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SLIT2, a Human Homologue of the Drosophila Slit2 Gene, Has Tumor Suppressor Activity and Is Frequently Inactivated in Lung and Breast Cancers1

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SLIT2, a Human Homologue of the Drosophila Slit2 Gene, Has Tumor Suppressor Activity and Is Frequently Inactivated in Lung and Breast Cancers1 [CANCER RESEARCH 62, 5874–5880, October 15, 2002] SLIT2, a Human Homologue of the Drosophila Slit2 Gene, Has Tumor Suppressor Activity and Is Frequently Inactivated in Lung and Breast Cancers1 Ashraf Dallol, Nancy Fernandes Da Silva, Paolo Viacava, John D. Minna, Ivan Bieche, Eamonn R. Maher, and Farida Latif2 Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, Birmingham B15 2TT, United Kingdom [A. D., N. F. D. S., E. R. M., F. L.]; CRC Renal Molecular Oncology Research Group, University of Birmingham, Birmingham B15 2TG, United Kingdom [N. F. D. S., E. R. M., F. L.]; Department of Oncology, Division of Pathology, University of Pisa, Pisa, Italy [P. V.]; Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390 [J. D. M.]; and Laboratoire d’Oncoge´ne´tique-INSERM E0017, Centre Rene´Huguenin, F-92210 St-Cloud, France [I. B.] ABSTRACT knot (Fig. 1; Ref. 4). These motifs are also found in other proteins and have been implicated in mediating protein-protein and ECM-protein Slit2 plays a vital role in axon guidance by signaling through Robo interactions. The SLIT2 protein can be present in three forms. The receptors. Recent evidence suggests that Slit2 protein may function in full-length protein (M ϳ200,000) is proteolytically cleaved into an other settings because human and Xenopus Slit2 has been shown to inhibit r leukocyte chemotaxis. SLIT2 protein is a putative ligand for the ROBO NH2-terminal fragment and a smaller COOH-terminal fragment. It is receptors. We recently demonstrated that ROBO1 is inactivated by pro- thought that different axons have specific responses to particular moter region hypermethylation in <20% of human cancers; furthermore, SLIT2 fragments (5). tumor suppressor activity has not been shown. Thus, the importance of SLIT2 is expressed in neuronal as well as nonneuronal tissues. Rat ROBO1 inactivation in human cancer is uncertain. Therefore, we inves- Slit2 is expressed in lung, breast, kidney, and heart (6). In situ tigated the status of SLIT2 located at 4p15.2 in lung and breast cancers. hybridization of human kidneys revealed the expression of SLIT2 in Although somatic SLIT2 mutations were not detected, epigenetic inacti- mesangial and epithelial cells in the glomeruli, in epithelial cells in the vation was common. SLIT2 promoter methylation was detected in 59% of tubules, and in endothelial cells of both arterioles and venules in the breast cancer, 77% of non-small cell lung cancer, and 55% of small cell kidney (6). In addition, Unigene cluster Hs.29802 shows that SLIT2 is lung cancer cell lines. In these tumor lines, SLIT2 expression was restored expressed in adult lung and kidney in addition to heart. This broad by treatment with a demethylating agent. SLIT2 promoter methylation was detected in 43% of breast cancer, 53% of non-small cell lung cancer, pattern of SLIT2 expression suggests that it may have other functions. and 36% of small cell lung cancer primary tumors. The majority of Slit2 has been shown to inhibit leukocyte chemotaxis (6), possibly methylated tumors demonstrated allelic loss at 4p15.2. In addition, SLIT2 through the Slit2 effect on actin cytoskeleton organization mediated expression was down-regulated in methylated breast tumors, relative to through cdc42 (7). Slit protein triggers Robo-DCC (deleted in colon normal control, as demonstrated by quantitative real-time reverse tran- cancer) interaction with the subsequent loss of responsiveness of DCC scription-PCR. Overexpression of SLIT2 suppressed >70% of colony to its ligand, netrin-1 (8). This loss may activate apoptotic pathways growth in each of three breast tumor lines (with either absent or low through caspase 3 and caspase 9 (9). Exon 2 of ROBO1 is homozy- SLIT2 expression). Because SLIT2 is primarily a secreted protein, SLIT2- gously deleted in two lung tumor cell lines and one breast tumor cell conditioned medium suppressed the growth of several breast cancer lines line (10). Most mice with deletion of exon 2 of Robo1 receptor die (with absent or weak SLIT2 expression) by 26–51% but had no significant effect on a breast tumor cell line that expresses normal levels of SLIT2. because of delayed lung maturation, whereas the surviving mice These findings demonstrate that SLIT2 is frequently inactivated in lung develop bronchial hyperplasia (11). and breast cancer by promoter region hypermethylation and allele loss The SLIT2 gene has been mapped to chromosome 4p15.2 (12). and is an excellent candidate for the lung and breast tumor suppressor Microsatellite markers from this region show LOH in 63% of me- gene previously mapped to 4p15.2. sothelioma, 60% of SCLC, and 25% of NSCLC (13). Around 63% of