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    • In our prior studies we

    2018-10-20

    In our prior studies, we showed that K-RasG12D promotes proliferation in the intestinal epithelium by activating MEK, but that K-RasG12D promotes proliferation of colorectal cancer buy ap4 through a MEK-independent mechanism (Haigis et al., 2008). To determine whether K-RasG12D promotes quiescent stem cell proliferation through a MEK-dependent or -independent mechanism, we treated control and mutant animals with PD0325901, an orally active MEK inhibitor (MEKi), (Bain et al., 2007) and then repeated label retention studies. Our pilot experiments indicated that PD0325901 was highly effective in suppressing MEK activity in vivo in the colonic epithelium (Supplemental Fig. 1). Similar to what we observed in our initial experiment, control and mutant animals treated with MEKi did not differ in label retention in the proximal small intestine (Fig. 2F). By contrast, while MEK inhibition did not alter label retention in the small intestine and colon of control animals, MEKi reverted the K-RasG12D label retention phenotype to that of control animals (Fig. 2G,H). These data strongly suggest that mutant K-Ras promotes hyperproliferation of quiescent intestinal stem cells via a MEK-dependent pathway. Quiescent intestinal stem cells are also known to play a central role in mucosal regeneration upon tissue injury (Potten & Grant, 1998). Following injury induced by cytotoxic or genotoxic agents, the quiescent stem cells become mitotically active, leading to restoration of normal epithelial architecture and barrier function (Roth et al., 2012). In order to determine whether mutant K-Ras can affect quiescent stem cells following injury, animals expressing WT or mutant K-Ras were exposed to 3% dextran sodium sulfate (DSS) in their drinking water for 6days and then sacrificed, or else allowed to recover for 6days and then sacrificed. Exposure to DSS induces mucosal injury and subsequent inflammation in the distal colon of rodents (Vowinkel et al., 2004). Significant weight loss was evident in both experimental groups following acute DSS treatment, although animals expressing K-RasG12D tended to lose less weight (Fig. 3A,B). To assess whether the weight loss phenotypes correlated with epithelial damage, we performed a histologic analysis of the colonic epithelium following DSS exposure. Both groups demonstrated alterations in the epithelial structure, neutrophil and lymphocyte infiltration into the mucosal and submucosal areas, and crypt loss (Fig. 3C), with control animals displaying a 1.6 fold increase in the amount of initial damage in the distal colon relative to animals expressing K-RasG12D (Fig. 3D). Following a 5-day recovery from DSS treatment, both experimental groups exhibited features of tissue repair, with K-Ras mutant animals exhibiting a more dramatic recovery in epithelial morphology than animals expressing WT K-Ras (Fig. 3D). These results suggest that animals expressing mutant K-Ras are able to recover from epithelial damage more efficiently than are control animals.
    Discussion Proper intestinal homeostasis requires tight regulation of proliferation in epithelial stem and progenitor cells. Nevertheless, the mechanisms controlling cell division in distinct populations of epithelial cells are not completely deciphered. We previously demonstrated that mutant K-Ras, a monomeric GTPase that functions as binary switch to control proliferation, differentiation, apoptosis, cell adhesion, and cell migration, increases the proliferative index in TACs (Haigis et al., 2008). Activating missense mutations are common in cancer and they impair GTPase activity, locking K-Ras into its GTP-bound (active) state and allowing it to constitutively activate downstream signaling (Ahearn et al., 2012). Expression of mutant K-Ras also accelerates the division rates of Lgr5+ stem cells, leading to clonal expansion of crypts expressing the oncoprotein (Snippert et al., 2014), suggesting that isolated mutational activation of K-Ras could lead to field effects prior to neoplastic initiation. Here, we have found that K-RasG12D also enhances proliferation of intestinal stem cells that are normally quiescent. These quiescent cells have been shown to differentiate into Paneth cells and enteroendocrine cells under homeostatic conditions, and then to give rise to all epithelial lineages following damage (Buczacki et al., 2013). Interestingly, small intestinal crypts expressing mutant K-Ras do not develop Paneth cells (Feng et al., 2011), suggesting that the quiescent state may be important for maturation into this differentiated cell type.