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Matrix Metalloproteinase-9 Expression in Lung Cancer Patients and Its Relation to Serum MMP-9 Activity, Pathologic Type, and Prognosis

El-Badrawy, Mohamed K. MD*; Yousef, Aida M. MD*; Shaalan, Dalia PhD; Elsamanoudy, Ayman Z. PhD

Journal of Bronchology & Interventional Pulmonology: October 2014 - Volume 21 - Issue 4 - p 327–334
doi: 10.1097/LBR.0000000000000094
Original Investigations
Free

Background: Matrix metalloproteinase-9 is closely associated with the invasive and metastatic potential of most types of solid cancers. Our objective was to investigate the MMP-9 expression in lung cancer and to evaluate their relations to histopathologic types and prognosis.

Methods: Bronchoscopic samples were obtained from tumor and normal bronchial mucosa in 25 patients with lung cancer. Total RNA was isolated from the tissues, and the relative expression as well as the activity of MMP-9 was evaluated.

Results: Non–small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) showed significantly higher MMP-9 expression (P<0.0001) compared with normal tissues. MMP-9 activity in tissue and serum samples from both cancer groups were significantly higher than normal tissue and serum controls (P<0.0001). Also, MMP-9 expression and tissue and serum activity were significantly higher in NSCLC than in SCLC (P=0.0167, 0.0454, and 0.004, respectively). As regards the pathologic types of NSCLC, similar results were found for the adenocarcinoma subgroup versus squamous cell lung cancer (P=0.0015, 0.0052, and 0.0011, respectively). MMP-9 expression and tissue activity were higher in stage III-IV NSCLC cases compared with early tumor stages (P=0.0120 and 0.0271, respectively).

Conclusions: The expression and activity of MMP-9 are upregulated in NSCLC and are related to the pathologic type and clinical stage of NSCLC. Significantly higher expression and activity of MMP-9 in tumor tissue than in the surrounding tissue supports the important role of this metalloproteinase in the growth of lung cancer, and it could be used as a suggested therapeutic target.

Departments of *Chest Medicine

Medical Biochemistry, Faculty of Medicine, Mansoura University, Mansoura, Egypt

Disclosure: There is no conflict of interest or other disclosures.

Reprints: Aida M. Yousef, MD, Faculty of Medicine, Mansoura University, Mansoura, mail 3115, Egypt (e-mail: aymanhusen2002@yahoo.com).

Received November 2, 2013

Accepted July 15, 2014

Worldwide, lung cancer is the most common cancer in terms of both incidence and mortality.1 Endobronchial ultrasound is an evolving diagnostic tool in respiratory medicine that allows the bronchoscopist to see beyond the airway. It can assist transbronchial biopsies and staging of tumors especially peripheral lesions without exposure to radiation,2 thus helping in understanding the disease. Although recent advances in chemotherapy and radiation therapy have yielded modest improvements in patient outcomes, the overall survival remains poor in advanced lung cancer patients.3 The discovery of multiple molecular mechanisms underlying the development, progression, and prognosis of lung cancer has created new opportunities for targeted therapy and improved outcome.4 The development of effective chemotherapeutics that target the EGFR pathway, for example, gefitinib,5 took place over 15 years, whereas the targeting of the EML4-ALK translocation with crizotinib has progressed steadily in less than a third of that time.6 However, as researchers become interested in identifying key molecular changes and developing targeted therapies for these mutations, the development process will only continue to find therapies that target other aberrations, including those pathways relevant to other NSCLC histologies and SCLC. The result will be the effective management of patients on the basis of the molecular profiles of their cancers.4 With regard to tumor metastasis, it is a complex process involving loss of cell-cell adhesion, invasion of the extracellular matrix (ECM), spread through lymphatic or vascular channels, and colonization at the metastatic site where secondary tumors must continue to grow and cause clinically significant disease.7 Therefore, new therapeutic targets to block the metastatic process at its earliest stage are urgently needed. Matrix metalloproteinases might be involved in various processes of cancer progression such as destruction of the basement membrane, entry and exit of tumor cells from the circulation, local migration, and angiogenesis of tumors.8,9 In humans, the MMP-9 has been the most consistently detected member of the MMP family in malignant tissues and is associated with tumor aggressiveness and metastatic potential.10,11 MMP-9 (gelatinase B, 92 kDa type IV collagenase) can degrade denatured collagen (gelatin) and collagen types IV, V, VII, IX, X, elastin, fibrin, fibrinogen, and plasminogen. In addition, it is responsible for the processing of cytokines, for example, prointerleukin-1β and protumor necrosis factor-α, into their active form.12 Immunohistochemical expression and increased plasma levels of MMP-9 have been demonstrated in non–small cell lung cancer (NSCLC) patients.13,14 The activation of MMPs is regulated by many factors, such as tissue inhibitors of metalloproteinases (TIMPs). The balance of MMPs to TIMPs, therefore, determines the matrix turnover, wherein either an excess of MMPs or a deficit of TIMPs may result in excess ECM degradation.15,16 Our hypothesis suggests that further investigation of MMP-9 in lung cancer patients may be useful for understanding its role in the disease progression. Also, it may be helpful for the development of new therapeutic modalities that might target MMP-9 protein.

