Down regulation of ADAM33 as a Predictive Biomarker of Aggressive Breast Cancer

Abstract

Breast cancer is a heterogeneous disease with differences in its clinical, molecular and biological features. Traditionally, immunohistochemical markers together with clinicopathologic parameters are used to classify breast cancer and to predict disease outcome. Triple-negative breast cancer (TNBC) is a particular type of breast cancer that is defined by a lack of expression of hormonal receptors and the HER2 gene. Most cases of TNBC also have a basal-like phenotype (BLBC) with expression of cytokeratin 5/6 and/or EGFR. A basal marker alone is insufficient for a better understanding of the tumor biology of TNBC. In that regard, the ADAM33 gene is silenced by DNA hypermethylation in breast cancer, which suggests that ADAM33 might be useful as a molecular marker. In the present study, we have produced monoclonal antibodies against the ADAM33 protein and have investigated the role of ADAM33 protein in breast cancer. We used 212 breast tumor samples and lower levels of ADAM33 were correlated with TNBC and basal-like markers. A lower level of ADAM33 was also correlated with shorter overall survival and metastasis-free survival and was considered an independent prognostic factor suggesting that ADAM33 is a novel molecular biomarker of TNBC and BLBC that might be useful as a prognostic factor.

Figure 1. ADAM33 expression in breast cancer cell lines.

(A) RT-PCR of breast cancer cell lines: PMC42, MCF7 and SKBR3 cells show the amplification of a 612-bp fragment while no amplification is observed in MDA-MB-231 and MDA-MB-436 cells. (B) Western blotting of breast cancer cell lines using an anti-ADAM33 antibody, which shows a positive signal in PMC42 (line 1), MCF7 (line 2), SKBR3 (line 3) and no signal in MDA-MB-231 (line 4) and MDA-MB-436 (line 5). Beta-actin was used as the Western blotting control. Immunocytochemistry of breast cancer cell lines using an anti-ADAM33 antibody shows positive staining in PMC42 (C), MCF7 (D), SKBR3 (E) and negative staining in MDA-MB-231 (F) and MDA-MB-436 (G) cells. The IHC results are shown at X100 magnification.

Figure 2. Characterization of the Monoclonal Antibody anti-ADAM33.

(A) Dilution of the monoclonal antibody and its performance in ELISA: ADAM33-Rec (10 μg/mL) was immobilized onto plates, and the purified monoclonal antibody anti-ADAM33 was diluted from 0.14 μg/mL. (B) Diluted monoclonal antibody performance by Western blotting analysis with ADAM33-Rec (10 μg/mL), the purified monoclonal antibody anti-ADAM33, which was diluted from 0.14 μg/mL. (C) Identification of CDRs responsible for antigen-binding specificity.

Figure 3. ADAM33 expression in paraffin-embedded breast cancer tissue samples.

(A) ADAM33 scores: score of 2, low expression of ADAM33 protein; score of 3, intermediate expression of ADAM33 protein; score of 4, high expression of ADAM33 protein. The IHC results are shown at X100 magnification. (B) Correlation between ADAM33 gene methylation and the ADAM33 protein score. Unmethylated promoter gene (U); Methylated promoter gene (M). Chi-square test was performed, statistical significance was assumed when p < 0.05.

Figure 4. Correlation between the ADAM33 Score and biomolecular markers.

Percentage of patient samples that showed a correlation between the ADAM33 score and (A) a ER (p < 0.001); (B) PR (p < 0.001); (C) HER2 (p = 0.045); (D) EGFR (p = 0.042); (E) CK 5/6 (p = 0.046); (F) CK17 (p = 0.040); (G) c-KIT (p = 0.023) and (H) tumor subclasses (p < 0.001).

Figure 5. Kaplan-Meier curves for the time to breast cancer progression according to the ADAM33 Score.

(A) Kaplan-Meier estimates are shown for overall survival and (B) metastasis-free survival using the ADAM33 scores. Symbols on the graph lines represent censored data; p values are given for log-rank tests.