conceived the experiments; M.J.K., H.N.S., S.H.L., S.J., E.M.L., and J.Z. intestinal tumorigenesis with reduced tumor cell stemness and suppressed Wnt/-catenin signaling activity, supported by transcriptome profiling. Collectively, our results unveil that PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively regulating self-renewing cells. transactivation during intestinal regeneration and tumorigenesis. INTRODUCTION Stem cells play key roles in tissue homeostasis and regeneration by self-renewing and repopulating progenitor cells (Fuchs et al., 2004; Morrison and Spradling, 2008). In the small intestine, two major ISCs co-exist. Crypt base columnar MELK-IN-1 cells (CBC) ISCs marked by the high Lgr5 expression are highly proliferative and essential for the intestinal homeostasis (Barker et al., 2007). The other ISCs located at position 4 (+4) and labeled by Hopx, Lrig1, Bmi1, and Tert are quiescent during intestinal homeostasis, whereas conditionally activated upon tissue damage. (Montgomery et al., 2011; Powell et al., 2012; Sangiorgi and Capecchi, 2008; Takeda et al., 2011). Accumulating evidence suggests that +4 ISCs function as a reservoir of ISCs during regeneration (Buczacki et al., 2013; Tian et al., 2011). Additionally, the committed progenitor cells (Dll1+, Alpi+, and Krt19+) located above the position +4 cells also dedifferentiate into ISCs for intestinal regeneration (Asfaha et al., 2015; Tetteh et al., 2016; Van Es et al., 2012), implying the involvement of the cell plasticity in rebuilding intestinal epithelium. Although the extensive lineage tracing studies have been used Hbg1 to identify ISCs or reservoir ISCs/IPCs populations, still the underlying mechanisms how these ISCs/IPCs cells are activated and expanded during regeneration remain elusive. It was proposed that cancer stem cells (CSCs) are a subpopulation of tumor cells, which drives tumor growth by self-renewing and giving rise to the daughter cells (Nguyen et al., 2012). The identities of CSCs are still controversial, however, it is plausible that CSCs might be related to therapeutic resistance and tumor recurrence (Dean et al., 2005; Kreso and Dick, 2014). Expression of CD44, CD133, and Lgr5 have been suggested as a maker for stemness of colorectal cancer (CRC) cells (OBrien et al., 2007; Ricci-Vitiani et al., 2007; Schepers et al., 2012; Zeilstra et al., 2008; Zhu et al., 2009). Nonetheless, how CSCs are maintained and expanded were not fully understood. PAF (also known as expression is significantly upregulated in many human cancers (Cheng et al., 2013; Hosokawa et al., 2007; Jain et al., 2011; Jung et al., 2013; Kais et al., 2011; Kato et al., 2012; Mizutani et al., 2005; Wang et al., 2016; Yu et al., 2001; Yuan et al., 2007). In pancreatic cancer cells, overexpression is necessary for pancreatic cancer cell proliferation (Hosokawa et al., 2007). Additionally, PAF is associated with MAPK hyperactivation transcriptional activation of the late endosomal/lysosomal adaptor, MELK-IN-1 MAPK and mTOR activator 3 (expression is associated with controlling ISCs/IPCs. Further comprehensive and genetic approaches revealed that PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively controlling the expansion of stem cells. RESULTS Upregulation of Expression upon Radiation Injury To identify essential genes associated with DNA repair during tissue regeneration, we conducted qRT-PCR array for DNA repair gene collections from irradiation (IR) treated mouse small intestine (1 day post-injury [1 dpi], 10 Gy) (Figures 1A and 1B). Among differentially expressed 79 genes, expression was highly upregulated by IR (7th ranked). Additionally, IR upregulated expression in the mouse small intestine in a dose- and time-dependent manner (Figures 1C and 1D), which led us to hypothesize that PAF plays crucial roles in intestinal regeneration. Open in a separate window Figure 1 Upregulation of PAF Expression upon Radiation Injury(A, B) Gene expression profiling of DNA repair genes upon radiation injury in mouse small intestine. After treatment of 10 Gy irradiation (1 day post-injury [1 dpi]), the whole small intestine samples were analyzed by qRT-PCR (N=3). is the fourth and are the seventh upregulated genes among the 79 genes related to DNA repair. (C) Time-dependent upregulation of expression upon IR injury. At 0, 1, 2, 4, and 7 dpi, the small intestine samples were collected and analyzed by MELK-IN-1 qRT-PCR (N=3). (D) Dose-dependent upregulation of expression upon injury. 6 and 10 Gy irradiation were used (1 dpi). Students roles of PAF in intestinal regeneration, we generated KO mouse model using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 gene targeting system (Wang et al., 2013; Yang et al., 2013)(Figure S1). Of note, KO mice are viable without any discernible phenotypes. To examine PAF expression in the crypt, we conducted immunohistochemistry (IHC) using two different PAF monoclonal antibodies (Figures 2A and S2B). We confirmed the specificity of.