of chemotherapy with or without VIS for advanced gastric cancer were analyzed for CD44 expression. In the chemotherapy alone group, high CD44 expressionwas associated with decreased survival, whereas in the chemotherapy plus VIS group, high CD44 expression was associated with improved survival. Conclusions:HH signaling maintains CSC phenotypes and malignant transformation phenotypes in CD44(þ) gastric cancer cells, and HH inhibition can reverse chemotherapy resistance in CD44(þ) cells. Gastric cancer is a heterogeneous disease, and the strategy of combining chemotherapy with HH inhibition may only be effective in tumors with high CD44 levels. Clin Cancer Res; 20(15); 3974–88.