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Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231 [D. M. L., E. E., C. B. P., D. K., S. S.]; Department of Medicine, University of California, San Diego, California 92161 [P. M. S.]; Division of Medicine, Imperial College School of Medicine, United Kingdom W12 0NN [B. N., L. B.]; and Northwestern University Medical School, Chicago, Illinois 60611 [S. A. W.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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The role of WT1 in breast cancer remains unclear. Using immunohistochemistry, Silberstein et al. (4) demonstrated WT1 expression in normal breast tissue, particularly in the myoepithelial cells that overlie the polygonal cells lining the ductal lumen. Reduced or absent WT1 staining was seen in 60% of breast tumors, leading these authors to conclude that loss of WT1 expression might be correlated with tumorigenesis. Furthermore, by RT-PCR analysis, they reported that the isoform of WT1 lacking the fifth exon and the one lacking the nine bases (KTS) between exons 9 and 10 were specifically expressed in tumors. Subsequently, Huang et al. (5) and Laux et al. (6) , using methylation-sensitive restriction endonucleases, demonstrated aberrant methylation of CpG islands associated with the promoter and first intron of WT1 in 5 of 20 (25%) breast carcinomas; however, their studies did not include a concurrent evaluation of gene expression. In the present study, we evaluated the extent of methylation of the WT1-associated CpG islands in normal mammary epithelium, in breast cancer cell lines, and in primary mammary tumors and correlated the findings with expression of the WT1 mRNA and protein in the same samples.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Tumors and Organoids.
Primary breast tumors were obtained from the Johns Hopkins frozen tumor
bank. Mammary organoids were prepared from reduction mammoplasty
specimens of women with benign or no abnormalities in the breast as
described (8)
. Briefly, the specimens were enzymatically
digested into duct-like structures (organoids), filtered,
histologically confirmed to contain >80% epithelial cells, and frozen
at -70°C until used (8)
. We also used highly purified
(95–99%) myoepithelial and luminal epithelial cells isolated by
differential centrifugation and fluorescence-activated cell sorting of
enzymatically digested normal mammoplasty specimens (9)
.
RT-PCR for WT1 mRNA.
Methods for RNA extraction and RT-PCR have been described previously
(7)
. The sequences of the primers used are as follows: for
amplifying the 555-bp region surrounding WT1 exon 5,
5'-GCGGCGCAGTTCCCCAACCA-3' (sense, nucleotides 882–901) and
5'-ATGGTTTCTCACCAGTGTGCTT-3' (antisense, nucleotides 1416–1437); for
amplifying the 382-bp region surrounding the KTS insert,
5'-GCATCTGAAACCAGTGAGAA-3' (sense, nucleotides 1320–1339) and
5'-TTTCTCTGATGCATGTTG-3' (antisense, nucleotides 1685–1702).
Amplification was performed using a hot-start protocol; samples were
heated to 94°C for 4 minutes and then cooled to 80°C prior to the
addition of Taq polymerase (RedTaq; Sigma Chemical Co., St. Louis, MO).
Samples were then heated to 94°C for 30 s, followed by either
50°C for 30 s (for the KTS primers) or 56°C for 30 s (for the
exon 5 primers) and then 72°C for 1 min for 40 cycles. PCR products
were resolved by electrophoresis, using a 2% agarose gel for the exon
5-splice variants and a 12% polyacrylamide gel to resolve the KTS
insert variants. Coamplification of the ribosomal RNA 36B4 was
performed as an internal control using the following primers:
5'-GATTGGCTACCCAACTGTTGCA-3' (sense) and
5'-CAGGGGCAGCAGCCACAAAGGC-3' (antisense).
Northern Blots.
Total RNA was extracted as described (7)
. After
electrophoresis through a 1.5% agarose gel in
4-morpholinepropanesulfonic acid buffer with 6.7% formaldehyde, RNA
was transferred to nitrocellulose. Blots were probed with a PCR product
corresponding to the WT1 zinc finger region, amplified using the
primers described above, and labeled with [
-32P]dCTP
by random priming using standard techniques.
