Overexpression of Matrix Metalloproteinase 11 in Thai Prostatic Adenocarcinoma is Associated with Poor Survival

Prostate cancer is a major problem in elderly men and is the second most common male cancer worldwide, especially in developed countries (Jemal et al., 2011). In Thailand, this cancer is the fourth most common cause of male cancer, but its incidence is increasing annually (Khuhaprema et al., 2010). Almost all patients with prostate cancer are advanced and at metastatic stage at the time of initial diagnosis and have been related to poor survival time more than early stage (Tuamsuk et al., 2011). Currently, serum prostatic-specific antigen (PSA) is used for both screening and monitoring of prostate cancer during treatment. However, PSA is more sensitive, but not more specific and for diagnosis because common pathologic condition such as benign prostatic hyperplasia (BPH) and prostatitis can also present abnormality of PSA levels (Madu and Lu, 2010). Consequently, there are requirements for accurate prognostic markers, that could be combined with recently those available in order to improve survival time and management to the most effective treatment of prostate cancer.


Introduction
Prostate cancer is a major problem in elderly men and is the second most common male cancer worldwide, especially in developed countries (Jemal et al., 2011). In Thailand, this cancer is the fourth most common cause of male cancer, but its incidence is increasing annually (Khuhaprema et al., 2010). Almost all patients with prostate cancer are advanced and at metastatic stage at the time of initial diagnosis and have been related to poor survival time more than early stage (Tuamsuk et al., 2011). Currently, serum prostatic-specific antigen (PSA) is used for both screening and monitoring of prostate cancer during treatment. However, PSA is more sensitive, but not more specific and for diagnosis because common pathologic condition such as benign prostatic hyperplasia (BPH) and prostatitis can also present abnormality of PSA levels (Madu and Lu, 2010). Consequently, there are requirements for accurate prognostic markers, that could be combined with recently those available in order to improve survival time and management to the most effective treatment of prostate cancer.

Overexpression of Matrix Metalloproteinase 11 in Thai Prostatic Adenocarcinoma is Associated with Poor Survival
Nongnuch Nonsrijun 1 , Jumphol Mitchai 2 , Kamoltip Brown 3 , Ratana Leksomboon 4 , Panya Tuamsuk 1 * Va r i o u s s t u d i e s h a v e s h o w n t h a t m a t r i x metalloproteinase 11 (MMP-11) was significant factor in progression of such cancers as pulmonary cancer (Kren, 2006), head (Thorns, 2003), neck carcinoma (Wasenius, 2003), and breast cancer (Mellick, 2003). In addition, MMP-11 mRNA and protein were correlated with a poor clinical outcome in breast cancer (Chenard et al., 1996;Ahmad et al., 1998). Moreover, the expression of MMP-11 in prostate cancer tissues was associated with high probability of biochemical recurrence that means PSA level at >0.2 ng/ml (Escaff et al., 2010). However, there has been a lack of correlation data between survival time of patients and prostate cancer. Therefore, the aim of this study was to quantify MMP-11 expression in normal and prostatic adenocarcinoma tissues and to determine whether overexpression correlates with survival outcome and so might serve as a new prognostic marker.

Human prostatic adenocarcinoma tissue
Paraffin embedded human prostatic adenocarcinoma

Immunohistochemical detection of MMP-11
The paraffin-embedded prostatic tissues were deparaffinized with xylene, and the rehydrated with stepwise descending concentration of ethanol. The sections were submerged into 10 mM citrate buffer (pH 6.0) and boiled for 5 min for antigen retrieval, and were the treated for 10 min in 3% (v/v) hydrogen peroxide in methanol to block endogenous hydrogen peroxide activity. Sections were incubated for 30 min with 3% normal horse serum (NHS) to block non-specific binding, then incubated with anti-MMP-11 (rabbit polyclonal, 1:500; Abcam, USA) overnight at 4̊C. NHS and normal human prostate tissue were used as internal and external negative controls, respectively. After washing, tissue sections were treated with 50% horseradish peroxidase (HRP) polymer detection (Thermo, USA). The color was developed with 3, 3'-diaminobenzidine tetrahydrochloride (DAB) as substrate, then counterstained with Mayer's hematoxylin, dehydrated, and mounted (Jamnongkan et al., 2012).

Statistical analysis
All statistical analyses were performed using IBM SPSS 19.0 software. Data were shown as median±SD. The survival curves were established using the Kaplan-Meier method. The correlation coefficient between MMP-11 expression and clinicopathological data of patients were analyzed by the Pearson correlation method. Univariate and multivariate analyses were considered by the Cox proportional hazards model to assess the prognosis values of MMP-11 expression. Statistical significance was set at P<0.05 (Zhao et al., 2010;Jamnongkan et al., 2012).

