Cancer Research Meeting Calendar Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xian, J.
Right arrow Articles by Rabbitts, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xian, J.
Right arrow Articles by Rabbitts, P.
[Cancer Research 64, 6432-6437, September 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Targeted Disruption of the 3p12 Gene, Dutt1/Robo1, Predisposes Mice to Lung Adenocarcinomas and Lymphomas with Methylation of the Gene Promoter

Jian Xian1, Alan Aitchison1, Linda Bobrow2, Gerard Corbett1, Richard Pannell3, Terence Rabbitts3 and Pamela Rabbitts1

1 Department of Oncology, University of Cambridge, Medical Research Council Centre, Cambridge, United Kingdom; 2 Department of Histopathology, Addenbrookes Hospital, Cambridge, United Kingdom; and 3 Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene’s promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The identification of genes associated with consistent tumor-specific chromosomal abnormalities has been an effective method of isolating genes important in cancer development (1) . Deletion mapping of chromosome 3 has indicated that somatic genetic loss to the short arm is a frequent event in a variety of common carcinomas and raised the expectation that tumor suppressor genes will be found located within the minimally deleted regions (2) . Lung cancer has received the most attention because many studies have indicated a very high incidence of loss on 3p (91% for small-cell lung carcinoma, 95% for squamous cell carcinoma, and 71% for adenocarcinoma) and because 3p loss is detectable in low-grade preinvasive lesions, implying an early, possibly initiating, role for 3p loss in lung tumorigenesis (3) . Several distinct regions along the chromosome have been delineated, and their boundaries have been reduced by the discovery of homozygous deletions nesting within the region of heterozygous loss (2) . A number of genes within these deletions have been examined for frequent point mutations in tumors, but none has shown this classical feature of tumor suppressor genes (4 , 5) , although reduced expression due to promoter hypermethylation is common (6) .

Our search for a tumor suppressor gene has focused on proximal 3p and identified three nested homozygous deletions, one of which is within a gene, DUTT1 (deleted in U-twenty-twenty; ref. 7 ). This intragenic deletion removes a segment encompassing exon 2 of the gene, creating a mutant form of the protein lacking the first immunoglobulin domain. The DUTT1 gene was independently isolated as the human homologue (ROBO1) of the Drosophila gene Roundabout (Robo; ref. 8 ). Dutt1/Robo1 is a trans-membrane receptor responding to the Slit family of ligands and mediates signals that control cell migration and cell position in a range of cell types from Drosophila to mammals (9) . It is not known whether it performs a similar role in epithelial cells, but abrogation of this function in tumors could explain the contribution of DUTT1/ROBO1 to malignancy because inappropriate migration and incorrect cell position are such defining features of the malignant phenotype (10) . However, although positional cloning indicates that DUTT1/ROBO1 is a candidate tumor suppressor gene, like other candidates on 3p, tumor-associated point mutations have not been detected (11) . If the mechanism of DUTT1/ROBO1 gene inactivation is epigenetic, a functional demonstration is required to conclusively confirm its involvement in tumor development. Tumor formation in mice with targeted inactivation of the candidate gene is recognized to fulfill this requirement (12) .

