Matrix metalloproteinase-19 promotes metastatic behavior in vitro and is associated with increased mortality in non-small cell lung cancer

Abstract

Rationale: Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Matrix metalloproteinases (MMPs) have been implicated in the development and progression of lung cancer, but their role in the molecular pathogenesis of lung cancer remains unclear. We have found that MMP19, a relatively novel member of the MMP family, is overexpressed in lung tumors when compared with control subjects.

Objectives: To test the hypothesis that MMP19 plays a significant role in the development and progression of non-small cell lung cancer (NSCLC).

Methods: We have analyzed lung cancer gene expression data, immunostained lung tumors for MMP19, and performed in vitro assays to test the effects of MMP19 in NSCLC cells.

Measurements and main results: We found that MMP19 gene and protein expression is increased in lung cancer tumors compared with adjacent and histologically normal lung tissues. In three independent datasets, increased MMP19 gene expression conferred a poorer prognosis in NSCLC. In vitro, we found that overexpression of MMP19 promotes epithelial-mesenchymal transition, migration, and invasiveness in multiple NSCLC cell lines. Overexpression of MMP19 with a mutation at the catalytic site did not impair epithelial-mesenchymal transition or expression of prometastasis genes. We also found that miR-30 isoforms, a microRNA family predicted to target MMP19, is markedly down-regulated in human lung cancer and regulates MMP19 expression.

Conclusions: Taken together, these findings suggest that MMP19 is associated with the development and progression of NSCLC and may be a potential biomarker of disease severity and outcome.

Keywords: MMP19; epithelial–mesenchymal transition; metastasis; miR-30; non–small cell lung cancer.

Figure 1.

Increased expression of matrix metalloproteinase (MMP) 19 in non–small cell lung cancer (NSCLC). (A) Quantitative reverse-transcriptase polymerase chain reaction was performed for MMP19 in NSCLC (adenocarcinoma and squamous cell carcinoma) compared with corresponding nonneoplastic lung tissues. The differences in mRNA levels of MMP19 were compared by the Wilcoxon two-sample test. Data represent the mean ± SD (*P < 0.05). (B) Immunostaining was performed for MMP19 on NSCLC tissue sections as described in the Methods section (scale bar = 100 μm).

Figure 2.

Increased matrix metalloproteinase (MMP) 19 gene expression in non–small cell lung cancer portends a worse prognosis. Overall survival analysis by Kaplan-Meier curves of MMP19 microarray gene expression levels in lung adenocarcinoma tissue obtained from 408 subjects from the Director’s Challenge Consortium for the Molecular Classification of Lung Adenocarcinoma (A) and 46 non–small cell lung cancer subjects obtained from the University of Pittsburgh (B). Adjusted overall survival differences in the (C) Challenge cohort (adjusted for age, sex, adjuvant chemotherapy, and radiotherapy) and (D) Pitt cohort (adjusted for age, sex, adjuvant chemotherapy, and radiotherapy as well as intraoperative brachytherapy). Blue line = patients with MMP19 microarray expression levels above the median value. Green line = patients with MMP19 microarray expression levels below the median value. Dotted black lines = the median survival in months for both groups.

Figure 3.

Matrix metalloproteinase (MMP) 19 expression promotes colony formation and motility in vitro. (A) Immunoblotting for MMP19 protein was performed on lysates from A549 cells stably expressing MMP19 compared with vector control. (B) Quantification of band intensity was performed with National Institutes of Health ImageJ as described in the Methods section (*P < 0.05). (C) Representative image of colony formation of A549 cells transfected with MMP19 full length cDNA and vector control. Colonies were allowed to form on a hard surface over 10 days. (D) Promotion of colony formation and anchorage-independent growth of A549 cells transfected with MMP19 compared with control subjects. Colonies were allowed to form on soft agar over 21 days and quantified as described in the Methods section (*P < 0.05). (E) Following transfection of A549 cells with MMP19, cell migration and invasion into matrigel was quantified as described in the Methods section (*P < 0.05). Data represent mean of five experiments with SEM. Five high-power fields were counted for each experiment.

