Discussion
It has been previously demonstrated that N-cadherin had significant effect on cell signalling, cell cycle regulation and, in consequence, proliferation of melanoma cells, as the use of siRNA against N-cadherin led to significant reduction of number of such treated cancer cells [1].
The current study confirmed that melanoma cells transfected with N-cadherin specific siRNA successfully and transiently decreased its expression at mRNA and protein levels. This resulted in inhibition of metalloproteinase 2 and 9 activities and reduced cell invasion.
In various cancer cells abnormal expression of N-cadherin correlates with induction of cell motility. Expression of N-cadherin induces cell migration in e.g. breast cancer, melanoma, prostate cancer, and gastric cancer [21, 24, 25]. Furthermore, the E- to N-cadherin switch is often observed in aggressive cancers [26]. The up-regulation of N-cadherin in aggressive carcinomas suggests that the level of its expression is a critical parameter for cancer cell invasion.
Melanoma cell invasion.
The results of the study on the effects of N-cadherin knock down on cell invasion confirm the role of this adhesion molecule in the process of invasion of melanoma cells. In all tested melanoma cell lines a statistically significant decrease number of invading cells in the range between 20-25% was observed. Furthermore, impact of protein kinase inhibitors: LY294002, U126, Everolimus, and combination of siRNA for N-cadherin and protein kinase inhibitors on the process of melanoma cell invasion reported here suggests involvement of all three kinases in N-cadherin initiated signalling. Simultaneous administration of any of protein kinase inhibitors and siRNA for N-cadherin resulted in a decrease of approximately 30%, whereas with the use of a combination of siRNA for N-cadherin and ERK MAPK pathway inhibitors-U126 diminished cells invasion up to 38%.
MMPs in tumour progression
MMPs play an important role in many biological and pathological processes. Uncontrolled, activity of metalloproteinases may well lead to development of many diseases such as arthritis, atherosclerosis, aneurysms, nephritis, tissue ulcers or fibrosis and cancer [27]. The MMPs have been present in the discussion on cancer for more than 40 years, being overexpressed in a wide range of malignant tumours in response to oncogenic cellular transformation, cytokines and several growth and angiogenic factors [28].
Initial observations on the role of MMPs in the cancer biology have suggested that the ability of tumour cells to metastasize correlates with increased levels of metalloproteinase activity. Elevated levels of gelatinases, MMP-2 and MMP-9 are often observed in malignant cancers. Among human melanoma cells, MMP-2 and -9 have attracted attention in the recent years, especially with regard to cutaneous, eye and oral melanomas [28]. Expression of MMP-2 has frequently been associated with malignant progression and poor prognosis [29]. Particularly high levels of MMP-2 were observed in WM793 melanoma cell line from the primary vertical growth phase (VGF).
Results of studies using tissue microarray, immunohistochemistry of melanoma biopsies of primary and metastatic lesions, as well as nevi, confirmed that MMP-2 is predictive of primary and metastatic stages [28]. High MMP-2 expression in the primary lesion contributes to the invasiveness of primary tumour cells, leading to metastases and poor survival outcomes [30].
Similarly to other publications [31], this study showed a high level of the MMP-2 activity in all tested melanoma cell lines.
The second gelatinase MMP-9 proved to be much less active than MMP-2, except for the Lu1205 from metastatic cell line, where activity of MMP-9 was at a high level. Here, monomeric and dimeric forms of MMP-9 were detected in all tested lines except for Lu1205. MMP-9 also exists as a monomeric and homodimeric molecule, in both its latent and active forms. Both monomeric and dimeric forms of MMP-9 have been identified in a variety of cells (normal and tumour cells alike) and in biological fluids and tissues, indicating that both forms are physiologically relevant. Dimerization or multimerization is mediated by the carboxyterminal domains of MMP-9, and occurs intracellularly [32]. The functional biological role of the MMP-9 dimer has not yet been elucidated, however, dimerization significantly decreases the activation rate of pro-MMP-9 by stromelysin (MMP-3) [32].
The herein reported here significant decrease of MMP-2 and MMP-9 activities that were observed after knockdown of N-cadherin remains in agreement with the view on the important role of this adhesion molecule in activation of matrix metalloproteinases, and in effect stimulation of invasion and metastasis. The largest decreases of MMP-2 activity were observed in the Lu1205 and line WM793 (by about 47% and 40% respectively). Concurrently, MMP-9 activity in the cell lines dropped by about 62% and 52% respectively.
Decreases of MMP-2 and MMP-9 activities were also observed upon using a combination of siRNA for N-cadherin and protein kinases inhibitors: LY294002 (PI3K), U126 (ERK1/2) and Everolimus (mTOR). The largest decrease in activity of metalloproteinase, MMP-2 in particular, was observed upon using a combination of siRNA for N-cadherin and PI3K inhibitor LY294002 after 48h treatment, while at shorter incubation time (24h) application of a combination of siRNA for N-cadherin and inhibitor U0126 produced best results. Using the combination of siRNA for N-cadherin and inhibitor of mTOR – Everolimus gave similar results regardless of the incubation time.
Hazan et al. 2004 [21] suggested that N-cadherin functionally interacts with the FGF receptor, causing sustained downstream signalling by PI3K, and through MAPK-ERK promotes cell survival, migration and invasion. Stabilization of FGF-1 receptor by N-cadherin followed by MAPK-ERK activation, may result in increased transcription of the extracellular matrix-degrading enzyme MMP-9 and hence, increased cellular invasiveness. The FGF-1 may interact with the fourth extracellular domain (EC4) of N-cadherin based on the fact that transfer of EC4 domain from N-cadherin onto E-cadherin reconstituted the invasive function of N-cadherin [21].
Current results show important role of N-cadherin in melanoma cell invasion. In light of our former studies, where we observed significant reduction of melanoma cell proliferation in effect of N-cadherin silencing the hypothesis that MMP-9 expression and cellular invasion are governed by at least two, but possibly more, distinct intracellular signalling pathways, when stimulated by N-cadherin-FGFR signalling [21], seems to be sound.
Melanoma cells react differently to the MEK kinase inhibitors, as Ferguson et al. 2013 [22] observed a slight increase in the activity of MMP-2 in the use of ERK1/2 inhibitor – U126. Preclinical data presented by Catalanotti et al. 2013 [33]
suggest that patients with B-RAF mutant melanomas and PI3K/AKT pathway activation are less sensitive to MEK inhibition. 50% of melanoma cases showed presence of B-RAF mutation. E.g. All studied cell lines exhibit B-RAF mutations: Lu1205 and WM793 – V600E, WM115 and WM266-4 V600D.
Particularly promising are the results with regard to the decrease of melanoma cells invasiveness after N-cadherin gene silencing and the use of Everolimus inhibitor of the mTOR pathway. Everolimus is a low toxicity drug in use for several years as an immunosuppressant in organ transplant patients, in particular in cancer-related cases [34]. In recent years, use of Everolimus and other inhibitors of the mTOR pathway for antitumor therapy has attracted some interest [35]. The fact that their inhibitory effect may be significantly enhanced by simultaneous use of siRNA for N-cadherin may be worth considering as a potentially new approach to effective treatment of melanoma.