Methylation of CpG dinucleotides in the promoter regions of tumour suppressor genes
(TSGs) producing transcriptional silencing is a major mechanism of TSG inactivation
in many human cancers (Baylin et al, 1998; Jones, 1999; Tycko, 2000; Costello and
Plass, 2001). The frequency of TSG inactivation by de novo methylation varies between
TSGs and between cancers. Initially, epigenetic silencing of TSGs was reported as
an alternative, but a minor mechanism of inactivation for TSGs such as RB1 and VHL
(Greger et al, 1994; Herman et al, 1994). However, recently a new class of TSGs has
been recognised for which epigenetic silencing is the overwhelming mechanism of inactivation
and somatic mutations are rare. This paradigm is exemplified by the RASSF1A 3p21.3
TSG, which is methylated in a wide range of human cancers (Dammann et al, 2000; Agathanggelou
et al, 2001; Astuti et al, 2001; Burbee et al, 2001; Dreijerink et al, 2001; Morrissey
et al, 2001).
Recently, we identified frequent promoter region hypermethylation of SLIT2 in lung
and breast cancers (Dallol et al, 2002a). SLIT2 maps to 4p15.2 and encodes a human
orthologue of the Drosophila Slit protein, a secreted glycoprotein, which regulates
axon guidance, branching and neural migration during development of the central nervous
system (Brose et al, 1999). Slit exerts its effects as a diffusible chemorepellent
via its receptor Roundabout (Robo) (Stein and Tessier-Lavigne, 2001). In mammals,
three distinct slit genes (slit1, slit2, and slit3) and three distinct robo genes
(robo1, robo2, rig-1) have been identified. In lung and breast cancers, SLIT2 mutations
were not detected despite frequent promoter methylation (similar to RASSF1A) (Dallol
et al, 2002a). Methylation of RASSF1A was first reported in lung and breast cancers,
but subsequently we and others demonstrated RASSF1A methylation in renal cell carcinoma
(RCC) and in common childhood tumours such as neuroblastoma (NB) and Wilms' tumour
(Astuti et al, 2001; Dreijerink et al, 2001; Lo et al, 2001; Morrissey et al, 2001;
Wagner et al, 2002). These findings prompted us to extend our studies of SLIT2 inactivation
by studying SLIT2 promoter methylation in NB, Wilms' tumour and RCC.
MATERIALS AND METHODS
Patients and samples
A total of 134 tumour samples were analysed (49 NBs, 37 primary Wilms' tumours and
48 adult RCC). Details of the tumours have been published previously (Astuti et al,
2001; Wagner et al, 2002). DNA was extracted from tumour and normal tissues (blood
or matched kidney) by standard methods.
Sodium bisulphite modification
Sodium bisulphite modification was carried out using an adapted method (Herman et
al, 1996). Genomic DNA (0.5–1 μg) was denatured at 37°C for 10 min in 0.3 M NaOH.
Unmethylated cytosines were sulphonated by incubation in 3.12 M sodium bisulphite/1 M
hydroquinone (pH 5) at (95°C (30 s) 50°C (15 min)) × 20 cycles. The resulting sulphonated
DNA was purified using the Wizard DNA clean-up system (Promega, Southampton, UK),
according to the manufacturer's instructions, except that DNA was eluted with distilled
water (50 μl) at room temperature. Following elution, DNA was desulphonated in 0.3 M
NaOH for 5 min at room temperature, then the DNA was precipitated with NaOAc (5 μl
of 3 M) and ethanol (125 μl of 100%) overnight at −20°C and resuspended in 50 μl distilled
water.
Methylation-specific polymerase chain reaction (PCR)
The CpG island within the putative SLIT2 promoter has been described in detail previously
(Dallol et al, 2002a). Methylation Specific PCR (MSP) analysis was performed essentially
using primers and conditions as described in Dallol et al, 2002a). Combined Bisulphite
Restriction Analysis (CoBRA) analysis was carried out as follows. In brief, the putative
promoter region from nt −761 to −212 (relative to the translation start site) was
amplified with primers reported previously (Slit2MODF-(5′-GGGAGGTGGGATTGTTTAGATATTT-3′
and Slit2MODR2 (5′-CAAAAACTCCTTAAACAACTTTAAATCCTAAAA-3′). From this reaction, one
out of 50 of the volume was used as a template for a nested PCR with primers Slit2MODF
as above and Slit2MODR (5′-ACTAAAACTTCCAACAACTACTAAAATACAAAAA-3′) to produce a 418 bp
product (PCR conditions: 95°C for 10 min, followed by 30–40 cycles of 1 min denaturation
at 95°C, 1 min annealing at 54°C, and 2 min extension at 74°C). PCR products were
then digested by BstU1 restriction enzyme to assess the methylation status of samples.