To investigate the possibility that inactivation of this gene predisposes to tumor development, we generated a targeted intragenic deletion within the Dutt1/Robo1 gene in the mouse germ line, recapitulating the loss of exon 2 detected in a human small-cell lung carcinoma (13) . Frequent perinatal mortality is observed in mice homozygous for the disrupted Dutt1/Robo1 gene. The few surviving homozygous mice die within 12 months, having developed extensive bronchial hyperplasia. We now demonstrate that heterozygous mice, with this deletion within only one allele, spontaneously develop lymphomas and carcinomas in their second year of life: invasive carcinoma of the lung is by far the predominant epithelial tumor. Dutt1/Robo1 expression is substantially reduced in these tumors and is accompanied by substantial methylation of the gene’s promoter. We conclude that Dutt1/Robo1 acts as a classic tumor suppressor gene with inactivation of both alleles in the development of malignant tumors in these mice.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Observation of Abnormal Mice Phenotypes.
The Dutt1/Robo1 mutant mice (mixed C57BL/6 and 129/SV genetic background) were generated by gene targeting (13) . Dutt1/Robo1 genotype was determined by standard Southern blot hybridization analysis of tail DNA as described previously (13) . The mice were housed in a pathogen-free environment, and their care was subject to United Kingdom Home Office regulations. They were observed frequently and sacrificed on decline in their health or obvious tumor burden. Complete necropsy was carried out on all mice. Histopathological examination was performed on all tissues found with visible abnormalities in mutant mice and controls. Tissues were fixed in buffered 4% paraformaldehyde and then embedded in paraffin wax, and the wax blocks were sectioned at 5 to 8 µm and stained with hematoxylin and eosin (H&E). Large tumors were also frozen for extraction of DNA, RNA, and protein using standard procedures.

Southern Blotting.
Genotyping of tumors was performed as described previously for genotyping of tail DNA (13) .

Reverse Transcription-Polymerase Chain Reaction.
Total RNA was prepared from mouse tumors, fibroblasts, and normal lung with Trizol reagent (GIBCO/BRL, Gaithersburg, MD). Reverse transcription-polymerase chain reaction (RT-PCR) was performed as described previously (13) . Mouse Gapdh mRNA was amplified as a template control. For polymerase chain reaction (PCR) of Dutt1/Robo1, the primers used were as follows: (a) exon 1 forward (5'-AGGGATTGACAAGCCTCCGG-3') and exon 2 reverse (5'-AGCTACCTCCAGCGATGCGT-3'); this PCR fragment includes exon 1 and exon 2 (527 bp); and (b) exon 3 forward (5'-ATACTACGGGATGACTTCAG-3') and exon 5 reverse (5'-CTGGATTTGGGCAGCTCTCC-3'); this PCR fragment includes exon 3 to exon 5 (230 bp). For PCR of mouse Gapdh mRNA, the primers used were as follows: forward, 5'- TGAACGGATTTGGCCGTATT-3'; and reverse, 5'-TGCCGTGAGTGGAGTCATAC-3'. The products were visualized by ethidium bromide staining after electrophoresis on agarose gels.

Bisulfite Modification and Methylation Analysis.
Bisulfite DNA sequencing was performed as described previously (14) . One microgram of genomic DNA from normal or tumor tissues in a volume of 50 µL was denatured in 0.2 mol/L NaOH for 15 minutes at 37°C. Thirty microliters of 10 mmol/L hydroquinone (Sigma, St. Louis, MO) were added to each sample, mixed gently, and left at room temperature for 5 minutes; 520 µL of freshly prepared 3 mol/L sodium bisulfite (pH 5; Sigma) were added; and samples were incubated under mineral oil at 50°C for 16 hours. Modified DNA was purified using Wizard DNA purification resin according to the manufacturer’s instructions (Promega, Madison, WI) and eluted with 60 µL of water. Modification was completed by incubation with NaOH to 0.25 mol/L for 15 minutes at 37°C, followed by ethanol precipitation. DNA was resuspended in Tris-EDTA and used immediately or stored at –20°C.

Bisulfite-modified DNA (100 ng) was amplified using the mouse Dutt1/Robo1 promoter region-specific primers designed by the MethPrimer program. The promoter region was amplified in two parts. CpG island region 1 from position –793 to –616 was amplified using forward primer 5'-GAAGTTTGGGTTGTAGTTTG-3' and reverse primer 5'-AACCCTTATTCCCATCAC-3'. CpG island region 2 from position –443 to the beginning of exon 1 (position –310) was amplified using forward primer 5'-GAGGAGTATTTTTTGGGGTG-3' and reverse primer 5'-TTTCCTACCTCCACAATCC-3'. PCR conditions were 95°C for 12 minutes and 30 cycles of 95°C for 50 seconds, 55°C for 50 seconds, and 72°C for 50 seconds. PCR products were purified using QIAquick PCR purification kit (Qiagen). Methylated cytosine residues were identified by direct sequencing of PCR products with the same primers used for PCR.