Expression of MMP-11 in prostatic adenocarcinoma tissue
The median age of 103 patients with prostatic adenocarcinoma under study was 70±9 years (range of 42-91 years). Positive immunostaining for MMP-11 was mainly localized in the cytoplasm of stromal cells, not shown in cancer cells (Figure 1). High expression of MMP-11 was detected in 48 (46.6%) cases, and low expression was detected in 55 (53.4%) cases. More than 50% samples with a classification of pT4 and a high Gleason grading also show high staining of MMP-11, while samples with a grading of pT1 and a low Gleason grading showed low expression levels of MMP-11 (Table  1).

MMP-11 and patient prognosis
The time of follow-up of patients was January 2003 to December 2012. The overall median of survival time was 69 months. A total of 43 of 103 cases died within the follow-up period, due to prostate cancer. Generally, patients whose prostate samples showed high levels of MMP-11 expression had significantly shorter survival (p<0.001) (Figure 2). The Kaplan-Meier analysis established that the survival rates of patients with high expression of MMP-11 levels was 41.7%, but 72.7% in patients with low expression of MMP-11 levels, after five years of follow-up (p<0.01, log rank analysis; Figure 2). For univariate analysis, MMP-11 expression, Gleason grading, pT, and bone metastasis were significantly associated with survival, whereas age and PSA levels were not associated with survival (Table 2). Multivariate analysis  (Table 2).
MMP-11 or stromelysin-3 was first characterized from stromal cells of breast cancer (Basset et al., 1990). It was unique among the MMPs since it was shown in stromal fibroblast cells adjacent to tumor cells, rather than within tumor cells themselves (Basset et al., 1990(Basset et al., , 1993. MMP-11 is also different from other MMPs in that it is secreted from cells in potential active form, while its inactive form is produced by furin-dependent proteolytic cleavage intracellularly (Pei and Weiss, 1995). Interestingly, MMP-11 was not able to degrade in major component of extracellular matrix when compared with other MMPs (Murphy et al., 1993;Pei et al., 1994;Manes et al., 1997). This study clearly showed that immunoreactivity of MMP-11 was present within stromal cell of prostate cancer tissue but did not occur in samples of normal prostate, which seem to involve in an important mechanism in the molecular biology of malignancy. In addition, MMP-11 protein was only shown in stromal cells while it was not present in tumor epithelial cells, similarly to the study of Basset et al. (1990). This phenomenon may indicate that MMP-11 promotes cancer progression by tumor microenvironment inducement that association with embedding in the extracellular host tissue at the time of cancer cells invasion (Masson et al., 1998;Boulay et al., 2001).
Our results reveal that MMP-11 expression is positively correlated with Gleason grading, pT, and bone metastasis. Further high expression of MMP-11 correlated significantly with advanced stage of prostate cancer (poor differentiation, pT4, and positive-bone metastasis categories). Our data thus reflects several studies of other human cancers, which showed that high expression of MMP-11 were found in human solid cancers including oral cancers (Arora, et al., 2005), desmoid tumors (Denys et al., 2004), non-small cell lung cancer (Kettunen et al., 2004), esophageal adenocarcinoma (Hourihan et al., 2003), and skin cancer (Chen et al., 2012). In addition, Sunil Kumar et al. (2013) showed that MMP-11 could play a significant role in the degradation of extracellular matrix in canine mammary tumor. Moreover, some studies demonstrated that MMP-11 protein related with cancer progression including gastric cancer (Zhao et al., 2009), oral cancer (Soni et al., 2003). Escaff et al. (2010) showed that prostate cancer with high MMP-11 expression was significantly associated with higher probability of biochemical recurrence of the disease.
Subsequently, we found that patients with low-level MMP-11 expression had significantly longer survival  P-value was determined using the log-rank test (p<0.01) time compared with high levels of MMP-11. Accordingly, overexpression of MMP-11 gene is related with increased aggressiveness of cancers and a poor clinical outcome (Boulay et al., 2001;Andarawewa et al., 2003). Yan et al. (2011) demonstrated that serum levels of MMP-11 in patients with gastric adenocarcinoma correlated with those advanced stages. Clinical studies have shown that high expression of MMP-11 is correlated with a lower survival time among patients with breast, non-small cell lung cancer, and colon cancer (Têtu et al., 2006;Cheng et al., 2010). Moreover, Boulay et al. (2001) stated that the cellular function of MMP-11 is closely related to decreasing cancer cells death via apoptosis and necrosis during malignancy. Inactivation of MMP-11 function may represent a new strategy for preventing cancer cell invasion and metastasis. In conclusions, MMP-11 expression in prostatic adenocarcinoma correlated positively with Gleason grading, pT, and bone metastasis and is closely associated with survival time. Therefore, high levels of MMP-11 expression may potentially be used to predict decreased survival in prostatic adenocarcinoma and be used for new prognostic marker in combination with currently available markers. Further studies are required to investigate whether serum MMP-11 levels correlate with survival outcome in patient with prostatic adenocarcinoma to determine whether the molecule should be developed as a prognostic marker.