Sequencing of PCR products
MSP and CoBRA products were excised from agarose gels and extracted using the QIAquick
Gel Extraction Kit (Qiagen, Crawley, UK, West Sussex), according to the manufacturer's
instructions. Products were confirmed by direct sequencing from the forward PCR primer
using ABI Prism® BigDye™Terminators Cycle Sequencing Kit according to the manufacturer's
instructions and run using ABI 377 automatic sequencers.
5-aza-2′-deoxycytidine treatment of cell lines
5-aza-2′-deoxycytidine (5-aza-dC, Sigma, Poole, UK) was freshly prepared in ddH2O
(at 2 mg ml−1) and filter-sterilised. A total of 0.5–1 × 106 cells were plated in
75 cm2 flask in RPMI 1640 media supplemented with 10% foetal calf serum and left to
settle for 24 h (day 0). Kidney and NB cell lines were treated with 2 μ
M 5-aza-dC for a total of 5 days. Kidney and NB have a different rate of growth. Kidney
tumour cell lines were treated with 5-aza-dC on days 1, 3 and 5 and harvested on day
6, with a medium change in days 2 and 4 (Nojima et al, 2001). Neuroblastoma cell lines
were treated on days 1 and 4. The medium was changed 24 h after treatment and then
every 3 days. RNA was prepared after treatment using the RNeasy kit (Qiagen) according
the manufacturer's guidelines. SLIT2 gene expression was ascertained by RT–PCR using
the primers 5′-GGTGTCCTCTGTGATGAAGAG-3′ and 5′-GTGTTTAGGAGACACACCTCG-3′, resulting
in a product size 387 bp. As a control, the GAPDH primers used were: 5′-GACCCCTTCATGACCTCAACTACA-3′
and 5′-CTAAGCAGTTGGTGGTGCAGGA-3′, resulting in a PCR product of 369 bp.
Microsatellite repeat analysis – loss of heterozygosity
By searching GDB and the UCSC assembly of the human genome sequence, we identified
D4S1546 as the closest marker to SLIT2 (within 100 kb). A 4p15.2 allele loss was assessed
with the D4S1546 marker. (PCR conditions: 95°C for 5 min followed by 35 cycles of
95°C for 30 s, 52°C (55°C) for 30 s, and 72°C for 30 s and a final extension of 10 min
at 72°C).
Statistical analysis
Fisher's exact test was used as appropriate. P-values of <0.05 were considered to
be statistically significant.
RESULTS
SLIT2 methylation status in NB
SLIT2 promoter methylation status was analysed in 49 primary NB tumours and 29% (14
out of 49) demonstrated SLIT2 CpG island promoter methylation (Figure 1A
Figure 1
Methylation analysis of SLIT2 by MSP in neuroblastoma tumours (A) and Wilm's tumours
(B) and by CoBRA in RCC cell lines and primary kidney tumours and corresponding normal
tissue (C). (A and B). Bisulphite-modified DNA was amplified with primers specific
for unmethylated (U) and methylated (M) DNA. Tumours positive for SLIT2 methylation
are depicted by the presence of a 160 bp product when using specific primers for methylated
DNA. N=blood; T=tumour tissue. (C) Bisulphite-modified DNA was amplified by nested
PCR and then digested with BstU1 restriction enzyme for 4 h at 60°C, uncut (Un) and
cut (C). SKRC 18 is partially methylated while SKRC 47 is unmethylated. The RCC tumour
shown above is completely methylated.
). Promoter CpG island methylation was confirmed by direct sequencing of five clones
from one tumour (Figure 4B). We also analysed eight NB cell lines for SLIT2 methylation
by restriction digestion and two cell lines (SK-N-F1 and SK-N-SH) were found to be
partially methylated. SLIT2 methylation was detected in one out of 49 corresponding
normal blood samples.
