Matrix metalloproteinase‑1 expression in breast cancer and cancer‑adjacent tissues by immunohistochemical staining

Introduction

Breast cancer is a pathologically and clinically heterogeneous disease, and is the most frequent malignancy among females (1). In addition, distant metastases are the most common type of breast cancer recurrence and are often the cause of fatality in breast cancer patients; metastases result in >40,000 fatalities per year in the USA alone (2). In a previous study, it was demonstrated that most complications of breast cancer are attributed to metastasis to distant organs, including lymph nodes, bone, lung and liver (3).

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases that digest components of the extracellular matrix (ECM) and cell surface receptors for soluble factors and junctional proteins involved in cell-cell and cell-ECM interactions. Due to their matrix-degrading abilities and high expression in advanced tumors, MMP activity has been shown to be required for breast cancer cell invasion and angiogenesis (4,5). MMP-1, a member of the MMP family, is upregulated in breast cancer cell lines with an enhanced ability of tumor growth, invasion and distant metastasis (6,7). However, the expression of MMP-1 in breast cancer and cancer-adjacent tissues remains to be established. To address this issue, immunohistochemical staining was used to detect the MMP-1 expression in breast cancer and cancer-adjacent normal breast tissue. An extended understanding of the expression of MMP-1 may provide a novel insight into the role of MMP-1 in pathological process of breast tumorigenesis.

Materials and methods

Tissue microarray and immunohistochemical analysis

Tissue microarray purchased from Chaoying Bio-Technology Co., Ltd., (BR1002a; Xi'an, Shanxi, China). All the specimens had detailed information including age, gender, organization, pathological diagnosis, clinical grade, tumor-node-metastasis classification, clinical stage, specimen type and results.

Immunohistochemical analysis

To detect the expression of MMP-1, immunohistochemical staining was performed. Sections were heat-immobilized at 60˚C for 30 min and were deparaffinized in xylene and rehydrated through a series of graded ethanol solutions. Antigen retrieval was performed in a pressure cooker at 95˚C for 2 min using 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 15 min at room temperature. Sections were subsequently incubated with anti-MMP-1 mouse monoclonal antibody (1:50; sc-21731; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 60 min at 37˚C in a humidified chamber. Subsequently, the sections were rinsed and incubated with a biotinylated secondary antibody for 30 min, followed by horseradish peroxidase-conjugated streptavidin for 30 min using the UltraSensitive™ Universal (anti-mouse/rabbit) Detection reagent (Fuzhou Maixin Biotechnology Development Co., Ltd., Fuzhou, China). Finally, the tissue microarray sections were stained with freshly prepared 3,3′-diaminobenzidine and counterstained lightly with hematoxylin. As a negative control, the primary antibody was replaced with normal rabbit or mouse immunoglobulin G at the same dilution. MMP-1 stain intensity was scored on a scale of 0 (negative) to 2+ (intense staining).

Results

Expression of MMP-1 in breast cancer and cancer-adjacent normal tissue

The MMP-1 expression in breast cancer and cancer-adjacent normal tissue was detected by immunohistochemical staining. As shown in Table I, and Figs. 1 and 2, MMP-1 had a positive expression in normal lymph node tissue and lymph node metastatic non-specific invasive ductal carcinoma. MMP-1 had partially negative expression in non-specific invasive ductal carcinoma of the breast and cancer-adjacent normal breast tissue. The MMP-1 negative expression rate was only 6.1% in non-specific invasive ductal carcinoma of the breast and 2.9% in cancer-adjacent normal breast tissue, respectively.

Figure 1. Positive and negative percentage analysis for matrix metalloproteinase-1 (MMP-1) expression in breast cancer tumors from 86 subjects.

Figure 2. Representative images of immunohistochemical staining of matrix metalloproteinase-1 (MMP-1).

Table I. Matrix metalloproteinase-1 (MMP-1) expression in breast cancer and cancer-adjacent normal tissue.

Table I.

Matrix metalloproteinase-1 (MMP-1) expression in breast cancer and cancer-adjacent normal tissue.

