ADAM33 gene silencing by promoter hypermethylation as a molecular marker in breast invasive lobular carcinoma

<p>Abstract</p> <p>Background</p> <p>ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, <it>ADAM23</it>, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer.</p> <p>Methods</p> <p>First, we analysed <it>ADAM33 </it>expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR) treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP). Finally, <it>ADAM33 </it>promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test.</p> <p>Results</p> <p>The expression analysis of <it>ADAM33 </it>in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR) demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to <it>ADAM33 </it>promoter hypermethylation. Using MSP, we detected <it>ADAM33 </it>promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM); tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC) was 76.2% compared with 25.5% in invasive ductal carcinoma (IDC), and this difference was statistically significant (p = 0.0002).</p> <p>Conclusion</p> <p><it>ADAM33 </it>gene silencing may be related to the discohesive histological appearance of ILCs. We suggest that <it>ADAM33 </it>promoter methylation may be a useful molecular marker for differentiating ILC and IDC.</p

Epigenetic Changes of CXCR4 and Its Ligand CXCL12 as Prognostic Factors for Sporadic Breast Cancer

Chemokines and their receptors are involved in the development and cancer progression. The chemokine CXCL12 interacts with its receptor, CXCR4, to promote cellular adhesion, survival, proliferation and migration. The CXCR4 gene is upregulated in several types of cancers, including skin, lung, pancreas, brain and breast tumors. In pancreatic cancer and melanoma, CXCR4 expression is regulated by DNA methylation within its promoter region. In this study we examined the role of cytosine methylation in the regulation of CXCR4 expression in breast cancer cell lines and also correlated the methylation pattern with the clinicopathological aspects of sixty-nine primary breast tumors from a cohort of Brazilian women. RT-PCR showed that the PMC-42, MCF7 and MDA-MB-436 breast tumor cell lines expressed high levels of CXCR4. Conversely, the MDA-MB-435 cell line only expressed CXCR4 after treatment with 5-Aza-CdR, which suggests that CXCR4 expression is regulated by DNA methylation. To confirm this hypothesis, a 184 bp fragment of the CXCR4 gene promoter region was cloned after sodium bisulfite DNA treatment. Sequencing data showed that cell lines that expressed CXCR4 had only 15% of methylated CpG dinucleotides, while the cell line that not have CXCR4 expression, had a high density of methylation (91%). Loss of DNA methylation in the CXCR4 promoter was detected in 67% of the breast cancer analyzed. The absence of CXCR4 methylation was associated with the tumor stage, size, histological grade, lymph node status, ESR1 methylation and CXCL12 methylation, metastasis and patient death. Kaplan-Meier curves demonstrated that patients with an unmethylated CXCR4 promoter had a poorer overall survival and disease-free survival. Furthermore, patients with both CXCL12 methylation and unmethylated CXCR4 had a shorter overall survival and disease-free survival. These findings suggest that the DNA methylation status of both CXCR4 and CXCL12 genes could be used as a biomarker for prognosis in breast cancer

MSP analysis in breast cancer cell lines and primary breast tumors.

<p>(A) Primer standardization for methylated and unmethylated conditions in tumor cell lines. (B) MSP analysis of primary tumors. Thirteen samples are represented. MW, Molecular Weight; NC, Negative Control.</p

Kaplan Meier curves for overall survival and disease-free survival according to the methylation status of <i>CXCR4</i> and <i>CXCL12</i>.

<p><i>CXCR4</i> methylation status and the correlation with (A) overall survival (OS) and (B) disease-free survival (DFS) are shown. <i>CXCL12</i> methylation status and association to <i>CXCR4</i> methylation for (C) OS and (D) DFS are shown.</p

<i>CXCR4</i> expression analysis using semi-quantitative RT-PCR in breast cancer tumor cell lines and <i>CXCR4</i> expression after 5-aza-2′-deoxycytidine (D-Aza) treatment.

<p>(A) The bands represent <i>CXCR4</i> expression in the PMC-42, MCF7, MDA-MB-436 cell lines and (B) MDA-MB-435 mock or MDA-MB-435 D-Aza represent the MDA-MB-435 cell line before and after treatment with 5-aza-2′-deoxycytidine, respectively. The <i>GAPDH</i> gene was used as a positive control in both experiments. MW, Molecular Weight, NC represents the PCR reaction without DNA (negative control).</p

Clinicopathological features of 69 patients with primary breast carcinomas and methylation status of <i>CXCR4</i> gene.

<p><b>Abbreviations:</b><i>p</i>, value from statistical analysis <i>χ²</i> test and Fisher's exact test; M, methylated; U unmethylated; significant data are in bold.</p><p>*<i>CXCL12</i> and <i>ESR1</i> methylation data were used from a previous study published by our group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029461#pone.0029461-Ramos1" target="_blank">[17]</a>.</p

Bisulfite sequencing of the <i>CXCR4</i> gene promoter in the breast cancer cell lines.

<p>The cell lines used are shown. The nineteen dinucleotides are numbered in agreement with the sequence. The open circles represent the unmethylated dinucleotides while the gray to black portion represents the percentage of methylation. On the right side methylation pattern are represented according to data of RT-PCR and the absolute percentage value. The arrows below the CpG dinucleotides represent the MSP primers that were used.</p

Sequence of the primers used for RT-PCR, nested-PCR and MSP.

<p><b>Abbreviations:</b> M, specific for methylated condition; U, specific for unmethylated condition.</p