Protein Detection by Western Blotting and Immunohistochemistry.
Immunohistochemical staining with a polyclonal antibody raised against the COOH-terminal peptide of DUTT1/ROBO1 (CYERGEDNNEELEETES) was performed as described previously (15) . Western blotting, using the same antibody, was as described previously (13) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor Incidence in Dutt1/Robo1 Heterozygous Mice.
Dutt1/Robo1–/– mice die at birth or within the first year of life, on average at 7 months, preventing evaluation of the effect of the homozygous deletion within this gene, which results in a truncated protein, on tumor development (13) . To assess whether tumors develop in heterozygotes, a cohort of 154 Dutt1/Robo1+/– mice was aged together with 106 wild-type littermates. Eight control mice (7.5%) developed tumors (three mice developed lung tumors, and five mice developed lymphomas), on average at 20.2 months. The incidence of lung tumors (2.8%) and lymphomas (5%) is within the range expected from the genetic background of the mice used in these studies.4 In the Dutt1/Robo1+/– group, 34 mice (22.0%) developed a total of 53 tumors (Fig. 1A)Citation . This is a significant increase in tumor predisposition compared with wild-type mice; (P < 0.01, {chi}2 test). Other than lymphomas, the tumors (both benign and malignant) were almost all epithelial in origin (Fig. 1B)Citation .



View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Tumor incidence and histopathology in Dutt1/Robo1+/– mice.

 
The germ-line disruption in mice of other tumor suppressor genes associated with lung cancer development, such as p53, p16INK4a, and Rb, does not show increased incidence of lung cancer development (16) . In contrast, 26.5% of the tumors that developed in the Dutt1/Robo1+/– mice were adenocarcinomas of the lung (Fig. 1B)Citation . Their development was spontaneous and not dependent on any guidance of genetic damage to the bronchial epithelium.

Phenotypic Characteristics of Lung Tumors Developing in Dutt1/Robo1+/– Mice.
The lung tumors that developed in the Dutt1/Robo1+/– mice were all adenocarcinomas, ranged in size from 2 to 10 mm2, and generally had an irregular, distorted appearance (Fig. 2A)Citation and showed clear invasion (Fig. 2B)Citation with pleiomorphic nuclei (Fig. 2C)Citation . Histologic subtypes were typical, papillary, and glandular (data not shown). In contrast, the three lung tumors arising in wild-type mice were very similar to each other and were small (<2 mm2) with a regular pebble-like appearance (Fig. 2D)Citation , well circumscribed (Fig. 2E)Citation , and with near normal nuclear morphology (Fig. 2F)Citation . Direct comparison of tumor incidence (Fig. 1A)Citation in wild-type and mutant mice masks these important differences in the degree of malignancy observed in the lung tumors. Although invasion was clearly detected in the lung adenocarcinomas that developed in the Dutt1/Robo1+/– mice (Fig. 2B)Citation , secondary deposits were not observed macroscopically. Examples of the other malignant tumors and preinvasive lesions occurring in the Dutt1/Robo1+/– group are illustrated in Fig. 2G–NCitation .



