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

Abstract

BackgroundADAM33 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, ADAM23, 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.MethodsFirst, we analysed ADAM33 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, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test.ResultsThe expression analysis of ADAM33 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 ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 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).ConclusionADAM33 gene silencing may be related to the discohesive histological appearance of ILCs. We suggest that ADAM33 promoter methylation may be a useful molecular marker for differentiating ILC and IDC.

ADAM33 as a New Biomarker for Invasive Lobular Breast Carcinoma

“…All the IDC samples were ADAM33 protein positive in IHC. However, 11 out of 38 tumoral samples (Table 1) showed to be hypermethylated in the ADAM33 promoter as seen in our previous work [13]. This result could be explained by other epigenetic mechanisms involved in the silencing of this gene as discussed below.…”

“…The results showed that mouse sera have high specificity for ADAM33 recombinant protein since no signal was detected in the MDA-MB-231 cell line, which does not express the ADAM33 gene [13] (Figure 2). Moreover, MCF7 and PMC42 cell lines that express the ADAM33 gene showed a single and strong signal of approximately 37 kDa for this protein.…”

“…Tumor types of 38 IDC (78%) and 11 ILC (22%) were used in the study. The analysis of the ADAM33 gene hypermethylation was done previously by our group, using MSP analysis [13] and used here to correlate the molecular silencing to the presence or absence of ADAM33 protein staining (Table 1). All the IDC samples were ADAM33 protein positive in IHC.…”

“…ADAM33 belongs to the ADAM family of transmembrane glycoprotein and play diverse roles in cell surface remodeling, ectodomain shedding of growth factors and receptors, and mediation of cell-cell and cell-matrix interactions [11,12]. Seniski et al [13] showed that the ADAM33 promoter hypermethylation was present in 76.2% of ILC compared with 25.5% in IDC (p=0.0002). Based on these results, the ADAM33 gene silencing may be related to the discohesive histological nature of ILCs.…”

“…The breast cancer cell lines were all obtained from the Ludwig Institute for Cancer Research (São Paulo, Brazil) and were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 . The cell lines MCF7 and PMC42 have positive ADAM33 expression and MDA-MB-231 was negative for this gene [13], and therefore, was used as positive and negative controls to test the polyclonal antibody. Breast cancer cell line protein lysates were obtained from 1.2 × 107 cells in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.5% Triton X-100 and 0.2% sodium deoxycholate) containing protease inhibitors (Kit Halt TM Thermo Scientific) and1 µl EDTA 0.5 M. The cell pellets were subjected to sonication and protein samples were used in the Western Blot protocol [14].…”