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

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.