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Molecular Biology and Genetics |
Section of Medical and Molecular Genetics, Division of Reproductive and Child Health [A. D., E. R. M., F. L.], and Department of Surgery [D. M.], University of Birmingham, Birmingham B15 2TT, United Kingdom, and Cancer Research United Kingdom, Renal Molecular Oncology Research Group, University of Birmingham, Birmingham B15 2TT, United Kingdom [E. R. M., F. L.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have recently shown that SLIT2 is frequently inactivated in breast and lung tumors and that the gene exhibited tumor suppressor activity (4) . The Slit family of large extracellular matrix secreted and membrane-associated glycoproteins are known to be important mediators of the repulsive cues on axons and growth cones during neural development. The first clue of SLIT2 importance outside a neural context came from being expressed in neuronal as well as nonneuronal tissues, and it has also been shown to inhibit leukocyte chemotaxis (5) . This inhibition could be mediated through the Slit2 effect on actin cytoskeleton organization mediated through cdc42 (6) . Slit protein triggers Robo/DCC interaction with the subsequent loss of responsiveness of DCC to its ligand, netrin-1 (7) . This loss may activate apoptotic pathways through caspase 3 and caspase 9 (8) .
SLIT2 gene has been mapped to chromosome 4p15.2 (9) . Deletion of the 4p15.1–15.3 region has been shown to occur early in colorectal carcinoma (10) . Microsatellite markers from this region also show LOH in mesothelioma, small cell lung cancer, non-small cell lung cancer, and breast tumors (11 , 12) . We already have shown that SLIT2 is a good candidate for a TSG (tumor suppressor gene) in lung and breast cancer, two cancer types exhibiting LOH at 4p15. SLIT2 could also be implicated in CRC pathogenesis based on the frequency of 4p15 deletions in CRC. Therefore, we have analyzed a panel of colorectal tumor cell lines and primary tumors with matching histologically normal tissue for the presence of SLIT2 methylation. We used direct sequencing of a region in the putative SLIT2 promoter in an attempt to distinguish better between the levels of SLIT2 methylation in normal versus tumor DNA. We also screened the same panel for ROBO1 methylation and looked for a correlation between SLIT2 methylation and the inactivation of ROBO1/RASSF1A/p16/K-RAS. We also sought to determine whether SLIT2-induced growth suppression is mediated, at least in part, by the process of apoptosis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bisulfite Modification and Methylation Analysis.
Bisulfite DNA sequencing was performed as described previously (14)
. Briefly, 0.5–1.0 µg of genomic DNA was denatured in 0.3 M NaOH for 15 min at 37°C, and then unmethylated cytosine residues were sulfonated by incubation in 3.12 M sodium bisulfite (pH 5.0; Sigma)/5 mM hydroquinone (Sigma) in a thermocycler (Hybaid) for 30 s at 99°C and for 15 min at 50°C for 20 cycles. The sulfonated DNA was recovered using the Wizard DNA cleanup system (Promega) in accordance with the manufacturer’s instructions. The conversion reaction was completed by desulphonating in 0.3 M NaOH for 10 min at room temperature. The DNA was ethanol precipitated and resuspended in water.
The SLIT2 putative promoter region was predicted by Promoter Inspector software (http://www.genomatix.de). This region is from -761 to -212 relative to the translation start site. The region was amplified from cell lines or tumors using the primers Sli2MOD4F (5'-GGGAGGTGGGATTGTTTAGATATTT-3') and primer Sli2MOD4R2 (5'-CAAAAACTCCTTAAACAACTTTAAATCCTAAAA-3') as we have described previously (8) . One-fiftieth volume of the PCR reaction (with primers Sli2MOD4F and Sli2MOD4R2) was used in a nested PCR reaction with 30 cycles using primer SL2-SP-F (5'-AGTTTAGAGTYGTGYGTTTTTAGAAT-3') and the primer SL2-SP-R (5'-CCRATCAAAATAAACTCCRTAAACTAA-3'), where Y is C+T and R is A+G. These primers amplify a region where we have found most of the methylated CpGs in the putative SLIT2 promoter to be concentrated. The PCR conditions for both the first and second PCR were 95°C for 10 min, followed by 30–40 cycles of 1 min denaturation at 95°C, 1 min annealing at 52–54°C, and 2 min extension at 74°C. The PCR products were concentrated and purified using QIAquick PCR Purification columns (Qiagen). The purified products were directly sequenced to obtain average methylation levels using ABI BigDye cycle sequencing kit. The ratio of methylated alleles was estimated from the relative peak heights of the G and A peaks at methylated CpG sequences after normalizing the A peak for the average peak height along the entire sequence and subtracting the background G peak. The values obtained were further normalized to the ratio of A and G peak heights in the primer sequence. The final values obtained were given an arbitrary maximum value of 2. DUTT1 methylation status was determined as described previously (15) .