To investigate the 4p15.2 allelic status of NBs with SLIT2 methylation, we typed 13
methylated tumours for loss of heterozygosity (LOH) at D4S1546 that maps close to
SLIT2. In all, 33% (four out of 11) of informative tumours demonstrated allele loss
consistent with homozygous SLIT2 inactivation (Figure 2
Figure 2
Genotyping of marker D4S1546 in neuroblastoma tumours. N and T, matched DNA samples
from blood (N) and tumour tissue (T). Tumours 125 and 162 showed LOH, while tumour
133 shows retention of allele. The position of the lost allele is indicated by the
arrows.
).
SLIT2 promoter methylation is associated with transcriptional silencing
To determine the relationship of SLIT2 promoter region CpG island methylation and
SLIT2 transcript expression in the NB cell lines SK-N-F1 and SK-N-SH, we treated the
cells with the demethylating agent, 5-aza-dC, for 5 days. The 5-aza-dC treatment significantly
increased SLIT2 expression in both cell lines, but there was little or no change in
the expression of GAPDH expression after the 5-aza-dC treatment (Figure 3
Figure 3
SLIT2 expression by RT–PCR in neuroblastoma (SK-N-SH) and renal cell carcinoma cell
lines (SKRC 39, SKRC 18 and SKRC 47), without (−) and with (+) 5-aza-2′-deoxycytidine
(5-aza-dc) treatment. Cells were treated for up to 5 days with 5-aza-dc and expression
was analysed by RT–PCR. The methylated cell lines (SK-N-SH, SKRC 39, SKRC 18) show
an increase in SLIT2 expression after 5-aza-dc treatment, while the unmethylated cell
line SKRC 47 presents no changes in SLIT2 expression before and after 5-aza-dc treatment.
).
SLIT2, CASP8 and RASSF1A methylation status in NB
Previously, we reported that RASSF1A and caspase 8 (CASP8) promoter methylation occurred
in 55 and 40% of NBs, respectively. To determine whether there was any relationship
between SLIT2 promoter methylation and de novo methylation of RASSF1A and CASP8 promoters,
we compared the frequencies of RASSF1A and CASP8 methylation in tumours with and without
SLIT2 methylation (using previously published RASSF1A and CASP8 methylation data (Astuti
et al, 2001). CASP8 methylation was detected in 36% of SLIT2 methylated and 41% SLIT2
unmethylated tumours (P=1.0). RASSF1A promoter methylation was more frequent in tumours
with SLIT2 promoter methylation (77 vs 59%), but this did not reach statistical significance
(P=0.32).
Neuroblastoma clinicopathological features and SLIT2 methylation status
We compared the results of SLIT2 methylation status in our tumour series to the previously
reported results for allelic loss of 1p or 3p loss, N-myc amplification and 17q gain
(Martinsson et al, 1997). There was no correlation between SLIT2 methylation and 1p
allele loss (22% of SLIT2 methylated and 22% of unmethylated tumours, P=1.0), 3p allele
loss (25 vs 12%, P=0.57), 17q gain (50 vs 65%, P=0.62) or N-myc amplification status
(15 vs 13%, P=1.0). There was no association between tumour stage and SLIT2 methylation
status: SLIT2 methylation was present in 33% of stage 1, 2 and 4S tumours and in 26%
of stage 3 and 4 tumours (P=0.73).
Methylation analysis of SLIT2 in primary Wilms' tumours
Next, we proceeded to analyse SLIT2 promoter methylation status in 37 Wilms' tumours
that had been investigated previously for RASSF1A and CASP8 promoter methylation status
(Wagner et al, 2002 and unpublished observations). In total, 38% (14 out of 37) Wilms'
tumours demonstrated SLIT2 CpG island promoter methylation (Figure 1B). Promoter CpG
island methylation was confirmed by direct sequencing in one tumour (Figure 4B). All
SLIT2 methylated tumours contained unmethylated SLIT2 alleles that might be attributable
to the presence of contaminating normal tissue (tumour samples were not microdissected).
SLIT2 methylation was detected in zero of six normal tissue samples corresponding
to the methylated tumours.
To investigate the 4p15.2 allelic status of Wilms' tumours with SLIT2 methylation,
we typed six methylated tumours for LOH at D4S1546. None of three informative tumours
demonstrated D4S1546 allele loss.
