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Although anoikis resistance has been considered a hallmark of the malignant phenotype, the causal relation between neoplastic transformation and anchorage-independent growth remains undefined. To study this correlation, we developed an experimental model of murine melanocyte malignant transformation, where different melanoma cell lines (4C3, 4C11, Tm1, Tm5) were established after submitting a non-tumorigenic melanocyte lineage (melan-a) to sequential substrate anchorage impediment cycles. Melan-a sublines, submitted to 2, 3 and 4 deadhesion cycles (2C, 3C and 4C), were also established, showing progressive anoikis-resistance, and representing distinct phases of tumor progression. Previous gene expression analysis comparing adherent melan-a, melan-a maintained in suspension for 24h, adherent Tm1 and Tm5 cells, showed an up-regulation of Timp1 in melanoma cell lines. Although initially described as a MMP inhibitor, this protein has been recently associated with anoikis resistance in human breast tumor cells. Timp1 protein increases along melanocyte tranformation and its possible association with anoikis resistance prompted us to investigate whether Timp1 expression is regulated by DNA methylation and whether it is associated with the acquisition of the anoikis-resistant phenotype. While melan-a cells express low levels of Timp1, all cell lines derived from melan-a express high levels of this protein. After 5AZAdC treatment, increased levels of Timp1 mRNA were observed in melan-a cells, suggesting that in these cells Timp1 expression is regulated by promoter hypermethylation. Ms-SNuPE analysis has shown an increased demethylation in Timp1 promoter and first exon along melan-a carcinogenesis. Overexpression of Timp1 in melan-a cells resulted in increased survival in anchorage-independent conditions, but Timp1 overexpression by itself was unable to transform non-tumorigenic melanocytes into tumor-forming cell lines. In addition, latency time for tumor appearance was reduced in Timp1-transfected Tm5 melanoma cells, indicating that this molecule may be associated with tumor aggressiveness. In conclusion, our results show a progressive increment in Timp1 expression along the malignant transformation process, resulting from progressive promoter demethylation, which is causally associated with an increase in anoikis-resistance but not with the acquisition, by itself, of a fully transformed malignant phenotype.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA