A case-control study of Metallothionein-1 expression in breast cancer and breast fibroadenoma

The overexpression of Metallothionein-1 (MT-1) may play an important role in breast cancer; however, few studies have compared MT-1 expression between breast cancer and fibroadenoma. A cross-sectional controlled study was performed in 66 premenopausal women, aged 20–49 years, who had been histologically diagnosed with breast fibroadenoma or breast cancer. The patients were divided into two groups: group A, control (fibroadenoma, n = 36) and group B, study (breast cancer, n = 30). Immunohistochemistry was performed on tissue samples of fibroadenoma and breast cancer patients to evaluate the expression of metallothionein using an anti-MT-1 polyclonal antibody (rabbit polyclonal anti-metallothionein-Catalog Number biorbyt-orb11042) at a dilution of 1:100. The data were analyzed using NOVA (p < 0.05). Microscopic analysis showed a higher concentration of anti-MT-1-stained nuclei in breast cancer tissues than in fibroadenoma tissues. The mean proportion of cells with anti-MT-1-stained nuclei was 26.93% and 9.10%, respectively, in the study and control groups (p < 0.001). Histological grade 3 tumors showed a significantly higher MT-1 expression than hitological grade 1 (p < 0.05), while breast tumors negative for estrogen-, progesterone- and HER2- receptors had a significantly higher MT-1 expression than positive breast tumors positive for these parameters (p < 0.05). MT-1 protein in women of reproductive age was significantly higher in breast cancer than in fibroadenoma in this study. Furthermore, there was higher MT-1 immunoreactivity in more aggressive tumors.

Immunohistochemistry for MT-1. Breast tissue samples fixed in buffered formalin were cut into 3 μm-thick sections. Sections were deparaffinized in xylol for 5 minutes, dehydrated in absolute ethanol, washed in buffered saline solution at pH 7.4 for 5 minutes and then treated for 5 minutes with 3% hydrogen peroxide (H 2 O 2 ) in buffered solution to block endogenous peroxidase activity. For antigen retrieval, the slides were placed in racks containing 0.21% citric acid (pH 6.0) and heated in a microwave oven for 15 minutes at maximum power. The slides were cooled, and phosphate-buffered saline was added for a cooling period of 20 minutes. Tissue samples were incubated overnight at 4-8 °C with monoclonal antibody metallothionein. A polyclonal antibody against MT-1 (rabbit polyclonalanti-metallothionein -Catalog Number biorbyt-orb11042) was used at a dilution of 1:100.
The slides were rinsed with PBS-Tween and incubated with secondary antibody (anti-mouse BA 2000, Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature. After being washed again with PBS-Tween, the slides were incubated with reagents from the ABC Elite detection system (PK 6100, Vector Laboratories) for 45 minutes at room temperature. The samples were rinsed once more with PBS-Tween and incubated with DAB (1.0 ml EnVision FLEX DAB for one drop of chromogen) and for 5 minutes. Finally, the slides were washed with distilled water, counterstained with hematoxylin, stained with ammoniacal solution, dehydrated with absolute ethanol, passed through Coplin jars containing xylol and mounted in Permount resin. Cells expressing metallothionein protein were identified by dark brown staining in the nucleus and cytoplasm.
Quantitative method. Two observers, blinded to patient identity and previously unaware of any of the cases, performed quantification of protein expression using a light microscope (Eclipse E-400, optic microscope, Nikon, Tokyo, Japan) connected to a color video camera (CHC-370 N digital camera, Samsung, Seoul, South Korea), where image was captured and transmitted to a computer equipped with the Image Lab software program, version 2.3 (SOFTIUM Informatica Ltda, São Paulo, Brazil) for image analysis.
To determine metallothionein expression, we counted nuclei of stained cells under a microscope with a magnification of 400X. At least 500 cells of the breast epithelium were counted on each slide, in random fields, starting in the area of highest MT-1 concentration in the cell nucleus, using Processing Software and Image Analysis-Image Lab ® (SOFTIUM Informatica Ltda, São Paulo, Brazil).

Statistical analysis.
Statistical analyses for this study were conducted using the software R, version 3.2.2.
Data were expressed as frequencies, percentages, measures of central tendency and dispersion. The normality of the data was tested with the Kolmogorov-Smirnov test. The Levene test was used to verify data homogeneity. To compare more than two means between normal and homogenous data, we used Student's t-test and ANOVA. Significant levels were set at p values ≤ 0.05.