breast tumors also show LOH at 4p15.1–15.3 (14). Deletion of the INTRODUCTION 4p15.1–15.3 region has also been shown to occur in colorectal carci- noma (15), invasive cervical cancer (16), head and neck squamous The Slit family comprises large ECM3 secreted and membrane- cell carcinoma (17), and bladder cancer (18). associated glycoproteins. Human Slits (SLIT1, SLIT2, and SLIT3) are Along with mutation screening of candidate TSGs, methylation candidate ligands for the repulsive guidance receptors, the ROBO analysis of promoter region CpG islands (if present) is required to gene family. The Slit-Robo interactions mediate the repulsive cues on study tumor-specific inactivation. Aberrant methylation of promoter axons and growth cones during neural development. The Slit2-Robo1 region CpG islands has been largely associated with gene silencing in interaction has been shown to be enhanced by cell surface heparan human cancers. This has been reported for several TSGs, most notably sulfates including glypican-1 (1, 2). The Slit proteins are highly for the new TSG RASSF1A located at 3p21.3 (19, 20). conserved in evolution and contain a putative signal peptide, four We have shown that the putative SLIT2 receptor, DUTT1/ROBO1, tandem arrays of leucine-rich repeats required for the Slit-Robo in- is methylated in Ͻ20% of breast tumors and clear cell renal cell teraction and axon repulsion (3), seven to nine EGF repeats, an carcinomas. Methylation of the putative DUTT1 promoter was a rare agrin-laminin-perlecan-Slit conserved spacer motif, and a cysteine event in lung carcinomas (21). The coding region of the DUTT1 gene lacked any inactivating mutations, and the gene was expressed in the Received 5/29/02; accepted 8/20/02. majority of tumor cell lines examined. However, LOH of DUTT1 The costs of publication of this article were defrayed in part by the payment of page occurred frequently in lung, breast, and kidney tumors, prompting us charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. to hypothesize that haploinsufficiency of DUTT1 coupled with hap- 1 Supported in part by Cancer Research UK (E. R. M. and F. L.) and by Specialized loinsufficiency/inactivation of its ligand could be detrimental for Programs of Research Excellence P50 CA70907 (to J. D. M.). tumor progression and metastasis. Thus ROBO1 remains a candidate 2 To whom requests for reprints should be addressed. Phone: 121-627-2741; Fax: 121-627-2618; E-mail: [email protected]. for the lung and breast 3p12 TSG (22). We reasoned that if ROBO1 3 The abbreviations used are: ECM, extracellular matrix; NSCLC, non-small cell lung inactivation was a significant event in human breast and lung cancers, cancer; SCLC, small cell lung cancer; RT-PCR, reverse transcription-PCR; EGF, epider- mal growth factor; LOH, loss of heterozygosity; TSG, tumor suppressor gene; 5-aza-dC, then other components of the signaling pathway might also be in- 5-aza-2Ј-deoxycytidine; MSP, methylation-sensitive PCR; Ct, control. volved in cancer. Therefore, we undertook genetic, epigenetic, and 5874 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2002 American Association for Cancer Research. SLIT2 HAS TUMOR SUPPRESSOR ACTIVITY Fig. 1. Genomic structure of SLIT2. The SLIT2 gene spans a genomic region of approximately 300 kb and is composed of 37 coding exons. The CpG island fulfilled the criteria of GC content Ͼ 0.5 and an observed:expected CpG ratio Ͼ 0.6 (CpG Plot program; http://www.ebi.ac.uk/Tools/). The exon sizes ranged from 24 to 383 bp. Thirteen exons share the same size of 72 bp. The exons are colored according to the domains they code for. Green indicates a cysteine-rich NH2-terminal flanking region; blue indicates a cysteine-rich COOH-terminal flanking region. Gray indicates the leucine-rich tandem repeats. Dark purple indicates the EGF-like domains, whereas light purple indicates the laminin G domains. Orange indicates the cysteine knot. The arrows indicate the relative position of the proteolytic cleavage site and the relative position of the marker D4S1546. functional analysis of the SLIT2 gene in lung and breast cancers. We GGGATTGTTTAGATATTT-3Ј) and Sli2MOD4R2 (5Ј-CAAAAACTCCT- demonstrate that the promoter region of SLIT2 is frequently hyper- TAAACAACTTTAAATCCTAAAA-3Ј). We used 0.02 volume of the first methylated in breast and lung cancer and that this mode of gene PCR reaction (with primers Sli2MOD4F and Sli2MOD4R2) in another PCR inactivation is the main cause of loss of function in these tumors. We reaction using Sli2MOD4F and the nested primer, Sli2MOD4R (5Ј-ACTA- Ј also demonstrate that overexpression of exogenous wild-type SLIT2 AAACTTCCAACAACTACTAAAATACAAAAA-3 ). The PCR conditions causes loss of colony formation ability in vitro and that conditioned for both the first and second PCR were 95°C for 10 min, followed by 30–40 cycles of 1 min of denaturation at 95°C, 1 min of annealing at 54°C, and 2 min medium from COS-7 cells transfected with SLIT2 also suppresses of extension at 74°C.
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