Organ	Pathology diagnosis	No. of tumors	MMP-1 (-, +, ++)
Breast	Non-specific invasive ductal carcinoma grade I	13	1, 6, 6
Breast	Non-specific invasive ductal carcinoma grade II	12	0, 4, 8
Breast	Non-specific invasive ductal carcinoma grade III	8	1, 3, 4
Breast	Cancer-adjacent normal breast tissue	34	1, 26, 7
Lymph node	Lymph node metastatic non specific invasive ductal carcinoma	9	0, 3, 6
Lymph node	Normal lymph node tissue	9	0, 9, 0

[i] Staining was scored using a 0–2⁺ scale. 0 means no staining; 1⁺ and 2⁺ indicate increased intensity of the staining. Sub-regions excluding necrosis, macrophages and infiltrated neutrophils and lymphocytes were selected and scored. The intensity score for an array spot is the average of all its sub-regions.

MMP-1 expression is higher in non-specific invasive ductal carcinoma compared to cancer-adjacent normal breast tissue

The MMP-1 expression rate in non-specific invasive ductal carcinoma and cancer-adjacent normal breast tissue, lymph node metastatic non-specific invasive ductal carcinoma and normal lymph node tissue was analyzed. As shown in Table II, the MMP-1 positive expression rate (+ and ++%) in non-specific invasive ductal carcinoma was 39.4 and 54.5%, respectively. The MMP-1 positive expression rate (+ and ++%) in cancer-adjacent normal breast tissue was 76.5 and 20.6%, respectively. The MMP-1 positive expression rate (++%) was significantly higher in non-specific invasive ductal carcinoma compared to cancer-adjacent normal breast tissue. In addition, the MMP-1 expression rate (++%) in lymph node metastatic non-specific invasive ductal carcinoma was 66.7%, significantly more than in the normal lymph node tissue.

Table II. Different matrix metalloproteinase-1 (MMP-1) expression rates in non-specific invasive ductal carcinoma and cancer-adjacent normal breast tissue.

Table II.

Different matrix metalloproteinase-1 (MMP-1) expression rates in non-specific invasive ductal carcinoma and cancer-adjacent normal breast tissue.

Pathology diagnosis	No. of tumors	MMP-1 expression rate (-, +, ++%)
Non-specific invasive ductal carcinoma	33	(6.1, 39.4, 54.5)
Cancer-adjacent normal breast tissue	34	(2.9, 76.5, 20.6)
Lymph node metastatic non-specific invasive ductal carcinoma	9	(0, 33.3, 66.7)
Normal lymph node tissue	9	(0, 100, 0)

Discussion

MMPs are expressed in nearly all tumors, where they facilitate tumor growth, invasion and metastasis (4,5). The study by Kang et al (8) used microarray analysis to show that overexpression of MMP-1 enhances MDA-MB-231 cell bone metastasis. Additionally, in breast cancer, short hairpin RNA-mediated stable knockdown of MMP-1 or induction of MMP-1 by overexpression of δ-crystallin enhancer factor 1 significantly regulated the invasive ability of MDA-MB-231 (6,7). Since MMP-1 has been confirmed as a factor that facilitates breast cancer metastasis, the present study provides a novel discovery that MMP-1 expression is higher in non-specific invasive ductal carcinoma and lymph node metastatic non-specific invasive ductal carcinoma compared to cancer-adjacent normal breast tissue and normal lymph node tissue. The high expression of MMP-1 in breast cancer may be closely associated with its invasion and metastasis.

Acknowledgements

The present study was supported by grants from the National Natural Science Foundation of China (no. 81302323), the Natural Science Foundation of Hebei province (nos. C2014209140 and C2013209024), General Higher Education Young Talents Program of Hebei province (no. BJ2014027), Science and Technology Support Program of Tangshan city (no. 14120208a) and Students' Innovation and Entrepreneurship Training Program of Hebei United University (no. X2014083) and the Educational Reform Project of Tangshan Normal University (no. 2011001012).

References