Making the Case for DCC and UNC5C as Tumor-Suppressor Genes in the Colon

December 2007

16. Van der Bij AK, Kloosterboer N, Prins M, et al. GB Virus C coinfection and HIV-1 disease progression: The Amsterdam cohort study. J Infect Dis 2005;191:678 – 685. 17. Berzsenyi MD, Bowden DS, Kelly HA, et al. Reduction in hepatitis C–related liver disease associated with GB virus C in human immunodeficiency virus coinfection. Gastroenterology 2007;133: 1821–1830. 18. Flynn J, Dore G, Hellard M, et al. Australian Trial in Acute HCV Study Group. Early predominant IL–10 production without IFN-g in individuals with acute HCV that progress to chronic infection. 14th International Symposium on Hepatitis C Virus and Related Viruses 2007; Abstract O-19. 19. Barbarin V, Xing Z, Delos M, et al. Pulmonary overexpression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles. Am J Physiol Lung Cell Mol Physiol 2005;288:841– 848. 20. Nelson DR, Tu Z, Soldevila-Pico C, et al. Long-term interleukin 10 therapy in chronic hepatitis C patients has a proviral and antiinflammatory effect. Hepatology 2003;38:859 – 868 21. Nunnari G, Nigro L, Palermo F, et al. Slower progression of HIV-1-infection in persons with GB virus C co-infection correlates with an intact T-helper 1 cytokine profile. Ann Intern Med 2003; 139:26 –30. 22. Xiang J, Rydze RA, Chang Q, et al. GB virus C infection and NS5A protein promote a Th1 cytokine profile in lymphocytes. General 107th Meeting of the American Society of Microbiology, 2007. 23. Clerici M, Shearer G. The Th1-Th2 hypothesis of HIV infection: new insights. Immunol Today 1994;15:575–581.

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24. Stapleton JT, Williams CF, Xiang J. GB virus C: a beneficial infection? J Clin Microbiol 2004;42:3915–3919. 25. Maidana Giret MT, Silva TM, Levi JE, et al. GBV-C infection is associated with less T cell activation in recently HIV-infected subjects and is independent of HIV-1 viral load. 4th IAS Conf HIV Pathog Treat 2007; Abstract No. MOAA105. 26. Xiang J, McLinden JH, Chang Q, et al. Identification of a 16 amino acid fragment within GBV-C NS5A protein that is required for HIV inhibition and downregulation of CD4 and CXCR4 in primary and transformed CD4⫹ T cells. 4th IAS Conf HIV Pathog Treat 2007; Abstract No. TUPDA05. 27. Brau N, Salvatore M, Rios-Bedoya CF, et al. Slower fibrosis progression of hepatitis C virus-related chronic liver disease in human immunodeficiency-coinfected patients with successful HIV suppression using antiretroviral therapy. J Hepatol 2006;44:47–55. 28. Margolis L. Cytokines—strategic weapons in germ warfare? Nat Biotechnol 2003;21:15–16.

Address requests for reprints to: Jack Stapleton, MD, Director, Division of Infectious Diseases, Department of Internal Medicine, The University of Iowa and Iowa City VA Medical Center. Iowa City, Iowa 52242. e-mail: [email protected] Supported by NIAID grant RO1 AI58740. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.10.032

Making the Case for DCC and UNC5C as Tumor-Suppressor Genes in the Colon

See “Epigenetic and genetic alterations in Netrin-1 receptors UNC5C and DCC in human colon cancer” by Shin SK, Nagasaka T, Jung BH, et al on page 1849; and “Inactivation of the UNC5C Netrin-1 receptor is associated with tumor progression in colorectal malignancies” by Bernet A, Mazelin L, Coissieux M-M, et al, on page 1840.