Methylation-specific PCR.
Genomic DNA was isolated using standard techniques and treated with
sodium bisulfite as described (10)
. Methylation-specific
PCR was performed using the following primers: to detect methylated
promoter DNA, 5'-TTTGGGTTAAGTTAGGCGTCGTCG-3' (sense, nucleotides -267
to -243) and 5'-ACACTACTCCTCGTACGACTCCG-3' (antisense, nucleotides +33
to +59); to detect unmethylated promoter DNA,
5'-TTTGGGTTAAGTTAGGTGTTGTTG-3' (sense) and
5'-ACACTACTCCTCATACAACTCCA-3' (antisense); to detect methylated intron
1 DNA, 5'-CGTCGGGTGAAGGCGGGTAAT-3' (sense) and
5'-CGAACCCGAACCTACGAAACC-3' (antisense); to detect unmethylated intron
1 DNA, 5'-TGTTGGGTGAAGGTGGGTAAT-3' (sense) and
5'-CAAACCCAAACCTACAAAACC-3' (antisense). The PCR reaction was as above,
except that the annealing temperature was 59°C, and the extension
time was 45 s.
Western Blots.
Total protein from cell lines was obtained from material harvested in
TriReagent (Molecular Research Center, Cincinnati, OH) and initially
used for RNA isolation. Protein purification was according to the
manufacturer’s protocol. After separation by SDS-PAGE and
electrophoretic transfer to nitrocellulose membranes, proteins were
incubated with an anti-WT1 antibody [WT (C-19); Santa Cruz
Biotechnology, Santa Cruz, CA], diluted 1:1000 in the blocking
solution. Horseradish peroxidase-conjugated antibody against rabbit IgG
(Amersham, Arlington Heights, IL) was used at 1:1000, and binding was
revealed using enhanced chemiluminescence (Amersham, Arlington Heights,
IL).
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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. Among the three immortal breast epithelial cell lines, WT1 expression
was observed in HMECs HBL-100 and MCF-10A but not in H16N (Fig. 1A)
. WT1 mRNA expression was examined in nine breast cancer
cell lines; expression was easily detectable in five: HS578T, T47D,
MDA-MB-468, 21MT, and 21PT, and undetectable in the remaining four:
SKBR3, MDA-MB-435, MCF-7, and MDA-MB-231 (Fig. 1B)
.
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and the two KTS isoforms
(Fig. 1C)
, were present.
To confirm the results of our RT-PCR experiments, Northern blot
analysis was performed using total RNA isolated from a number of breast
cancer cell lines. Similar to the results obtained by RT-PCR (Fig. 1, A–C)
, WT1 mRNA expression was readily detected in HBL-100,
HS578T, T47D, and MDA-MB-468 cells but was not detected in MDA-MB-435,
MDA-MB-231, SKBR3, or MCF-7 cells (Fig. 1D)
. Thus, WT1 mRNA
expression was undetectable in finite lifespan primary breast
epithelial cell cultures but was easily detectable in the neoplastic
and immortalized HMECs and in 7 of 12 breast cancer cell lines. Also,
the striking correlation between results of our Northern blot and
RT-PCR experiments validated the RT-PCR protocol as an accurate
reflection of WT1 mRNA expression in the breast cancer cells.
Methylation of the WT1 Locus in Breast Cancer Cell
Lines.
The promoter and first intron of the WT1 gene contain dense
CpG islands. These sequence elements are frequently sites of DNA
methylation and play a role in transcriptional silencing (11
, 12)
. To determine whether methylation silences gene expression
in the WT1-negative cell lines, we investigated the status of the WT1
promoter in the breast cancer cell lines. We found that the promoter
was methylated in the four cell lines that did not express WT1 (Fig. 2A)
but not in the five cell lines that did (Fig. 2A
and data
not shown), consistent with the idea that methylation is a critical
determinant of WT1 expression. There was one exception to this
correlation. T47D cells contained methylated, as well as unmethylated,
WT1 sequences but nevertheless expressed WT1 mRNA, suggesting that in
this case, methylation alone is insufficient to silence expression.