Cell Lines and 5-aza-dC Treatment.
Colorectal carcinoma cell lines (SW48, LoVo, DLD1, LS411, HCT116, and LS174T) were routinely maintained in DMEM growth medium (Invitrogen) supplemented with 10% FCS at 37°C, 5% CO2. The demethylating agent, 5-aza-dC (Sigma) was freshly prepared in double-distilled H2O and filter-sterilized. Cells (5–10 x 105) were plated in a 25-cm2 flask in DMEM supplemented with 10% FCS. Twenty-four h later, cells were treated with 10 µM 5-aza-dC. The medium was changed 24 h after treatment and then every 3 days. RNA was prepared 10 days after treatment using the RNeasy kit (Qiagen) according to the manufacturer’s guidelines. SLIT2 expression was detected by reverse transcription-PCR using the primers 5'-GGTGTCCTCTGTGATGAAGAG-3' and 5'-GTGTTTAGGAGACACACCTCG-3'. The expected product size was 387 bp. Expression of GAPDH was used as a control. The GAPDH primers were described previously (15)
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Microsatellite Repeat Analysis.
PCR amplification of the dinucleotides repeats microsatellite sequence of marker D4S1546 was performed on the methylated colorectal carcinomas. The D4S1546 marker was the nearest possible marker (within 100 kb) available to the SLIT2 gene on 4p15.2 according to Genome Databases and the UCSC assembly of the human genome sequence. The PCR conditions were 96°C for 5 min followed by the addition of 0.2 units of Taq polymerase (Invitrogen) followed by 35 cycles of 96°C for 30 s, 52°C for 30 s, and 72°C for 30 s. A final extension step of 5 min at 72°C was performed.
Plasmid Constructs and Growth Suppression Analysis.
The SLIT2 expression construct was made by cloning the full length of the human Slit2 coding region into the BamHI-XbaI sites of pSecTagB vector (Invitrogen) and, thus, contains both myc and His epitope tags at its COOH terminus (16)
. Transfection with Fugene6 reagent (Roche) used 2 µg of each plasmid per 25-cm2 flask containing 5 x 105 cells seeded 24 h before transfection. Forty-eight h after transfection, 5 x 104 transfected cells were seeded and maintained in DMEM-10% fetal bovine serum supplemented with 50 µg/ml Zeocin (Invitrogen). Surviving colonies were counted 14–21 days later after staining with crystal violet. SLIT2-conditioned medium (with 
10 nM SLIT2 protein) was prepared by transient transfection of COS-7 cells of SLIT2- or vector-only plasmids. Forty-eight h after transfection, the presence of SLIT2 was confirmed by Western blotting, and medium was cleared by centrifugation and added to 5 x 104 cells seeded 24 h earlier in each well of 6-well plates. After 4 days, the viable cells (as determined by trypan blue exclusion) in each indicated treatment were counted using a hemocytometer.
Detection of Apoptosis.
SW48 cells undergoing early stages of SLIT2-induced apoptosis were detected by their increased ability to bind Annexin V-phycoerythrin conjugate (PharMingen). Cell necrosis was detected by the incorporation of 7-amino-actinomycin D dye. Labeled cells were detected by fluorescence-activated cell sorting (FACS) analysis on a Coulter Epics flow cytometer. TUNEL activity was detected in SW48 cell line exposed to SLIT2-conditioned medium for 48 h using the In Situ Cell Death Detection kit (Roche) according to the manufacturer’s instruction. These experiments were done in replicates and repeated three times.
Statistical Analysis.
Comparisons were made by Fisher’s exact test or t test when appropriate. P values of <0.05 were taken as statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, left panel). To confirm that methylation was responsible for silencing SLIT2 gene expression, we treated three CRC cell lines with the demethylating agent 5-aza-dC. SLIT2 expression was restored in cell lines with absent or weak SLIT2 expression after 5-aza-dC treatment (Fig. 1A
, right panel), whereas the unmethylated glioma cell line demonstrated no difference in SLIT2 expression before or after treatment.