I

t is well established that DNA alterations in epithelial cells in the colon play a pivotal role in the pathogenesis of colorectal cancer (CRC). The discovery of frequent and recurring allelic imbalance events (termed loss of heterozygosity [LOH]) and oncogenic mutations in colorectal neoplasms firmly established the concept that the accumulation of DNA mutations is a fundamental aspect of CRC formation that is true of cancer in general. One of the most common LOH events originally discovered in CRC is the loss of a region of chromosome 18q21.1 The 18q21 LOH is commonly found in advanced CRCs, and the genes in this locus have been considered to be important tumor-suppressor genes because of the frequency of LOH at this locus.

One of the first genes discovered in this region was DCC (which is Deleted in CRC), which led to intense research activity to determine whether this gene was the tumorsuppressor gene that was targeted by the LOH events on chromosome 18. However, a relative dearth of mutations in DCC and the lack of a CRC phenotype in mice carrying heterozygous inactivating mutations in Dcc, even when crossed with mice carrying Apc mutations, raised questions about DCC’s role in CRC.2– 4 Indeed, these data and the discovery of other plausible tumor-suppressor genes in the 18q21 locus, namely SMAD2 and SMAD4, led to waning enthusiasm for DCC as a legitimate tumor-suppressor gene in the colon. However, interest in DCC as a tumor-suppressor gene was revived with the discovery that DCC is a netrin-1– dependence receptor that can regulate apoptosis and also likely plays a role in directing cell motility.5,6 This discovery of DCC’s role in apoptosis led to an appreciation that DCC was most likely involved in tumor suppression through its function as a netrin receptor rather than as a modulator of cell– cell adhesion, which had been predicted by its similarity to NCAM family members.3 In fact, in retrospect, it appears as though at least some of the controversy related to DCC’s status as a tumor-suppressor gene

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is the consequence of a lack of understanding related to its biological role in epithelial cells. In this issue of GASTROENTEROLOGY, studies by Shin et al7 and Bernet et al8 provide more evidence that makes the case for DCC and UNC5C (another member of the netrin receptor family) being tumor suppressors in the colon. In independent studies, both groups found that the netrin receptors are often inactivated in colon cancer. Through a careful analysis of genetic and epigenetic alterations in DCC and UNC5C, the research teams led by Goel and by Mehlen have discovered that DCC LOH and loss of expression of UNC5C through LOH or aberrant methylation occur commonly in colorectal neoplasms. Moreover, and perhaps of most interest, they provide evidence that inactivation of the netrin receptors may affect both the establishment and progression phases of colon cancer formation. This is exciting news because DCC loss/18q21 LOH has been predominantly found in advanced CRCs and metastases, which has led to the belief that the netrin receptors are most likely playing a role in the invasive and metastatic behavior of CRC and not in the initiation of CRC formation.3,9 –11 The results of the study by Shin et al7 and the demonstration that netrin-1 may be a regulator of cell proliferation and apoptosis in the intestinal epithelium provides evidence that targeting netrin signaling may not only have therapeutic potential in advanced CRC but also in early cancers or even advanced adenomas.7,12,13 The netrins and netrin receptors. To appreciate the significance of the studies by Shin et al7 and Bernet et al,8 it is essential to understand the biology of netrin signaling in epithelial cells. The discovery of the netrin family was made over a decade ago through the genetic analysis of nematode mutants with defects in the unc-6 gene, which led to the identification of netrin-1, the vertebrate homolog of UNC6, as a diffusible protein that could attract commissural axons.14,15 Netrin-1 is 1 member of a family of laminin-related secreted proteins that include netrin-1, netrin-2, netrin-G1, netrin G2, and netrin-4/␤-netrin, which all signal through a class of immunoglobulin-like transmembrane receptors.16 These receptors are divided into 2 main families of type I receptors, which include DCC and its homolog neogenin, and the UNC5H (UNC5 homolog) receptors, which include UNC5A, UNC5B, UNC5C, and UNC5D (also known as UNC5H1-4). The identification of this family of ligands and receptors in the developing nervous system suggested that they mainly regulated neuron outgrowth. However, it is now clear netrin signaling can affect sprouting angiogenesis, cell motility, and apoptosis and that the netrin ligands and receptors are expressed widely throughout the body demonstrating that they are involved in a broad variety of cellular processes in epithelial cells as well as endothelial cells.17,18 Indeed, the netrin receptors have been shown to regulate morpho-

