Mol Cell Biochem (2012) 366:269–275 DOI 10.1007/s11010-012-1305-z
Clinical relevance of cyclooxygenase-2 and matrix metalloproteinases (MMP-2 and MT1-MMP) in human breast cancer tissue Mohammad A. Mohammad • Ahmed A. Zeeneldin • Zakaria Y. Abd Elmageed • Ebtsam H. Khalil • Said M. E. Mahdy • Hayat M. Sharada • Sabry K. Sharawy • Abdel-Hady A. Abdel-Wahab
Received: 12 September 2011 / Accepted: 3 April 2012 / Published online: 18 April 2012 Ó Springer Science+Business Media, LLC. 2012
Abstract Breast cancer (BC) is the most common neoplasm among women in most developed countries, including Egypt. Elevated levels of certain proteins in human BC are associated with unfavorable prognosis and progressive stages of the disease. The aim of our study was to evaluate the protein expression profile and prognostic significance of cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP-2), MMP-9 and membrane type 1-MMP (MT1-MMP) and their interaction in operable BC patients. The protein expression of COX-2, MMP-2 and MT1-MMP were evaluated by western blot technique, whereas enzymatic activity of MMP-2 and MMP-9 was determined by zymography in 47 breast cancer patients as well as normal adjacent tissues. Also, the correlation between these proteins and age, tumor size, LN stage, TNM stage, estrogen receptor, progesterone receptor, disease-free survival, and overall survival (OS) has been investigated. As compared to adjacent normal tissues, COX-2, MMP-2 and MT1-MMP were over-expressed in
M. A. Mohammad S. K. Sharawy A.-H. A. Abdel-Wahab (&) Department of Cancer Biology, National Cancer Institute, Cairo University, 1 Kasr El-Aini St, Cairo, Egypt e-mail:
[email protected] A. A. Zeeneldin Department of Medical Oncology, National Cancer Institute, Cairo University, Cairo, Egypt Z. Y. Abd Elmageed Department of Urology and Oncology, Tulane University Medical Center, New Orleans, LA 70112, USA E. H. Khalil S. M. E. Mahdy H. M. Sharada Department of Chemistry, Faculty of Science, Helwan University, Helwan, Egypt
43, 64, and 60 % of tumor tissues, respectively. In the same pattern, the activity of MMP-2 (62 %) and MMP-9 (45 %) was elevated in BC tissues. Multivariate analysis showed a positive correlation between the protein expression of COX-2, MMP-2, and MT1-MMP and the activity of MMP2 and MMP-9 in BC patients. However, the enzymatic activity showed no correlation with clinicopathological features. This study confirms the preclinical evidence that COX-2 increased the expression of MT1-MMP, which in turn activates MMP-2. The lack of correlation with clinicopathological features, OS or disease-free survival ascertains the complexity of tumor progression and metastasis with many pro- and counter regulatory factors. Keywords Breast cancer Cox-2 MMPs MT1-MMP Western blot Zymography
Introduction Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide with an estimated 1.4 million new BC cases and 458,000 deaths in 2008 [1]. The main contributing factor to the rising mortality rate is the spread of distant metastases to different organs [2]. Higher incidence of BC in urban and more developed populations in Egypt was recently reported and this problem is usually linked to continuous exposure to xenoestrogens, endocrine disruptors or genotoxic substances [3]. It is reported that BC represents about 37 % of all female cancers hospitalized in the National Cancer Institute of Egypt [4]. Cyclooxygenase (COX) is proved to convert membrane arachidonic acid into prostaglandins where their target effects were determined by G protein-coupled receptors [5]. COX-2 is well known to be involved in precursor