View larger version (134K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Abnormal phenotypes in lung tumors from Dutt1/Robo1+/– mice. A, a pair of lungs from a Dutt1/Robo1+/– mouse sacrificed at 18 months. The arrowhead indicates the lung distorted by tumor. B, H&E-stained section of lung adenocarcinoma from a Dutt1/Robo1+/– mouse showing stromal invasion (arrowheads). C, higher power view of B showing pleiomorphic nuclei. D, a pair of lungs from a wild-type mouse sacrificed at 18 months; tumor is indicated by the arrow. E, H&E staining of a lung tumor from a wild-type mouse; tumor is indicated by the arrow. F, higher power view of E showing virtually normal nuclei. G, H&E-stained section of lymphoma. H–N are H&E-stained sections of other mainly epithelial malignant tumors and preinvasive lesions. H, hepatocellular carcinoma; I, sweat gland carcinoma; J, renal cell carcinoma; K, thecal cell tumor; L, hepatoctye severe dysplasia; M, ovarian epithelial atypia; N, gallbladder metaplasia (bars, 50 µm).

 
Dutt1/Robo1 Expression in Tumors from Dutt1/Robo1+/– Mice.
Because the mutant allele is an intragenic deletion, transcription could occur from both mutant and wild-type alleles (Fig. 3A and B)Citation . Primers for RT-PCR were used to detect transcription from either allele (exons 3–5) and specifically from the wild-type allele (exons 1–2; Fig. 3BCitation ). Although transcripts were detectable when mouse lung from wild-type mice was the source of template RNA (Fig. 3CCitation , Lane 1), products were barely detectable when RNA from the Dutt1/Robo1+/– tumors was used as template (Fig. 3C)Citation . In parallel with apparent low transcription levels of the Dutt1/Robo1 gene, using immunohistochemistry, very low levels of Dutt1/Robo1 protein were detected in 9 of 12 lung tumors examined that developed in Dutt1/Robo1+/– mice in contrast to surrounding normal tissue (Fig. 4A and B)Citation and to normal bronchial epithelium (Fig. 4C)Citation . Dutt1/Robo1 protein was also virtually undetectable by immunohistochemistry in the three other carcinomas listed in Fig. 1BCitation (data not shown) and in 12 of 17 lymphomas tested (data not shown), but the benign neoplasms tested showed positive staining (Fig. 4D–F)Citation , implying that inactivation was associated with the malignant phenotype. The low levels of Dutt1/Robo1 protein in the malignant tumors (lymphomas and lung adenocarcinomas) were confirmed by Western blotting using mouse brain and a human tumor cell line as positive controls (Fig. 4G)Citation . Nonmicrodissected tumor samples contain variable amounts of normal cells (e.g., blood vessels, stroma, lymphocytes, and so forth) that may express Dutt1/Robo1 protein, presumably resulting in the trace of protein detectable by Western blotting (Fig. 4GCitation , Lanes 4 and 8).



View larger version (48K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Partial structure and expression of wild-type and mutant Dutt1/Robo1 gene. A, wild-type allele exons 1 to 5 and mutant allele with exon 2 replaced by a neo construct (13) . B, the transcripts that arise from the mutant and wild-type genes and the priming sites and PCR products that distinguish the transcripts. C, examination of expression of Dutt1/Robo1 alleles in mutant mice and controls. RT-PCR analysis of RNA from lung tumors and lymphomas amplified with primers from exons 1 and 2 (only RNA from the wild-type Dutt1/Robo1 allele can act as a template) and from exons 3 and 5 (RNA from both wild-type and mutant Dutt1/Robo1 alleles can act as a template). Amplification with primers from Gapdh acts as a template control. Lane 1, RNA from normal lung from a wild-type littermate; Lane 2, RNA from uninvolved lung of Dutt1/Robo1+/– mice; Lanes 3–6, RNA from lung tumors; Lanes 7–10, RNA from lymphomas; Lane 11, RNA from fibroblasts from Dutt1/Robo1 homozygous mice lacking exon 2; Lane 12, no template control.