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. Interestingly, this treatment did not cause WT1
expression in MCF-7 cells (Fig. 2B)
, nor did TSA restore
expression in either cell line (Fig. 2C)
. In the same
samples, these treatments restored expression of another gene,
14-3-3
, frequently hypermethylated in breast cancers
(7)
. These findings suggest that although promoter
methylation correlates with gene silencing in breast cancer cells, it
may not play a causal role.
Expression of WT1 in Primary Breast Tissue.
We expanded our findings from cell lines to patient samples, including
normal breast epithelium and primary breast tumors. Breast carcinomas
arise from luminal epithelial cells in the mammary duct. Normal breast
ducts also contain a layer of myoepithelial cells that overlie the
luminal epithelium. To ensure that our normal samples contained luminal
epithelial cells, we used three different types of epithelial cell
preparations including (a) three short-term cultures of
HMECs; (b) nine organoid preparations of mammary ducts; and
(c) eight samples of highly purified luminal and
myoepithelial cells (isolated from four patient samples).
WT1 expression was undetectable by RT-PCR in three HMEC samples (Fig. 1A)
in eight of nine breast organoid preparations (Fig. 3A)
nor in any of eight purified epithelial cell preparations (Fig. 3B)
. By Western blotting, WT1 protein was not detected in
three organoid samples or in two HMECs (Fig. 3C)
. In
contrast, WT1 expression was easily detectable by Western blotting in
27 of 31 (87%) primary breast carcinomas (Fig. 3C
and data
not shown).
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. We also detected KTS (+) and KTS (-) isoforms in all nine tumors
examined (Fig. 4B)
. Thus, in agreement with the published
data, a majority of the tumors expressed both exon 5 splice variants of
WT1, and all of the tumors expressed both splice variants involving the
KTS insert. Interestingly, the sole breast organoid sample that
expressed WT1 (B44; Fig. 3A
) expressed all four splice
variants as well (data not shown).
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Methylation-specific PCR was performed using DNA extracted from 19
primary tumors and 9 breast organoid preparations. The WT1 promoter CpG
island was unmethylated in DNA from all nine organoid samples (Fig. 5A
and data not shown). In contrast, 6 of 19 tumors contained methylated
DNA, and the remaining 13 were completely unmethylated (Fig. 5B
and data not shown). This rate of promoter methylation
(32%) is similar to the 25% incidence reported by Laux et
al. (6)
. Thus, methylation of the WT1 promoter is a
tumor-specific phenomenon. Contrary to expectation, however, each of
the six tumors that contained methylated WT1 also expressed WT1 protein
(Table 1)
. Specifically, tumor 7103 clearly expresses WT1, as judged by
Western blotting (Fig. 3C)
, despite the presence of
methylated CpG dinucleotides in the promoter (Fig. 5B)
. WT1
promoter methylation, therefore, was not effective in silencing gene
expression. Next, we examined the CpG island in the first intron of the
WT1 gene, a region where tumor-specific methylation has also
been reported previously (5)
. We detected methylation of
WT1 in three of three breast organoid preparations (Fig. 5C)
and in 9 of 10 tumor samples evaluated (Fig. 5D
and data not
shown). Thus, the 1st intron of WT1 is methylated in both normal and
malignant breast tissue, and this methylation is unrelated to
tumorigenesis.
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We also found evidence of tumor-specific methylation of the CpG
islands. Surprisingly, we detected expression of WT1 mRNA and protein
in the majority of breast cancer samples that we evaluated, including
in every sample that contained methylated DNA (Table 1)
. Our finding that breast carcinomas express WT1 despite tumor-specific
gene methylation emphasizes the importance of evaluating methylation
and gene expression concurrently in the same tissue.