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lesions of several solid tumors [6] and contributes to tumorigenesis by blocking the signaling pathways of proand anti-apoptotic proteins. Also, COX-2 might induce the secretion of tumor growth factors to promote angiogenesis, which accompanied with an increase in protein expression of matrix metalloproteinases (MMPs) [7]. Eventually, invasive BC exerts high protein expression of COX-2, which is associated with large tumor size, high tumor grade, negative estrogen receptor (ER) status, high p53 expression, and unfavorable prognosis [8]. It is reported that overexpression of COX-2 in BC patients and it was associated with poor prognosis and exerted no effect on disease-free survival (DFS) or overall survival (OS). MMPs belong to a zinc-dependent endopeptidases family that is involved in various aspects of tumor progression. MMPs act as enzymes that degrade structural components of the extracellular matrix and they divided into two types; the soluble-type and the membrane-type of MMPs [9]. Currently, 28 human MMPs have been identified and classified according to their substrate specificities and structural similarities [10]. MMP-2 is a 72 kDa type IV collagenase secreted as pro-active form that is processed into the active form through interaction with MT1-MMP (MMP14) on the cell surface at the target site [11]. MMP-2 is associated with the progression of malignancy of several types of carcinoma [12, 13]. MT1-MMP participates in the activation of other soluble MMPs such as MMP-2 [14]. Recently, clinical studies showed that the expression of MT1-MMP and MMP-2 is associated with poor prognosis in patients suffering cancer including BC [15, 16]. In the light of the previous evidence, the aim of the current study was to investigate the expression of COX-2, MMP-2 and MT1-MMP and whether they correlate with the enzymatic activity of enzymes of MMP-2 and MT1MMP in BC tissues.
Table 1 Clinical features of BC patients
Adjuvant hormone therapy (tamoxifen)
35
75
Clinical features of BC patients
Adjuvant radiotherapy
37
79
Distant metastases
8
17
The study was conducted in compliance with the guidelines of good clinical practice (GCP) and the ethical principles rooted in the Declaration of Helsinki. All patients consented to participate in the study. All treatments and interventions were according to the Egyptian National Cancer Institute (NCI-Egypt) practice guidelines. This study comprised 47 female BC patients presented to NCIEgypt from August 2006 to December 2008. The average age was 52 years (Table 1). Premenopausal women reported about 53 % of all patients. Grade II invasive duct carcinoma was the common histologic grade of the tumor. The average size of the primary tumor was 5 cm3 with T2 stage and counting 66 %. The average number of positive axillary lymph nodes (LNs) was 5.7 % and with N0–1 was 53 %. About 66 % of the tumors samples were positive to
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Parameter
Number
Age (year ± SD)
52.4 ± 12.4
(%)
Menopausal status Premenopausal
25
53
Postmenopausal
22
47
Invasive duct carcinoma (IDC)
38
81
Invasive lobular carcinoma (ILC)
4
9
Mixed (IDC & ILC)
3
6
Mucoid/tubular carcinoma
2
4
T1 T2
1 31
2 66
T3
7
15
T4
8
17
Histology type
Tumor stage
Positive LN (mean ± SD)
5.7 ± 6.4
ER Positive
31
66
Negative
13
28
Unknown
3
6
Positive
33
71
Negative
11
23
Unknown
3
6
28
60
PR
Chemotherapy use Adjuvant Neo-Adjuvant No Chemotherapy regimen
13
27
6
13
FEC
21
45
FAC
14
30
CMF
6
12
SD standard deviation, LN lymph node, CMF cyclophosphamide, methotrexate, fluorouracil, CAF cyclophosphamide, doxorubicin, fluorouracil, CEF cyclophosphamide, epirubicin, fluorouracil
estrogen (ER) or progesterone receptor (PR). Mastectomy was the common surgical procedure used in our study and chemotherapy, tamoxifen and radiotherapy were applied in 87, 75 and 79 % of BC patients. Sample collection and processing After surgical excision, tumor tissues along with the corresponding normal adjacent tissues were snap frozen at -80 °C until used in western blot or zymographic analysis (ZA).
