The mechanisms underlying the development and

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1 mtor Regulation by JNK: Rescuing the Starving Intestinal Cancer Cell? See JNK signaling promotes intestinal tumorigenesis through activation of mtor complex 1inAPC 716 mice, by Fujishita T, Aoki M, and Taketo MM, on page The mechanisms underlying the development and progression of colorectal cancer (CRC) are being unraveled using a combination of novel genetic and biochemical approaches. Recent studies of human CRCs have revealed that large numbers of genes are mutated and involve the crosstalk of numerous cooperating signaling pathways. 1 Dysregulation of Wnt signaling through loss of APC and the subsequent failure of a protein complex comprising Apc/Axin/GSK-3 to target -catenin for degradation by the proteasome is clearly the key initiating event for approximately 60% 80% of sporadic CRCs and provides the context in which these additional pathways must act to promote tumor growth. One such recognized pathway is the PI3KCA/ AKT/mammalian target of rapamycin (mtor) pathway, of which the FRAP gene product (mtor) acts as the effector arm to mediate oncogenic transformation. Cancers show genetic dysregulation of this pathway at a number of points. Around 25% of CRCs show gain-offunction, oncogenic mutations of PI3KCA. 2 The tumor suppressor gene PTEN antagonizes PI3K and is downregulated or lost in CRCs. Moreover, in the familial hamartomatous Peutz Jeghers and tuberous sclerosis syndromes, germline mutations in the LKB1 and TSC2 genes respectively, act to inhibit mtor. mtor is a member of the family of phosphoinositide- 3-kinase (PI3K)-related kinases and responds to many different stresses including nutrient, energy, and oxygen deprivation. mtor exists in 2 complexes, mtorc1 and mtorc2. mtorc1 is the most well-characterized and resides as a complex of mtor, Raptor, Deptor, PRAS40, and mlst8. mtor is directly activated by Rheb, a small GTPase, which is in turn regulated by the TSC1/2 tumor suppressor complex (Figure 1A). TSC2 inhibits mtor function under both hypoxic and energy-deprived conditions, and activation of TSC1/2 is determined by its phosphorylation status. Depending on the sites that have been phosphorylated, the ability of TSC2 to act as a GTPase-activating protein (GAP) toward Rheb can be either inhibited or activated. Rheb is a member of the small GTPase family and shuttles between a GDP-bound form and a GTP-bound form. Inhibition of TSC2 GAP activity maintains the active form of Rheb (GTP), which stimulates the mtorc1 complex. Phosphorylation of TSC1 at S939 and S981 by AKT generates binding sites for interaction with the protein, and subsequent binding of TSC2 with triggers its interaction with Dishevelled, preventing its interaction with TSC1. 3 Raptor has a variety of functions, including regulating the assembly of mtorc1 and recruiting substrates such as 4EBP1. 4EBP1 represses cap-dependent protein translation by sequestering eif4e, an essential subunit of the eif4f complex that binds the mrna cap. 4 Proliferative stimuli trigger phosphorylation of 4EBP1 by mtor and additional kinases, resulting in the dissociation of eif4e and promoting translation. Activated mtor also phosphorylates S6 kinase (S6K) at T389, which enables it to phosphorylate the 40S ribosomal protein S6 (Figure 1A). 5 In adverse conditions, mtorc1 signaling is inhibited, and autophagy, the process whereby cellular components are recycled for new synthesis, is initiated. mtor is also involved in transcription; inhibition of mtor has been shown to block Pol I and III, thus inhibiting the synthesis of rrnas and trnas. 6,7 In addition to the upstream regulators P13KCA and PTEN, mutations in individual components in the mtor signaling pathway have been noted, for example, in TSC2 in renal cell carcinoma and S6K in breast carcinomas. 8,9 4EBP1 and eif4e are frequently overexpressed in CRC; nonetheless, 4EBP1 levels are markedly higher in patients with little or no metastatic disease, possibly suggesting that this antagonist of cap-dependent translation is up-regulated as part of a negative feedback mechanism that is partially able to restrict cancer progression. 