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genesis of endothelial cells, induce cytoskeletal reorganization of vascular smooth muscle cells, and control adhesion and migration of epithelial cells in the lungs, pancreas, and colon.18,19 Netrin receptors and cancer: The complicated story of dependence receptors and their role in cancer. A major question related to the studies of DCC in

CRC is why has it been so difficult to establish the role of DCC and the netrin receptor family members in the pathogenesis of colon neoplasms. This is in part because of the complex and fascinating biology of this family of ligands and receptors. Unlike with classic receptor biology in which a receptor is in the “off” position when not bound with a ligand, the netrin receptors are a class of receptors, called dependence receptors, that are biologically active in both the ligand bound (“on”) and unbound (“off”) state.20 Dependence receptors include not only the netrin receptors, but also the androgen receptor AR, RET, and PTCH as well as others.21–23 These dependence receptors all share the quality that, when bound to their specific ligand, they typically transmit positive signals of proliferation, differentiation, migration, and so on, and when unbound induce apoptosis13 (Figure 1). For instance, when DCC is not bound to netrin-1, it triggers apoptosis in epithelial cells through caspase-dependent mechanisms.24 Interestingly, both DCC and UNC5H appear to require caspase cleavage to expose a proapoptotic domain named addiction dependence domain (ADD) in the intracellular domains of the proteins; however, DCC appears to trigger the activation of caspase-9, whereas UNC5C appears to regulate caspase-mediated apoptosis through other mechanisms such as death-associated protein kinase (DAPK) or NRAGE, a member of the MAGE (melanoma antigen) family of apoptosis regulators.20,25–27 In contrast and in keeping with the Jekyll-and-Hyde nature that characterizes this family of receptors, when the netrin receptors are engaged, they mediate a set of effects that would be considered oncogenic, such as the induction of cell migration and cytoskeleton reorganization.16,19 In retrospect, it is now clear that the initial studies that focused attention strictly on DCC neglected key aspects that affect the biological consequences of DCC inactivation. It has only been through study of the bound and unbound receptors that we have begun to understand how DCC and its family members might act as tumorsuppressor genes in the colon. The evolving understanding of the role of netrins and the netrin receptors in CRC. Support for DCC as a

tumor-suppressor gene in the colon has been tumultuous at best and definitive evidence for DCC as a colon cancer tumor-suppressor gene remains to be shown. The initial discovery of DCC in the 18q21 locus led to the expectation that it would be quickly proven a bona fide tumor-suppres-

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Figure 1. Schematic representation of the signaling pathways and biological effects induced when DCC and UNC5C are bound and not bound by netrin. The receptors can activate a variety of pathways when bound to netrin that regulate different biological events. In the unbound state, both DCC and UNC5C can induce apoptosis through caspase-mediated cleavage and exposure of the ADD. Both receptors have extracellular domains that are composed of immunoglobulin-like repeats (half circles). In addition, DCC has 6 extracellular fibronectin type III-like repeats (rectangles) and a cytoplasmic dependence domain (cylinder). UNC5C has 2 extracellular thrombospondin-like repeats (rectangles), a cytoplasmic zona occludens-1 domain (oval), and a death domain (cylinder). Abbreviations: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; PI3K, phosphoinositide 3 kinase. APPL, adaptor protein containing pH domain, PTB domain. (Color version of this figure available online at www.gastrojournal.org)