In this study, our objectives were to determine the expression and the activity of MMP-9 in normal lung tissues, NSCLC tissues, and small cell lung cancer (SCLC) tissues and to investigate their relations to histopathologic types and prognosis. We also attempted to clarify a possible relationship between serum and cancer tissue MMP-9 activity.

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PATIENTS AND METHODS

Patients

The study group consisted of 25 patients with lung cancer (24 men, 1 woman), with a mean age of 62.4±1.86 years (mean±SEM) (range, 40 to 80 y). A control group comprised 25 completely normal, nonsmokers (21 men, 4 women) with a mean age of 59.12±1.33 years (range, 46 to 71 y). All selected controls were nonsmokers because smoking was found to enhance the production of MMP-917 and we need to detect the normal level of MMP-9 in the serum. Demographic data in both groups were similar regarding the age, sex, and body mass index. Patients’ characteristics in both groups were shown in Table 1. The patients were mild and moderate smokers. Between May 2012 and April 2013, patients who underwent diagnostic bronchoscopy with cytologically or histologically proven lung cancer were included in the study. None of the patients had received radiotherapy, chemotherapy, or immunotherapy. Patients with a history of bronchial asthma, chronic obstructive pulmonary disease, or interstitial pulmonary fibrosis were excluded according to chest X-ray, the pulmonary function tests, and computed tomography (CT). For the pretreatment evaluation, complete history taking and physical examination were performed with evaluation of the Karnofsky performance status, complete blood cell count, serum chemistry analysis, and chest X-rays in posteroanterior and lateral views. Cranial CT, radionuclide bone scan spirometry, and abdominal ultrasound were performed in all patients to evaluate distant metastasis. A CT scan of the chest was performed to evaluate the size of the tumor, the stage, presence or absence of lymph nodes, and possible metastasis in relation to the tumor. Fiberoptic bronchoscopy with endobronchial biopsy was performed under sedation (midazolam) and local anesthesia (lidocain). A fiberoptic bronchoscope (Pentax FB-19 TV) was used in this study: (a) 3 to 5 forceps biopsies preserved in formalin 10% and bronchoalveolar lavage were taken for histopathologic diagnosis; (b) 3 to 5 forceps biopsies were taken for endobronchial tumors and 3 to 5 mucosal biopsies were taken from the contralateral lung. In the right lung, mucosal biopsies were taken from the carina between the right upper lobe and the intermediate bronchus. In the left lung, mucosal biopsies were taken from the carina between the left upper lobe and left lower lobe. The biopsies taken were preserved in 2 mL of RNA later solution (Sigma) to conserve RNA till the time of extraction. Patients proven to have lung cancer were included in the study and further investigations were carried out.

TABLE 1

TABLE 1

In addition, 2 mL of venous blood from all patients and controls were collected and serum was separated. Blood samples were obtained from all patients on the same day before doing the bronchoscope and they were stored at −80°C until time of analysis.

The protocol was approved by the local ethics committee, and the written informed consent from each patient was obtained before the bronchoscopy. Histologic type, pathologic stage, and tumor-node-metastasis classification were established according to the criteria of the American Joint Committee on Cancer.18

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Total RNA Extraction From Tissue Samples

Total RNA extraction from tissue samples was carried out using TriFast reagent (PeqLab. Biotechnology GmbH, Erlongen, Germany, Cat. No.30-2010) according to the manufacturer’s instructions. The remaining DNA was removed by digestion with DNase I (Sigma). The concentration of isolated RNA was determined spectrophotometrically by measuring the optical density at 260 nm (Jenway, Genova Model, UK). A volume of 10 µL of each sample (about 2 ng of DNA) was added to 990 µL of DEPC-treated water and quantified by measuring the absorbance at 260 nm as RNA yield (µg/mL)=A260×40×100 (dilution factor).19 The purity of RNA was determined by gel electrophoresis using formaldehyde agarose gel electrophoresis and ethidium bromide staining to show 2 sharp purified bands representing 28S and 18S ribosomal RNA.

