The DCC Protein -- NEURAL DEVELOPMENT AND THE MALIGNANT PROCESS

Kimberly M. Rieger-Christ, Karina L. Brierley and Michael A. Reale

Department of Internal Medicine/Oncology, Yale School of Medicine/West Haven Veterans Administration Medical Center, 333 Cedar St., P.O. Box 208032, New Haven, CT 06520-8032

Received 8/25/97 Accepted 9/5/97

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. DCC and Development

3.1. DCC guides axonal migrations

3.2 DCC guides cell migrations

3.3. Summary

4. DCC and Cancer

4.1. 18q Allelic loss and DCC expression studies

4.2. Experimental approaches

4.3. Summary

5. Perspective

6. Acknowledgments

7. References

1. ABSTRACT

The deleted in colorectal cancer (DCC) gene encodes a neural cell adhesion family molecule that was originally identified as a candidate tumor suppressor target of 18q allelic loss in colorectal cancer. However, the importance of the DCC protein has been most clearly demonstrated in neural development. Mutational and subsequent biochemical studies in C. elegans, Drosophila and vertebrates have shown that DCC functions in the guided migration of cells and cell processes in response to stimuli from netrins, a family of secreted laminin-like proteins. It appears that DCC may act in this signal transduction pathway as a netrin receptor or a component of the receptor complex, though a definitive receptor:ligand relationship has not yet been demonstrated. It is also clear that DCC can affect migrations in a netrin-independent manner, implying the existence of other DCC ligands. Though the loss of DCC expression appears to be a later event in several malignancies and is associated with disease dissemination, it has not been adequately demonstrated that DCC is the tumor suppressor gene targeted by 18q allelic loss. However, DCC expression does have potential clinical utility as it stratifies an important group of colorectal cancer patients into good and poor prognosis subgroups.

2. INTRODUCTION

The DCC (deleted in colorectal cancer) gene was originally identified through studies of the progressive stages of human colorectal tumor development. These studies demonstrated allelic loss involving a region of the long arm of chromosome 18 in approximately 50% of class III adenomas, greater than 70% of carcinomas and nearly 100% of hepatic metastases (1,2). The common region of loss at 18q21.3 was found to contain the extraordinarily large DCC gene (1.35 megabases, 29 exons) (3-4). The sequence of the DCC cDNA predicts a 1447 amino acid transmembrane protein with extracellular immunoglobulin and fibronectin type III domains typical of the neural cell adhesion molecule (NCAM) family of proteins (4-6). DCC and neogenin, a protein whose expression is dynamically regulated in chicken neural development, define an NCAM subfamily on the basis of their unique constellation of extracellular domain motifs (six immunoglobulin and four fibronectin type III domains) and cytoplasmic domain (7). Their approximately 325 amino acid cytoplasmic domains do not share similarity with any other proteins in the data base (Figure 1). Several DCC homologs have been isolated and cloned from both invertebrates and vertebrates (8-12). DCC has been highly conserved in vertebrate evolution as the sequence predicted by the human cDNA shares co-linearity and greater than 75% overall identity at the amino acid level to the cDNA for a Xenopus laevis homologue of DCC (12) (Figure 1).

Figure 1. The DCC family of proteins. Percent identity with human DCC at the amino acid level is represented for each of the three major domains. (TM - transmembrane).

The expression pattern of the DCC gene in adult tissues is informative as levels of expression in the central and peripheral nervous system far exceed those of non-neural tissues (5,6,11,12). Alternative splicing involving both the extracellular and cytoplasmic domains of the DCC protein has been described, though the functional significance of these splicing events is not yet understood (6,11,13).

3. DCC AND DEVELOPMENT

Studies in the developing chicken have implicated DCC in the epithelial-mesenchymal interactions of feather bud formation and have begun to characterize its expression in developing epithelia (14). However, the majority of studies have focused on DCC in neural differentiation and development. DCC antisense and overexpression studies in the rat pheochromocytoma (PC12) model system have suggested a role for DCC in the induction of neuronal differentiation (15-16). Work in the mouse (11) and Xenopus laevis (12) have demonstrated that DCC expression is highest in the neural tube and developing forebrain, midbrain and hindbrain, and this expression is developmentally regulated. Moreover, DCC was expressed as a consequence of neural induction in the Xenopus system. These studies suggested an important functional role for DCC in developing neural tissue, a role that would be characterized in the studies described below.

3.1. DCC guides axonal migrations

The guided migration of axons to connect with a specific target cell is a fundamental process in developmental neurobiology. It has become increasingly clear that this process involves both attractive and repulsive environmental cues that act via signal transduction mechanisms to assist the growth cone in finding its proper pathway (17-18). The netrins are one such family of guidance molecules. Netrins were originally identified as the activity in rat floor plate cells that can promote the outgrowth of commissural axons from rat dorsal spinal cord explants (19-20). These proteins constitute a family of highly phylogenetically conserved molecules related to the extracellular matrix protein, laminin (20-21).

Figure 2. Summary of mutational studies of commissural axon migration in nematodes (C. Elegans). Thick horizontal bars indicate a major inhibitory effect, while the thin horizontal bar indicates a lesser effect of DCC/unc-40 on dorsal axonal migrations [adapted from Drescher (26) and Goodman ].

The netrin response pathway has begun to be clarified with mutational studies primarily in C. Elegans (8,22). These studies have demonstrated that the products of the unc-5, unc-6 and unc-40 ("uncoordinated") genes are required for proper dorsoventral migrations of commissural axons (Figure 2). unc-40 is now known to be a C. elegans homolog of DCC (8), while unc-6 is a netrin homolog (20,23). unc-5 is a transmembrane protein characterized by extracellular immunoglobulin and thrombospondin-1 domains and a cytoplasmic domain that is similar to the ZO-1 tight junction protein (24,25).