 


View larger version (155K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Protein expression in tumors from Dutt1/Robo1 mice. Immunohistochemistry of various tissues using anti-DUTT1/ROBO1 antibody. A, Dutt1/Robo1+/– lung adenocarcinoma showing the tumor with no detectable staining (pink) surrounded by weakly positive (purple-gray) stroma. B, enlargement of the boxed area in A. C, normal mouse bronchus with occasional strongly stained cells in the bronchial epithelium. D, thecal cell tumor, a benign tumor of the ovary, with strongly positive cells. E, hepatocyte dysplasia. F, positively stained hyperplastic sweat glands. G, Western blotting with anti-DUTT1/ROBO1 antibody of the following tissues: Lane 1, newborn mouse brain; Lanes 2–5, lymphomas from Dutt1/Robo1+/– mice; Lane 6, NCI-H69 small-cell lung carcinoma cells; and Lanes 7–10, lung tumors from Dutt1/Robo1+/– mice.

 
Dutt1/Robo1 Gene Structure in Tumors from Dutt1/Robo1+/– Mice.
The RNA and protein expression data suggested that Dutt1/Robo1 gene silencing had occurred in the etiology of the malignant tumors. When tumors develop in mice with targeted disruption of a tumor suppressor gene, the remaining allele is often lost or inactivated, mimicking the behavior of tumor suppressor genes in spontaneously arising human tumors. However, Southern blotting of DNA from lung tumors and lymphomas showed that the wild-type allele was retained in tumors developing in these Dutt1/Robo1 heterozygous mice (Fig. 5A)Citation .



View larger version (54K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Analysis of Dutt1/Robo1 gene structure and epigenetic modification in Dutt1/Robo1+/– mice. A, Southern blot analysis of the Dutt1/Robo1 gene in lung tumors and lymphomas indicates that both the wild-type allele (6.9 kb) and the mutated allele (7.4 kb) are detectable in all tumors examined. Lane 1, normal mouse DNA; Lane 2, mouse tail DNA from a Dutt1/Robo1+/– mouse; Lanes 3–7, DNA from Dutt1/Robo1+/– lung tumors; Lanes 8–12, DNA from Dutt1/Robo1+/– lymphomas. B, a CpG map of the promoter region of the mouse Dutt1/Robo1 gene (accession number NW_0000108). The blue-shaded areas indicate the CpG islands predicted using CpG plot program, +1 indicates the translation start site. The primer pairs for CpG islands 1 and 2 are F1 and R1 and F2 and R2, respectively. C, the sequence within CpG region 1 (–653 to –708) before bisulfite treatment (unconverted) and after treatment if the CpG islands are methylated (converted methylated) or unmethylated (converted unmethylated). Chromatographs highlighting the different methylation patterns of normal lung (unmethylated), benign tumor (unmethylated), lung tumor (methylated), and lymphoma (methylated). The arrows indicate the locations of nucleotides that can be methylated.

 
To determine whether this apparent gene silencing was linked to hypermethylation of the promoter region of the Dutt1/Robo1 gene, we identified the related CpG islands by referring to the location of those associated with the human DUTT1/ROBO1 gene (11) . DNA from lymphomas, lung adenocarcinomas, and controls was treated with sodium bisulfite and amplified with primers flanking two potentially methylated regions (Fig. 5B)Citation , and the PCR products were sequenced. DNA from normal lung was unmethylated, as was that from a benign tumor control. In contrast, DNA from the six lymphomas and six lung tumors assessed for methylation of the Dutt1/Robo1 promoter region was all found to be methylated (Fig. 5C)Citation . Because the antibody to the COOH-terminal peptide detects both mutant and wild-type protein, we conclude that the very low levels of any Dutt1/Robo1 protein in those malignant tumors tested imply that both alleles are methylated in the malignant tumors of Dutt1/Robo1+/– mice.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Before the observations described here, only circumstantial evidence linked the human DUTT1/ROBO1 gene to tumor formation (7 , 11) . In our earlier work, we used gene targeting in mice to recapitulate a specific human intragenic deletion that occurs within the DUTT1/ROBO1 gene. The abnormal phenotypes that developed in mice homozygous for the deletion provided a direct link between this gene and epithelial lung abnormalities in both developing and adult mice (13) . In this study, we demonstrate that tumors arise in the heterozygous mutant mice as a direct consequence of the specific genetic disruption of the Dutt1/Robo1 gene, providing the first unequivocal demonstration of a functional association between this candidate tumor suppressor gene and tumor formation. No other candidate tumor suppressor gene mapping within regions of homozygous deletion on 3p has been directly linked in this way to spontaneous epithelial tumor development.