Mol Cell Biochem (2012) 366:269–275
Western blot analysis After homogenization of tissue samples, the total protein content of each sample was estimated by Bradford method according to the manufacture’s protocol (Bio-Rad, CA). Samples were resolved on SDS-PAGE under reducing conditions followed by semi-dry protein transfer into nitrocellulose membranes (Bio-Rad, CA). The membranes were exposed to primary antibodies raised against COX-2, MMP-2 and MT1-MMP and b–actin as a control (Santa Cruz Biotechnology, Inc.). After extensive washing, the membranes were incubated with a secondary horseradish peroxidaseconjugated goat anti-mouse antibody (Sigma Chemical Co., St. Louis, Mo), hydrogen peroxide (BDH) and diaminobenzidine (Sigma-Aldrich). Protein bands were evaluated using Scion Image software (Scion Corporation, MD). Zymographic analysis The pro-enzyme and activated forms of MMP-2 and MMP9 were detected by zymography using 10 % SDS-PAGE copolymerized with 1 mg gelatin according to the procedures described before [17]. Statistical analysis The patients were described using descriptive statistics functions. Nominal and categorical variables were summarized as percentages while numerical data as means ± SD. Sub-groups were compared using v2 test when dealing with nominal or categorical variable and independent t test or ANOVA when comparing numerical values. Comparisons of each parameter in the tumor and adjacent normal tissues were done using paired t test. Correlation coefficient between protein expressions (numerical) was performed by linear correlation and regression. Survivals were plotted using the Kaplan–Meier method. Groups were compared using the log-rank test. Data are considered significant at p \ 0.05. The software SPSS (SPSS Corporation, IL, USA) was used in data analysis.
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of MMP-2 and MMP-9 was increased in 62 and 45 % of BC patients, respectively (Table 2). The expression of COX-2, MMP-2 and MT1-MMP in tumor tissues was 131, 141 and 183 % compared to normal adjacent tissues (Fig. 1a). Although this increase was not significant, MT1-MMP instead showed a significant change (p = 0.050). The activity of MMP-2 and MMP-9 in tumor tissues was 342 and 211 % compared to the normal tissues (Fig. 1b). Only, MMP-2 was found significant (p = 0.002) (Table 3). Protein expression of MMP-2 was positively correlated with COX-2 (p = 0.001) and MT1-MMP (p \ 0.088). Also, a positive correlation was found between the activity of MMP2 and MMP-9 (p = 0.001, Table 4). There was a positive correlation between N2–3 (p = 0.032) and stage III (p = 0.022) with high expression of MMP-2. The protein expression of COX-2, MMP-2 or MT1-MMP along with activity of MMP-2 or MMP-9 showed no significant correlation with tumor size, ER, PR, HR, LN status, distant metastases or tumor stage (data not shown). Our results also showed that COX-2 expression was higher in premenopausal than postmenopausal, Grade 3 (G3) than G1–2, tumor (T) stage 3–4 than T1–2, lymph node (N) stage 2–3 than N0–1, TNM stage III than II. The same pattern was found in non-metastatic than metastatic, ER, PR and HR positive than hormone-negatives samples. However, a positive correlation was found between COX-2 expression and menopausal status (p = 0.047). Both MMP-2 and MT1-MMP had the same pattern of correlation similar to COX-2. However, there