SummaryTranscription factors initiate programs of gene expression and are catalysts in downstream molecular cascades that modulate a variety of cellular processes. Pax3 is a transcription factor that is important in the melanocyte and influences melanocytic proliferation, resistance to apoptosis, migration, lineage specificity and differentiation. In this review, we focus on Pax3 and the molecular pathways that Pax3 is a part of during melanogenesis and in the melanocyte stem cell. These roles of Pax3 are emphasized during the development of diseases and syndromes resulting from either too much or too little Pax3 function. Due to its key task in melanocyte stem cells and tumors, the Pax3 pathway may provide an ideal target for either stem cell or cancer therapies.

Melanoma is a highly aggressive disease that is difficult to treat due to rapid tumor growth, apoptotic resistance, and high metastatic potential. The MET tyrosine kinase receptor promotes many of these cellular processes, and while MET is often overexpressed in melanoma, the mechanism driving this overexpression is unknown. Since the MET gene is rarely mutated or amplified in melanoma, MET overexpression may be driven by to increased activation through promoter elements. In this report, we find that transcription factors PAX3 and ETS1 directly interact to synergistically activate MET expression. Inhibition of PAX3 and ETS1 expression in melanoma cells leads to a significant reduction of MET receptor levels. The 300 bp 5′ proximal MET promoter contains a PAX3 response element and two ETS1 consensus motifs. While ETS1 can moderately activate both of these sites without cofactors, robust MET promoter activation of the first site is PAX-dependent and requires the presence of PAX3, while the second site is PAX-independent. The induction of MET by ETS1 via this second site is enhanced by HGF-dependent ETS1 activation, thereby MET indirectly promotes its own expression. We further find that expression of a dominant negative ETS1 reduces the ability of melanoma cells to grow both in culture and in vivo. Thus, we discover a pathway where ETS1 advances melanoma through the expression of MET via PAX-dependent and independent mechanisms.

Glycogen Synthase Kinase-3 (GSK-3) is a serine/threonine kinase involved in a diverse range of cellular processes. GSK-3 exists in two isoforms, GSK-3α and GSK-3β, which possess some functional redundancy but also play distinct roles depending on developmental and cellular context. In this report we found that GSK-3 actively promoted cell growth and survival in melanoma cells, and blocking this activity with small molecule inhibitor SB216763 or gene-specific siRNA decreased proliferation, increased apoptosis and altered cellular morphology. These alterations coincided with loss of PAX3, a transcription factor implicated in proliferation, survival and migration of developing melanoblasts. We further found that PAX3 directly interacted with and was phosphorylated in vitro on a number of residues by GSK-3β. In melanoma cells, direct inhibition of PAX3 lead to cellular changes that paralleled the response to GSK-3 inhibition. Maintenance of PAX3 expression protected melanoma cells from the anti-tumor effects of SB216763. These data support a model wherein GSK-3 regulates proliferation and morphology of melanoma through phosphorylation and increased levels of PAX3.

Background: CXCR4 promotes melanoma metastasis. Results: FOXD3 and PAX3 drive CXCR4 expression. Blocking these factors inhibits cell migration, but this is rescued by ectopic CXCR4 expression. Conclusion: PAX3-and FOXD3-mediated melanoma cell migration is dependent on promoting the expression of CXCR4. Significance: This study identifies a new pathway and targets for therapeutic strategies to treat invasive melanoma.

Several key transcription factors regulate cell growth, survival, and differentiation during neural crest and melanoblast development in the embryo, and these same pathways may be reactivated in tumors arising from the progenitors of these cells. The transcription factors PAX3 and FOXD3 have essential roles in melanoblasts and melanoma. In this study, we define a regulatory pathway where FOXD3 promotes the expression of PAX3. Both factors are expressed in melanoma cells and there is a positive correlation between the transcript levels of PAX3 and FOXD3. The PAX3 gene contains two FOX binding motifs within highly conserved enhancer regulatory elements that are essential for neural crest development. FOXD3 binds to both of these motifs in vitro but only one of these sites is preferentially utilized in melanoma cells. Overexpression of FOXD3 upregulates PAX3 levels while inhibition of FOXD3 function does not alter PAX3 protein levels, supporting that FOXD3 is sufficient but not necessary to drive PAX3 expression in melanoma cells. Here, we identify a molecular pathway where FOXD3 upregulates PAX3 expression and therefore contributes to melanoma progression.