Hypermethylation Leads to Silencing of the SYK Gene in Human Breast Cancer

INTRODUCTION

The SYK gene encodes a protein tyrosine kinase, Syk, that is highly expressed in hematopoietic cells. Its critical roles in T-cell and B-cell development and activation have been established (1 , 2) . SYK expression is evident in the mammary gland (3) . A recent study suggested that Syk functions as a tumor suppressor in breast cancers (4) , and several lines of evidence support this hypothesis. Syk is expressed in normal breast ductal epithelial cells but not in a subset of invasive breast carcinoma. Also, the loss of Syk expression seems to be associated with malignant phenotypes such as increased motility and invasion. Additionally, cells expressing transfected SYK cDNA exhibit decreased tumorigenicity. Despite strong biological evidence, no mutations, translocation, or homozygous deletions involving the SYK gene have been reported in naturally occurring neoplasm. It could be argued that the loss of expression is not considered inheritable genetic information when cell division occurs, and that this loss merely reflects the transient adaptation to culture conditions by the cancer cell. The absence of Syk protein is reflected by the loss of its mRNA expression in invasive breast cancers, which suggests that the loss of Syk expression occurs at the transcriptional level (4) . The mechanism(s) responsible for the loss of expression have remained unclear. A few mechanisms could explain the loss of expression when the chromosomal rearrangement or exonic mutation appears to be minimal, such as aberrant methylation of the 5′-regulatory sequences, promoter mutations, loss of transcriptional activators, or binding of suppressor proteins to the promoter.

A growing body of evidence indicates methylation to be a major mechanism of silencing tumor suppressor genes (5 , 6) . An estimated 50% of human genes have clusters of CpG dinucleotides (CpG islands) in their 5′-regulatory sequences. In a vast majority of these genes, however, the CpG islands are unmethylated. Gene silencing through methylation of these sites has been observed in early developmental stages (7) and aging (8 , 9) . Aberrant methylation may lead to deregulation of gene expression. In human cancer, alterations of methylation pattern have been observed, including global hypomethylation, increased DNA methyltransferase activity, and local DNA hypermethylation of CpG islands (10 , 11) . Most notably, tumor suppressor genes (p16/CDKN2, Rb, VHL, and BRCA1), mismatch repair genes (hMLH1), and others such as estrogen receptor α, E-cadherin, 14–3-3 ς, death-associated protein kinase, and Thrombospodin-1, are repressed by CpG island hypermethylation in cancer tissues (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) . Inhibition of the transcription of these genes provides an epigenetic mechanism of clonal selection during tumorigenesis.

In this study, we explored whether methylation of SYK CpG sites is associated with the loss of SYK expression in malignant breast tumors. We also evaluated non-neoplastic, morphologically normal breast parenchyma and breast tumors and identified high-frequency hypermethylation of the SYK gene in malignant breast tumors.

MATERIALS AND METHODS

Cell Lines and Tissues.

All of the breast cell lines were purchased from the American Type Culture Collection (Manassas, VA) and maintained in recommended culture conditions. Normal and neoplastic tissues were obtained from breast carcinomas resected at the M. D. Anderson Cancer Center and archived in the breast tumor bank. All of the normal and tumor samples were histologically confirmed.

Patient records were reviewed to determine the age of onset, size of tumor, lymph node metastasis, pathological grading, and expression profiles of estrogen receptor, progesterone receptor, and HER2/neu. The data were then used to correlate with the SYK methylation status using the SPSS statistical program.

Bisulfite Modification and Sequencing.

Genomic DNA from cell lines or frozen breast tissues was extracted by using a DNeasy kit (Qiagen, Valencia, CA). Genomic DNA was treated with sodium bisulfite (Sigma Chemical Co., St. Louis, MO) as reported previously (23) . Briefly, 1 μg/50 μl DNA was denatured by NaOH (final concentration 0.2 m) for 10 min at 37°C. Thirty μl of 10 mm hydroquinone (Sigma Chemical Co.) and 520 μl of 3 m sodium bisulfite (pH 5.0) were added, followed by incubation at 50°C for 16 h. The modified DNA was purified using Wizard DNA purification columns (Promega, Madison, WI). The purified DNA was treated again with NaOH and precipitated. DNA was resuspended in 30 μl of TE buffer [3 mm Tris (pH 8.0)/0.2 mm EDTA] and subjected to PCR amplification using a primer set (forward 5′-GATTAAGATATATTTTAGGGAATATG-3′; reverse 5′-CACCTATATTTTATTCACATAATTTC-3′) that spanned the SYK CpG island. A 15-μl reaction that contained 30 ng of bisulfite-treated DNA and 1 × RDA buffer [67 mm Tris (pH 8.8)/16 mm (NH₄)₂SO₄, 100 mm 2-mercaptoethanol, 1 μg/μl BSA] were processed in 30 thermal cycles of 94°C for 45 s, 58°C for 45 s, and 72°C for 45 s. The resultant 664-bp PCR product was subcloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA), and plasmids were subjected to sequencing by the M. D. Anderson Cancer Center Core DNA Analysis Facility using vector-specific primers (T7 or M13 reverse).