sor gene. However, the discovery that only 10%–15% of colon cancers carry mutations in DCC and the lack of an effect of inactivation of Dcc in mouse models to affect tumor formation suggested that DCC had little to no biological role in colon cancer. The elegant studies from the research groups led by Goel and by Mehlen in this issue of GASTROENTEROLOGY as well as other studies of netrin and UNC5C in CRC have garnered evidence in support of DCC and the netrins and netrin receptors as being biologically important tumor-suppressor genes in the colon.7,10,12,16,28 Shin et al7 have shown that the aberrant methylation of UNC5C silences its expression in cell lines and that UNC5C methylation is common in colon cancer and occurs in adenomas, as well as adenocarcinomas. Indeed, the studies by both Shin et al7 and Bernet et al8 as well as prior studies showing the reduced expression of UNC5A, UNC5B, and UNC5C in 48%, 27%, and 74%–77% of CRCs, respectively, reinforce the concept that DCC and UNC5C are legitimate tumor-suppressor genes.28 Furthermore, Mazelin et al12 found that approximately 7% of colon cancers overexpress the DCC ligand, netrin-1, and that induced overexpression of netrin-1 in the colon results in decreased apoptosis and

histologically advanced adenomas when crossed with mice that carry a germline mutation in Apc (Apc1638N/wt).12 These studies are in stark contrast to those in which mice that carry inactivating mutations in both Apc and Dcc do not develop colon neoplasms.4 Our current understanding of netrin biology suggests that the differences in tumor formation between these mouse models may be the result of compensation by other netrin receptor family members. Indeed, netrin-1 may affect tumor formation through activation of alternative receptors, such as A2br or integrins or possibly through effects on the UNC5H receptors.13 Consistent with the concept that UNC5C is the critical compensating netrin receptor, Bernet et al8 have shown mice that carry an Apc1638N germline mutation and that are heterozygous or homozygous for mutant Unc5c develop adenomas that progress to adenocarcinoma at a higher frequency than seen in the Apc1638N mice.8 A2b receptor activation is another compelling possibility given that this receptor can activate a variety of pathways, including RhoROCK and PI3K, which have been shown to be activated by netrin-1. Netrin-1 can activate a variety of other pathways that can induce proinvasive behaviors, including FAK and

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PKA, with the determination of which pathways are activated likely depending on the specific netrin receptors that are present on the cell membrane.29,30 Particularly interesting findings by Shin et al7 are that DCC and UNC5C inactivation occur concurrently in roughly half of all colon cancers and that UNC5C loss appears to precede DCC loss in the polyp¡cancer progression sequence.7 Questions that these findings raise are as follows: (1) What is the selective pressure for inactivating multiple receptors that bind netrins? and (2) Why does UNC5C loss occur early and DCC loss occur late in colon cancer formation if both are netrin receptors? One answer to these questions can be found by assessing the biological consequences of activating the UNC5 receptors vs. DCC receptor. Genetic and biochemical studies indicate that DCC usually mediates attractive responses to netrin, whereas UNC5 mediates repulsive responses to netrin.31 Even of potentially more interest, it appears that the 2 receptors can interact at a biological level and that DCC only mediates attractive responses in the absence of UNC5 and that UNC5 can convert DCC-mediated attraction to repulsion.32 Thus, the finding that UNC5C is silenced by aberrant methylation in the adenoma phase of colon cancer formation suggests that DCC can then mediate attractive responses until it is eventually lost in the advanced cancers. Furthermore, the UNC5 receptors appear to have some unique biological effects not observed with DCC, such as on angiogenesis, creating the potential for differential and potentially additive tumor-promoting effects by inactivating UNC5C and DCC.13,17,31 In addition, the UNC5 receptors and DCC appear to induce different signaling pathways and to be regulated by different pathways, which could result in a growth advantage for a developing tumor to inactivate both netrin receptor family members.32 What remains to be proven about the role of DCC and UNC5 in CRC? The studies by Bernet et al8 and by

Shin et al7 provide clear evidence that UNC5C is inactivated in colon cancer, but they also raise several questions. Given that DCC and UNC5C are dependence receptors, it would be interesting to know the concurrent expression levels of the whole family of netrin ligands as well as the expression of the other netrin receptors UNC5A, UNC5B, and neogenin. It would also be interesting to know what effect inactivation of the receptors has on apoptosis and proliferation in primary tumors in humans as well as on markers of invasive behavior of the tumors. Furthermore, determination of which of these effects requires inactivation of both or just one of the receptors is needed to understand how netrin signaling deregulation affects colon cancer formation. The studies by Bernet et al8 and Shin et al7 have provided compelling evidence to justify further study of the pathophysiologic effects of UNC5C and DCC inactivation in the colon. These studies are critical if the netrins and their receptors are ever to be successfully used for targeted therapies.