Although at first glance our findings seem to contradict those of Silberstein et al. (4) , who reported that breast tumors underexpress WT1, in fact they are probably in agreement. These authors stated that 15 of 21 carcinomas were WT1 negative by immunohistochemistry. However, they defined as negative a tumor with less than half of the cells staining for WT1. Six of their 15 WT1-negative tumors had between 10 and 50% positive cells and would certainly be positive by either of our assays. The other nine tumors may have had up to 10% of their cells expressing WT1, and these, too, may have been WT1 positive in our RT-PCR assays but negative by Western analysis. However, we were unable to replicate their finding of WT1 expression in the majority of nontransformed mammary epithelial samples. Interestingly, their paper reports the use of nested PCR to detect WT1 mRNA. We readily detected WT1 mRNA in tumor samples using a single-step PCR protocol. Although it is possible that we would have detected WT1 expression in normal epithelium using a nested PCR, this would not alter our basic finding that the gene is overexpressed in tumors compared with normal tissue, a finding strongly borne out by data obtained by Western blotting of normal and breast tumor samples. This finding suggests that WT1 may play a functional role in breast cancer.
Our data also reveal a discrepancy between gene regulation in tissue culture and in vivo. We found that methylation of the WT1 promoter is associated with gene silencing in breast cancer cell lines. In contrast, the promoter-associated CpG island was methylated in 32% of the tumors we examined; contrary to expectation, these tumors express WT1. These findings are reminiscent of those of Costello et al. (15) , who reported data suggesting that there is no correlation between methylation and expression of WT1 in human gliomas and in glioma cell lines. Very similar results were also reported recently for the CpG island associated with the human telomerase gene promoter. Dessain et al. (16) reported that methylation silences expression of hTERT in some cell lines, and that treatment with 5-aza-deoxyC induces expression, but that the CpG island is methylated in many tumors that are telomerase positive. These data, and ours, highlight the fact that there are multiple mechanisms for gene silencing, of which hypermethylation of a CpG island is only one. More importantly, these findings emphasize the idea that cell lines do not necessarily reflect the situation in vivo. They also serve to point out that hypermethylation of a CpG island may be insufficient to silence expression, demonstrating the importance of assessing gene expression as well as promoter methylation status when evaluating the role of a particular gene in a particular tumor type.
The precise function of WT1 outside of the genitourinary system is unclear. A number of WT1 target genes have been identified, some of which may be relevant for tumorigenesis, such as E-cadherin (17) , expression of which has been associated with improved cell survival in metastatic foci of breast cancer. Another group demonstrated that WT1 can transcriptionally activate bcl-2 (18) , a gene associated with resistance to apoptosis. Our laboratory has identified cyclin E as another WT1 target,4 and there is accumulating evidence implicating this gene in mammary carcinogenesis as well (19) . The growing number of identified WT1 target genes implicated in mammary carcinogenesis strengthens the concept that this gene may play a crucial role in this disorder.
In summary, our data demonstrate that WT1 is not expressed in normal breast epithelium and is overexpressed in the majority of primary breast tumors. Tumor-specific methylation of the CpG island occurs in breast cancer but appears to be inconsequential to gene expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grants R01-CA48493
and P50-CA88843 (to S. S.) and Grant T32 CA60441-06 (to D. M. L.) from the NIH. 
2 To whom requests for reprints should be
addressed, at Johns Hopkins Oncology Center, BBCRB Room 410, 1650
Orleans St., Baltimore, MD 21231. Phone: (410) 614-2479; Fax: (410)
614-4073; E-mail: saras{at}jhmi.edu
3 The abbreviations used are: WT1, Wilms’ tumor
suppressor 1; MSP: methylation specific PCR, RT-PCR, reverse
transcription-PCR; HMEC, human mammary epithelial cells; 5-aza-deoxyC,
5-aza-2'-deoxcytidine; TSA, trichostatin A; HDAC, histone
deacetylase.
4 D. M. Loeb and S. Sukumar, manuscript in
preparation.
Received 8/ 9/00. Accepted 12/ 6/00.
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