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Semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Semiquantitative RT-PCR was performed using the QIAGEN One-step RT-PCR kit (Qiagen Inc. Valencia) according to the method of McPherson and Moller.20 QIAGEN One-step RT-PCR utilizes omniscript and sensiscript reverse transcriptase enzymes and HotStarTaq DNA polymerase to generate a PCR product from RNA template. Each test is optimized to allow the first strand cDNA synthesis and PCR reaction to proceed sequentially as a single-tube, single-step reaction. The reaction passed as follows:

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Synthesis of cDNA

A reaction mix of 40 μL/reaction was prepared as follows: 2 μL of first strand primer, 3 μL containing 30 pmol of PCR gene-specific primer (sense), 3 μL containing 30 pmol of PCR gene-specific primer (antisense), 2 μL One-step RT-PCR enzyme mix, 10 μL 5× One-step RT-PCR buffer, and 20 μL of DEPC-treated water to obtain a total volume of 40 μL. It was mixed thoroughly, and then 10 μL of each template RNA was added in each tube and mixed well. One tube was prepared as a negative control reaction to test for DNA contamination. The reactions were transferred to the thermal cycler (TECHNE C-312) and incubated at 50°C for 30 minutes for synthesis of cDNA, followed by incubation at 95°C for 15 minutes to inactivate the reverse transcriptase and completely denature the template. Gene-specific primers were purchased from Biolegio (Nijmegen, Netherlands). The oligonucleotide primers for MMP-9; forward: 5′-TTCATCTTCCAAGGCCAATC-3′ and reverse: 5′-CTTGTCGCTGTCAAAGTTCG-3′ were designed on the basis of the human MMP-9 (287 bp).21 Two oligonucleotide primers: forward 5′-CGTGGAAGGACTCAT GACCA-3′ and reverse 5′-TCCAGG GGTCTT ACTCCTTG-3′ were also used to amplify GAPDH as an internal control (509 bp).22

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PCR Conditions

Thermal cycling reaction was performed using a thermal cycler (Techne C-312) with the following program: 30 cycles of denaturation at 95°C for 1 minute, annealing at 60°C (MMP-9) and at 58°C (GAPDH) for 1 minute, and extension at 72°C for 1 minute then final extension at 72°C for 10 minutes.

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Detection of Amplified RT-PCR Products

Specific PCR products were subjected to agarose gel electrophoresis using 2% agarose stained with ethidium bromide and visualized by means of light UV Transilluminator (Model TUV-20, OWI. Scientific Inc. 800 242-5560) and photographed under fixed conditions (the distance, the light, and the zooms). Minus RT controls were permitted to rule out genomic contamination. Similarly, no products were detected when the RT-PCR step was carried out with no added RNA, indicating that all reagents were free of target sequence contamination. The intensity of each product band in pixels was obtained by analysis using image J software. Assessment of relative MMP-9 expression was performed using GAPDH expression as an internal control by calculating the MMP-9/GAPDH ratio.

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Gelatin Zymography

MMP-9 activity in serum and tissue samples was determined by gelatin zymography as described previously.23 Protein concentration in the tissue homogenates and serum samples was measured by the Bradford protein assay. A sample containing 20 mg of total protein was loaded for the gelatinolytic activity of MMP-9 onto sodium-dodecyl sulphate polyacrylamide gel under nonreducing conditions using 8% polyacrylamide gel containing 0.1 mg/mL gelatin. After electrophoresis, the gels were washed 3 times in 2.5% (wt/vol) and treated with Triton X-100 for 30 minutes at room temperature to remove the SDS and then incubated in a reaction buffer containing 50 mm Tris (pH 7.4), 5 mm CaCl2, and 150 mm NaCl for 24 hours at 37°C, followed by staining with 2.5% (wt/vol) Coomassie blue at 30% (vol/vol) methanol and 10% (vol/vol) acetic acid. Gelatinase activity was revealed by negative staining with Coomassie brilliant blue R-250 (0.1% Coomassie brilliant blue R-250, 45.5% methanol, 9% acetic acid). The MMP-9 activity was detected as clear, noncolored bands (digested gelatin) on a blue background of stained undigested gelatin. The intensity in pixels of the gelatinolytic bands was determined using Image J software. The activity of MMP-9 in both tissue samples and serum samples was determined as the percentage ratio between the intensity of the gelanolytic bands in the samples in relation to the standard MMP-9 (92 kDa) (NBP1-99195-Novus Biologicals).