Further support for the Dutt1/Robo1 gene as a tumor suppressor gene is provided by comparison with HIC1 (17) . After confirmation of HIC1 as a tumor suppressor gene, not only by the observation of tumor formation in mice heterozygous for a targeted deletion of Hic1, but also by virtue of methylation of its promoter in these mouse tumors, Chen et al. (18) have proposed that the detection of methylation arising in the course of tumor development in genetically modified mice provides a "prime strategy" for confirming tumor suppressor gene status on genes without somatic mutations as a defining feature.

However, although we believe we have provided compelling evidence that Dutt1/Robo1 behaves as a tumor suppressor gene in the development of these mouse tumors, we cannot at present extrapolate these observations directly to human lung carcinomas because Northern blotting has shown that the DUTT1/ROBO1 gene is expressed in a variety of human tumor cell lines (19) . Furthermore, protein is easily detectable by immunohistochemistry in tissue sections of primary human tumors.5 The function of DUTT1/ROBO1 in normal epithelial cells is not known, making it currently impossible to investigate whether the DUTT1/ROBO1 protein detected in human tumors is performing some aberrant function. The apparent disparity between the observed DUTT1/ROBO1 protein expression in these experimental mouse tumors and human tumors may be accounted for by reduced or absent expression at the early stage of lung tumor development and increased expression at a later stage of malignancy, possibly performing some oncogenic function. Transforming growth factor ß, which suppresses the early stages of tumorigenesis but promotes tumor progression and invasion, is an example of such a dual function (20) . The expression pattern of E-cadherin in tumors also has similar parallels to DUTT1/ROBO1. Loss of E-CAD expression is associated with promotion of invasion, but subsequent reexpression of E-CAD may encourage survival of metastatic deposits. In a study of heterogeneity of expression of this gene in breast cancer, the fluctuation was shown to be modulated by variation in methylation of its promoter (21) . During the active selection processes that are part of tumor development, cells with partial reactivation of tumor suppressor genes may be selected because of the growth advantage conferred by reexpression of the gene. For genes with pleiomorphic and opposing functions at different stages of tumor progression, inactivation by an epigenetic process rather than mutation provides a selective advantage during tumor development because reactivation is possible.

SLIT2, a likely ligand for DUTT1/ROBO1, also appears to have both tumor-suppressing and -promoting roles. Dallol et al. (22 , 23) consider the SLIT2 gene to be an excellent candidate tumor suppressor due to its growth-suppressing properties and frequent methylation in several human tumors including colorectal, lung, and breast carcinomas. In contrast, Wang et al. (24) detected SLIT2 expression in a wide variety of tumor cell lines and primary tumors and showed in a series of colon preinvasive lesions of increasing grade that the degree of SLIT2 expression correlated with the grade of malignancy, consistent with tumor-promoting properties. Clearly, the roles of DUTT1/ROBO1 and its ligand(s) in tumorigenesis require further detailed investigation, but these observations, together with this report, suggest that a new signaling pathway in tumorigenesis has been identified. Because signaling mediated by this pathway is likely to affect cell motility, an understanding of the abrogation of this pathway in tumorigenesis may provide insight into the mechanisms of tumor invasion and metastatic spread.


   ACKNOWLEDGMENTS
 
Thanks to Anindo Banerjee and Adeline Nicholas for help with preparation of the manuscript, to Mark Arends and Linda Ko Ferrigno for helpful comments on the manuscript, and to the Medical Research Council Laboratory of Molecular Biology Biomedical Facilities staff for animal husbandry.