MSP. 3

Bisulfite-treated DNA was PCR-amplified as described above. One aliquot (2 μl) of diluted PCR product (40-fold) was subjected to nested duplex PCR amplification in a 15-μl volume. Methylation-specific primers were chosen to cover 9 CpG dinucleotides numbered 17–21 (forward) and 47–50 (reverse), both of which were consistently found heavily methylated. Similarly, unmethylation-specific primers covered 8 CpG dinucleotides numbered 18–22 (forward) and 35–37 (reverse). Primers specific for methylated DNA (forward 5′-CGATTTCGCGGGTTTCGTTC-3′; reverse 5′-AAAACGAACGCAACGCGAAAC-3′) and unmethylated DNA (forward 5′-ATTTTGTGGGTTTTGTTTGGTG-3′; reverse 5′-ACTTCCTTAACACACCCAAAC-3′) were added to the same reaction and expected to generate 243- and 140-bp products, respectively. PCR conditions were 24 cycles of 94°C for 30 s, 67°C for 30 s, and 72°C for 30 s.

RT-PCR.

Total RNA was prepared using an RNeasy kit (Qiagen). Five μg of total RNA was reverse-transcribed using 18-mer oligodeoxythymidylate and Superscript II (Life Technologies, Inc., Rockville, MD) in a volume of 20 μl. Reactions lacking RT were used to verify the absence of amplification from genomic DNA contamination. The cDNA templates were subjected to PCR amplification. As a control of cDNA integrity, β₂-microglobulin expression was analyzed as well. The primer sets were 5′-TGTCAAGGATAAGAACATCATAG-3′ (forward) and 5′-CACCACGTCATAGTAGTAATTG-3′ (reverse) for SYK or 5′-ACCCCCACTGAAAAAGATGA-3′ (forward) and 5′-GCATCTTCAAACCTCCATGAT-3′ (reverse) for β₂-microglobulin, respectively. PCR conditions were 35 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 40 s for SYK; and 25 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s for β₂-microglobulin. All of the RT-PCR experiments were repeated at least once.

In some experiments, cells were treated with a final concentration of 2.0 μm of 5-aza-dC (Sigma Chemical Co.) for 3–5 days or 1.0 μm of TSA (Sigma Chemical Co.) for 1 day before cells were harvested for RNA extraction.

RESULTS

Loss of SYK Expression in Breast Cancer Cell Lines.

Using RT-PCR, we evaluated SYK mRNA expression in 20 randomly selected breast cancer lines. Representative data are shown in Fig. 1 ⇓ . Consistent with an earlier report (4) , we found that BT-549, MDA-MB-231, and MDA-MB-435S lines did not express SYK mRNA. Three other breast cancer lines, i.e., Hs854T, MDA-MB-134VI, and MDA-MB-453, were also found to be SYK-negative (Fig. 1) ⇓ . In total, we found 6 (30%) of 20 breast lines tested to be SYK-negative (Table 1) ⇓ .

Hypermethylation Leads to SYK Silencing.

We surmised that the loss of SYK expression in these lines was attributable to gene hypermethylation. To obtain the genomic structure of the SYK gene, we used the 5′-most sequences of the SYK cDNA (GenBank accession no. Z29630) to search the draft human genome database using the BLAST program. A clone on chromosome 9 (Hs9_19655) that covers both exons 1 and 2 was obtained. The ATG translation start site is located on exon 2. A 600-bp region surrounding the exon 1 of the SYK gene contains 62 CpG sites (Fig. 2) ⇓ . The high density of CpG sites in the 5′-regulatory region suggested that methylation may influence the transcriptional regulation of SYK expression.

To explore the potential role of methylation in silencing SYK gene expression, we determined the nucleotide sequence of this region after treating the genomic DNA with sodium bisulfite. PCR primers were designed to amplify a region spanning the 62 CpG dinucleotides. The PCR product was then subcloned and sequenced. We compared the methylation status of three SYK-negative (MDA-MB-231, MDA-MB-435S, and MDA-MB-453) and two SYK-positive (T47D and BT-483) cell lines. As shown in Fig. 2 ⇓ , the CpG island exhibited extensive methylation in three SYK-negative lines. In contrast, no methylation was observed in DNA from two SYK-positive cell lines.

On the basis of the striking differences in methylation status between SYK-positive and -negative cell lines, we then used MSP to evaluate the breast cancer cell line panel. Consensus methylation sites were chosen to design the methylation-specific primers. Duplex PCR was used to detect both methylated and unmethylated DNA in the same reaction. The MSP results correlated with the genomic sequencing information (Fig. 3A) ⇓ , and are summarized in Table 1 ⇓ . As we examined SYK expression and methylation, it became evident that two categories of SYK expressers were entirely differentiated by MSP (Table 1) ⇓ . These results assured the reliability of MSP in determining SYK methylation status and consequent SYK expression.