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Statistical Analysis

The data were expressed as mean±SEM. The data were processed and analyzed using the Statistical Package of Social Science version 10.0 (SPSS, version 10.0). Results were compared by using the 2-tailed Student t test. The Pearson correlation coefficient was used to study the correlation between different variables in all groups. Differences were considered to be statistically significant at P value ≤0.05.

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RESULTS

The MMP-9 gene was expressed in normal lung tissue samples. Its expression was significantly increased in lung cancer tissues (P<0.0001). Also, MMP-9 tissue and serum activities were significantly increased in lung cancer samples (P<0.0001) when compared with control samples. As regards tumor type, MMP-9 gene expression was significantly elevated in NSCLC and SCLC tissue samples (P<0.0001). Also, MMP-9 tissue and serum activities were significantly higher in NSCLC and SCLC samples (P<0.0001) when compared with control samples (Table 2). MMP-9 tissue expression and MMP-9 tissue and serum activities were significantly higher in NSCLC samples than in SCLC samples (P=0.0167, 0.0454, and 0.004, respectively). The predominant histologic type of NSCLC in the cancer group was squamous cell carcinoma (10 patients, 50%), followed by adenocarcinoma (6 patients, 30%). The study showed a highly significant increase in MMP-9 tissue expression and activity as well as MMP-9 serum activity in both adenocarcinoma and large cell carcinoma when compared with squamous cell carcinoma samples. Pathologic staging revealed that among the 20 patients with NSCLC at the time of diagnosis, 5 (25%) had stage I/II (early) disease and 15 (75%) had stage III/IV (late) disease. Lymph nodes were present in 9 (45%) NSCLC and absent in 11 (55%) NSCLC patients. Distant metastasis was present in 10 (50%) NSCLC patients. Both MMP-9 expression and tissue activity were significantly higher in stage III-IV (late) NSCLC cases compared with early tumor stages (I-II) (P=0.0120 and 0.0271, respectively), whereas MMP-9 activity in the serum did not differ significantly between early and late stages (P>0.05). Also, MMP-9 expression and activity (either tissue or serum) were significantly higher in NSCLC patients with distant metastasis when compared with others without metastasis (Table 3). Neither age nor body mass index has an effect on MMP-9 expression and activity (Table 4; Figs. 1, 2).

TABLE 2

TABLE 2

TABLE 3

TABLE 3

TABLE 4

TABLE 4

FIGURE 1

FIGURE 1

FIGURE 2

FIGURE 2

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DISCUSSION

In the current study, the gene expression of MMP-9 in lung cancer tissue samples was analyzed in comparison with normal lung tissues from the same patients as an internal control. The results showed that MMP-9 was expressed in normal lung tissue, but its expression was significantly higher in lung cancer samples (P<0.001) as shown in Table 2. Our results were in agreement with Safranek et al24 who reported that MMPs are produced not only by tumor cells (MMP-7), but even by surrounding, stromal tissue (MMP-9), including fibroblasts and inflammatory cells. The role of MMPs is not only the proteolytic degradation of the ECM and basal membrane, but also includes the influence upon changes in the growth, apoptosis, and migration of healthy cells.25 Safranek et al24 demonstrated a statistically significantly increase in the expression of MMP-9 mRNA in NSCLC tumor tissue and in both the histologic subgroups (squamous carcinoma and adenocarcinoma) in comparison with the normal nontumor lung tissue of the same patients. This coincides with the results of our study as in Table 2 in which there were significant increases in the expression and the activity of MMP-9 in lung cancer (either NSCLC or SCLC) tissues than normal. Also, Table 3 showed that there was a significant increase of MMP-9 expression and activity in NSCLC sample than that of SCLC sample, a finding to be studied on a larger scale of samples.