10 mtor was found to be up-regulated in CRC and adenomas, and mtor and S6K were significantly over-expressed in CRCs compared with matched normal tissue. 11,12 Indeed, in 1 study 40% of CRC patients exhibited activation of the mtorc1 pathway. 13 Furthermore, FBXW7, a ubiquitin ligase that binds to mtor to promote its targeting for degradation by ubiquitination, is inactivated in CRC and may result in mtor protein stabilization. 14,15 Treatment with the mtorc1 inhibitor RAD001, a rapamycin analogue, dramatically inhibits intestinal tumorigenesis and extends survival times in both the Apc min and Apc 716 mouse models. Wnt inhibition of GSK-3 may activate mtor signaling in certain contexts (Figure 1A). Inoki et al 16 demonstrated that AMPK primes TSC2 at S1345 for subsequent phosphorylation by GSK-3. They also showed that GSK-3 inactivation was required for mtor activity by over-expressing constitutively active GSK-3 in actively 1387

2 Figure 1. Network interactions between Wnt, AKT/mTOR, and JNK pathways. (A) The activated mtor complex promotes translation and ribosome biosynthesis, and lies downstream of Akt regulation of the TSC1/2 complex. There is substantial modulation of this pathway by components of the Wnt pathway, including GSK-3 and Dvl2. Note the positive feedback provided by the S6 Kinase (S6K) activated by mtor. The newly recognized role for JNK in regulating the mtor pathway is both direct (via Raptor) and indirect (via S6K). (B) As well as collaborating to regulate mtor, the Wnt and JNK pathways operate synergistically to regulate common transcription targets. proliferating cells to block S6K activation, and found that knockdown or chemical inhibition of GSK-3 was sufficient to stimulate mtor/s6k. Further suggestion of a role for Wnt signaling in the regulation of mtor came from the demonstration by the Taketo laboratory that mtor signaling was elevated in the adenomas of mice carrying the Apc 716 mutation. 17 The authors demonstrated that knockdown of -catenin in SW480 human colon carcinoma cells reduced both mtor expression and S6 phosphorylation. Mediators of Wnt signaling such as the scaffolding protein Dishevelled (Dvl2) may regulate mtor. Over-expressed Dvl2 seems to transduce Wnt signals to -catenin via a receptor complex at the plasma membrane, and is able to activate -catenin independently of Wnt ligand by recruitment of Axin and inhibition of GSK3. Moreover, the deletion of Dvl2 in mice phenocopies attenuation of mtor signaling to cause a reduction of gut length and cell size, and inhibits adenoma formation on Apc min mice. 18 Importantly, Wnt signaling can activate several noncanonical pathways, such as the c-jun N-terminal kinase (JNK) signaling cascade. 19,20 The JNK proteins are members of the superfamily of mitogen-activated protein kinases, and are phosphorylated and activated by upstream kinases MKK4 and MKK7 in response to cellular stresses, proinflammatory cytokines, and heat shock. JNK serves to regulate a variety of processes including proliferation, differentiation, migration, and cell death. JNK was first identified by its ability to phosphorylate specific sites on the N-terminal transactivation domain of the transcription factor c-jun after exposure of cells to ultraviolet irradiation or expression of transforming oncogenes, and c-jun is routinely used as the model substrate for JNK signaling. 21 Upstream activators of the JNK pathway remain open to investigation and some debate, but include the Rho subfamily of small GTPases and Ste20-related protein kinases. Scaffold proteins have also been implicated in activation of the JNK pathway. The number and size of polyps in Apc min intestine were reduced by the introduction of c-jun with mutated phosphorylation sites at the N-terminus (S63/73A). 22 This either could be due to inhibition of c-jun transcriptional activity or increased c-jun turnover after protein destabilization, resulting in an inability of c-jun to form heterodimers with other AP-1 proteins. With respect to the Wnt pathway, Axin induces JNK activity through binding to MEKK1 via a domain distinct from those involved in Wnt signaling, termed the MEKK1-interacting domain (Figure 1B). 23 High JNK activity has also been shown to increase the strength of Wnt signaling by stimulating the Wnt target genes Axin2 and Lgr5. 24 ChIP analysis has demonstrated that c-jun binds several Wnt promoters, and the TCF4 promoter region has been identified as a target for 1388