was a positive correlation of MMP-2 but not MT1-MMP with LN and TNM stages (p = 0.032). The activity of MMP-2 and MMP-9 was high in premenopausal than postmenopausal, G3 than G1–2, T3–4 than T1–2, N 2–3 than N0–1, TNM stage III than II, metastatic than non-metastatic, ER, PR and HR positive compared to hormone negative. However, none of these enzymatic activities were found significant. After a median follow up of 17 months, four patients were dead and eight developed relapse. Acknowledging that a single patient had more than one relapse site; lung metastasis was reported in 4 patients, nodal and soft tissue Table 2 Percentage change in the expression of COX-2 and MMPs (and activity) in tumor tissues relative to adjacent normal tissues
Results Our results showed that the percentage of protein expression of COX-2, MMP-2 and MT1-MMP in tumor versus adjacent normal tissues was observed in 70 % versus 40 %, 89 % versus. 77 % and 70 % versus 38 %, respectively. Enzymatic activity of MMP2 and MMP9 in tumor versus adjacent tissues was detected in 74 % versus 53 % and 72 % versus 60 %, respectively. COX-2, MMP-2 and MT1-MMP were over expressed in 43, 64, and 60 % of all cases. The activity
Protein
Min
Max
Mean
SD
COX-2 (WB)
-88
1,243
131
303
MMP-2 (WB)
-91
1,328
141
335
MT1-MMP (WB)
-93
1,348
183
334
MMP-2 (ZA)
-93
2,871
341
652
MMP-9 (ZA)
-96
2,090
212
507
SD standard deviation, COX-2 cyclooxygenase-2, WB western blot, MMP-2 matrix metalloproteinase 2, MT1-MMP membrane type-1 MMP, ZA Zymographic analysis
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Fig. 1 Over expression of Cox-2, MMP-2 and MT1-MMP (a) and enzymatic activity (zymography) of MMP-2 and MMP-9 (b) in human normal and tumor tissue samples (n = 47). *Data are significant at p \ 0.05 in comparison with normal adjacent tissues
Table 3 Protein expression in Egyptian BC tissues using western blot (WB) and zymography (ZA) technique Protein
Negative expression N (%)
Nonexpression N (%)
Positive expression N (%)
COX-2 (WB)
17 (36)
10 (21)
20 (43)
MMP-2 (WB)
14 (30)
3 (6)
30 (64)
7 (15)
12 (25)
28 (60)
MT1-MMP (WB) MMP-2 (ZA)
11 (23)
7 (15)
29 (62)
MMP-9 (ZA)
16 (34)
10 (21)
21 (45)
Table 4 Correlation between protein expressions (activity) in BC tissues MMP2 (WB) COX-2 (WB) MMP-2 (WB) MT1-MMP (WB) MMP-2 (ZA)
MT1-MMP (WB)
MMP2 (ZA)
MMP9 (ZA)
r = 0.52
r = 0.2
r = 0.15
r = 0.09
p \ 0.001
p = 0.088
p = 0.158
p = 0.282
r = 0.44 p = 0.001
r = 0.22 p = 0.067
r = 0.07 p = 0.322
r = -0.06
r = 0.01
p = 0.338
p = 0.484 r = 0.54 p \ 0.001
COX-2 cyclooxygenase-2, WB western blot, MMP-2 matrix metalloproteinase 2, MT1-MMP membrane type-1 MMP, ZA zymographic analysis, r correlation coefficient
relapse in 4 patients, bone metastasis in 2 patients, while liver and brain metastasis were observed in one patient. Expression of COX-2, MMP-2 and MT1-MMP and the activity of MMP-2 and MMP-9 showed no significant
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differences in patients who developed relapse compared to others. The median of OS was not recognized (Fig. 2a). Oneyear and two-year survival rates were 93 and 90 %, respectively. Similarly, the median of DFS was not identified (Fig. 2b). One-year and two-year DFS rates were 87 and 81 %, respectively. No significant change was found between OS and DFS and the level of protein expression or enzymatic activity of the above mentioned protein variables.
