Targeting MAD2 modulates stemness and tumorigenesis in human Gastric Cancer cell lines.

Gastric cancer (GC) is a solid tumor that contains subpopulations of cancer cells (CSCs), which are considered drivers of tumor initiation and metastasis; responsible for therapeutic resistance; and promoters of tumor relapse. The balance between symmetric and asymmetric division is crucial for cell maintenance. The objective of this study is to evaluate the role of MAD2, a key protein for proper mitotic checkpoint activity, in the tumorigenesis of GC. Gastric cancer cells (GCSCs) were obtained from MKN45, SNU638 and ST2957 cell lines. Pluripotency and markers were evaluated by RT-qPCR and autofluorescence and membrane markers by flow cytometry. Relevant signal transduction pathways were studied by WB. We analysed cell cycle progression, migration and invasion after modulation of MAD2 activity or protein expression levels in these models. assays were performed in a nude mouse subcutaneous xenograft model. We found that , CXCR4 and autofluorescence are common and consistent markers for the GCSCs analysed, with other markers showing more variability. The three main signalling pathways (Wnt/β-catenin; Hedgehog and Notch) were activated in GCSCs. Downregulation of MAD2 in MKN45 decreased the expression of markers CXCR4, CD133, CD90, and , without affecting cell cycle profile or therapy resistance. Moreover, migration, invasion and tumor growth were clearly reduced, and accordingly, we found that metalloprotease expression decreased. These results were accompanied by a reduction in the levels of transcription factors related with epithelial-to-mesenchymal transition. We can conclude that MAD2 is important for GCSCs and its downregulation in MKN45 plays a central role in GC tumorigenesis, likely through CXCR4--MMP1. Thus, its potential use in the clinical setting should be studied as its functions appear to extend beyond mitosis.