Methylation of SLIT2 and other cancer genes in primary Wilms' tumours
To investigate the relationships between SLIT2 promoter methylation and de novo methylation
of RASSF1A and CASP8, we compared the frequencies of CASP8 and RASSF1A methylation
in tumours with and without SLIT2 methylation. In tumours with SLIT2 methylation,
CASP8 and RASSF1A were methylated in 43% (six out of 14) and 36% (five out of 14),
respectively. In tumours without SLIT2 methylation, CASP8 and RASSF1A promoter methylation
was detected in 39% (nine out of 23) and 70% (16 out of 23), respectively. Thus, although
there was no association between SLIT2 and CASP8 methylation, there was an inverse
relationship between SLIT2 and RASSF1A methylation, although this did not reach statistical
significance (P=0.09).
SLIT methylation status and clinicopathological status
The frequency of relapse in Wilms' tumours with SLIT2 methylation was similar to that
without SLIT2 methylation (21% (three out of 14) and 17% (four out of 23), respectively),
and there was no significant association between SLIT2 methylation and advanced stage
tumours (the frequency of stage 3 and 4 tumours in the SLIT2 methylated and unmethylated
groups was 45% (five out of 11) and 63% (12 out of 19), respectively).
Methylation analysis of SLIT2 in primary RCC
We detected SLIT2 promoter methylation in 25% (12 out of 48) primary RCC and in 75%
(six out of eight) RCC cell lines (Figure 1C). Promoter CpG island methylation was
confirmed by direct sequencing of five clones from RCC cell lines and 2 tumours (Figure
4A). SLIT2 promoter methylation was also detected in one out of 12 of the matching
normal kidney tissue samples for methylated tumours. All RCC with SLIT2 methylation
also contained unmethylated SLIT2 alleles, which might be attributable to the presence
of contaminating normal tissue (tumour samples were not microdissected). Loss of heterozygosity
at D4S1546 was not detected in 10 informative RCC with SLIT2 methylation.
SLIT2 promoter methylation is associated with transcriptional silencing in an RCC
cell line
We investigated the possible association between the SLIT2 promoter region CpG island
methylation and SLIT2 transcript expression in a panel of RCC cell lines (SKRC 18,
SKRC 39, SKRC 45, SKRC 47, SKRC 54, KTCL 26,UMRC-2 and 786-0). Cells were treated
with the demethylating agent 5-aza-dC for 5 days. Except for SKRC 45 and SKRC 47 (both
unmethylated for SLIT2), SLIT2 expression was significantly increased in the kidney
tumour cell lines after 5-aza-dC treatment. GAPDH expression levels were equal in
both 5-aza-dC-treated and untreated cell lines (Figure 3
Figure 4
SLIT2 methylation profile. Illustration of the SLIT2 methylation pattern detected
in (A) kidney tumour cell lines (SKRC 39, SKRC 18 and SKRC 47) and (B) neuroblastoma
tumours (NB107) and Wilm's tumours (WT244). The CpG island numbered according to Dallol
et al (2002a).
).
Methylation status of SLIT2 and inactivation of RASSF1A and VHL in primary RCC
Previously, we analysed primary RCC for RASSF1A methylation and inactivation of the
VHL tumour suppressor gene. There was no association between SLIT2 methylation and
the presence of VHL mutation in clear cell RCC, and the frequency of RASSF1A methylation
was similar in RCC with and without SLIT2 methylation (25 and 21%, respectively).
SLIT2 methylation status and clinicopathological status
The frequency of SLIT2 methylation in clear cell RCC (24%, nine out of 37) was similar
to that found in all tumour types. There was no significant association between SLIT2
methylation status and grade or TNM status.
DISCUSSION
Previously, we (a) identified SLIT2 promoter methylation in 53% non-small-cell lung
cancer, 36% small-cell lung cancer and 43% of breast cancers, (b) demonstrated that
promoter methylation is associated with reversible transcriptional silencing and (c)
determined that restoration of SLIT2 expression suppressed tumour growth in in vitro
studies (Dallol et al, 2002a). Thus having established SLIT2 as a lung and breast
cancer suppressor gene, we have now identified frequent SLIT2 hypermethylation in
paediatric cancers and in RCC and, recently, in 59% of gliomas (Dallol et al, 2003).