3 phosphorylated c-jun. 25,26 Reciprocally, TCF4 and -catenin have been identified as novel partners of c-jun and form a complex in a JNK-dependent manner to induce the expression of common target genes such as CD44, c-jun, and MMP7 (Figure 1B). 22,27 Thus, the Wnt and JNK signaling pathways operate both in parallel and synergistically. The role of JNK signaling in CRC has been welldocumented in recent years, and there is mounting evidence in support of up-regulated JNK activation in intestinal tumors. Conditional activation of JNK1 specifically in the intestine increased cell proliferation. 24 Induction of JNK activity is antagonized by FBXW7, a tumor suppressor known to be inactivated in CRC (Figure 1B). 28 Similarly, PDCD4, also a putative tumor suppressor gene, inhibits the activity of JNK. 29 PDCD4 is frequently down-regulated in human CRC and HT29 cells and results in -catenin, TCF, and AP-1 dependent transcription. 30 Pharmacologic inhibition of JNK reduced the growth of HCT116 adenocarcinoma cells. 24 Conversely, JNK1 / mice developed spontaneous intestinal tumors in 1 study; however, this knockdown was not localized to the intestinal epithelium and could have caused organism-wide defects that resulted in intestinal tumors. 31 In this issue of GASTROENTEROLOGY, Fujishita et al 32 have applied multiple approaches to demonstrate a clear chain of causality that links mtor activation to the JNK pathway and independently of constitutive Wnt signaling in intestinal tumors. The authors noted that the phenotype of JNK-deficient mice resembled that of S6K mutants and set out to investigate the role of JNK in mtordriven intestinal tumorigenesis. Fujishita et al 32 report that the mtorc1 inhibitor RAD001 that is known to restrict tumor growth in mice also inhibited the elevated JNK activation found in intestinal tumors. Additionally, treatment with the JNK inhibitor SP suppressed tumor formation. Fujishita et al 32 convincingly demonstrate that knockdown of JNK significantly affects translation by combining this pharmacologic inhibition with kinase strategies, shrna knockdown, and site-specific mutation of key phosphorylation sites in Raptor. JNK activation was associated with up-regulated mtorc1 signaling in human colorectal adenocarcinoma cell lines, SW480 and HT29. In Apc min mice, activation of Wnt occurred in both early and more developed adenomas, whereas S6K and c-jun activation was found only in larger polyps. This is suggestive of stepwise progression in the molecular events occurring during the development of intestinal tumorigenesis. Indeed, tumor stagespecific roles have been proposed for JNK and may determine if proliferation or apoptosis is the resulting output. 33 However, these data are inconsistent with findings reported by Metcalfe et al, 18 who found that the mtor substrate 4EBP1 is active in normal intestinal crypts of Apc min mice, and up-regulated in both nascent polyps and larger intestinal tumors. This may indicate different thresholds in the activation of various mtor substrates. Fujishita et al 32 found that knockdown of JNK decreased the phosphorylation level of S6K but did not completely ablate it. These data suggest that pathways may be operative at a low level alongside the JNK pathway. Furthermore, JNK can phosphorylate S6K directly at S411, whereas interferon- stimulates mtor activation of JNK. 34 JNK was shown to mediate mtor signaling directly by phosphorylating a component of the mtorc1 complex, Raptor, to induce kinase activity of the complex (Figure 1A). This event occurred downstream of the mtorc1 regulator, Rheb, perhaps indicating that JNK signaling could function in parallel with mtor signaling rather than by directly regulating it. In early lesions, mtor is likely to be inhibited by the absence of nutrient and oxygen delivery owing