Gamma-secretase inhibitor IX (GSI) impairs concomitant activation of Notch and wnt-beta-catenin pathways in CD44+ gastric cancer

Abstract

Background & Aims: Gastric cancer is the second most common cause of cancer related death in the world. Advanced stages of gastric cancer are often associated with limited and marginal chemotherapeutic regimes. This fact further emphasises on the need of new advances in gastric cancer treatement. Cancer stem cells (CSCs) known to be one of the main candidates responsible for tumor recurrence, metastasis and resistance to cancer therapy are identified and charecterised in different types of gastric cancers. Studies have indicated that Notch and wnt-beta-catenin pathways are crucial for CSC development. Here in this study we higlight on the concomitant activation of Notch and wnt-beta-catenin pathway in CD44+ gastric cancer. We also showed effective inhibition of Notch and wnt-beta-catenin pathway in CD44+ gastric tumor initiating cells using the γ-secretase inhibitor IX (GSI IX)

Methods: We analysed the effect of GSI IX on growth, epithelial plasticity and tumorogenicity of gastric tumor intiating CD44+ cells. We have used gastric cancer cell line MKN45 in our experiments. For in vitro analysis proliferation,wound healing, invasion, adhesion and tumorsphere assays were performed to analyse the migration ,invasive, adhesive and tumorogenic potential of CD44+ sorted gastric tumor initaiting cells after GSI IX treatment. Western blot analysis of downstream signaling targets of Notch and wnt-beta-catenin were tested after gamma-secretase inhibitor IX (GSI IX) treatment. Immunohistochemistry, immunofluorescence and flowcytometric analysis were used to determine CD44 and Hes1 expression in human GC tissues, cells and blood. For in vivo analysis sorted CD44+ cells were subcutaneously injected into the nude mice (NMRI-nu/nu) and were treated with vehicle or GSI IX.

Results: GSI IX treatment effectively inhibits cell growth, migration, invasion, adhesion and induced apoptosis in CD44+ gastric cancer tumor initiating cells. Moreover, complete inhbition of notch and beta catenin downstream signaling targets were observed. Gastric cancer patientswith expression of CD44 and Hes1 showed overall reduced survival. CD44+CSCs showed high expression of Hes1 as compared to the CD44-subpopulation. Interestingly, amongst all Notch receptors, Notch1 was found to be important in mediating a crosstalk between Notch and wnt-beta-catenin signaling cascades in CD44+CSCs. Silencing of both CD44 and Notch1 by siRNA inhibited downstream targets and reconfirmed the proposed hypothesis of CD44 mediated signaling crosstalk in GC cells. GSI IX treatment effectively inhibits the concomitant activation of both (Notch and wnt-beta-catenin) pathways in gastric cancer via specific targeting of CD44+ gastric CSCs.

Conclusion: Thus, our study supports the presence of previously indentified CD44+ CSC in human gastric cancer cells. Our study also highlights the activation of Notch and beta catenin in gastric tumor initiating CD44+ cells and its effective inhibiton after GSI IX treatment. Moreover expression of CD44 and Hes1double positive cells showed a strong correlation with overall patient survival. Therefore, GSI IX could be a possible effective,alternative, treatment option for gastric cancer patients.