Netrin/unc-6 function requires either unc-5 or DCC /unc-40, and mutations in netrin/unc-6 affect both ventral and dorsal migrations of commissural axons. unc-5 mutations affect only dorsal axonal migrations, and there has been no demonstration as yet that unc-5 can function independently of netrin/unc-6. DCC/unc-40 acts cell autonomously to predominantly affect ventral migrations, though it is also required along with unc-5 for dorsal migrations (Figure 2). It is noteworthy that the phenotype of DCC/unc-40 null mutations is less severe than mutations which result in truncated DCC/unc-40 proteins. These nematode studies have been supported by work in Drosophila where the DCC homolog, frazzled, has been shown to be important primarily in ventral axonal migrations (10). Recent studies of netrin-1 (28) and DCC deficient (29)mice have demonstrated defects in commissural axon projections essentially identical to those of invertebrates. Moreover, these knockout studies have extended the effects of DCC and netrin to the developing brain by showing specific defects in the corpus callosum, hippocampal commissure and anterior commissure.

These mutational studies clearly suggest a ligand:receptor relationship for netrins and DCC and/or unc-5, and there is now biochemical evidence in support of this hypothesis. Keino-Masu et al. (9) have shown that netrins bind specifically to DCC expressing cells and that a monoclonal antibody directed to the DCC extracellular domain can abrogate netrin-mediated axonal outgrowth in vitro. Interestingly, this antibody blockade did not affect binding of netrin to the DCC expressing cells. Similar studies have also shown the ability of netrin to bind specifically to unc-5 expressing cells. A simplistic model consistent with these findings is that DCC mediates attractive responses to a netrin gradient, while unc-5 mediates a repulsive response. Refinement of this model awaits further definition of the actual receptor complex(es), since one interpretation of the above data is that DCC and unc-5 are modifiers of the actual receptor (24,25). Also, this model does not adequately explain the findings that unc-5 repulsive function requires DCC/unc-40 and the more severe phenotypes seen with truncated forms of DCC/unc-40. A heteromeric receptor in which truncated DCC/unc-40 acts in a dominant negative fashion has been hypothesized (26).

3.2. DCC guides cell migrations

While DCC has not had any apparent effects on cell proliferation in developmental models, it has been shown to provide pathway guidance for mesodermal and ectodermal cell migrations. Ventral migrations of the mesodermal male linker cell are abnormal in C. elegans DCC/unc-40 mutants, as are migrations of the ectodermal Q neuroblasts and P ectoblasts. The latter two defects are netrin-independent. The Q neuroblast defect is of particular interest because it has been linked to defects in wingless/wnt signaling in C. elegans (8). The wingless/wnt cell fate determination pathway is mediated by beta-catenin (armadillo), an oncogenic protein that is required for cadherin-mediated cell adhesion in addition to its role in signaling (30). The antagonism of beta-catenin signaling function by an interaction with the APC tumor suppressor protein further emphasizes the role of the developmentally important beta-catenin protein in cancer (31,32). The significance of the potential relationship between DCC/unc-40 mediated Q neuroblast migration and wingless/wnt signaling remains to be elucidated (8).

In DCC^-/- mice the pontine nuclei at the base of the rostral midbrain are absent (29), a defect that is also seen in netrin-1 deficient mice (28). Though it has not yet been rigorously shown that this represents a mismigration, this seems likely as these nuclei arise in a manner similar to that of spinal commissural axons. They must undergo a ventral, circumferential migration of cells from the lateral recess of the IVth ventricle to their final position in the pons (33).

3.3. Summary

The DCC protein is of unequivocal importance in development, particularly of neural tissue. It functions in the guided migration of cells and cell processes as a component of the netrin response pathway. It appears that DCC may act in this signal transduction pathway as a netrin receptor or a component of the receptor complex, though a definitive receptor:ligand relationship has not yet been demonstrated. It is also clear that DCC can affect migrations in a netrin-independent manner, implying the existence of other DCC ligands.

4. DCC AND CANCER

4.1. 18q Allelic loss and DCC expression studies

Allelic loss involving the long arm of chromosome 18 is a frequent event in a variety of malignancies and is therefore likely the site of a tumor suppressor gene(s). Tumors of diverse tissue origins are affected by these deletions--bladder, breast, colon, endometrium, esophagus, germ cell, neural crest (neuroblastoma), osteosarcoma, ovary, pancreas, prostate and stomach (Table 1). The DCC gene is a candidate target for this 18q deletion event on the basis of its location within the minimally lost region (18q21.3), some evidence of mutations within the remaining DCC allele and frequent loss of DCC expression.

In colorectal and esophageal cancer the remaining DCC allele has been shown to be affected by localized somatic mutations in only a subset of cases. Single point mutations have been found in intron 5, intron 13, exon 3 and exon 28 (3,4,52) and approximately 10-15% of colorectal cancer cases have expansions in a dinucleotide repeat tract immediately downstream of one of the exons (4). Although the effect of this expansion on DCC expression is unclear, Campuzano et al. (79) have found that a large expansion of a trinucleotide repeat sequence within the first intron of the Friedreich’s Ataxia gene accounts for the majority of the germline mutations causing this syndrome and that the mutant alleles fail to express stable transcripts. No familial mutations in DCC that confer a predisposition to tumor formation have yet been described. It is noteworthy that less than 1% of the 1.35 megabase DCC gene has been analyzed for mutations to date. There have also been no significant studies to address the possibility of methylation silencing of the DCC gene, a mechanism now known to achieve tumor suppressor gene inactivation in several cases (80,81).