to lack of vasculature. However, these same stresses up-regulate JNK activity, bypassing the inhibitory signals on mtor by phosphorylating and activating Raptor to promote protein translation. Hence, the current findings provide significant evidence for the role of JNK stimulation in providing a partial rescue of mtormediated restriction on tumor cell proliferation, and signal a need for further exploration as to the mode and context of JNK signaling in developing colon cancers. Activated JNK also increased cyclin E protein levels via mtorc1 activation and cyclin E mrna levels through activation of c-jun. Interestingly, whereas genetic ablation of TSC2 in fibroblasts does not affect cyclin E levels, much lower levels of the cyclin-dependent kinase (CDK) inhibitor p27 are expressed, resulting in increased proliferation. 35 p27 binds to cyclin E/CDK2 complexes to arrest the cell cycle at G1. SGK1, an mtorc1 substrate, phosphorylates p27 at T157 to prevent its nuclear import and thus promote cell-cycle progression. 36 Therefore, the mtor and JNK pathways may act in synergy to inhibit p27 while increasing cyclin E to drive cell division. JNK signaling may also contribute to CRC progression independently of mtor by up-regulating 2 proteins implicated in angiogenesis and metastasis, namely, proliferin and osteopontin, respectively. Ultimately, the increased translation arising from activated mtor causes cellular transformation because there is nonrandom translation of mrna species that favors expression of oncogenic proteins owing to competition between mrnas at the point of initiation. Perhaps, counterintuitively, many growth factors and oncogenes arise from transcripts with long, GC rich and highly structured 5= untranslated regions that render them at a disadvantage under conditions that are limiting for translation (low mtor) compared to the bulk of cellular 1389

4 mrnas. 37 Understanding that mtor is regulated in synergy by several upstream signaling pathways, now including JNK, reinforces the suitability of mtor as a therapeutic target. However, current inhibitors are of a single type rapalog derivatives of rapamycin that act by promoting a ternary complex between rapalog, the FKBP1A protein, and mtor. (Notably, 2 inhibitors are approved by the US Food and Drug Administration for the treatment of renal cell carcinoma.) This single axis may provide a relatively nondiscriminatory intervention in manipulating the selective translation of transcripts with different properties by the highly regulated mtorc complexes. Paradoxically, the existence of rapalogs may have provided a disincentive to developing other pharmacologic inhibitors. It remains to be determined whether more imaginative manipulations of mtor complexes can improve on the current rapalogs, with JNKmediated Raptor interaction with mtorc1 providing an intriguing target. H. NIKKI MARCH DOUGLAS J. WINTON Stem Cell Biology of the Intestine Group Cancer Research-UK Cambridge Research Institute Li Ka Shing Centre Cambridge, United Kingdom References 1. Wood LD, Parsons DW, Jones S, et al. The genomic landscapes of human breast and colorectal cancers. Science 2007;318: Samuels Y, Ericson K. Oncogenic PI3K and its role in cancer. Curr Opin Oncol 2006;18: Cai SL, Tee AR, Short JD, et al. Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning. J Cell Biol 2006;173: Pain VM. Initiation of protein synthesis in eukaryotic cells. Eur J Biochem 1996;236: Park IH, Bachmann R, Shirazi H, et al. Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin. J Biol Chem 2002; 277: Mahajan PB. Modulation of transcription of rrna genes by rapamycin. Int J Immunopharmacol 1994;16: Leicht M, Simm A, Bertsch G, et al. 