With the demonstration by Cavenee et al. (1) of loss of alleles in familial tumors, validating the Knudson et al. (2) two-hit tumor suppressor hypothesis, it became possible to look for loss of regions of chromosomes in sporadic cancers of all types. Among the earliest reports of such losses were these reporting loss of regions of the short arm of chromosome 3 in lung and kidney cancers (3, 4). Since 1987, the losses on this chromosome have been under refinement, so that by 1993 it was clear that there were likely to be multiple tumor-suppressor genes on 3p, most pinpointed by homozygous deletions observed in cancer cell lines (5–7) or by familial cancer loci (8, 9). The search has been on for 3p tumor-suppressor genes, genes whose loss contributes to cancer development, since the early 1990s when the availability of markers from human chromosomes began to increase. At the same time, alterations of chromosome region 3p in other common human cancers such as cervical, breast, and stomach were increasingly reported (see Fig. 1). The 10-year search for the lung cancer-suppressor gene at chromosome region 3p12 (Fig. 1) has now culminated in a report (10) that says recreation in a knockout mouse of a naturally occurring cancer-associated homozygous exon 2 deletion of a gene called DUTT1/ROBO1 causes lung-specific developmental defects, and in surviving mice causes bronchial hyperplasia and dysplasia.
The 3p21.3 region, which is among the most frequently lost in many types of cancer, has been the most difficult region in which to find tumor-suppressor genes.
Figure 1.
Chromosome 3p tumor-suppressor genes. The four tumor-suppressor genes discussed are shown in their approximate locations on the short arm of chromosome 3. Known or hypothetical functions associated with the genes are listed in blue. The RASSF1A gene may be involved in negative regulation of cell growth (19, 20) through interaction with a RAS signaling pathway. The human DUTT1/ROBO1 gene has been implicated in migration of axons, myoblasts, and leukocytes and its absence could lead to uncontrolled or inappropriate cell migration, and thus hyperplasia and dysplasia, in cancer. A prominent function of the Vhl protein is in regulation of proteins involved in angiogenesis, and Fhit overexpression induces apoptosis. Inactivation of several of these genes occurs in large fractions of several types of cancer, possibly as hypothesized in this figure, because inactivation of each provides an essential hallmark (21) contributing to malignant growth—uncontrolled growth (RASSF1A), evasion of apoptosis (FHIT), sustained angiogenesis (VHL), and cell migration/invasion (DUTT1/ROBO1).
DUTTI/ROBO1 is the most recent addition to the list of long-sought 3p tumor-suppressor genes in four loss of heterozygosity (LOH) target regions (see Fig. 1). The VHL gene at 3p25.3, associated with familial kidney cancer and multiple benign tumors of the retina, brain, and pancreas, was cloned in 1993 (8) and its function has been studied extensively (11). As expected for a “classic” tumor-suppressor gene, both VHL alleles are inactivated through a combination of deletion, mutation, or promoter methylation mechanisms in the familial kidney cancers and in the majority of sporadic kidney cancers. Mice with two inactivated Vhl alleles die early in embryogenesis; mice with one inactivated allele have no observable phenotype (12), whereas mice with inactivation of Vhl specifically in the liver show cavernous hemangiomas of the liver (13). The second 3p-tumor suppressor to be isolated was the FHIT gene at 3p14.2. The position of this gene was also pinpointed by a feature of inherited renal cancer. In this family, renal cancer developed in individuals who inherited a balanced reciprocal chromosome translocation (14), thus the position of the translocation at 3p14.2 marked the location of a gene that might contribute to the development of renal cancer. The FHIT gene was cloned and found to be interrupted by the 3p14.2 translocation and to exhibit intragenic homozygous deletions in many cancers (9, 15). Fhit knockout mice are exquisitely sensitive to carcinogen induction of tumors but they do not exhibit developmental abnormalities nor do they spontaneously develop kidney or lung tumors (16, 17). The 3p21.3 homozygous deletion target was perhaps the most ardently pursued tumor-suppressor gene. Large regions of the 3p21.3 region were positionally cloned, and the region of homozygous deletion narrowed to about 600 completely sequenced kilobase pairs (18). Genes were identified and mutations were sought. Last year, one isoform, RASSF1A, of the RASSF1 gene in this region was shown to be epigenetically silenced by methylation of its promoter in lung cancers (19) and this isoform also suppressed tumorigenicity (19, 20). Inactivation of this gene in mice has not yet been reported.