Two other candidate target genes, both members of the MAD-related family of proteins essential in TGF-beta signaling, have been identified in the minimally lost region of 18q21 (70,82). The DPC4 /Smad4 gene has been shown to be a target of 18q deletions and localized mutations in pancreatic cancer (70), and this study showed that DCC was likely not targeted by allelic loss in this malignancy. However, more recent studies have shown that DPC4 /Smad4 mutations can account for, at most, one third of the 70% 18q allelic loss seen in colorectal cancer (83,84), and that DPC4 is seldom mutated in a variety of other cancers studied(85-88). The second target candidate, JV18-1/MADR2, has been shown to be mutated in 6% of colorectal tumors studied, but was not altered in a large panel of breast carcinomas and sarcomas (82,89). It appears that the majority of 18q allelic loss in colon cancer and other malignancies cannot presently be accounted for by alterations in DPC4 /Smad4 and/or JV18-1/MADR2.

Despite the limited direct evidence for mutational or epigenetic inactivation of the remaining DCC allele, there have been several independent demonstrations of the apparent loss of DCC expression in primary tumors and metastatic deposits. These studies have included most malignancies in which 18q allelic loss has been demonstrated, but have also implicated DCC in other non-epithelial malignancies such as gliomas and leukemias (Table 1).

Using RT-PCR based assays, expression of DCC transcripts has been found to be reduced/absent in 17/20 (85%) colorectal cancer cell lines as compared to normal colonic mucosa (4). Similar studies have established that DCC gene expression is reduced/absent in 40/68 (59%) primary colorectal cancers and in 9/9 (100%) colorectal metastases to the liver (44-46). The recent retrospective analysis of Shibata et al. (42) in which DCC expression was examined by immunohistochemistry in 132 paraffin-embedded specimens from curatively resected colorectal cancer patients is of particular clinical relevance. These investigators showed that DCC expression in primary tumors stratified stage II and III patients into good and poor prognosis subgroups. Stage II patients with DCC(+) primary tumors had a 94.3% 5 year survival rate as compared to 61.6% for those with DCC(-) tumors, and stage III patients were also stratified according to their DCC expression status [5 year survival: DCC(+) - 59.3%, DCC(-) - 33.2%]. Similar results had been obtained in an earlier study that assessed 18q allelic loss in an equivalent patient population (43). Stage II colorectal cancer patients have represented a management dilemma for clinicians. While most are cured by surgery alone, a significant proportion will die of their disease and there has previously been no effective means of defining this biologically aggressive subset. DCC expression may fill this void and certainly will be an important stratifying variable in future studies of adjuvant chemotherapy in stage II and III patients (90).

DCC expression has also been examined by immunoblot and immunohistochemistry in a large panel of primary neuroblastomas (n=84), a pediatric tumor of neural crest origin (65, unpublished data). Allelic loss involving the DCC locus had previously been shown to be the second most frequent site of allelic loss in this malignancy (66). The DCC protein was absent in 36% of the primary tumors overall, and tumors from patients with advanced stage or disseminated disease were much more frequently DCC-negative. Consistent with this association between the absence of the DCC protein and disease dissemination, metastatic deposits were more frequently DCC-negative [9/11 (82%)] than primary tumors.

The study of Reyes-Mugica et al. (60) is particularly noteworthy as it overcame a fundamental problem in all studies that attempt to compare precursor cells with their malignant counterpart -- the uncertain cellular origin of most tumors. Though many glioblastomas arise rapidly in a de novo fashion (i.e. without demonstrable precursor lesions), a subset arise from precursor astrocytomas/anaplastic astrocytomas (91-93). Moreover, sequential resections are often performed in these patients so that matched pair tumors from the same site in the same patient provide an opportunity to directly assess the molecular basis of tumor progression. These investigators exploited the astrocytoma --> glioblastoma sequence to examine the role of the DCC gene in glioma progression. Their examination of paired astrocytoma-glioblastoma specimens directly demonstrated the loss of DCC expression with glioma progression in approximately 50% of cases. This study provided further evidence that alterations in DCC expression are generally a later event in the malignant process and also linked DCC loss of expression to the development of the highly invasive glioblastoma multiforme.

4.2. Experimental approaches

Three studies have directly addressed the question of a tumor suppressor function for the DCC protein. Narayanan et al. (93) stably transfected Rat-1 fibroblasts with an inducible DCC antisense construct. Their data was consistent with a tumor suppressor role in that antisense DCC-expressing Rat-1 cells demonstrated a faster growth rate, anchorage independence and tumorigenicity in nude mice. Klingelhutz et al. (94) transfected tumorigenic human keratinocytes with the DCC cDNA and demonstrated inhibition of tumor growth in nude mice. This effect was not seen when a truncated DCC cDNA that lacked most of the cytoplasmic domain was used, and tumorigenic reversion of initially suppressed transfectants was associated with loss of DCC expression and loss/rearrangement of transfected DCC sequences. The recent DCC knockout study of Fazeli et al. (29) was not consistent with a tumor suppressor function. DCC^+/- mice did not demonstrate an increased frequency of tumor formation. DCC^-/- mice were not evaluable as they died within 24 hours of birth due to neurologic maldevelopment.

While these three studies come to different conclusions regarding the putative tumor suppressor function of DCC, the Fazeli et al. (29) study clearly utilizes a model system of greater biologic significance. All three studies can be criticized as their endpoint of cell growth may not be the most relevant endpoint for a molecule such as DCC. The developmental studies described above have demonstrated DCC’s role in the guidance of cell migrations and failed to show any affect on cell proliferation. Moreover, the reduction in DCC expression in tumors appears to be a later event in the malignant process and is associated with the process of dissemination (44,45,52,60, unpublished data). Correlates of the complex processes important in disease dissemination - attachment, invasion, angiogenesis - may be more appropriate endpoints.