Slit protein binds to the roundabout receptor and acts as a midline repellent to guide
axonal development during embryogenesis (Rajagopalan et al, 2000; Simpson et al, 2000;
Wong et al, 2001) In humans, three SLIT orthologues have been identified, but to date
only SLIT2 has been implicated in cancer. In mice, Robo1 inactivation produces delayed
lung maturation and bronchial hyperplasia (Xian et al, 2001) and we have demonstrated
de novo ROBO1 promoter methylation in 19% primary invasive breast carcinomas and 18%
primary clear cell RCC, although somatic mutations were not identified (Dallol et
al, 2002b).
The characterisation of tumour-specific TSG methylation profiles provides insights
into the molecular pathology of the tumour and can be used to establish tumour-specific
methylation profiles (Esteller et al, 2001a). It is anticipated that such profiles
will be useful for diagnosis and clinicopathological classification. In colorectal
cancer, the identification of a subset of tumours with widespread de novo methylation
of TSG and mismatch repair genes (‘the methylator phenotype’) (Toyota et al, 1999)
has been of considerable interest and it is unclear as to whether similar subtypes
will be a feature of other cancers.
The molecular pathology of sporadic NB has been investigated extensively by molecular
cytogenetic and LOH analysis. Frequent alterations include N-myc amplification (20–25%)
and gain of genetic material at 17q23–qter (∼50% of tumours). Neuroblastoma TSGs have
been mapped by LOH studies to 1p36 (∼35% in primary tumours show LOH), 11q23 (44%),
14q23–qter (22%) and 3p (15%) (Ejeskar et al, 1998; Maris and Matthay, 1999). However,
specific gene mutations have not been defined, although we did identify RASSF1A methylation
in 55% of NBs and that CASP8 methylation occurs in ∼50% of tumours (Teitz et al, 2000;
Astuti et al, 2001). Caron et al (1996) reported 4p allele loss in ∼20% of NB and
we have now identified promoter methylation of the 4p15.2 candidate TSG SLIT2 in 29%
of NBs. Thus, epigenetic inactivation of SLIT2 is a common feature of NB, although
less frequent than methylation of CASP8 and RASSF1A. Harada et al (2002b) reported
that NB patients with CASP8 methylation were older than those without tumour methylation,
but to date, CASP8, RASSF1A or SLIT2 methylation has not been associated with specific
clinicopathological, cytogenetic or molecular features of NB. However, very large
studies may be needed to identify significant prognostic correlations in the presence
of a large number of potential variables. We did not find clear evidence of a ‘methylator
phenotype’, although methylation of RASSF1A was more common in tumours with SLIT2
methylation than in those without SLIT2 methylation and Harada et al (2002b) reported
an association between RASSF1A and CASP8 methylation in NB tumours. However, we found
no association between SLIT2 and CASP8 methylation. The frequency of SLIT2 methylation
in NB was less than that for RASSF1A and CASP8, but is still a significant finding
as other candidate TSGs that demonstrate frequent promoter methylation in some cancers
(e.g. p16INK4A, MGMT, RARâ, DAPK, APC, GSTP1, CDH1 and CDH13) are rarely methylated
in NB (Harada et al, 2002a).
Although de novo methylation and silencing of H19 in Wilms' tumours was first reported
some years ago (Steenman et al, 1994; Taniguchi et al, 1995), epigenetic changes have
not been investigated in great detail in Wilms' tumour. Recently, we reported frequent
CASP8 (43%) and RASSF1A (56%) promoter methylation in Wilms' tumours and the present
study has demonstrated that SLIT2 methylation represents a further frequent epigenetic
change in Wilms' tumours. To date, we have not identified an association between CASP8,
RASSF1A and SLIT2 methylation in individual tumours, so there is little evidence of
a methylator phenotype in a subset of Wilms' tumours. Indeed there was a negative,
albeit statistically insignificant, correlation between RASSF1A and SLIT2 methylation.
This finding merits further investigation as it could indicate that the simultaneous
inactivation of specific TSGs might be disadvantageous in specific cancer types. Although
p16
INK4a
(CDKN2a) promoter methylation has been reported in advanced stage Wilms' tumours (Arcellana-Panlilio
et al, 2000), this trend was not significant in our series, and to date no specific
clinicopathological features have been associated with SLIT2, CASP8 and RASSF1A methylation.
