Abstract 3873: Hedgehog signaling maintains gastric cancer stem cells and promotes chemotherapy resistance: results from laboratory and clinical studies

Abstract

Abstract Introduction: Gastric cancers may harbor a small subset of cancer stem cells (CSCs) with the exclusive ability to self-renew and differentiate into heterogenous cell types. These CSCs may also contribute to chemotherapy resistance. The Hedgehog (HH) pathway is a key developmental pathway that can be subverted by CSCs during tumorigenesis. However in a recent randomized phase II trial of chemotherapy with or without the small molecule HH inhibitor vismodegib for advanced gastric cancers, the addition of vismodegib did not increase progression-free survival (PFS) or overall survival (OS). In this study, we examine the role of HH signaling in gastric CSC maintenance and chemotherapy resistance. Methods and Results: Gastric cancer cell lines AGS, MKN-45, and NCI-N87 were grown as spheroids to enrich for CSCs. Spheroid cells were found to have upregulation of the putative gastric CSC marker CD44 along with HH pathway proteins Shh, Ptch1, Smo, and Gli1. Inhibition of the HH signaling using Smo shRNA or vismodegib decreased spheroid formation by 70.3-78.4% or 66.9-70.8%, respectively, and attentuated another CSC phenotype, single cell colony formation, by 66.9-78.4%. Transformation phenotypes such as migration, invasion, and anchorage-independent colony formation were also inhibited in gastric CSCs by 50.2-65.6%, 57.4-66.3%, and 3.8-4.6 fold, respectively. CD44(+) gastric CSCs from all 3 cell lines were resistant to 5-fluorouracil or cisplatin chemotherapy, and this resistance was reversed with the addition of Smo shRNA or vismodegib. The combination of Smo shRNA and cisplatin synergistically blocked the growth of MKN-45 xenografts, and treated tumors demonstrated a 1.8-2.6 fold increase in tumor cell apoptosis compared to tumors treated with cisplatin alone. Clinical tumor samples from the phase II vismodegib trial were analyzed for CD44 expression (as a surrogate to levels of CSCs). In the chemotherapy alone group, high CD44 expression was associated with worse PFS and OS. However in the chemotherapy with vismodegib group, high CD44 expression was associated with improved PFS and OS. For two patients in the vismodegib arm of the study who had a complete response, CD44 levels were 6.1-fold higher than the other patients in the group (p=0.001). Conclusions: HH signaling is required to maintain gastric CSC phenotypes such as spheroid formation and colony formation from single cells as well malignant transformation phenotypes such as migration, invasion, and anchorage-independent growth. Gastric CSCs are resistant to chemotherapy compared to unselected cells, and HH inhibition can reverse this resistance. Given gastric cancer is a heterogeneous disease, the strategy of combining chemotherapy with HH inhibition may only be effective in a subset of gastric cancer patients with high levels of CD44(+) gastric CSCs. Citation Format: Changhwan Yoon, Do Joong Park, Benjamin Schmidt, Nicholas J. Thomas, Hae-June Lee, Teresa S. Kim, Yelena Y. Janjigian, Deirdre J. Cohen, Sam S. Yoon. Hedgehog signaling maintains gastric cancer stem cells and promotes chemotherapy resistance: results from laboratory and clinical studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3873. doi:10.1158/1538-7445.AM2014-3873