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WILLIAM M. GRADY Clinical Research Division Fred Hutchinson Cancer Research Center Department of Medicine University of Washington Medical School R&D Service VA Puget Sound Health Care System Seattle, Washington References 1. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319: 525–532. 2. Cho KR, Oliner JD, Simons JW, et al. The DCC gene: structural analysis and mutations in colorectal carcinomas. Genomics 1994;19:525–531. 3. Mehlen P, Fearon ER. Role of the dependence receptor DCC in colorectal cancer pathogenesis. J Clin Oncol 2004;22:3420 – 3428. 4. Fazeli A, Dickinson SL, Hermiston ML, et al. Phenotype of mice lacking functional Deleted in colorectal cancer (Dcc) gene. Nature 1997;386:796 – 804. 5. Pierceall WE, Reale MA, Candia AF, et al. Expression of a homologue of the deleted in colorectal cancer (DCC) gene in the nervous system of developing Xenopus embryos. Dev Biol 1994; 166:654 – 665. 6. Keino-Masu K, Masu M, Hinck L, et al. Deleted in colorectal cancer (DCC) encodes a netrin receptor. Cell 1996;87:175–185. 7. Shin SK, Nagasaka T, Jung BH, et al. Epigenetic and genetic alterations in Netrin-1 receptors UNC5C and DCC in human colon cancer. Gastroenterology 2007;133:1849 –1857. 8. Bernet A, Mazelin L, Coissieux M-M, et al. Inactivation of the UNC5C Netrin-1 receptor is associated with tumor progression in colorectal malignancies. Gastroenterology 2007;133: 1840 –1848. 9. Vogelstein B, Fearon ER, Kern SE, et al. Allelotype of colorectal carcinomas. Science 1989;244:207–211. 10. Fearon ER, Cho KR, Nigro JM, et al. Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 1990;247:49 –56. 11. Watanabe T, Wu T-T, Catalano PJ, et al. Molecular Predictors of survival after adjuvant chemotherapy for colon cancer. N Engl J Med 2001;344:1196 –1206. 12. Mazelin L, Bernet A, Bonod-Bidaud C, et al. Netrin-1 controls colorectal tumorigenesis by regulating apoptosis. Nature 2004; 431:80 – 84. 13. Mehlen P, Llambi F. Role of netrin-1 and netrin-1 dependence receptors in colorectal cancers. Br J Cancer 2005;93:1– 6. 14. Hedgecock EM, Culotti JG, Hall DH. The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 1990; 4:61– 85. 15. Serafini T, Kennedy TE, Galko MJ, et al. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 1994;78:409 – 424. 16. Rodrigues S, De Wever O, Bruyneel E, et al. Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis. Oncogene 2007;26: 5615–5625. 17. Larrivee B, Freitas C, Trombe M, et al. Activation of the UNC5B receptor by Netrin-1 inhibits sprouting angiogenesis. Genes Dev 2007;21:2433–2447. 18. Wilson BD, Ii M, Park KW, et al. Netrins promote developmental and therapeutic angiogenesis. Science 2006;313:640 – 644.

