Tumoral migration and invasion RECK-mediated: melanoma as a model

Abstract

Melanomas have a highly chemoresistence and worse prognosis. The survival rate is 6 to 8 months, therefore, new therapeutic strategies are needed. The tumor cells accumulate such alterations in gene expression that contribute to uncontrolled proliferation, evasion of senescence and inhibition of cell death in multiple intracellular routes. The RECK gene is expressed in various normal human tissues, but its expression is repressed during cellular transformation. Studies have been shown an inverse correlation between the expression of RECK and MMPs, and their relationship to cell motility and invasiveness in tumor lines. All literature generated to date reports on the possibility of using this gene as a molecular marker for prognosis of pancreatic carcinoma-type tumors, breast, hepatocellular, lung, colorectal and even as genetic therapy in degenerative diseases. Our laboratory has explored the RECK gene in various tumors such as gliomas and uterine cervical carcinomas (Sasahara et al. 2002; Cardinal da Silva et al., 2006, Correa et al., 2006 and 2010). In addition, we rely on models that reorganize the tumor microenvironment in vitro, a valuable tool, which skin reconstructed with melanoma can simulated tumor invasion into the dermis. Our proposed study therefore aims to characterize the mechanism of action of RECK monolayer cultures and in artificial skin. This 3D model, also known as organotypic culture, already used in other models of different types of tumors, including breast, prostate and melanoma. Through this model, it is possible to estimate the therapeutic potential of some drugs, manipulation of genes in addition to conventional therapies. We aimed therefore to assess the effects of RECK in human metastatic melanoma lines (SK-Mel) when monolayers or artificial skin model to evaluate the parameters involved in cell migration and invasion. The methodology used for the functional study of these genes in human metastatic melanoma will by using shRNA lentiviral transfection and also overexpress RECK genes. Zymography assay will be used to verify the activity of MMPs 2 and 9, important in the process of invasive tumors. Transwell invasion assay in Boyden chamber will be used to verify the modulation of in vitro invasion by these genes. Evaluation of gene expression and protein targets of interest will be conducted by means of Real Time PCR and Western blotting, respectively. In sections of artificial skin, immunohistochemistry to indentify invaopodio components will be used to verify the role of this gene in the invasive process. Immunocytochemistry for components of the cytoskeleton and focal adhesion will be used to clarify the function of RECK in cell motility. Tumorigenicity in vivo tests verify the formation, growth, progression and ability to induce metastasis of cells that are overexpressed or inhibited RECK. Thus, given the importance of this gene and the lack of markers in the progression of melanoma, as well as the inefficiency of existing treatments, we propose to evaluate the expression of RECK as a possible marker in melanomas, and also its importance in tumor progression and invasion, can be used in future gene therapies for the treatment of melanomas. (AU)