4.3. Summary

Though DCC was originally implicated as a suppressor of tumor initiation or formation, the present data, particularly the lack of demonstrated mutational inactivation of the remaining allele and the absence of a predisposition to tumor formation in DCC-deficient mice, argue that DCC may not be a tumor suppressor. Further studies of the minimally lost region at 18q21 should be informative in terms of identifying another target gene or elucidating the mechanism of apparent DCC gene inactivation.

The reduction or loss of DCC expression appears to be a later event in several malignancies and is associated with disease dissemination. Taken together with DCC’s role in responding to extracellular matrix cues during development, future studies should focus on a possible role for DCC in the metastatic process. DCC expression has potential clinical utility as it can stratify an important subgroup of colorectal cancer patients in terms of survival. DCC expression should be a stratifying variable in future therapeutic trials in colorectal cancer, and its ability to stratify patient populations should be further addressed in malignancies such as gliomas and neuroblastomas.

5. PERSPECTIVE

The importance of DCC in the migration of cells and their processes during neural development and cellular differentiation is evident. While reduction/loss of DCC expression appears to be a later event in the malignant process and is associated with disease dissemination, a definitive role for DCC as an inhibitor of the malignant process is less clear. In light of the frequent crosstalk between developmental studies and cancer biology (30,31,95), it is intriguing to speculate that the developmental role of DCC in cell/cell process pathfinding indicates a similar role in the analogous process of tumor dissemination.

6. ACKNOWLEDGMENTS

This work was supported by grants to M.A.R. from the National Institutes of Health (CA63297), the American Cancer Society (MGO-86203) and the Brain Tumor Society.

7. REFERENCES

1. Vogelstein, B., E. R. Fearon, S. R. Hamilton, S. E. Kern, A. C. Preisinger, M. Lepper, Y. Nakamura, R. White, A. M. M. Smits, & J. L. Bos: Genetic alterations during colorectal tumor development. N. Engl. J. Med. 319, 525-532 (1988)

2. Ookawa, K., M. Sakamoto, S. Hirohashi, Y. Yoshida, T. Sugimura, M. Terada, & J. Yokota: Concordant p53 and DCC alterations and allelic losses on chromosomes 13q and 14q associated with liver metastases of colorectal carcinoma. International J Cancer 53, 382-387 (1993)

3. Cho, K. R., J. D. Oliner, J. W. Simons, L. Hedrick, E. R. Fearon, A. C. Preisinger, P. Hedge, G. A. Silverman, & B. Vogelstein: The DCC gene: structural analysis and mutations in colorectal carcinomas. Genomics 19, 525-531 (1994)

4. Fearon, E. R., K. R. Cho, J. M. Nigro, S. E. Kern, J. W. Simons, J. M. Ruppert, S. R. Hamilton, A. C. Preisinger, G. Thomas, K. W. Kinzler, & B. Vogelstein: Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 247, 49-56 (1990)

5. Hedrick, L., K. R. Cho, E. R. Fearon, J. D. Oliner, K. W. Kinzler, & B. Vogelstein: The DCC gene product in cellular differentiation and colorectal tumorigenesis. Genes Dev 8, 1174-1183 (1994)

6. Reale, M. A., G. Hu, R. H. Getzenberg, A. I. Zafar, S. M. Levine, & E. R. Fearon: Expression of the deleted in colorectal cancer (DCC) tumor suppressor gene in cell lines and normal and malignant tissues -- evidence for alternative splicing. Cancer Res 54, 4493-4501 (1994)

7. Vielmetter, J., J. F. Kayyem, J. M. Roman, & W. J. Dreyer: Neogenin, an avian cell surface protein expressed during terminal neuronal differentiation, is closely related to the human tumor suppressor molecule deleted in colorectal cancer. J Cell Biol 127, 2009-20 (1994)

8. Chan, S. S., H. Zheng, M. W. Su, R. Wilk, M. T. Killeen, E. M. Hedgecock, & J. G. Culotti: UNC-40, a C. elegans homolog of DCC (Deleted in Colorectal Cancer), is required in motile cells responding to UNC-6 netrin cues. Cell 87, 187-95 (1996)

9. Keino-Masu, K., M. Masu, L. Hinck, E. D. Leonardo, S.-Y. Chan, J. G. Culotti, & M. Tessier-Lavigne: Deleted in colorectal cancer (DCC) encodes a netrin receptor. Cell 87, 175-185 (1996)

10. Kolodziej, P. A., L. C. Timpe, K. J. Mitchell, S. R. Fried, C. S. Goodman, L. Y. Jan, & Y. N. Jan: frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance. Cell 87, 197-204 (1996)

11. Cooper, H. M., P. Armes, J. Britto, J. Gad, & A. F. Wilks: Cloning of the mouse homologue of the deleted in colorectal cancer gene (mDCC) and its expression in the developing mouse embryo. Oncogene 11, 2243-54 (1995)

12. Pierceall, W. E., M. A. Reale, A. F. Candia, C. V. E. Wright, K. R. Cho, & E. R. Fearon: Expression of a Xenopus laevis homologue of the deleted in colorectal cancer (DCC) tumor suppressor gene is developmentally regulated. Devel. Biol. 166, 654-665 (1994)

13. Ekstrand, B. C., T. Mansfield, S. Bigner, & E. R. Fearon: DCC expression is altered by multiple mechanisms in human brain tumors. Oncogene 11, 2393-2402 (1995)

