The Involvement Of Protease Inhibitors OVOS2and TIMP-4in Pathogenesis Of Melanoma

Cutaneous malignant melanoma (CMM) is characterized by aggressive invasion, early metastasis, and resistance to chemotherapy or radiotherapy, accounting for the highest lethality of all dermatological cancers. The molecular mechanisms leading to melanoma are still poorly understood. Protease inhibitors are multifunctional proteins, which display an abnormal expression in several kinds of tumors. Recent data indicate that protease inhibitors have both inhibitory effect and stimulatory effect on tumorigenesis. Due to its dual regulation on tumor progression, protease inhibitors become one of the top areas of cancer research. In the current study, functions of two protease inhibitors OVOS2and TIMP-4in pathogenesis and progression of melanoma were investigated for the first time.Part1:Involvement of proteinase inhibitor Ovostatin2(OVOS2) in pathogenesis of cutaneous malignant melanoma1) Expression of Ovostatin2(OVOS2) in human cutaneous malignant melanoma tissues and cellsWe previously found the expression of OVOS2gene was apparently higher in acral-melanoma tissues of Chinese patients through an advanced whole-genome oligonucleotide microarray. To confirm results of the preliminary microarray analysis, and to see if expression of OVOS2is associated with the development of CMM, real-time PCR and Western blot analysis were used to detect mRNA and protein expression of OVOS2. The results revealed that OVOS2was overexpressed in mRNA and protein level in CMM compared with paired normal skin and in melanoma cells compared with primary melanocytes. Of four kinds of melanoma cells, A375presented the highest expression level. To investigate the relationship of OVOS2expression with the progression of CMM, immunohistochemical staining was further performed on114tissue samples taken from CMM and benign nevi, followed with clinicopathological significance analysis. We confirmed OVOS2was significantly up-regulated in CMM again, and found the expression of which positively correlated with the known prognostic variables including clinical stage, Clark level and Breslow depth, and significantly associated with the status of ulcer, positivity of Ki-67and expression of VEGF in primary melanoma. These results proved the over-expression of OVOS2in melanoma tissues and cells, and suggested a relationship of OVOS2expression and aggressive behaviours of melanoma.2) Inhibition of the expression of OVOS2by VshRNAWe chosed A375as target cells as they presented the highest expression level of OVOS2. Four shRNA fragment for OVOS2were synthetized and subcloned into pGMLV-SB1RNAi lentiviral vectors respectively(VshRNA-OVOS2). Lentivirus package was conducted in293T cells, followed by transfection with the four OVOS2-shRNA into A375cells. The optimum shRNA fragment against OVOS2was determined by Real-time PCR and Western blot and the corresponding stably transfected A375cells (A375/OVOS2-shRNA) were chosen for further study. A mock lentiviral vector was transfected into A375cells as a negative control (A375/NC-shRNA). Immunocytochemistry confirmed OVOS2expression was obviously inhibited in OVOS2-shRNA transfected A375cells compared with negative control-shRNA transfected A375cells and untreated A375cells. The stably transfectant clones were used for further study.3) Effect of OVOS2on biological behaviours of A375cell and mechanism research To evaluate the effect of inhibition of OVOS2on biological behaviours of A375cell and investige the possible involved mechanisms, biological behaviours of untreated A375cell, A375/NC-shRNA and A375/OVOS2-shRNA were evaluated and compared with each other. Cell proliferation ability was observed by cell number counting, MTT assay and Colony formation assay; Cell cycle and apoptosis were detected by Flow cytometer; Cell migration and invasive ability was tested by wound-healing and Transwell study. We also observe the subcutaneous transplanted tumor growth in nude mice to study the proliferation capability in vivo. Western blot was used to detect expression of p-FAK, p-AKT, p-ERK, MMP-2, E-cadherin, N-cadherin, β-catenin and cell cycle regulatory proteins to investigate the possible mechanisms of OVOS2involving in biological behaviours of melanoma cells. These in vivo and in vitro results manifested that inhibition of OVOS2had significantly suppressive effects on the growth of A375, with a G2/M phase arrest in cell cycle process, but affected cell apoptosis rate weakly, indicating high expression of OVOS2can promote cell cycle process and enhance proliferation of melanoma cells. The involved mechanisms may be associated with its over-activation of FAK/MAPK/ERK and FAK/PI3K/AKT signals. Furthermore, down-regulation of OVOS2can reduce motility and migration capability of melanoma cells significantly, accompanied with up-regulated epithelial phenotype E-cadherin and β-catenin and down-regulated mesenchymal phenotype N-cadherin expression, suggesting an important role of OVOS2in process of cell motality and migration, and the underlying mechanisms may related to the epithelial-mesenchymal transition of melanoma cells. Part2:Expression of tissue inhibitor of metalloproteinase-4(TIMP-4) in CMM tissue and cellsWestern blot revealed that TIMP-4was overexpressed in CMM tissues compared with paired normal skin and benign nevi and in melanoma cells compared with primary melanocytes. To investigate the relationship of OVOS2expression with the development and progression of CMM, immunohistochemical staining was further performed on94cases tissue samples taken from CMM and benign nevi, followed with clinicopathological significance analysis. We confirmed TIMP-4was significantly up-regulated in CMM again, and found the expression of which was positively correlated with the progression of CMM. Furthermore, it was associated with the expressions of VEGF and MMP-2. These results suggest TIMP-4protein may be closely associated with tumor initiation and progression, especially with the process of metastasis and angiogenesis of CMM.ConclusionsProteinase inhibitors OVOS2and TIMP-4were significantly overexpressed in melanoma tissues and cells, and the expression of which was positively correlated with the aggressive progression of CMM. The in vivo and in vitro results manifested that inhibition of OVOS2had significantly suppressive effects on the proliferation, motility and migration capability of A375, and can blocked cell cycle process, suggesting a promotive role of OVOS2in pathogenesis of melanoma. The involved mechanisms may be associated with the over-activation of FAK/MAPK/ERK and FAK/PI3K/AKT signals and the enhancement of epithelial-mesenchymal transition caused by over-expressed OVOS2. These results suggest proteinase inhibitors OVOS2and TIMP-4may play an important role in development and progression of CMM, as OVOS2mainly effects cell proliferation and migration, while overexpression of TIMP-4may be associated with the potential of metastasis and angiogenesis in CMM. The current study enriched the concept that protease inhibitors are multifunctional with key regulation on tumor progression, which represents new insights and areas for diagnostic and therapeutic research of melanoma, and provided valuable theoretical basis and experimental data for further investigation on the precise function and molecular mechanisms of these two proteinase inhibitors.