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19. Shekarabi M, Kennedy TE. The netrin-1 receptor DCC promotes filopodia formation and cell spreading by activating Cdc42 and Rac1. Mol Cell Neurosci 2002;19:1–17. 20. Mehlen P, Bredesen DE. The dependence receptor hypothesis. Apoptosis 2004;9:37– 49. 21. Ellerby LM, Hackam AS, Propp SS, et al. Kennedy’s disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity. J Neurochem 1999;72:185–195. 22. Bordeaux MC, Forcet C, Granger L, et al. The RET proto-oncogene induces apoptosis: a novel mechanism for Hirschsprung disease. EMBO J 2000;19:4056 – 4063. 23. Thibert C, Teillet MA, Lapointe F, et al. Inhibition of neuroepithelial patched-induced apoptosis by sonic hedgehog. Science 2003;301:843– 846. 24. Mehlen P, Rabizadeh S, Snipas SJ, et al. The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis. Nature 1998;395:801– 804. 25. Llambi F, Lourenco FC, Gozuacik D, et al. The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase. EMBO J 2005;24:1192–1201. 26. Williams ME, Strickland P, Watanabe K, et al. UNC5H1 induces apoptosis via its juxtamembrane region through an interaction with NRAGE. J Biol Chem 2003;278:17483–17490. 27. Forcet C, Ye X, Granger L, et al. The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation. Proc Natl Acad Sci U S A 2001;98:3416 – 3421.

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28. Thiebault K, Mazelin L, Pays L, et al. The netrin-1 receptors UNC5H are putative tumor suppressors controlling cell death commitment. Proc Natl Acad Sci U S A 2003;100:4173– 4178. 29. Nguyen QD, De Wever O, Bruyneel E, et al. Commutators of PAR-1 signaling in cancer cell invasion reveal an essential role of the Rho-Rho kinase axis and tumor microenvironment. Oncogene 2005;24:8240 – 8251. 30. Yebra M, Montgomery AM, Diaferia GR, et al. Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration. Dev Cell 2003;5:695–707. 31. Geisbrecht BV, Dowd KA, Barfield RW, et al. Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin. J Biol Chem 2003;278:32561–32568. 32. Hong K, Hinck L, Nishiyama M, et al. A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell 1999;97:927–941.

Address requests for reprints to: William M. Grady, MD, Fred Hutchinson Cancer Research Center 1100 Fairview Ave N. D4-100, Seattle, Washington 98109. e-mail: [email protected]; fax: (206) 6672917. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.10.034

Colonic Neutrophils in Inflammatory Bowel Disease: Double-Edged Swords of the Innate Immune System With Protective and Destructive Capacity

See “Aggravation of different types of experimental colitis by depletion or adhesion blockage of neutrophils” by Kühl AA, Kakirman H, Janotta M, et al, on page 1882.

S

everal forms of active inflammatory bowel disease (IBD) feature the presence of a mixed inflammatory infiltrate that includes neutrophils. A large influx of neutrophils can lead to the formation of crypt abscesses, a characteristic histologic lesion resulting from activated neutrophils transmigrating through the paracellular space separating enterocytes, breaching tight junctions at the apical aspect of the lateral surface of epithelial cells, and accumulating in the crypt lumen. Because such lesions and the presence of neutrophils in stool samples correlate with the presence of active IBD, neutrophils have been proposed to be effector cells capable of conspiring with lymphocytes to bring about the epithelial injury and dysfunction that are associated with IBD. Neutrophils clearly have a major contributing role to the development of many types of tissue damage; one

example is ischemia–reperfusion injury. Tissue damage results when neutrophils are activated to release granule contents into the local microenvironment in concert with elaboration of reactive oxygen species generated through the action of a membrane-associated NADPH oxidase. Chemokine-mediated recruitment of neutrophils to a specific tissue site without concurrent activation of the recruited neutrophils is not sufficient to cause tissue damage. This was demonstrated in vivo using a transgenic mouse model featuring doxycycline-inducible expression of human interleukin (IL)-8 in intestinal epithelium in the ileum and cecum.1 Foci of dense neutrophil infiltration in the mucosa were achieved after doxycycline induction, but these areas were not associated with epithelial damage, presumably due because the neutrophils were not activated sufficiently by the attracting chemokine (IL-8) alone and had not breached epithelial tight junctions. The development of in vitro model systems to study the interactions of neutrophils with intestinal epithelial cells has contributed to the identification of novel mechanisms by which neutrophils and epithelial cells communicate when they are brought into proximity.2 For