Targeted therapy represents a tremendous leap forward in cancer treatment. The mutation of epidermal growth factor receptor is a highlight of lung cancer diagnosis and therapy . Therefore, elucidating the mechanisms of novel molecular targets may contribute to improving lung cancer treatment. Previous reports have demonstrated that CypA is overexpressed in cancers such as lung, pancreatic, colorectal, and breast cancer. CypA overexpression was considered to play important roles in cancer pathogenesis and act like a "molecular switch" , since it is known to regulate signaling via prolyl isomerization. Nonetheless, the mechanism underlying the effect of CypA on the biological behavior of lung cancer cells has not been fully elucidated.
In this study, we showed that the knockdown of CypA in human NSCLC cells inhibited cell proliferation, increased sensitivity to density-dependent inhibition, and down-regulated anchorage-independent cell growth. This is in agreement with a previous report showing that CypA is a key promoter of tumor cell growth and tumorigenesis . Notably, CypA knockdown dramatically inhibited cell migration and invasion by NSCLC cells, suggesting that CypA has a significant impact on the metastasis of NSCLC cells. Furthermore, we investigated the mechanism of action of CypA in NSCLC cells, and detected enhanced MMP9 activity. To our knowledge, this study for the first time correlates CypA with metastasis and MMP9 in NSCLC cells. Our data indicate that CypA plays a crucial role in the proliferation, motility, and invasionof NSCLC cells.
The expression of CypA in lung cancer tissue was approximately seven-fold higher than that in adjacent nonmalignant tissue . Herein, we showed that compared to MRC5 cells, CypA expression was higher in several lung cancer cell lines, including five NSCLC (95C, A549, H460, A2, and H1299) and one SCLC (H446) cell lines. Interestingly, proliferation and wound healing assays indicated that 95C had a greater proliferative and migratory capacity than A549, suggesting that elevated CypA expression in NSCLC cells might influence cell growth and metastasis.
It has been thought that CypA accelerates cell growth by stimulating cell proliferation, tumorigenesis, and metabolism, and by inhibiting apoptosis . Our proliferation and tumorigenesis data are consistent with those of previous reports, but the mechanism by which CypA acts on cell growth remains unclear. We also checked whether cell apoptosis was regulated by CypA and cell apoptosis was not affected by the suppression of CypA expression (data not shown). Recent studies have pointed out that PPI activity is required for CypA-induced cell proliferation, and that several growth-related signaling molecules, including ERK1/2, Jak2, p38, and Stat5 [10, 16, 20], are stimulated by CypA in cancer cells. In present research, some important regulatory molecules of MAP kinase and JAK2 signaling pathways were determined. Our data indicate that CypA enhanced cell growth by up-regulating MAPK kinase pathway (ERK1/2 and p38) in NSCLC cells. But JAK2/STAT5 was not involved in the CypA regulating pathway.
Metastasis is the primary cause of morbidity and mortality in cancer patients. Besides its role in cell growth, the involvement of CypA in metastasis has also been investigated. Stable CypA RNA-interfered breast cancer and osteosarcoma cells showed reduced migratory capacity [20, 29]. CypA is also involved in the attraction and migration of monocytes or vascular smooth muscle cells in rheumatoid arthritis and cardiovascular disease by irritating adhesion molecules and regulating MMP9 secretion [10, 30]. These reports inspired us to explore the effect of - CypA on metastasis in NSCLC cells. KD cells exhibited a deficiency in migratory capacity compared with WT and MOCK cells. Invasion is another characteristic of metastasis. To some extent, invasive ability is even more important because the first step in metastasis involves passing through a basement membrane (BM), which is the major physical obstacle to cancer cell metastasis . Our invasion assay suggested that KD cells could not pass through the Matrigel Matrix, which is similar to the BM; however, WT and MOCK cells could. Nevertheless, the mechanism of CypA in metastasis remains a mystery. It is thought that CypA might disrupt the F-actin structure in osteosarcoma cells or the regulation of JAK2 signaling in breast cancer cells and metastatic melanoma cell lines [19, 22]. Thus far, no studies have focused on NSCLC cells. In our study, the phosphorylation level of JAK2 was not changed when CypA expression was inhibited.
We hypothesized that a relationship exists between CypA and MMPs, in particular MMP2 and MMP9, as CypA stimulates MMP expression via the ligand CD147 [27, 32, 33] . The suppression of CD147 in breast cancer cells inhibited MMP2 and MMP9 production and cell invasion in vitro, while the invasive properties conferred on inflammatory cells by CypA are a result of MMP9 stimulation . We suppressed CypA in NSCLC cells without changing CD147 expression (data not shown). Gelatin zymography showed that MMP9 was down-regulated in CypA supressed cells compared to control cells, while the expression of MMP2 did not change significantly. This is consistent with a previous report showing that CypA increased MMP9, but not MMP2, expression via CD147 in rheumatoid arthritis . Therefore, CypA may promote metastasis by upregulating MMP9 activity in NSCLC cells.
The initial step of tumor cell invasion is characterized by BM breakdown, a process dependent on type IV collagen-degrading enzymes, mainly MMP2 and MMP9 . MMPs are a family of proteases that are required for the invasion of tumor cells into surrounding connective tissues, intravasation and extravasation from blood vessels, and metastasis to distant organs . MMP2 and MMP9, otherwise known as gelatinases, are strongly upregulated in cancers of the lung, colon, breast, skin, and prostate, which are correlated with enhanced tumor invasiveness and metastasis . MMP9 digests decorin; elastin; fibrillin; laminin; types IV, V, XI, and XVI collagen; and gelatin. Among these, laminin serves as an important component of the BM. MMP9 can also activate growth factors, such as proTGF-β and proTNF-α . The inhibition of MMP9 reduced the number of colonies formed in the lung of mice . As demonstrated in this study, CypA upregulated the activity of MMP9 in NSCLC cells, which could help elucidate the mechanism of CypA effect on lung cancer metastasis. Furthermore, MMP9 is widely accepted as a prognostic marker in NSCLC; its expression level is correlated with survival . Whether CypA could be used as a prognostic marker in NSCLC was evaluated by Howard using tissue microarray immunohistochemistry; however, the results were negative . Further research into CypA may change this using larger samples or alternate methods.