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    • In this study CD CD

    2018-10-22

    In this study, CD133−/CD44− CRC cells are considered important in tumor progression because they secrete EDA and can sustain EDA-integrin α9β1 pathway in a paracrine manner. CD133+/CD44+ cells may take advantage of CD133−/CD44− cells-secreted EDA via increasing expression of EDA receptor integrin α9β1. Interestingly, EDA silencing in SW480 cells changes relative abundance of CD133+/CD44+ and CD133−/CD44− subpopulations, indicating an EDA-dependent dynamic equilibrium between these two subsets of cells. A recent report found that CD133− relative to CD133+ human CRC cells are more resistant to 5-fluorouracil (FU) (Hongo et al., 2011). Collectively, these findings highlight the importance of cancer therapy targeting non-CSCs and tumor microenvironment in addition to CSCs. Silencing EDA strikingly reduces the formation of spheroids in SW480 cells, which is associated with reduced CD133+ and CD44+ subpopulations and expression of several embryonic stem cell markers including SOX2, NANOG, and OCT3/4 (Fig. 3). In contrast, the expression of differentiation-related marker CK7 and MUC2 are increased (Fig. 3). These observations demonstrated that EDA signaling is required for sustaining CD133+/CD44+ subpopulation and sphere formation of SW480 cells, and EDA may act through maintaining expression levels of stemness-associated proteins. We showed that EDA interacts with its receptor integrin α9β1 in SW480 cells (Fig. 4a). When integrin α9β1 function is blocked by an monoclonal antibody, the colony formation of EDA-overexpressing CD133+/CD44+ cells and the gnrh antagonist progression of bulk SW480 cells are substantially suppressed, which is associated with increased apoptosis (Figs. 4b–f). These findings support a central role of integrin α9β1 in EDA signaling, at least, in SW480 CRC cells. It is well established that integrins alone or their interactions with growth factors and chemokines in a paracrine and autocrine manner promotes the evolution, progression and metastasis of tumors (Desgrosellier and Cheresh, 2010). Given this, integrins are considered as potential therapeutic targets in cancer (Cox et al., 2010). What might be signal transduction pathways downstream of EDA-integrin α9β1 signaling? Wnt signaling plays important roles in sustaining stem cells in normal tissues and cancers (Logan and Nusse, 2004) and in the development of CRC (Kinzler and Vogelstein, 1996; Sancho et al., 2004). We observed a substantial downregulation of active β-catenin protein in fibronectin EDA silenced SW480 cells, indicating suppression of Wnt signaling by EDA silencing in these cells. To the best of our knowledge, this study is the first to show that EDA is required for maintaining Wnt/β-catenin activity. Our observation raised an interesting question of how EDA is linked to the regulation of β-catenin. We found that the phosphorylation of FAK, Akt and Erk is down-regulated in the EDA-silenced SW480 cells (Fig. 5a). FAK is an immediate downstream target of integrins. Interestingly, silencing of FAK also reduces activation of β-catenin (Fig. 5b). FAK may exert effects through Akt and/or Erk pathways (Cabodi et al., 2010). Additionally, Akt was shown to link FAK to β-catenin activation in breast cancer progenitor cells exposed to the interleukin 8 receptor CXCR1, playing a crucial role in maintaining the properties of these cells (Ginestier et al., 2010). It was also reported that the Erk pathway can regulate β-catenin activation (Kim et al., 2007). Using specific inhibitors of MAPK/Erk and PI3K/Akt pathways, we showed that activation of β-catenin requires MAPK/Erk pathway, but not PI3K/Akt pathway in our cells (Fig. 5c). Thus, we propose that EDA may sustain CD133+/CD44+ subpopulation in CRC cells via activating Wnt signaling through integrin α9β1-FAK-MAPK/ERK pathway (Fig. 5g). More than 20years ago, Gradl et al. identified fibronectin as a direct target of Wnt/β-catenin pathway (Gradl et al., 1999). Together with our finding that fibronectin EDA sustains β-catenin activity, it appears there is a feed-forward regulatory loop between fibronectin secretion and β-catenin activity. This loop may represent a vicious cycle for promoting cell growth. Thus, targeting this cycle in cancer cells might be effective in controlling cancer growth.