Each of the 3p tumor-suppressor genes has belied expectations in some way: The VHL gene, because it is inactivated mainly in kidney cancer, although other cancer types show LOH in the vicinity of the VHL locus, and because Vhl knockout mice have not proven to be models for VHL disease. The 3p21.3 region, which is among the most frequently lost in many types of cancer, has been the most difficult region in which to find tumor-suppressor genes. One difficulty was that there were many genes in a fairly small region and they all needed to be tested for loss and mutation, mostly in lung cancer. Bona fide point mutations were not observed in any of the identified genes at a frequency that would suggest an important tumor-suppressor gene (18). The RASSF1A isoform of the RASSF1 gene may be the first important tumor-suppressor gene mainly inactivated through methylation, which occurs in a sizable fraction of lung, breast, kidney, and other cancers. We do not yet know what phenotype a Rassf1a knockout mouse would exhibit and thus cannot speculate about whether loss of its expression might cause abnormalities in major epithelial organs. The FHIT gene at 3p14.2 is a large gene, encoding a small protein, in which mutations are almost never found in cancer. The gene encompasses the most active chromosome-fragile site and is inactivated through deletions and promoter methylation in cancers of many organs. FHIT was among the first tumor-suppressor genes to encounter strong resistance to its candidacy as a result of its lack of classical tumor-suppressor gene features (loss of one allele through LOH and point mutation of the other). Mice hemi or homozygous for inactivated Fhit alleles are susceptible to spontaneous and induced tumors at similar frequencies (17), suggesting that loss of one Fhit allele confers tumor susceptibility, but Fhit knockout mice also do not spontaneously show frequent abnormalities of epithelial organs. The DUTT1/ROBO1 gene has not been shown to be frequently inactivated in cancer, and tumor-suppressor activity has not been shown (Table 1). Thus, each of the 3p tumor-suppressor contenders except VHL exhibit “neoclassical” features—lack of inactivating point mutations, inactivation through epigenetic mechanisms, possible haploid insufficiency—but VHL, FHIT, and RASSF1A are completely inactivated in a large fraction of human cancers; such inactivation is the tumor-suppressor gene sine qua non. Surprisingly, alteration of expression of DUTT1/ROBO1 in cancer has not been reported (10).
Table 1.
Does DUTT1/ROBO1 qualify as a tumor suppressor gene?
Pro |
Homozygous deletion of exon 2 in lung cancer cell line. |
LOH in large fraction of lung, kidney, and other cancers. |
Expressed in bronchial epithelia of normal lung. |
Most mice with absence of DUTT1/ROBO1 exon 2 die because of delayed lung maturation. |
Surviving mice develop bronchial epithelial hyperplasia. |
Con |
No point mutations of the gene in lung cancers. |
Tumor suppression not shown. |
Aberrant expression in cancers not shown. |
The Dutt1/Robo1 gene is the first of the 3p neoclassical tumor-suppressor genes to actually cause a lung-specific phenotype when inactivated in mice. These mice show a phenotype compatible with a role for Dutt1/Robo1 in early stages of the multistep route to lung cancer. If indeed DUTT1/ROBO1 turns out to be the 3p12 tumor-suppressor gene, exploration of its function in normal and tumor cells will be facilitated by the growing knowledge of the ROBO1 pathway, from extracellular interaction with SLIT proteins to the intracellular regulation of actin polymerization (22). It will be especially interesting to observe the cancer phenotype of carcinogen-exposed Dutt1/Robo1 +/− mice. As pointed out by Xian et al. (10) “further genetic damage to these mutant mice … may result in progression toward a more malignant phenotype. These DUTT1/ROBO1 mutants would … be a useful starting point … for mouse models … driven by sequential … genetic changes in lung cancer.” Possible models for lung, kidney, and other cancers may be created by crossing mice with different inactivated 3p-suppressor genes as illustrated in Fig. 2. Such mice are very likely to be excellent models to study induction, therapy, and prevention of several types of important human cancers.
Figure 2.
Possible models for lung and kidney cancer. Vhl, Fhit, and Dutt/Robo1 knockout mice are available, and Rassf1a knockout mice may become available. It may not be surprising that no single knockout mouse provides a model for lung or kidney cancer, because we have seen that combinations of these genes (and other important tumor-suppressor genes) are invariably inactivated in the human cancers. By crossing the individual knockout mice to make the compound mutants illustrated, we may develop models for lung and kidney cancer that recapitulate the genetic changes observed in the human diseases, models that could contribute to prevention, and therapy strategies for these frequently fatal human malignancies.
Footnotes
See companion article on page 15062.
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