   FOOTNOTES
 
Grant support: Grant G9703123 from the Medical Research Council. T. Rabbitts and R. Pannell are funded by a Medical Research Council grant to the Laboratory of Molecular Biology.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Pamela Rabbitts, Department of Oncology, University of Cambridge, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom. E-mail: phr{at}mrc-lmb.cam.ac.uk

4 http://tumor.informatics.jax.org/straingrid.html. Back

5 G. Corbett, J. Xian, M. Arends, J. Catto, and P. Rabbitts, unpublished observations. Back

Received 7/19/04. Accepted 8/ 3/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Albertson DG, Collins C, McCormick F, Gray JW. Chromosome aberrations in solid tumours. Nat Genet, 2003;34:369-76, [CrossRef][Medline]
  2. Zabarovsky ER, Lerman MI, Minna JD. Tumour suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene, 2002;21:6915-35, [CrossRef][Medline]
  3. Wistuba II, Behrens C, Virmani AK, et al High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res, 2000;60:1949-60, [Abstract/Free Full Text]
  4. Lerman MI, Minna JD. The 630kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. Cancer Res, 2000;60:6116-33, [Abstract/Free Full Text]
  5. Huebner K. Tumor suppressors on 3p: a neoclassic quartet. Proc Natl Acad Sci USA, 2001;98:14763-5, [Free Full Text]
  6. Pfeifer GP, Yoon JH, Liu L, et al Methylation of the RASSF1A gene in human cancers. Biol Chem, 2002;383:907-14, [CrossRef][Medline]
  7. Sundaresan V, Chung G, Heppell-Parton A, et al Homozygous deletions at 3p12 in breast and lung cancer. Oncogene, 1998;17:1723-9, [CrossRef][Medline]
  8. Kidd T, Brose K, Mitchell KJ, et al Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell, 1998;92:205-15, [CrossRef][Medline]
  9. Rao Y, Wong K, Ward M, Jurgensen C, Wu JY. Neuronal migration and molecular conservation with leukocyte chemotaxis. Genes Dev, 2002;16:2973-84, [Free Full Text]
  10. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell, 2000;100:57-70, [CrossRef][Medline]
  11. Dallol A, Forgacs E, Martinez A, et al Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers. Oncogene, 2002;21:3020-8, [CrossRef][Medline]
  12. Baylin SB, Herman JG. Promoter hypermethylation: can this change alone ever designate true tumor suppressor gene function?. J Natl Cancer Inst (Bethesda), 2001;93:664-5, [Free Full Text]
  13. Xian J, Clark KJ, Fordham R, et al Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene. Proc Natl Acad Sci USA, 2001;98:15062-6, [Abstract/Free Full Text]
  14. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 1996;93:9821-6, [Abstract/Free Full Text]
  15. Clark K, Hammond E, Rabbitts P. Temporal and spatial expression of two isoforms of the Dutt1/Robo1 gene in mouse development. FEBS Lett, 2002;523:12-6, [Medline]
  16. Tulverson DA, Jacks T. Modelling lung cancer in mice: similarities and shortcoming. Oncogene, 1999;18:5318-24, [CrossRef][Medline]
  17. Wales MM, Biel MA, el Deiry W, et al p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3. Nat Med, 1995;1:570-7, [CrossRef][Medline]
  18. Chen WY, Zeng X, Carter MG, et al Heterozygous disruption of Hic1 predisposes mice to a gender-dependent spectrum of malignant tumours. Nat Genet, 2003;33:197-202, [CrossRef][Medline]
  19. Sundaresan V, Roberts I, Bateman A, et al The DUTT1 gene, a novel NCAM family member, is expressed in developing murine neural tissues and has an unusually broad pattern of expression. Mol Cell Neurosci, 1998;11:29-35, [CrossRef][Medline]
