PAX3 knockdown in metastatic melanoma cell lines does not reduce MITF expression

Abstract

PAX3 and MITF are important transcriptional activators in the melanocyte lineage and PAX3 is thought to control MITF expression during normal melanocyte differentiation. However, it is not clear whether this is still true in melanoma and whether the effects of knockdown of PAX3 on the inhibition of melanoma growth or survival are by its regulation of MITF. By western blot and quantitative real-time reverse transcription-PCR, we investigated the relationship between PAX3 and MITF expression in 27 metastatic melanoma and one immortalized melanocyte cell lines. All lines were found to express both PAX3 and MITF proteins but levels varied by 15 fold and more than 100 fold, respectively. The expression of PAX3 protein was correlated with that of MITF (r=0.75; P<0.001) but the expression of PAX3 protein and MITF mRNA was not. Immunofluorescence microscopy showed that individual cells expressed widely differing relative amounts of PAX3 and MITF protein. By MTT cell proliferation and flow cytometry assays, both MITF and PAX3 proteins seemed to be functional, as knockdown with siRNA led to reduced proliferation and induction of apoptosis. However, knockdown of PAX3 with small interfering RNA did not decrease MITF expression and vice versa. In one cell line (NZM15), silencing of PAX3 induced terminal differentiation whereas silencing of MITF induced expression of FOXD3, a repressor of melanogenesis. The results suggest that the melanoma lines used in this study show considerable phenotypic variation of expression of these two transcriptional activators and reflect a deregulation of the developmental process operating in the genesis of the melanocyte lineage, and that they probably function independently to enhance the survival of melanoma cells.