Elsevier

Acta Histochemica

Volume 116, Issue 8, October 2014, Pages 1367-1373
Acta Histochemica

Immunoexpression of ROCK-1 and MMP-9 as prognostic markers in breast cancer

https://doi.org/10.1016/j.acthis.2014.08.009Get rights and content

Abstract

Breast cancer is the most common tumor in women and it has high mortality mainly due to the occurrence of tumor metastasis. Both the processes of cell migration and anchorage to the substrate are essential for the development of metastasis. These processes occur by rearrangements of the actin cytoskeleton, regulated by Rho-associated protein kinase 1 (ROCK-1). The degradation of the extracellular matrix, influenced by metalloproteinase 9 (MMP-9) also exerts greater cell invasiveness. The present study evaluated the ROCK-1 and MMP-9 proteins using an immunohistochemical method through the selection of invasive ductal breast carcinoma. The protein expression was correlated to clinicopathological parameters and overall survival of the patients. High expression of the ROCK-1 protein was correlated statistically to the status of lymph nodes (p = 0.007) and showed variable expression in different clinical stages of the tumor. MMP-9 showed a strong immunostaining in patients with metastasis that had died, whereas there was no marker in normal breast tissues. In addition, 46.6% of patients classified as poor prognosis showed high expression of ROCK-1 and MMP-9 protein and another 40.0% just showed high expression of MMP-9. Thus, the differential expression of ROCK-1 and MMP-9 proteins suggests their potential use as prognostic markers in breast cancer.

Introduction

Breast cancer is the most common cancer among women (IARC, 2012) and for 2014 there is an estimated occurrence of 57,120 new cases of breast cancer in Brazil (INCA, 2014). Despite progress in treatment with chemotherapy in patients in the early stages, there is no effective therapy for the end-stage in metastatic breast cancer. Thus, the identification of biomarkers that allow early detection of metastasis is essential for therapeutic success in the treatment of breast cancer (Fan et al., 2012).

The most common type of breast cancer is ductal carcinoma, which affects the mammary ducts and can metastasize to other locations (Germano and O’Driscoll, 2009). Tumor invasion and metastasis affect more than 90% of patients with breast cancer and are the main factors that contribute to high mortality (Moreau et al., 2007, Fagan-Solis et al., 2012). These processes occur due to the weak cytoskeletal structure allowing the low anchorage of neoplastic cells (Ortíz-López et al., 2009).

Rho-associated protein kinase 1 (ROCK-1) is among the factors that regulate cell migration and anchorage to the substrate and controls the rearrangement of the actin cytoskeleton (Ortíz-López et al., 2009). This protein plays a key role in the regulation of cell morphology, adhesion and motility (Provenzano et al., 2008) and its inhibitors are capable of reducing cancer cell migration, proliferation and invasion (Deng et al., 2010). The increased expression of ROCK-1 is related to the presence of tumor metastasis, and its inhibition is a novel approach for treating breast cancer (Liu et al., 2009).

The degradation of the extracellular matrix (ECM) is essential for the initiation and progression of tumor invasion (Wang et al., 2011), allowing the tumor cells to invade tissue, blood vessels and affecting new metastatic sites. This process is mainly influenced by the activity of matrix metalloproteinases (MMPs) that are enzymes which degrade structural components of the ECM, molecules of cell–cell and cell–ECM interactions, and activate other MMPs (Sternlicht and Werb, 2001).

MMPs can release and activate growth factors and cytokines, facilitating tumor invasion and the metastatic processes (Ellerbroek and Stack, 1999), with many human tumors characterized by increased concentrations of MMPs (Hoekstra et al., 2001). One of the major MMPs involved in breast cancer is MMP-9 (Kato et al., 2002), which is present at high levels in malignant breast tumors (Jinga et al., 2006) and related to high numbers of distant metastases (Li et al., 2004, Vizoso et al., 2007) and poor prognosis (Ranogajec et al., 2012).

The aim of the study was to estimate the prognostic value of ROCK-1 and MMP-9 proteins in women with breast cancer and to correlate them with clinicopathological parameters and overall survival. The aim was to determine their potential use in routine examinations and diagnosis and to assist in developing novel protocols to prevent development of tumor growth and metastasis.

Section snippets

Sample characterization

Tumor fragments from 60 women treated at Hospital de Base at Faculdade de Medicina de São José do Rio Preto (FAMERP) between the years 2000–2005 with a histopathological diagnosis of invasive ductal carcinoma were selected and the clinicopathological parameters were evaluated. Among the clinicopathological features, women aged over 50 years (70.0%), those who underwent surgical treatment (90.0%), chemotherapy and radiotherapy (68.3%), absence of hormone replacement therapy (68.4%) and

Results

When the expression of ROCK-1 protein in comparison to clinicopathological characteristics was correlated, higher expression was found in women with lymph node involvement compared to those without lymph nodes metastases (p = 0.007; Fig. 1a) and, observed variation in expression of ROCK-1 between different clinical tumor stages, but without a statistically significant difference (p > 0.05; Fig. 1b; Table 1). Furthermore, there were strong labeling areas of ROCK-1 protein in the group of women who

Discussion

ROCK-1 and ROCK-2 are the major kinase effector proteins of Rho GTPase signaling, correlated to the processes of invasion, angiogenesis and tumor aggressiveness (Schackmann et al., 2011). The active ROCK-1 signaling results in cell resistance to apoptosis and subsequent growth of metastatic breast lobular tumors (Schackmann et al., 2011). Tang et al. (2012) confirmed the action of ROCK-1 in the reorganization of the actin cytoskeleton, and this induces morphological alterations that are the

Acknowledgement

This study was funded by the FAPESP/Brazil – Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (2011/13154-0).

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