Results
Under optical microscopy, breast cancer cells had a higher concentration of nuclei stained with anti-MT-1 than fibroadenomas (Fig. 1). The characteristics of both groups were similar, except for age and waist circumference ( Table 1).
Quantitative analysis showed mean percentages of nuclei stained with Metallothionein-1 per 500 breast epithelial cells in women at 9.10 ± 5.90 and 26.93 ± 15.87 in the control and study groups, respectively (Table 2).
Furthermore, Metallothionein-1 expression was statistically significant in histological grade 3 than in grade 1 tumors p < 0.05 (Fig. 2). MT-1 expression was statistically significant in breast cancers negative for HER2-, estrogen-and progesterone-receptors in comparison to tumors that were positive for these hormone receptors (p < 0.05), (Table 3).

Discussion
Metallothioneins participate in carcinogenesis by mechanisms promoting the development of tumor cells that are more resistant to chemotherapy or radiotherapy 15 . Elevated levels of this protein, with its antioxidant effect, can protect cancer cells against damage from free radicals. This protein has antiapoptotic and pro-proliferative effects, which support uncontrolled cellular growth in breast cancer [18][19][20] . www.nature.com/scientificreports www.nature.com/scientificreports/ There is accumulating evidence that metallothionein is an immunohistochemical biomarker due to its elevated expression in myoepithelial cells of invasive breast carcinoma 12 . However, very few studies have attempted to elucidate the behavior of this protein in fibroadenoma, a benign tumor that does not increase the risk of developing breast cancer 13 , and this characteristic makes this condition an ideal control to determine the effect of metallothionein expression on prognosis in breast cancer.
In the current study, overexpression of Metallothionein-1 was observed in cells of breast cancer tissues relative to that in the cells of fibroadenoma tissues. Based on our literature search, only El Sharkawy and Farrag 14 have investigated metallothionein expression in human breast fibroadenomas. According to those authors, higher MT-1 expression is related to more aggressive tumor behavior in ductal breast carcinoma. Other authors have   www.nature.com/scientificreports www.nature.com/scientificreports/ provided additional confirmation that a higher nuclear expression of MT-1 is more frequently observed in carcinomas than in benign tumors 15,21 .
Furthermore, it is noteworthy that a significant difference was found between the mean age of breast cancer patients and breast fibroadenoma patients. Nevertheless, fibroadenomas are known to be more common in younger women, while breast cancer occurs more frequently in older women 22 . Waist circumference was larger in women with cancer, consistent with the literature, since premenopausal women with excess visceral fat have a higher risk of developing triple-negative breast cancer, which has a relatively worse prognosis 23 .
Agresti et al. 24 observed that overweight premenopausal patients were at higher risk of developing triplenegative breast cancer than menopausal women. These findings suggest that obesity may play a role in the biological mechanisms underlying more aggressive types of breast cancer and the higher expression of metallothionein 25 .
Based on some authors, elevated MT expression blocks cellular apoptosis by sequestering zinc ions that stabilize p53, a gene that acts as a tumor suppressor by inducing apoptosis. Thus, MT enables the maintenance and integrity of the genome 26 . Recent studies indicate a strong relationship between p53 and MT, where overexpression of metallothionein is consistently associated with the presence of mutant p53, and in breast cancer, this relationship has been associated with a smaller number of apoptotic cells and a worse prognosis 27 .
Histologic tumor grade is associated with the immunoreactivity of metallothionein in breast cancer in addition to its anti-apoptotic effects. This discovery is consistent with previous observations in invasive breast carcinomas and in situ tumors reported by other researchers 10,12 . The current study showed a significant difference in MT-1 expression according to histologic tumor grade. Grade 3 tumors have a higher positivity for MT-1 protein, as well as HER2-overexpressing and negative hormonal receptors breast cancer. The association between these variables is relevant, indicating that metallothionein has a role in the differentiation, proliferation and progression of breast cancers. Proliferation is an important guide for the prognosis and therapy of malignant tumors 28,29 .
Therefore, studies have indicated that the expression of MT proteins in ductal breast cancer may represent an unfavorable prognostic index, since its highest expression is related to malignant cells with a higher histologic grade. Nevertheless, there is a need for further studies to obtain a better understanding of the behavior of metallothionein in tumorigenesis and to define the clinical significance of its expression in malignant and benign breast tumors.
Ethical approval. The internal review board of the Federal University of Piauí approved this protocol.
Informed consent was obtained from all individual participants included in the study. All the procedures performed in this study complied with current Brazilian laws and were in accordance with the ethical standards of the institutional and national research committees, as well as the 1964 Helsinki declaration and its later amendments.   Table 3. Metallothionein-1 meanexpression in positive/negative tumors for ER, PR, and Her2. *Teste ANOVA.