In our Wilms' tumour series, the frequency of SLIT2 promoter methylation in Wilms'
tumours was similar to that for CASP8 but higher than that for TSGs, which may show
frequent promoter methylation in other tumour types, for example, MGMT (30%), NORE1A
(15%), p14
ARF
(15%), p16INK4a (10%), DAPK (11%), CRBP1 (9%), RARB2 (0%), CDH13 (0%) and CDH1 (3%)
(Morris et al, 2003).
Renal cell carcinoma is the most common adult tumour and the majority (∼75%) of RCC
are classified as clear cell RCC, with papillary being the most frequent nonclear
cell histopathological subtype (∼15% of all cases) (Storkel and van den Berg, 1995).
The most frequent genetic change in RCC is somatic inactivation of the VHL TSG (usually
be mutation and loss, but promoter methylation may also occur), although VHL inactivation
is specific for clear cell RCC (Foster et al, 1994; Gnarra et al, 1994; Clifford et
al, 1998). Combining the results of the current study with previous investigations,
frequent epigenetic changes (⩾20%) in RCC include promoter methylation of RASSF1A,
TIMP3, DAPK, SLIT2, MT1G and GSTP1 (Esteller et al, 2001a; Nojima et al, 2001, Dreijerink
et al, 2001; Morrissey et al, 2001, Morris et al, 2003). In contrast, promoter methylation
not at (or rarely at) SDHB, RARB2, p16
INK4a
and CDH13 is uncommon (<5%). To date, apart from VHL, none of the epigenetic changes
in RCC have been associated with specific clinicopathological features.
The failure to detect an association between clinicopathological stage and SLIT2 methylation
status could indicate that SLIT2 methylation is an early event in tumorigenesis. In
tumours such as colorectal cancer, where there is a well-validated adenoma–carcinoma
sequence, it is possible to define the genetic changes associated with different stages
of tumorigenesis. However, in sporadic cases of NB, Wilms' tumour and RCC, there is
generally no well-defined pathway from precursor lesion to tumour (although nephroblastomatosis
may be present in patients with Beckwith–Widemann syndrome and ‘early lesion RCC’
has been described in von Hippel-Lindau disease). Hence, we are unable to precisely
define when SLIT methylation occurs in the pathogenesis of these tumours. However,
it is known that TSG inactivation may be an early event in tumorigenesis. Thus, methylation
may be the ‘second hit’ in familial cancer syndrome tumours (Prowse et al, 1997, Esteller
et al, 2001b). Furthermore, in sporadic and familial adenomatous polyposis coli, TSG
CpG island methylation may be detected in the earliest precursor lesion in colorectal
carcinogenesis, aberrant crypt foci (Chan et al, 2002). Similarly, Zöchbauer-Müller
et al (2001) suggested that TSG methylation may be a preneoplastic change in non-small-cell
lung cancer. We have analysed previously RASSF1A promoter methylation status in normal,
ductal-carcinoima-in situ (DCIS) and breast cancer trios. RASSF1A promoter hypermethylation
was detected in 65% of invasive cancers and in 42% of corresponding DCIS but in none
of the normal breast samples (Honorio et al, 2003). In all, 30% of DCIS without invasive
breast cancer also underwent RASSF1A promoter hypermethylation, suggesting that inactivation
of RASSF1A by CpG island methylation is an early event in breast tumorigenesis. Preliminary
unpublished data also reveal SLIT2 methylation in DCIS samples (RE Dickinson and F
Latif, unpublished). Thus, there is evidence that SLIT2 hypermethylation can be implicated
in early tumorigenesis.
In breast and lung cancers, TSG promoter methylation SLIT2 appears to resemble TSGs
such as RASSF1A, as epigenetic inactivation is more frequent than somatic mutations.
RASSF1A methylation has been reported in a wide range of human cancers. We have demonstrated
that SLIT2 methylation is common in paediatric and adult cancers, and further analysis
of additional tumour types seems indicated. Frequent 4p allele loss has been reported
in cancers that demonstrate SLIT2 methylation such as lung, breast and NB, and also
in cancers in which SLIT2 methylation status has not been investigated including colorectal,
bladder and head and neck cancers (Knowles et al, 1994; Rosin et al, 1995; Gryfe et
al, 1997; Pershouse et al, 1997; Yustein et al, 1999; Girard et al, 2000). During
development, the SLIT2 protein functions as a secreted chemorepellent so that restoration
of SLIT2 function by reversal of epigenetic inactivation or administration of SLIT2
agonists might provide novel therapeutic opportunities for human cancers.