14. Chuong, C.-M., T.-X. Jiang, E. Yin, & R. B. Widelitz: cDCC (chicken homologue to a gene deleted in colorectal carcinoma) is an epithelial adhesion molecule expressed in the basal cells and involved in epithelial-mesenchymal interaction. Develop Biol 164, 383-397 (1994)

15. Lawlor, K. G., & R. Narayanan: Persistent expression of the tumor suppressor gene DCC is essential for neuronal differentiation. Cell Growth & Differ 3, 609-616 (1992)

16. Pierceall, W. E., K. R. Cho, R. H. Getzenberg, M. A. Reale, L. Hedrick, B. Vogelstein, & E. R. Fearon: The deleted in colorectal cancer (DCC) tumor suppressor gene product stimulates neurite outgrowth in rat PC12 pheochromocytoma cells. J Cell Biol 124, 1017-1027 (1994)

17. Tanaka, E., & J. Sabry: Making the connection: cytoskeletal rearrangements during growth cone guidance. Cell 83, 171-6 (1995)

18. Keynes, R., & G. M. Cook: Axon guidance molecules. Cell 83, 161-9 (1995)

19. Tessier-Lavigne, M., M. Placzek, A. G. Lumsden, J. Dodd, & T. M. Jessell: Chemotropic guidance of developing axons in the mammalian central nervous system. Nature 336, 775-8 (1988)

20. Serafini, T., T. E. Kennedy, M. J. Galko, C. Mirzayan, T. M. Jessell, & M. Tessier-Lavigne: The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78, 409-24 (1994)

21. Harris, R., L. M. Sabatelli, & M. A. Seeger: Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila Netrin/UNC-6 homologs. Neuron 17, 217-28 (1996)

22. Hedgecock, E. M., J. G. Culotti, & D. H. Hall: 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 4, 61-85 (1990)

23. Ishii, N., W. G. Wadsworth, B. D. Stern, J. G. Culotti, & E. M. Hedgecock: UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans. Neuron 9, 873-81 (1992)

24. Leonardo, E. D., L. Hinck, M. Masu, K. Keino-Masu, S. L. Ackerman, & M. Tessier-Lavigne: Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors. Nature 386, 833-8 (1997)

25. Ackerman, S. L., L. P. Kozak, S. A. Przyborski, L. A. Rund, B. B. Boyer, & B. B. Knowles: The mouse rostral cerebellar malformation gene encodes an UNC-5-like protein. Nature 386, 838-42 (1997)

26. Drescher, U.: Netrins find their receptor [news]. Nature 384, 416-7 (1996)

27. Goodman, C. S.: The likeness of being: phylogenetically conserved molecular mechanisms of growth cone guidance. Cell 78, 353-6 (1994)

28. Serafini, T., S. A. Colamarino, E. D. Leonardo, H. Wang, R. Beddington, W. C. Skarnes, & M. Tessier-Lavigne: Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87, 1001-14 (1996)

29. Fazeli, A., S. L. Dickinson, M. Hermiston, R. V. Tighe, R. G. Steen, C. G. Small, K. Keinu-Masu, M. Masu, H. Rayburn, J. Simons, R. T. Bronson, J. I. Gordon, M. Tessier-Lavigne, & R. A. Weinberg: Phenotype of mice lacking functional Deleted in colorectal cancer (Dcc). Nature 386, 796 (1997)

30. Peifer, M.: Cell adhesion and signal transduction: the armadillo connection. Trends Cell Biol 5, 224-229 (1995)

31. Morin, P. J., A. B. Sparks, V. Korinek, N. Barker, H. Clevers, B. Vogelstein, & K. W. Kinzler: Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275, 1787-1790 (1997)

32. Rubinfeld, B., P. Robbins, M. El-Gamil, I. Albert, E. Porfiri, & P. Polakis: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 275, 1790-1792 (1997)

33. Bourrat, F., & C. Sotelo: Migratory pathways and selective aggregation of the lateral reticular neurons in the rat embryo: a horseradish peroxidase in vitro study, with special reference to migration patterns of the precerebellar nuclei. J Comp Neurol 294, 1-13 (1990)

34. Miyamoto, H., T. Shuin, I. Ikeda, M. Hosaka, & Y. Kubota: Loss of heterozygosity at the p53, RB, DCC and APC tumor suppressor gene loci in human bladder cancer. J Urol 155, 1444-7 (1996)

35. Brewster, S. F., J. C. Gingell, S. Browne, & K. W. Brown: Loss of heterozygosity on chromosome 18q is associated with muscle-invasive transitional cell carcinoma of the bladder. Br J Cancer 70, 697-700 (1994)

36. Dalbagni, G., J. Presti, V. Reuter, W. R. Fair, & C. Cordon-Cardo: Genetic alterations in bladder cancer [see comments]. Lancet 342, 469-71 (1993)

37. Presti, J. C., Jr., V. E. Reuter, T. Galan, W. R. Fair, & C. Cordon-Cardo: Molecular genetic alterations in superficial and locally advanced human bladder cancer. Cancer Res 51, 5405-9 (1991)

38. Huang, T. H., P. L. Yeh, M. B. Martin, R. E. Straub, T. C. Gilliam, C. W. Caldwell, & J. L. Skibba: Genetic alterations of microsatellites on chromosome 18 in human breast carcinoma. Diagn Mol Pathol 4, 66-72 (1995)

39. Thompson, A. M., R. G. Morris, M. Wallace, A. H. Wyllie, C. M. Steel, & D. C. Carter: Allele loss from 5q21 (APC/MCC) and 18q21 (DCC) and DCC mRNA expression in breast cancer. Br J Cancer 68, 64-8 (1993)