To determine whether methylation was responsible for the loss of SYK expression, we treated SYK-methylated cell lines with a methylation inhibitor, 5-aza-dC (24) . 5-aza-dC reactivated SYK expression in all of the six methylation-positive cell lines as detected by RT-PCR (Fig. 4A ⇓ and Table 1 ⇓ ). When their relative expression levels and an SYK-positive cell line, ZR-75.1, were evaluated by RT-PCR, it became evident that the methylation inhibition only partially restored SYK expression (Fig. 4A) ⇓ . One-day treatment with 1.0 μm TSA, a histone deacetylase inhibitor (25) , was unable to reactivate SYK gene expression (Fig. 4B) ⇓ . These results indicated that aberrant 5′ hypermethylation plays a causal role in silencing the SYK gene.

SYK is Hypermethylated in Primary Breast Tumors.

Having used the MSP method to establish a strong correlation between SYK 5′ CpG hypermethylation and its loss of expression in cell lines, we next examined whether this epigenetic alteration could be extrapolated to primary breast neoplasm. We used MSP to analyze the SYK methylation status of the primary tumors and their matched normal breast tissues. Thirty-seven nonselected primary breast tumors (35 invasive carcinomas and 2 sarcomas) were screened. Among the 35 carcinomas examined, 11 of them, namely TR365, TR616, TR647, TR758T, TR1108, TR1211, TR1458, TR1462, TR2061, TR2615, and TR2905, exhibited strong SYK methylation. Representative examples are shown in Fig. 3B ⇓ . The presence of unmethylation bands in the breast tumors reflects heterogeneity of the breast tumors, but it may also represent contaminated normal tissues or infiltrating lymphocytes. In contrast to their corresponding carcinomas, the SYK gene of the 11 matched neighboring normal breast tissues remained unmethylated (Fig. 3B) ⇓ . The remaining two sarcomas examined were a cystosarcoma (TR748) that was found SYK-methylated and an angiosarcoma (TR1098) that remained unmethylated. The matched normal tissue for TR748 was unavailable. However, based upon the apparent unmethylated status in normal breast tissue (100%, 11/11), the SYK methylation in TR748 was presumed to be tumor-specific. In total, methylation of the SYK CpG island occurred in 32% (12/37) of primary breast tumors.

There was no significant correlation between the SYK methylation status and clinical/pathological parameters with respect to age, tumor size, lymph node status, pathological grading, and expression statuses of estrogen receptor, progesterone receptor, and HER2/neu.

DISCUSSION

Loss of SYK expression, but not genetic alteration, has been identified in breast cancers (4) . The loss of expression occurs at the transcriptional level, and, as our findings indicated, as a result of DNA hypermethylation. Chemical inhibition of DNA methylation partially restored SYK expression (Table 1 ⇓ and Fig. 4 ⇓ ). Hypermethylated DNA is believed to interact with several methyl-CpG binding proteins, including MeCP2. The interaction helps to assemble a repressive complex, including histone deacetylase, and forms an inactive chromatin context that lead to gene silencing (26 , 27) . Indeed, the expression of a few methylation-repressed genes can be reactivated by TSA treatment (19 , 28) or demethylation-induced gene reexpression can be potentiated by TSA (29) . We found, however, that TSA did not restore SYK expression in SYK-negative cells (Fig. 4B) ⇓ . These results underscored the indispensable role of methylation in SYK gene inactivation.

Previous reports have indicated that cells grown in culture exhibit elevated DNA methylation by mechanisms that are not completely understood (30) . Our study, however, identified a similar SYK methylation rate (>30%) in breast cancer cell lines and primary breast tumors. The finding is evidence that silencing of the SYK gene occurs in primary breast neoplasia and has biological significance in tumorigenesis. No genetic alterations of the SYK gene have yet been identified in solid tumors. Thus, SYK joins a category of genes, including p15^INK4B and p73 (31 , 32) , that are inactivated in tumors primarily by frequent methylation. The >30% methylation rate in primary tumors may even be underestimated, given the fact that we were using duplex PCR in which both methylated and unmethylated DNA was amplified in a competitive fashion. Thus, limited methylation alleles that would otherwise be observed by conventional MSP method (23) might not be detected under our current experimental conditions.

By using in situ hybridization, it was found that, compared with the normal breast epithelial cells, SYK mRNA expression is reduced in low-grade breast carcinoma in situ and lost in high-grade invasive breast cancers. Furthermore, the SYK expression level was inversely associated with the invasiveness of breast cancer cell lines (4) . A prevalently higher SYK methylation rate in high-grade/late-stage tumors would support this hypothesis. In the present study, however, we did not observe a significant correlation between SYK methylation and tumor grade. This may be attributable to a relatively small sample size and the complexity of unselected patient population. Additional detailed studies using patient cohort are needed to examine the value of SYK methylation as a diagnostic or prognostic marker. Interestingly, the SYK methylation seems to be independent of other prognostic markers, such as estrogen receptor and HER2/neu expression status. This distinction and its clinical relevance need to be assessed.