Figure 4.

Matrix metalloproteinase (MMP) 19 induces epithelial–mesenchymal transition (EMT) in vitro. (A) Quantitative reverse-transcriptase polymerase chain reaction was performed for markers of EMT in A549, H1299, and H522 cells 24 hours after transient transfection with MMP19 siRNA and full length MMP19 cDNA compared with control subjects (ACTA2 = α-smooth muscle actin; CDH2 = N-cadherin; FN1 = fibronectin 1; VIM = vimentin). Data were analyzed by t test, *P < 0.05, MMP19 silencing or overexpression compared with the scrambled control, or MMP19 overexpression compared with the mock control. (B) Immunoblots for the epithelial marker (CDH1 = E-cadherin) and mesenchymal markers (ACTA2, VIM, FN1, and CDH2) in A549 cell lysates from MMP19 cDNA and siRNA transiently transfected A549 cells and mock control. (C) Immunofluorescent staining of A549 cells for ACTA2 (green, top) and VIM (green, middle), and CDH1 (red). Cells were counterstained with DAPI (blue; original magnification ×400). (D) Microarray heatmap of EMT-relevant gene expression in stable MMP19-transfected A549 cells and mock control as described in the Methods section. (E) Quantitative reverse-transcriptase polymerase chain reaction was performed for markers of EMT following silencing or overexpression of MMP19 in the presence of the MMP inhibitor GM6001. GM6001 did not block differential expression of these EMT markers (*P < 0.05 compared with mock transfection control).

Figure 5.

Effect of matrix metalloproteinase (MMP) 19 catalytic domain on A549 in vitro phenotypes: epithelial–mesenchymal transition, migration, and invasion. (A) Schematic representation of the E213A MMP19 point mutation in the catalytic domain. (B) Pitt cohort of MMP19 expression in protein in A549 cells transfected with wild-type (WT) and the E213A mutated MMP19 construct by immunoblotting. (C) Quantitative reverse-transcriptase polymerase chain reaction was performed for markers of epithelial–mesenchymal transition. Data represent the mean ± SEM (*P < 0.05 compared with mock transfection control). (D) Immunofluorescent staining of mesenchymal markers (anti-ACTA2 and anti-VIM, green) and epithelial marker (anti-CDH1, red). Cells were counterstained with DAPI (blue). (E) Induction of A549 cell migration and invasion with expression of MMP19 WT and MMP19E213A in matrigel. Data represent mean of five experiments with SEM. Five high-power fields were counted for each experiment (*P < 0.05 compared with mock transfection control). (F) Scratch assay was performed on A549 following transfection of the mock control, MMP19 WT, and the E213A MMP mutant. (G) Quantification of the gap coverage as described in the Methods section (*P < 0.05, compared with time = 0 control). ACTA2 = α-smooth muscle actin; CDH1 = E-cadherin; VIM = vimentin.

Figure 6.

Effect of matrix metalloproteinase (MMP) 19 overexpression on gene signatures associated with metastasis and prognosis gene in non–small cell lung cancer. Agilent microarray heatmaps of metastasis (A) and prognosis (B) gene expression signatures in A549 cells following stable transfection with MMP19 and vector control (P < 0.05).

Figure 7.

miR-30 regulates matrix metalloproteinase (MMP) 19 expression in A549 cells. (A) Expression of miR-30a, -30d, and -30e were down-regulated in human non–small cell lung cancer compared with corresponding normal control subjects as detected by Taqman quantitative reverse-transcriptase polymerase chain reaction (*P < 0.05; n = 15). (B) MMP19 gene expression was determined by reverse-transcriptase polymerase chain reaction in A549 cells 24 hours following transfection with antagomiR-30e oligonucleotides or controls. Data represent the mean ± SEM (*P < 0.05, n = 4). (C) MMP19 protein expression was determined by immunoblotting lysates of A549 cells 48 hours following transfection with antagomiR-30e oligonucleotides or control. (D) Quantification of MMP19 band intensity in C in ImageJ. Data represent mean ± SEM (*P < 0.05, n = 3).