Identification of the genomic DNA hydroxymethylation profile during the melanoma progression

Abstract

Our in vitro melanoma progression model employs substrate adhesion blockade as a malignant transforming factor. The cell lines corresponding to steps of melanoma progression are: melan-a (non-tumorigenic melanocytes), 4C (pre-malignant melanocytes), 4C11- (non-metastatic melanoma cells) and 4C11+ (metastatic melanoma cells). The gene and protein levels of TET2 and 5hmC levels are found to be increased in the 4C11+ cell line in comparison to the others. This cell line was chosen for stable silencing of TET2 and the obtained clones were evaluated for proliferation, colony formation, dacarbazine resistance, death by anoikis resistance and collective migration capabilities. They displayed increased anoikis resistance and collective migration capacities.In advanced stages of human melanomas, the loss of TET2 and 5hmC are credited to be markers of increased aggressiveness and worse prognosis. Since the metastatic 4C11+ cell line showed the highest levels of Tet2 and 5hmC, we hypothesized that the tumor microenvironment could lead to the loss of 5hmC. Therefore, we established cell lines originated from in situ tumor and lung metastasis (T and M cell lines) in mice. These cell lines showed a decrease of gene and protein levels of TET2, decrease in genomic 5hmC levels and increased colony formation, collective migration and anoikis resistance capacities, in comparison with the 4C11+ cell line and in a consistent pattern amongst them. Using the RT2 Profiler PCR Array kit (QIAGEN), we were able to obtain an expression profile of pro-metastatic genes in a TET2 silenced clone in comparison with the parental 4C11+ cell line. Some of the genes were chosen for validation by quantitative qPCR and also investigated in the T and M cell lines. The genes Cdkn2aip, Hmmr and Ncor1, involved in cell cycle control, motility and invasion, respectively, showed distinct profiles among the melanoma model, TET2-silenced clones and T and M cell lines. These profiles emulate different conditions amongst the lineages, suggesting a role for the microenvironment in 5hmC distribution and regulation of aggressive characteristics such as migration and anoikis resistance. Since the cell lines 4C11+, 4C11+shTET2 #3 (the clone silenced for TET2), 4C11+ T1 (cell line established from in situ tumor) and 4C11+ M2 (cell line established from lung metastasis) present similar aspects concerning anoikis resistance, collective migration and colony formation capabilities, but different levels of genomic 5hmC, we chose these cells to investigate what happens during the melanoma progression in advanced stages and also considering the tumor microenvironment role.The next step of this study will be the identification of the methylation and hydroxymethylation profiles in the chosen cell lines, their relation with the already obtained methylome and transcriptome of our original melanoma progression model cells (melan-a, 4C, 4C11- and 4C11+), differentially expressed genes and complex pathways evaluation, and the validation of the findings in human melanoma databanks. (AU)