Introduction Linear transcriptional pathways that govern

Introduction Linear transcriptional pathways that govern intestinal tumorigenesis have been extensively explored in mice using gene knock outs (KO) and to a limited extent hypomorphs as well as chemical inhibitors (Aoki and Taketo, 2007; Barker et al., 2009, 2010; Batlle et al., 2005; Cheasley et al., 2011; Fre et al., 2005, 2011; Malaterre et al., 2007; Muncan et al., 2006; Sansom et al., 2007; Taketo, 2006). Specifically these studies on the Wnt, Notch and Myb pathways confirm a dependency on their various downstream target genes but do not necessarily address the cross-talk between these pathways when they are instead activated. An additional consequence of gene deletion studies is that changes in disease course can be due to the actual loss of the very c-met inhibitor that are normally subject to transformation. To address these gaps we have focused on these three transcriptional programs that are activated in intestinal cancers and that have been shown to interact with each other (Cheasley et al., 2011; Ciznadija et al., 2009; Fre et al., 2009). To do this we have generated compound transgenic and mutant mice that modulate all three pathways and followed the earliest events in stem/progenitor cell (ISC) transformation in vitro and in vivo. ISCs are at the nexus of the hierarchical organization of the intestinal epithelium, producing cells which give rise to differentiated enterocytes, enteroendocrine cells, Paneth cells, goblet cells, Tuft cells and Peyer\'s Patch M cells (Barker et al., 2010). To guarantee the lifelong maintenance of the tissue, the ISC/progenitor cell compartment has to be tightly regulated by several molecular pathways that control ISC self-renewal, proliferation and differentiation. The Wnt pathway is active in ISCs and is essential for their self-renewal (Korinek et al., 1998) as well as Paneth cell differentiation (Andreu et al., 2005; Farin et al., 2012; van Es et al., 2005a). Activation of the Wnt pathway leads to stabilization of cytoplasmic and nuclear β-catenin, which interacts with other proteins to induce the expression of genes such as Lgr5 and Ascl2 in ISCs (Barker et al., 2007; van der Flier et al., 2009), Myc and Ccnd1 (Andreu et al., 2005), as well as Cryptidin, and Lysozyme genes in Paneth cells (Andreu et al., 2005; van Es et al., 2005a). Similarly, the Notch pathway (Kopan and Ilagan, 2009) is essential for ISC self-renewal and differentiation (Pellegrinet et al., 2011). Notch is clearly active in ISCs (Fre et al., 2011; Smith et al., 2012) and stimulates proliferation (Fre et al., 2005, 2009) while blocking secretory cell differentiation (Fre et al., 2005, 2011; van Es et al., 2005b). Furthermore, Notch is a direct activator of the ISC gene Olfm4 (VanDussen et al., 2012) and the bHLH transcriptional repressor Hes1, which in turn blocks expression of the driver of secretory cell differentiation, Math1 (Jensen et al., 2000; Yang et al., 2001). The co-operation between Notch and Wnt in the intestine results in the amplification of Wnt-driven proliferation and Wnt-driven tumor formation is potentiated by constitutive Notch activation (Fre et al., 2009). Conversely, Wnt target genes are repressed by Notch signaling and negatively correlate with the Notch target gene Nrarp expression in CRC (Kim et al., 2012). Myb is additionally active in ISCs where it is essential for self-renewal and blockade of goblet cell differentiation (Cheasley et al., 2011; Malaterre et al., 2007). Furthermore, we have demonstrated that Myb is crucially involved in the regulation of the Wnt target genes Myc and Lgr5 in ISCs (Cheasley et al., 2011; Ciznadija et al., 2009). Myb is over-expressed in human CRC (Ramsay and Gonda, 2008) and Myb heterozygous deletion prolongs survival of intestinal cancer-prone Apc mutant mice (Ciznadija et al., 2009), indicating that Myb is involved in Wnt-dependent intestinal tumor formation.
Materials and methods
Results
Discussion In summary, we have demonstrated that using an in vitro model of ApcWT-loss and using in vivo intestinal cancer model that activated Wnt, Notch and Myb are all essential to intestinal tumorigenesis. We systematically dissected within the same experimental milieu the influence of the Wnt, Notch and Myb pathways on optimal formation and growth of ISC intestinal organoids (or: self-renewal and proliferation of ISC) in vitro. Using this approach we deconstruct here how these three pathways interact during the earliest event of CRC, the initiation of intestinal adenomas, and report a remarkable shift in their functional and transcriptional interaction between ISCs and tumor-initiating cells.