40. Devilee, P., M. van Vliet, N. Kuipers-Dijkshoorn, P. L. Pearson, & C. J. Cornelisse: Somatic genetic changes on chromosome 18 in breast carcinomas: is the DCC gene involved? Oncogene 6, 311-5 (1991)

41. Cropp, C. S., R. Lidereau, G. Campbell, M. H. Champene, & R. Callahan: Loss of heterozygosity on chromosomes 17 and 18 in breast carcinoma: two additional regions identified. Proc Natl Acad Sci U S A 87, 7737-41 (1990)

42. Shibata, D., M. A. Reale, P. Lavin, M. Silverman, E. R. Fearon, G. Steele Jr., M. J. Jessup, M. Loda, & I. C. Summerhayes: Loss of DCC expression and prognosis in colorectal cancer. N Engl J Med 335, 1727-1732 (1996)

43. Jen, J., H. Kim, S. Piantadosi, Z. F. Liu, R. C. Levitt, P. Sistonen, K. W. Kinzler, B. Vogelstein, & S. R. Hamilton: Allelic loss of chromosome 18q and prognosis in colorectal cancer [see comments]. N Engl J Med 331, 213-21 (1994)

44. Iino, H., M. Fukayama, Y. Maeda, M. Koike, T. Mori, T. Takahashi, R. Kikuchi-Yanoshita, M. Miyaki, S. Mizuno, & S. Watanabe: Molecular genetics for clinical management of colorectal carcinoma. 17p, 18q, and 22q loss of heterozygosity and decreased DCC expression are correlated with the metastatic potential. Cancer 73, 1324-31 (1994)

45. Itoh, F., Y. Hinoda, M. Ohe, Y. Ohe, T. Ban, T. Endo, K. Imai, & A. Yachi: Decreased expression of DCC mRNA in human colorectal cancers. Int J Cancer 53, 260-3 (1993)

46. Kikuchi-Yanoshita, R., M. Konishi, H. Fukunari, K. Tanaka, & M. Miyaki: Loss of expression of the DCC gene during progression of colorectal carcinomas in familial adenomatous polyposis and non-familial adenomatous polyposis patients. Cancer Res 52, 3801-3 (1992)

47. Enomoto, T., M. Fujita, C. Cheng, R. Nakashima, M. Ozaki, M. Inoue, & T. Nomura: Loss of expression and loss of heterozygosity in the DCC gene in neoplasms of the human female reproductive tract. Br J Cancer 71, 462-7 (1995)

48. Gima, T., H. Kato, T. Honda, T. Imamura, T. Sasazuki, & N. Wake: DCC gene alteration in human endometrial carcinomas. Int J Cancer 57, 480-5 (1994)

49. Fujino, T., J. I. Risinger, N. K. Collins, F. S. Liu, H. Nishii, H. Takahashi, E. M. Westphal, J. C. Barrett, H. Sasaki, and M. F. Kohler: Allelotype of endometrial carcinoma. Cancer Res 54, 4294-8 (1994)

50. Imamura, T., T. Arima, H. Kato, S. Miyamoto, T. Sasazuki, & N. Wake: Chromosomal deletions and K-ras gene mutations in human endometrial carcinomas. Int J Cancer 51, 47-52 (1992)

51. Maesawa, C., G. Tamura, S. Ogasawara, Y. Suzuki, K. Sakata, J. Sugimura, S. Nishizuka, N. Sato, K. Ishida, K. Saito, & R. Satodate: Loss of heterozygosity at the DCC gene locus is not crucial for the acquisition of metastatic potential in oesophageal squamous cell carcinoma. Eur J Cancer 32A, 896-8 (1996)

52. Miyake, S., K. Nagai, K. Yoshino, M. Oto, M. Endo, & Y. Yuasa: Point mutations and allelic deletion of tumor suppressor gene DCC in human esophageal squamous cell carcinomas and their relation to metastasis. Cancer Res 54, 3007-10 (1994)

53. Shibagaki, I., Y. Shimada, T. Wagata, M. Ikenaga, M. Imamura, & K. Ishizaki: Allelotype analysis of esophageal squamous cell carcinoma. Cancer Res 54, 2996-3000 (1994)

54. Huang, Y., R. F. Boynton, P. L. Blount, R. J. Silverstein, J. Yin, Y. Tong, T. K. McDaniel, C. Newkirk, J. H. Resau, & R. Sridhara: Loss of heterozygosity involves multiple tumor suppressor genes in human esophageal cancers. Cancer Res 52, 6525-30 (1992)

55. Cho, J. H., M. Noguchi, A. Ochiai, & S. Hirohashi: Loss of heterozygosity of multiple tumor suppressor genes in human gastric cancers by polymerase chain reaction. Lab Invest 74, 835-41 (1996)

56. Schneider, B. G., D. R. Pulitzer, R. D. Brown, T. J. Prihoda, D. G. Bostwick, V. Saldivar, H. A. Rodriguez-Martinez, M. E. Gutierrez-Diaz, & P. O'Connell: Allelic imbalance in gastric cancer: an affected site on chromosome arm 3p. Genes Chromosomes Cancer 13, 263-71 (1995)

57. Uchino, S., H. Tsuda, M. Noguchi, J. Yokota, M. Terada, T. Saito, M. Kobayashi, T. Sugimura, & S. Hirohashi: Frequent loss of heterozygosity at the DCC locus in gastric cancer. Cancer Res 52, 3099-102 (1992)

58. Peng, H. Q., D. Bailey, D. Bronson, P. E. Goss, & D. Hogg: Loss of heterozygosity of tumor suppressor genes in testis cancer. Cancer Res 55, 2871-5 (1995)