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. We designed bisulfite-modified DNA-specific primers to amplify this short region and to use it in direct sequencing analysis. Using such a short region (146 bp) minimizes the problems associated with sequencing directly from bisulfite-modified DNA PCR products using the ABI BigDye chemistry. One major problem is the background G levels (obtained from the reverse sequence) caused by the ABI software compensating for the lower-than-expected G ratio. We have validated all of the sequencing results by a combination of COBRA (Combined Bisulfite Restriction Analysis) and sequencing of individual clones from PCR products cloned into pGEM T-easy vector. In our experience, we have found that direct sequencing allows more accurate estimation of the level of methylation of SLIT2 in comparison with using methylation-sensitive PCR or COBRA. The results of the sequencing analysis of the CRC cell lines are shown in Fig. 1C
. The CRC cell lines SW48, DLD1, and LoVo are heavily methylated for this region, whereas the CRC line LS174T shows partial methylation, and the glioma cell line U343 shows no methylation (Fig. 1C)
. These results correlate very well with our expression analysis (Fig. 1A)
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We have applied the same strategy to show the relative methylation levels between tumor tissues and neighboring histologically normal mucosa (Fig. 2)
. We found tumor-specific methylation of SLIT2 promoter region in at least 23 (72%) of 32 CRC tumors. We also found a weak level of SLIT2 methylation in almost all of the corresponding normal mucosa samples. No significant methylation could be found in the normal samples that had no methylation in the corresponding tumor. In our previous study, we found that there was no detectable SLIT2 methylation in lymphocytes from healthy individuals (4)
. The presence of SLIT2 methylation in tumor cases did not correlate with tumor grade or patient’s age. LOH analysis at D4S1546 (which maps within 100 kb of SLIT2) demonstrated allele loss in 39% of informative CRC cancers with SLIT2 methylation. We have previously shown that SLIT2 is mainly silenced by hypermethylation and that we did not find any somatic mutations in the coding region of SLIT2 in a panel of lung and breast cancers, and, therefore, it is unlikely that SLIT2 is inactivated by mutations in a significant manner in CRC.
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; Fig. 3
). This observation was similar to ROBO1/DUTT1 methylation levels that we have found previously in breast and kidney tumors (15)
. There was no significant correlation between SLIT2 methylation and ROBO1/DUTT1 methylation in the tumors analyzed. There was also no significant correlation with inactivation of the ROBO1/DUTT1 gene and tumor grade or patient’s age.
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; Fig. 3
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SLIT2 Induces Growth Suppression and Apoptosis.
Having demonstrated that epigenetic inactivation of SLIT2 is frequent in CRC; we investigated tumor suppressor activity of SLIT2 in in vitro colony formation assays. A wild-type SLIT2 expression plasmid or the pSecTagB empty vector were transfected into SW48 and LoVo tumor cell lines. Both constructs expressed a zeocin resistance gene. Transfection with SLIT2 significantly reduced (P < 0.0055) the number of zeocin-resistant colonies by >70% compared with transfection with empty vector in both of the cell lines tested (Fig. 4A)
. This effect was consistent through three independent experiments and using three independent plasmid DNA preparations. As additional evidence for the ability of SLIT2 to suppress growth, conditioned medium from COS-7 cells transfected with SLIT2 causes a significant growth inhibition (P < 0.0001) in relation to control. SLIT2-conditioned medium causes up to 65% growth inhibition in the SW48 cell line (Fig. 4B)
. In addition, SLIT2 induces apoptosis as detected by the increase in Annexin V-PE labeling in SW48 (up to 20%) after treatment with SLIT2-conditioned medium for 48 h (Fig. 5A)
. SLIT2-conditioned medium caused an average increase of 11.50 ± 1.79% of annexin V positive/7-AAD negative cells. This effect was significant and consistent throughout three experiments (P < 0.008). SLIT2-induced apoptosis could also be detected using TUNEL. The average increase in TUNEL positive cells after exposure to SLIT2 was 7.7 ± 0.65% compared with control (P < 0.0001; Fig. 5B