Discussion BC is a heterogeneous disease with multiple genetic alterations that can influence the tumor growth, progression, and metastasis. However, BCs with favorable clinical and pathological features are probably curable, given availability of several effective treatment regimens and early diagnosis [18]. Consequently, at the time of diagnosis, defining prognostic and predictive biological markers is pivotal in BC management. In this study, we investigated the protein expression of COX-2 and its correlation with MMPs involved in metastasis (MMP-2, MT1-MMP, MMP-9). The role of COX-2 in BC is still incompletely defined. One study has reported that COX2 was expressed in all types of human BC compared with normal tissues and it was correlated with the increase of tumor size [19]. However, another study reported that only 2 out of 44 tumor samples have shown a high expression of COX-2 [20]. On mRNA and proteins levels, COX-2 was highly expressed in non-tumor ductile epithelium as
Mol Cell Biochem (2012) 366:269–275
Fig. 2 OS (a) and disease-free survival (b) for Egyptian BC patients
compared with invasive ductile carcinoma [21]. In 2004, another study stated that COX-2 plays an important role not only for regulating MMPs during the normal physiological processes, but also during the metastatic stage of tumor cells by regulating the expression of MMPs [22]. We found 44 % of BC cases expressed COX-2 in accordance with our previous reports [23, 24] and other studies [25, 26]. However, others reports showed higher protein expression of COX-2 and this difference might be related to sampling, racial variation or the use of different methodologies [27–29]. According to our previous studies [23, 24] and to the others [27, 28], COX-2 expression was not correlated with ER, tumor grade, age, DFS or OS. Our records considered that the expression and activity of MMP2 was higher in tumor tissue relative to normal adjacent ones where the level of expression was varied between 30 and 80 %. Other studies reported the same observations and attributed these discrepancies to different methods used, races and experimental standardization [30–32]. Here, the protein expression of MMP-2 in tumor tissues was significantly correlated with COX-2 and MT1-MMP as
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previously described [31, 33]. This evidence suggests that COX-2 increased the secretion of prostanoids that could induce the expression of MMP-2 in human tumors. In vitro studies have shown that aberrant COX-2 expression could increase the invasiveness of colon cancer cells through the induction of MT1-MMP, which activates pro-MMP-2. MT1-MMP may act directly by degrading ECM components such as type III collagen or indirectly by activating pro-MMP-2 or by inducing tumor angiogenesis through vascular endothelial growth factor [31]. Our results are in accordance with Stankovic et al. [34] as we were able to show a positive correlation between the activity of MMP-2 and MMP-9. Our results showed that over-expression of MMP-2 was correlated with higher chance of having N2–3 or stage III [32]. However, such correlation was not reported in the study done by Li et al. (2004) because all of the patients had lymph node negative [35]. Similarly, the expression and activity of MMP2 had no correlation with other clinicopathological features or OS and DFS as described before [34, 35]. Also, the higher expression of MT1-MMP in tumor versus normal adjacent tissues was not significant or correlated with clinicopathological parameters including nodal stage, tumor stage, DFS and OS. A significant correlation with total but not the active form of MMP-2 unveiled the role of MT1-MMP in activation of MMP-2 [31]. In contrast, another study revealed a high expression of MT1MMP in cases with metastatic lymph node [36], poor clinical stage and large tumor size [31, 37]. The activity of MMP-9 was higher in tumor tissues and it was correlated with the clinicopathological characteristics, DFS and OS [34]. The unfavorable effect of MMP-9 on DFS might be explained by the lack of protective effect of the adjuvant therapy [35]. The lack of detrimental effects of the studied enzymes on metastasis and survival has many explanations. In addition to the relatively small sample size and the short follow up time period, tumor progression and metastasis are complex processes. In conclusion, our study elucidated the interplay between COX-2, MT1-MMP and MMP-2 in BC tissues and confirmed the preclinical evidence that COX-2 increases the expression of MT1-MMP followed by activation of MMP-2. The lack of correlation with clinicopathological features, OS or disease-free survival ascertains the complexity of tumor progression and metastasis with many pro- and counter regulatory factors. Acknowledgments This work was supported by grant from National Cancer Institute (NCI-48019), Cairo University, Cairo, Egypt. Conflict of interest We declare that we have no conflict of interest for any of the authors including financial, personal or other relationships with other people or organization.
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