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, as well as reduced cell invasion.
In various cancer cells, high expression of N-cadherin is associated with activation of cell motility. Expression of N-cadherin induces cell migration of cancer such as breast, prostate, gastric, and melanoma [
21,
24‐
27]. Furthermore, the E-cadherin to N-cadherin switch is often found in aggressive cancers [
28]. The up-regulation of N-cadherin in aggressive carcinomas suggests that the level of N-cadherin expression is a critical step for cancer cell invasion.
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 [
29]. The MMPs have been present in the discussion on cancer for more than 40 years, being overexpressed in a large range of malignant tumours in response to oncogenic transformation, and activation of cytokines, several growth and angiogenic factors [
30].
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 MMP-9 have attracted attention in the recent years, especially with regard to cutaneous, eye, and oral melanomas [
30]. Expression of MMP-2 has frequently been associated with malignant progression and poor prognosis [
31]. Particularly, high levels of MMP-2 were observed in WM793 melanoma cell line from the primary VGP.
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 [
30]. High MMP-2 expression in primary lesion contributes to invasiveness of primary tumour cells, leading to metastases and poor survival outcomes [
32].
Similarly to other publications [
33], 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 biological fluids and tissues, in a variety of normal and neoplastic cells [
34]. Dimerization or multimerization is mediated by the carboxyterminal domains of MMP-9 and occurs intracellularly [
34]. 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) [
34].
The herein reported significant decrease in MMP-2 and MMP-9 protein levels and activities that was observed after knock-down of N-cadherin remains in agreement with the view on the importance of this adhesion molecule for 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 48-h treatment, while at shorter incubation time (24 h) 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. [
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, an 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]. Other authors suggest the role of the cytoplasmic domain of N-cadherin [
35,
36] and soluble N-cadherin domain in regulating cell migration [
37].
Current results demonstrate the important role of N-cadherin in melanoma cell invasion. In light of our former studies, where observed was a significant reduction in 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 a sound one.
Melanoma cells react differently to the MEK kinase inhibitors, as Ferguson et al. [
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. [
38] 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. For example, all studied cell lines exhibit B-RAF mutations: Lu1205 and WM793—V600E, WM115 and WM266-4 V600D.
The need for search of effective multitargeted therapies or broad spectrum approach to cancer treatment [
39] fully justifies the attempts to recognize the effect of collective inhibition of the activity of N-cadherin and some crucial signal transducers, such as protein kinases: PI3K, ERK, and mTOR. The conducted studies demonstrated that inhibition of N-cadherin or one of kinases produces less satisfactory effects than parallel inhibition of at least two of them, as shown here and formerly [
23]. These results many open up new avenues for developing low-dose anticancer therapies, which would allow to reduce the negative side effects of the high doses often administered in case of individual anticancer agent [
40].
Particularly promising are the results with regard to the decrease in melanoma cells invasiveness after N-cadherin gene silencing and the use of everolimus inhibitor of the mTOR pathway. everolimus is a low-toxicity drug that has been used as an immunosuppressant in organ transplant patients, in particular in cancer-related cases for several years [
41]. In recent years, use of everolimus and other inhibitors of the mTOR pathway for antitumor therapy has attracted some interest [
41,
42]. 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.