  20. Derynck R, Akhurst RJ, Balmain A. TGF-ß signalling in tumour suppression and cancer progression. Nat Genet, 2001;29:117-29, [CrossRef][Medline]
  21. Graff JR, Gabrielson E, Fujii H, Baylin SB, Herman JG. Methylation patterns of the E-cadherin 5' CpG island are unstable and reflect the dynamic, heterogeneous loss of E- cadherin expression during metastatic progression. J Biol Chem, 2000;275:2727-32, [Abstract/Free Full Text]
  22. Dallol A, Da Silva NF, Viacava P, et al A human homologue of the Drosoplila Slit2 gene has tumor suppressor activity and is frequently inactivated in lung and breast cancers. Cancer Res, 2002;62:5874-80, [Abstract/Free Full Text]
  23. Dallol A, Morton D, Maher ER, Latif F. SLIT2 axon guidance molecule is frequently inactivated in colorectal cancer and suppresses growth of colorectal carcinoma cells. Cancer Res, 2003;63:1054-8, [Abstract/Free Full Text]
  24. Wang B, Xiao Y, Ding B, et al Induction of tumour angiogenesis by Slit-Robo signalling and inhibition of cancer growth by blocking Robo activity. Cancer Cell, 2003;4:19-29, [CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
R. E. Dickinson, M. Myers, and W. C. Duncan
Novel Regulated Expression of the SLIT/ROBO Pathway in the Ovary: Possible Role during Luteolysis in Women
Endocrinology, October 1, 2008; 149(10): 5024 - 5034.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
Q. Tao, J. Fujimoto, T. Men, X. Ye, J. Deng, L. Lacroix, J. L. Clifford, L. Mao, C. S. Van Pelt, J. J. Lee, et al.
Identification of the Retinoic Acid Inducible Gprc5a As a New Lung Tumor Suppressor Gene
J Natl Cancer Inst, November 21, 2007; 99(22): 1668 - 1682.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
W. Yue, S. Dacic, Q. Sun, R. Landreneau, M. Guo, W. Zhou, J. M. Siegfried, J. Yu, and L. Zhang
Frequent Inactivation of RAMP2, EFEMP1 and Dutt1 in Lung Cancer by Promoter Hypermethylation
Clin. Cancer Res., August 1, 2007; 13(15): 4336 - 4344.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. Fischer, A. Oberthuer, B. Brors, Y. Kahlert, M. Skowron, H. Voth, P. Warnat, K. Ernestus, B. Hero, and F. Berthold
Differential Expression of Neuronal Genes Defines Subtypes of Disseminated Neuroblastoma with Favorable and Unfavorable Outcome
Clin. Cancer Res., September 1, 2006; 12(17): 5118 - 5128.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
H. Ito, S.-i. Funahashi, N. Yamauchi, J. Shibahara, Y. Midorikawa, S. Kawai, Y. Kinoshita, A. Watanabe, Y. Hippo, T. Ohtomo, et al.
Identification of ROBO1 as a Novel Hepatocellular Carcinoma Antigen and a Potential Therapeutic and Diagnostic Target.
Clin. Cancer Res., June 1, 2006; 12(11): 3257 - 3264.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. C. Hollander, R. T. Philburn, A. D. Patterson, S. Velasco-Miguel, E. C. Friedberg, R. I. Linnoila, and A. J. Fornace Jr.
Deletion of XPC leads to lung tumors in mice and is associated with early events in human lung carcinogenesis
PNAS, September 13, 2005; 102(37): 13200 - 13205.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
N. Zanesi, R. Mancini, C. Sevignani, A. Vecchione, M. Kaou, M. Valtieri, G. A. Calin, Y. Pekarsky, J. R. Gnarra, C. M. Croce, et al.
Lung Cancer Susceptibility in Fhit-Deficient Mice Is Increased by Vhl Haploinsufficiency
Cancer Res., August 1, 2005; 65(15): 6576 - 6582.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xian, J.
Right arrow Articles by Rabbitts, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xian, J.
Right arrow Articles by Rabbitts, P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online