59. Murty, V. V., G. J. Bosl, J. Houldsworth, M. Meyers, A. B. Mukherjee, V. Reuter, & R. S. Chaganti: Allelic loss and somatic differentiation in human male germ cell tumors. Oncogene 9, 2245-51 (1994)

60. Reyes-Mugica, M., K. Rieger-Christ, H. Hogaki, M. Helie, G. Kleinman, B. C. Ekstrand, P. Kleihues, E. R. Fearon, & M. A. Reale: Loss of DCC expression and glioma progression. Cancer Res 57, 382-386 (1997)

61. Scheck, A. C., & S. W. Coons: Expression of the tumor suppressor gene DCC in human gliomas. Cancer Res. 53, 5605-5609 (1993)

62. Miyake, K., K. Inokuchi, K. Dan, & T. Nomura: Expression of the DCC gene in myelodysplastic syndromes and overt leukemia. Leuk Res 17, 785-8 (1993)

63. Porfiri, E., L. M. Secker-Walker, A. V. Hoffbrand, & J. F. Hancock: DCC tumor suppressor gene is inactivated in hematologic malignancies showing monosomy 18. Blood 81, 2696-701 (1993)

64. Miyake, K., K. Inokuchi, K. Dan, & T. Nomura: Alterations in the deleted in colorectal carcinoma gene in human primary leukemia. Blood 82, 927-30 (1993)

65. Reale, M. A., M. Reyes-Mugica, W. E. Pierceall, M. C. Rubinstein, L. Hedrick, S. L. Cohn, A. Nakagawara, G. M. Brodeur, & E. R. Fearon: Loss of DCC expression in neuroblastoma is associated with disease dissemination. Clin Cancer Res 2, 1097-1102 (1996)

66. Takita, J., Y. Hayashi, T. Kohno, M. Shiseki, N. Yamaguchi, R. Hanada, K. Yamamoto, & J. Yokota: Allelotype of neuroblastoma. Oncogene 11, 1829-1834 (1995)

67. Yamaguchi, T., J. Toguchida, T. Yamamuro, Y. Kotoura, N. Takada, N. Kawaguchi, Y. Kaneko, Y. Nakamura, M. S. Sasaki, & K. Ishizaki: Allelotype analysis in osteosarcomas: frequent allele loss on 3q, 13q, 17p, and 18q. Cancer Res 52, 2419-23 (1992)

68. Toguchida, J., K. Ishizaki, M. S. Sasaki, M. Ikenaga, M. Sugimoto, Y. Kotoura, & T. Yamamuro: Chromosomal reorganization for the expression of recessive mutation of retinoblastoma susceptibility gene in the development of osteosarcoma. Cancer Res 48, 3939-43 (1988)

69. Chenevix-Trench, G., J. Leary, J. Kerr, J. Michel, R. Kefford, T. Hurst, P. G. Parsons, M. Friedlander, & S. K. Khoo: Frequent loss of heterozygosity on chromosome 18 in ovarian adenocarcinoma which does not always include the DCC locus. Oncogene 7, 1059-65 (1992)

70. Hahn, S. A., M. Schutte, A. T. M. Shamsul Hoque, C. A. Moskaluk, L. T. da Costa, E. Rozenblum, C. L. Weinstein, A. Fischer, C. J. Yeo, R. H. Hruban, & S. E. Kern: DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271, 350-353 (1996)

71. Seymour, A. B., R. H. Hruban, M. Redston, C. Caldas, S. M. Powell, K. W. Kinzler, C. J. Yeo, & S. E. Kern: Allelotype of pancreatic adenocarcinoma. Cancer Res 54, 2761-4 (1994)

72. Simon, B., R. Weinel, M. Hohne, J. Watz, J. Schmidt, G. Kortner, & R. Arnold: Frequent alterations of the tumor suppressor genes p53 and DCC in human pancreatic carcinoma. Gastroenterology 106, 1645-51 (1994)

73. Hohne, M. W., M. E. Halatsch, G. F. Kahl, & R. J. Weinel: Frequent loss of expression of the potential tumor suppressor gene DCC in ductal pancreatic adenocarcinoma. Cancer Res 52, 2616-9 (1992)

74. Crundwell, M. C., S. Chughtai, M. Knowles, L. Takle, M. Luscombe, J. P. Neoptolemos, D. G. Morton, & S. M. Phillips: Allelic loss on chromosomes 8p, 22q and 18q (DCC) in human prostate cancer. Int J Cancer 69, 295-300 (1996)

75. Latil, A., J. C. Baron, O. Cussenot, G. Fournier, T. Soussi, L. Boccon-Gibod, A. Le Duc, J. Rouesse, & R. Lidereau: Genetic alterations in localized prostate cancer: identification of a common region of deletion on chromosome arm 18q. Genes Chromosomes Cancer 11, 119-25 (1994)

76. Brewster, S. F., S. Browne, & K. W. Brown: Somatic allelic loss at the DCC, APC, nm23-H1 and p53 tumor suppressor gene loci in human prostatic carcinoma. J Urol 151, 1073-7 (1994)

77. Gao, X., K. V. Honn, D. Grignon, W. Sakr, & Y. Q. Chen: Frequent loss of expression and loss of heterozygosity of the putative tumor suppressor gene DCC in prostatic carcinomas. Cancer Res 53, 2723-7 (1993)

78. Carter, B. S., C. M. Ewing, W. S. Ward, B. F. Treiger, T. W. Aalders, J. A. Schalken, J. I. Epstein, & W. B. Isaacs: Allelic loss of chromosomes 16q and 10q in human prostate cancer. Proc Natl Acad Sci U S A 87, 8751-5 (1990)