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5 31. Tong C, Yin Z, Song Z, et al. c-jun NH2-terminal kinase 1 plays a critical role in intestinal homeostasis and tumor suppression. Am J Pathol 2007;171: Fujishita T, Aoki M, Taketo MM. JNK signaling promotes intestinal tumorigenesis through activation of mtor complex 1 in APC 716 mice. Gastroenterology 2011;140: Engelberg D. Stress-activated protein kinases tumor suppressors or tumor initiators? Semin Cancer Biol 2004;14: Panaretakis T, Hjortsberg L, Tamm KP, et al. Interferon alpha induces nucleus-independent apoptosis by activating extracellular signal-regulated kinase 1/2 and c-jun NH2-terminal kinase downstream of phosphatidylinositol 3-kinase and mammalian target of rapamycin. Mol Biol Cell 2008;19: Soucek T, Yeung RS, Hengstschlager M. Inactivation of the cyclindependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2. Proc Natl Acad SciUSA1998;95: Hong F, Larrea MD, Doughty C, et al. mtor-raptor binds and activates SGK1 to regulate p27 phosphorylation. Mol Cell 2008; 30: De Benedetti A, Graff JR. eif-4e expression and its role in malignancies and metastases. Oncogene 2004;23: Reprint requests Address requests for reprints to: Douglas J. Winton, Stem Cell Biology of the Intestine Group, Cancer Research-UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 ORE, UK. doug.winton@cancer.org.uk; Fax: 44 (0) Conflicts of interest The authors disclose no conflicts by the AGA Institute /$36.00 doi: /j.gastro CRF and Urocortins: A Challenging Interaction Between Family Members See Activation of corticotropin-releasing factor receptor 2 mediates the colonic motor coping response to acute stress in rodents, by Gourcerol G, Wu SV, Yuan P Q, et al, on page Discord of varying degrees is common among family members, and now it seems that members of an ancient gene family are not immune to this trait either. In a study published in this issue of GASTROENTEROLOGY, Gourcerol et al 1 demonstrate that corticotropin-releasing factor (CRF) and its closely related family member urocortin 2 (Ucn2) are at odds with regard to activating their common receptor CRF 2, and their subsequent effect on colonic motility and fecal pellet output (FPO) in mice. 1 The CRF family comprises 4 ligands (CRF and Ucn1 3) and 2 known G-coupled protein receptors, CRF 1 and CRF 2. CRF binds to CRF 1 with 30-fold higher affinity than CRF 2, whereas Ucn1 binds to both receptors with equal affinity. Ucn1 binds to CRF 1 with a 6- to 10-fold higher affinity than CRF does, and Ucn2 and Ucn3 selectively bind CRF 2. 2 CRF is best known for regulating and/or initiating stress responses via activation of the hypothalamic pituitary adrenal (HPA) axis 3 and CRF 1. In contrast with CRF, the other 3 family members the recently discovered urocortins play a role in recovery responses after exposure to stress. 4 Chronic stress and stressful episodes can exacerbate symptoms of certain gastrointestinal (GI) diseases, including inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). 5 Many potential candidate gene targets for IBD have been suggested, and pharmacologic or genetic alteration of numerous genes and their encoded proteins is known to ameliorate histologic damage in animal models of experimental colitis. Are members of the CRF gene family potential game changers for GI diseases? Given the importance of CRF in regulating stress responses, it seems obvious that CRF and Ucns could be important players in IBD and IBS. Previous work has demonstrated that members of the CRF family influence gut function, including motility, permeability, and barrier function. 6 8 Physical restraint stress (that results in endogenous release of CRF and other hormones) or exogenous CRF injection are known to increase FPO and alter colonic contractions in animal models. Gourcerol et al 1 show that Ucn2 dampens FPO and colonic contractions induced by CRF or partial restraint stress (Figure 1). Ucn2 injection on its own is known to inhibit defecation, 9 and localized elimination of Ucn2 by RNA interference in the gut increases shortterm FPO 10 ; however, the current study suggests for the first time that CRF and Ucn2 can counteract each other s effects via an unknown mechanism to alter GI function. The authors show that stress and exogenous CRF injection activates neurons, as revealed by increased numbers of c-fos positive neurons in the longitudinal muscle myenteric plexus (LMMP) of proximal and/or distal colons from mice, whereas Ucn2 injection after CRF treatment decreased the numbers of activated c-fos neurons in the LMMP. Which receptor may be involved in each of 1391