Brn-2 expression controls melanoma proliferation and is directly regulated by beta-catenin.
Journal: 2004/May - Molecular and Cellular Biology
ISSN: 0270-7306
PUBMED: 15024079
Abstract:
Constitutive activation of the Wnt/beta-catenin signaling pathway is a notable feature of a large minority of cases of malignant melanoma, an aggressive and increasingly common cancer. The identification of target genes downstream from this pathway is therefore crucial to our understanding of the disease. The POU domain transcription factor Brn-2 has been implicated in control of proliferation and melanoma survival, and its expression is strongly upregulated in melanoma. We show here that in vivo Brn-2 is expressed in melanocytes but not in embryonic day 11.5 melanoblasts and that its expression is directly controlled by the Wnt/beta-catenin signaling pathway in melanoma cell lines and in transgenic mice. Moreover, silent interfering RNA-mediated inhibition of Brn-2 expression in melanoma cells overexpressing beta-catenin results in significantly decreased proliferation. These results, together with the observation that BRAF signaling also induces Brn-2 expression, reveal that Brn-2 is a focus for the convergence of two key melanoma-associated signaling pathways that are linked to cell proliferation.
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Mol Cell Biol 24(7): 2915-2922

Brn-2 Expression Controls Melanoma Proliferation and Is Directly Regulated by β-Catenin

Signaling and Development Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom, UMR146 CNRS, Institut Curie, Centre Universitaire, 91405 Orsay Cedex, France2
Corresponding author. Mailing address: Signaling and Development Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom. Phone: (44) 1883 722306. Fax: (44) 1883 714375. E-mail: ku.ca.ircm@gnidog.c.
Present address: The Institute of Cancer Research, London, SW3 6JB, United Kingdom.
Received 2003 Dec 4; Accepted 2004 Jan 2.

Abstract

Constitutive activation of the Wnt/β-catenin signaling pathway is a notable feature of a large minority of cases of malignant melanoma, an aggressive and increasingly common cancer. The identification of target genes downstream from this pathway is therefore crucial to our understanding of the disease. The POU domain transcription factor Brn-2 has been implicated in control of proliferation and melanoma survival, and its expression is strongly upregulated in melanoma. We show here that in vivo Brn-2 is expressed in melanocytes but not in embryonic day 11.5 melanoblasts and that its expression is directly controlled by the Wnt/β-catenin signaling pathway in melanoma cell lines and in transgenic mice. Moreover, silent interfering RNA-mediated inhibition of Brn-2 expression in melanoma cells overexpressing β-catenin results in significantly decreased proliferation. These results, together with the observation that BRAF signaling also induces Brn-2 expression, reveal that Brn-2 is a focus for the convergence of two key melanoma-associated signaling pathways that are linked to cell proliferation.

Abstract

Melanocytes originate in the neural crest as undifferentiated nonpigmented melanoblasts that migrate to the epidermis and hair follicles, where they differentiate and are responsible for skin and hair color. As melanocytes are not essential for survival and as mutations affecting the survival or differentiation of the melanocyte lineage are reflected in an obvious pigmentation phenotype, the melanocyte system represents an excellent model for understanding how signal transduction pathways coordinate the program of gene regulation underlying the genesis of a specific cell type. Importantly, constitutive activation of signaling pathways normally operating during melanocyte development is linked to the transformation of a melanocyte to a malignant melanoma (5, 16), a highly aggressive cancer, the incidence of which is increasing at an alarming rate (33).

The Wnt signaling pathway (for reviews of Wnt signaling, see references 3 and 12) is critically required for development of the melanocyte lineage; in both zebrafish and mice, overexpression of components of the Wnt signaling pathway result in an increase in the number of melanocytes at the expense of neurons and glia (9, 11), and disruption of the Wnt-1 and Wnt-3a genes leads to complete loss of melanoblasts (24). Wnt proteins interact with frizzled receptors and lead to the inhibition of serine-threonine kinase glycogen synthase kinase 3β. Phosphorylation of β-catenin by glycogen synthase kinase 3β is associated with the destabilization of β-catenin. Thus, increased Wnt signaling leads to stabilization of β-catenin and its translocation from the cytoplasm to the nucleus, where it can activate transcription via association with the Lef1 and Tcf transcription factors (1, 22, 29). A key role for Wnt signaling in melanocyte development is the activation of the promoter for the gene encoding the microphthalmia-associated transcription factor Mitf (10, 43). Mitf (19, 23) is essential for the development of the melanocyte lineage and has two key functions: in control of cell proliferation and survival and in differentiation (17).

The link between melanocyte development and melanoma is underscored by the fact that many melanomas exhibit constitutively elevated levels of nuclear β-catenin (34, 36). Given the key role played by β-catenin in development, it is likely that the elevated level of this protein observed in melanomas makes a substantial contribution to their transformed phenotype, and indeed, activation of the Wnt pathway has been linked to a metastatic or migratory phenotype (30). The identification of β-catenin target genes therefore represents an important goal if the link between melanocyte development and malignant melanoma is to be understood.

Using both cell lines and transgenic mice, we show here that the promoter for the gene encoding the POU domain transcription factor Brn-2 (also called N-Oct3 and POU3F2) that has been implicated in control of proliferation and survival of melanoma cells is a direct target for β-catenin/Lef1 and that reduction of Brn-2 expression by silent interfering RNA (siRNA) results in a decrease in proliferation. The results provide a novel insight into the link between melanocyte development and melanoma and β-catenin and regulation of proliferation.

Acknowledgments

We thank Rudi Grosschedl for the β-catenin expression vector, Kathy Jones for anti-LEF1 antibody, Marshall Nirenberg for a Brn-2 genomic clone, Ian Hart for the VUP melanoma cell line, Jean-Jacques Panthier for the newborn W/W-lacZ pups, and Hans Clevers for the Lef1 expression vector.

This work was supported by the Association for International Cancer Research (AICR), Candis and Marie Curie Cancer Care, the Fondation de France, the Ligue Nationale Contre le Cancer, and l'Association pour la Recherche contre le Cancer.

Acknowledgments

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