79. Campuzano, V., L. Montermini, M. D. Molto, L. Pianese, M. Cossee, F. Cavalcanti, E. Monros, F. Rodius, F. Duclos, A. Monticelli, F. Zara, J. Canizares, H. Koutnikova, S. I. Bidichandani, C. Gellera, A. Brice, P. Trouillas, G. De Michele, A. Filla, R. De Frutos, F. Palau, P. I. Pragna, S. Di Donato, J-L. Mandel, S. Cocozza, M. Koenig and M. Pandolfo.: Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion [see comments]. Science 271, 1423-7 (1996)

80. Merlo, A., J. G. Herman, L. Mao, D. J. Lee, E. Gabrielson, P. C. Burger, S. B. Baylin, & D. Sidransky: 5' CpG island methylation is associated with transcriptional silencing of the tumor suppressor p16/CDKN2/MTS1 in human cancers. Nature Med 1, 686-692 (1995)

81. Herman, J. G., F. Latif, Y. Weng, M. I. Lerman, B. Zbar, S. Liu, D. Samid, D. S. Duan, J. R. Gnarra, & W. M. Linehan: Silencing of the VHL tumor suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci USA 91, 9700-9704 (1994)

82. Eppert, K., S. W. Scherer, H. Ozcelik, R. Pirone, P. Hoodless, H. Kim, L.-C. Tsue, B. Bapat, S. Gallinger, I. L. Andrulis, G. H. Thomsen, J. L. Wrana, & L. Attisano: MADR2 maps to 18q21 and encodes a TGF-beta-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell 86, 543-552 (1996)

83. Takagi, Y., H. Kohmura, M. Futamura, H. Kida, H. Tanemura, K. Shimokawa, & S. Saji: Somatic alterations of the DPC4 gene in human colorectal cancers in vivo [see comments]. Gastroenterology 111, 1369-72 (1996)

84. Thiagalingam, S., C. Lengauer, F. S. Leach, M. Schutte, S. A. Hahn, J. Overhauser, J. K. V. Willson, S. Markowitz, S. R. Hamilton, S. E. Kern, K. W. Kinzler, and B. Vogelstein: Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nat. Genetics 13, 343-346 (1996)

85. Barrett, M. T., M. Schutte, S. E. Kern, & B. J. Reid: Allelic loss and mutational analysis of the DPC4 gene in esophageal adenocarcinoma. Cancer Res 56, 4351-3 (1996)

86. Kim, S. K., Y. Fan, V. Papadimitrakopoulou, G. Clayman, W. N. Hittelman, W. K. Hong, R. Lotan, & L. Mao: DPC4, a candidate tumor suppressor gene, is altered infrequently in head and neck squamous cell carcinoma. Cancer Res 56, 2519-21 (1996)

87. Lei, J., T. T. Zou, Y. Q. Shi, X. Zhou, K. N. Smolinski, J. Yin, R. F. Souza, R. Appel, S. Wang, K. Cymes, O. Chan, J. M. Abraham, N. Harpaz, & S. J. Meltzer: Infrequent DPC4 gene mutation in esophageal cancer, gastric cancer and ulcerative colitis-associated neoplasms. Oncogene 13, 2459-62 (1996)

88. Schutte, M., R. H. Hruban, L. Hedrick, K. R. Cho, G. M. Nadasdy, C. L. Weinstein, G. S. Bova, W. B. Isaacs, P. Cairns, H. Nawroz, D. Sidransky, R. A. Casero, Jr., P. S. Meltzer, S. A. Hahn, & S. E. Kern: DPC4 gene in various tumor types. Cancer Res 56, 2527-30 (1996)

89. Riggins, G. J., S. Thiagalingam, E. Rozenblum, C. L. Weinstein, S. E. Kern, S. R. Hamilton, J. K. Willson, S. D. Markowitz, K. W. Kinzler, & B. Vogelstein: Mad-related genes in the human. Nat Genet 13, 347-9 (1996)

90. Banerjee, A. K.: DCC expression and prognosis in colorectal cancer. Lancet 349, 968 (1997)

91. Louis, D. N., & J. F. Gusella: A tiger behind many doors: multiple genetic pathways to malignant glioma. Trends Genetics 11, 412-415 (1995)

92. Kleihues, P., F. Soylemezoglu, B. Schauble, B. W. Scheithauer, & P. C. Burger: Histopathology, classification, and grading of gliomas. Glia 15, 211-221 (1995)

93. Cavenee, W. K.: Accumulation of genetic defects during astrocytoma progression. Cancer (Suppl) 70, 1788-1793 (1992)

93. Narayanan, R., R. Q. J. Schaapveld, K. R. Cho, B. Vogelstein, P. B. V. Tran, M. P. Osborne, & N. T. Telang: Antisense RNA to the putative tumor-suppressor gene DCC transforms Rat-1 fibroblasts. Oncogene 7, 553-561 (1992)

94. Klingelhutz, A. J., L. Hedrick, K. R. Cho, & J. K. McDougal: The DCC gene suppresses the malignant phenotype of transformed human epithelial cells. Oncogene 10, 1581-1586 (1995)

95. Hahn, H., C. Wicking, P. G. Zaphiropoulous, M. R. Gailani, S. Shanley, A. Chidambaram, I. Vorechovsky, E. Holmberg, A. B. Unden, S. Gillies, K. Negus, I. Smyth, C. Pressman, D. J. Leffell, B. Gerrard, A. M. Goldstein, M. Dean, R. Toftgard, G. Chenevix-Trench, B. Wainwright, & A. E. Bale: Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85, 841-51 (1996)