According to the results of the current study, the predominant histologic type of NSCLC in the cancer group was squamous cell carcinoma (10 patients, 50%), followed by adenocarcinoma (6 patients, 30%) (Table 3). This result is in agreement with Safranek et al24 who stated that the expression of MMP-9 was significantly higher in the adenocarcinoma subgroup than in benign lung disease tissues. This result may be related to the hypothesis that MMP-9 is produced mainly by carcinoma cells and stromal cells.26

In our study, pathologic staging revealed that among the 20 patients with NSCLC at the time of diagnosis, 5 (25%) had stage I/II (early) disease and 15 (75%) had stage III/IV (late) disease. Lymph nodes were present in 9 (45%) NSCLC and absent in 11 (55%) NSCLC patients. Both MMP-9 expression and tissue activity were significantly higher in stage III-IV compared with earlier tumor stages (I-II) and in NSCLC patients with distant metastasis when compared with others without metastasis. In addition, MMP-9 expression was significantly higher in tumors associated with lymph node metastasis than in others with negative nodal metastasis (Table 3). This coincides with the results of Zheng et al27 who found that the expression of MMP-9 correlated with pathologic stage, lymph node metastasis, and survival. Also, they found that MMP-9 expression increased with tumor size and that its expression was significantly higher in NSCLC cases with metastasis compared with those without metastasis. In addition, MMP-9 expression was significantly higher in lymph node metastasis than primary lesions and these results are confirmed by the results of the current study in Table 3. This could be explained by the fact that MMP-9, as a member of MMPs, has been involved in the degradation of the ECM and growth of metastasis by its angiogenic properties. Also, MMPs may work during the processes of tumor cell migration28 and change the gene expressions and the activity of some growth factors and their receptors,24,29 leading to distant metastasis. In addition, Zheng et al27 found an inverse relationship between Kisspeptin-1 protein and MMP-9 protein expression. These data suggest that Kisspeptin-1 protein has an inhibitory effect on the metastatic process of NSCLC, whereas MMP-9 has a metastasis-promoting role. This Kisspeptin-1 gene has been reported to function as a metastasis-suppressor gene in a number of malignancies, including those of the thyroid gland, liver, esophagus, and urinary bladder.30 Roomi et al31 localized the major source of in vivo expression of MMP-9 in the stromal tissue adjacent to tumor cells, which implies a close cooperation between tumors and stromal cells. Also, they stated that a glycoprotein on the surface of cancer cells termed ECM metalloproteinase inducer (EMMPRIN) was found to stimulate these peritumor fibroblasts. Thus, EMMPRIN plays a role in invasion and metastasis of cancer cells by stimulating nearby fibroblasts to secrete increased amounts of interstitial collagenase, stromelysin-1, and gelatinase A.31 Ertan et al32 reported that angiogenesis is crucial in cancer development, and MMP-9 degrades type IV collagen associated with angiogenesis. Overexpression of MMP-9 will facilitate the invasive and metastatic spread. These finding and explanations are in accordance with the results of our study in which there is increased expression and enzyme activity of MMP-9 in late disease (stages III and IV) when compared with early disease (stages II and II), as well as in those with lymph node involvement compared with those with no lymph node involvement. Qian et al33 have linked the overexpression of MMP-9 with lung cancer propagation by the stimulatory effect of the overexpressed cyclophyllin A (CypA) protein. They hypothesized that a relationship exists between CypA and MMPs, in particular MMP2 and MMP-9, as CypA stimulates MMP expression by the ligand CD147.33,34

In conclusion, the results of our study revealed that MMP-9 may serve as a potential prognostic marker in lung cancer. It can be helpful in differentiating between different types of lung cancer. The expression and activity of MMP-9 were upregulated in NSCLC and were related to the pathologic type and clinical stage of NSCLC. Significantly higher expression and activity of MMP-9 in tumor tissue supports the important role of this metalloproteinase in the growth of lung cancer.

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LIMITATION AND FUTURE DIRECTIONS

The main limitation of this study is the small number of the included subjects. This small number was due to the restricted criteria in selection of our patient. To overcome this limitation, we recommend repeating that work on a larger scale in other areas and collecting the data for meta-analysis. Another limitation was that almost all of the collected cases were male individuals because in our communities, smoking is prevalent among men only. Also, this study was not funded and so we decided to use the cheapest methods for expression and MMP-9 activity. Recommended repetition of the same work with more expensive accurate techniques like enzyme-linked immunosorbent assay or western blotting as well as real-time quantitative PCR could be carried out to collect more accurate data for confirmation or not. Moreover, further studies should be conducted to evaluate the effect of treatment of lung cancer on MMP-9.

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Keywords:

MMP-9; expression; NSCLC; lung cancer

© 2014 by Lippincott Williams & Wilkins.