Functional role of MIA in melanocytes and early development of melanoma

Abstract

The protein MIA (melanoma inhibitory activity) is highly expressed in malignant melanomas but not in melanocytes. Furthermore, expression of MIA correlates with tumor progression in vivo. Here, MIA-dependent changes of gene expression after long-term inhibition of MIA expression in the human melanoma cell line HMB2 were investigated. Primarily, we observed characteristic changes in cell morphology, and also found re-established cell-cell contacts in MIA-deficient cell clones grown in monolayer culture. Real-time reverse transcription-polymerase chain reaction (RT-PCR) showed a downregulation of N-cadherin expression and a reinduction of E-cadherin expression in the MIA-deficient cell clones. Further, both cancer cDNA array and protein arrays verified a marked downregulation of several other melanoma-associated genes (e.g. membrane-type 1 matrix metalloproteinase tissue-type plasminogen activator integrin b3, secreted protein acidic and rich in cysteins and fibronectin) in the MIA-deficient melanoma cells, confirmed by real-time RT-PCR and Western blotting. As all these molecules are associated with migration, the effect of MIA on migration of human primary melanocytes was analysed. In the presence of MIA, we observed enhanced migratory ability of melanocytic cells, induction of melanoma-associated genes as well as inhibition of apoptosis due to anoikis. These results suggest that expression of MIA promotes melanoma progression by inducing further melanoma-associated genes.

Cell-type-specific regulation of distinct sets of gene targets by Pax3 and Pax3/FKHR

“…Altered Pax3 activity is associated with malignant transformation in muscle (Anderson et al, 1999;Xia et al, 2002) and in skin (Vachtenheim and Novotna, 1999;Blake and Ziman, 2003;Poser and Bosserhoff, 2004). For example, one reciprocal translocation (t(2;13)(q35;q14)) characteristic of human alveolar rhabdomyosarcomas (ARMS) results in fusion of the transactivation domain of the Forkhead-family protein, Forkhead-Box O1a (FOXO1a; formerly known as ForkHead (FKHR); Kaestner et al, 2000), to the Nterminus of Pax3 (Shapiro et al, 1993).…”

Integrin β3 expression is regulated by let-7a miRNA in malignant melanoma

“…By stable transfection of HMB2 melanoma cells with an antisense-melanoma inhibitory activity (MIA) cDNA expression plasmid, our group recently generated MIA-deficient melanoma cell clones (Poser et al, 2004;Tatzel et al, 2005). MIA-deficient melanoma cell clones HMB2-MIA5 and HMB2-MIA8 showed a melanocytic growth pattern and gene expression patterns similar to melanocytes as detected by cDNA and protein array experiments.…”

Specific c-Jun target genes in malignant melanoma

“…4). To further ensure that the predicted target genes are regulated by the transcription factor c-Jun in malignant melanoma, we transfected a melanocyte-resembling cell clone lacking cJun expression (HMB2-5 24 ) with a c-Jun expression construct (HA-JunMut1 11 ) and confirmed an increase in c-Jun accumulation by Western blotting with a transfection efficiency of about 80% ( Fig. 5 (a)).…”

“…HMB2-5 is a cell clone in our laboratory resembling melanocytes. 24 Once a week this cell line is treated with G418 (2 mg/ml) to ensure clone selection.…”