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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19% of breast and kidney cancers and in a small number of lung cancers. Analysis of the SLIT2 genetic and epigenetic status revealed a higher level of inactivation (>40%), mainly by hypermethylation, in lung and breast cancers, and we correlated this methylation with reduced expression in tumor cell lines and primary tumors. Therefore, we hypothesized that haploinsufficiency of DUTT1 coupled with haploinsufficiency/inactivation of its putative ligand SLIT2 could be promoting tumor progression and metastasis. In this study, we provide evidence for SLIT2 inactivation by promoter region hypermethylation in the majority of CRC samples analyzed. Our data support the revised Knudson two-hit theory (17) with one allele being inactivated by methylation and the other by LOH. Methylation of SLIT2 found in the neighboring normal mucosa, in which the tumor is methylated to a significantly higher degree, may indicate that SLIT2 methylation is an early event in CRC tumorigenesis. However, in view of the role of SLIT2 in controlling axon and leukocytes migration, it is possible that methylated SLIT2 from migrating tumor cells is being detected. All of our samples come from patients ages 47–96 years old with an average age of 74 and a median age of 76 years old. Therefore, all of the samples were collected from "older" patients. Although there is no statistical correlation between age and SLIT2 methylation, there were no samples from young patients in our study; therefore, we cannot exclude the possibility that the methylation of SLIT2 in normal mucosa is associated with aging, as has been described for the methylation of the estrogen receptor in CRC (18) . We also demonstrate ROBO1 methylation in CRCs at a frequency equal to that with breast and kidney tumors (19%). SLIT2 methylation did not correlate with the methylation of ROBO1/p16/RASSF1A or with K-RAS mutations.
The SLIT family is one of four conserved families of axonal guidance cues that have prominent developmental effects. The fact that these molecules are secreted provide a novel therapeutic potential. The other three are the netrins, the semaphorins, and the ephrins (19) . Evidence is growing for the involvement of these guidance cues and their receptors in carcinogenesis. SEMA3B, a member of the semaphorin family, suppresses growth of adenocarcinoma cancer cell lines (20) . SEMA3B also induces apoptosis and causes growth suppression of lung cancer cell lines (21) , possibly through the p53 pathway (22) . SEMA3B expression is frequently lost in lung cancer, and this loss is caused by hypermethylation of its promoter region (21) . The Netrin-1 receptor, DCC, is frequently inactivated in CRC (23) . DCC loss of expression is also associated with promoter region hypermethylation in primary gastric cancer (24) .
Ectopic expression of SLIT2 in CRC cell lines suppressed growth and reduced colony formation abilities. Additionally, secreted SLIT2, which consists predominantly of the smaller COOH-terminal fragment, in conditioned medium from SLIT2-transfected COS-7 cells, reduced cell number and growth of these CRC lines. We have shown previously that this effect could be seen in breast tumor cell lines and that it is not attributable to general SLIT2-induced cytotoxicity (4) . We have studied SLIT2-induced apoptosis in an attempt to investigate the mechanism of SLIT2-induced growth suppression. We show that SLIT2 induces apoptosis and cell death as indicated by the increased Annexin V binding and TUNEL staining. The mechanism by which SLIT2 induces apoptosis is unclear. If this apoptosis is induced through the DCC pathway, then it is possible to consider caspase 3 and caspase 9 as likely candidates for mediating SLIT2-induced apoptosis (8) .
In conclusion, aberrant promoter methylation and associated transcriptional silencing is now recognized as a major mechanism of TSG inactivation. SLIT2 methylation appears to be a very frequent and possibly early event in CRC and is independent from p16/RASSF1A/ROBO1 methylation and K-RAS mutation status. The demonstration that SLIT2 can actively induce apoptosis suggests that this gene may have an important role in controlling the growth and migration of colorectal cells. SLIT2 provides an excellent candidate for a 4p15.2 TSG for sporadic CRC.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Cancer Research United Kingdom. 
2 To whom requests for reprints should be addressed, at Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, Birmingham B15 2TT, United Kingdom. Phone: (44)(0)-121-627-2741; Fax: (44)(0)-121-627-2618; E-mail: flatif{at}hgmp.mrc.ac.uk
3 The abbreviations used are: CRC, colorectal cancer; CIMP, CpG island methylator phenotype; DCC, deleted in CRC; 5-aza-dC, 5-aza-2'-deoxycytidine; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; LOH, loss of heterozygosity.
Received 9/10/02. Accepted 1/ 2/03.
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REFERENCES |
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, unmethylated CpGs; 
, methylated CpGs; partially filled squares, partially methylated CpGs.
