The Brn-2 Transcription Factor Links Activated BRAF to Melanoma Proliferation

Abstract

Malignant melanoma, an aggressive and increasingly common cancer, is characterized by a strikingly high rate (70%) of mutations in BRAF, a key component of the mitogen-activated protein (MAP) kinase signaling pathway. How signaling events downstream from BRAF affect the underlying program of gene expression is poorly understood. We show that the Brn-2 POU domain transcription factor is highly expressed in melanoma cell lines but not in melanocytes or melanoblasts and that overexpression of Brn-2 in melanocytes results in increased proliferation. Expression of Brn-2 is strongly upregulated by Ras and MAP kinase signaling. Importantly, the Brn-2 promoter is stimulated by kinase-activating BRAF mutants and endogenous Brn-2 expression is inhibited by RNA interference-mediated downregulation of BRAF. Moreover, silent interfering RNA-mediated depletion of Brn-2 in melanoma cells expressing activated BRAF leads to decreased proliferation. The results suggest that the high levels of Brn-2 expression observed in melanomas link BRAF signaling to increased proliferation.The mitogen-activated protein (MAP) kinase signaling pathway, comprising the RAS-RAF-MEK-ERK cascade, plays a key role in the cellular response to extracellular signals, and deregulation of the pathway, for example, as a result of activating mutations in Ras, is a feature of many cancers. Three Raf genes have been described (ARAF, BRAF, and CRAF) that are regulated by interaction with Ras, and RAF protein can phosphorylate and activate the MAP kinase kinase MEK. The importance of this signaling pathway in cancer has recently been highlighted by the observation that almost 70% of melanomas and primary nevi exhibit kinase-activating mutations in BRAF (11,26). Understanding how signaling by the MAP kinase pathway affects the underlying program of gene expression is therefore a particularly important issue for melanoma, and it is presumed that deregulation of MAP kinase signaling results in altered expression or activity of key transcription factors, leading to aberrant growth control. The identification of BRAF target genes currently represents a major goal in melanoma biology.The POU domain transcription factor Brn-2 (also called N-Oct3 and POU3F2) (see reference 28 for a review) has been implicated in neuronal differentiation (16) and activation of the corticotropin-releasing hormone gene (22,29). Targeted disruption of the Brn-2 gene in the mouse results in loss of specific neuronal lineages in the hypothalamus and consequent loss of the posterior pituitary gland (25, 29). Brn-2-negative mice therefore die within 10 days after birth, although the specific cause of death is not apparent. Although Brn-2 clearly plays a major role in neuronal development, evidence also implicates it in melanoma growth and survival. Thus, Brn-2 mRNA is overexpressed in melanoma compared to normal melanocytes (15,33,34), and Brn-2 expression is substantially downregulated by differentiating agents (32). Importantly, downregulation of Brn-2 with an antisense strategy resulted in ...

Regulation of iNOS by the p44/42 mitogen-activated protein kinase pathway in human melanoma

“…However, other pathways and transcription factors that are relatively more specific to melanoma should also be explored. One such factor is the octamer (OCT)-binding protein BRN-2, which is expressed by melanoma cells in response to ERK activation and is associated with melanoma proliferation and survival (Goodall et al, 2004). The iNOS promoter contains an OCT binding site and can be activated in vitro by BRN-3, another member of the OCT family (Gay et al, 1998).…”

BRAFE600 in benign and malignant human tumours

“…BRN2 is a transcription factor often overexpressed in melanoma (reviewed in Vance and Goding, 2004). Loss of BRAF E600 expression reduces BRN2 levels, and downregulation of BRN2 leads to decreased proliferation of melanoma cell lines (Goodall et al, 2004). In contrast, in some of the melanoma cell lines studied silencing of BRAF E600 expression results in stabilization of MITF (a key transcription factor in melanocyte biology; see below), leading to upregulation of tyrosinase and the tyrosinase-related protein 1 (TRP-1).…”

BRN2 is a transcriptional repressor of CDH13 (T-cadherin) in melanoma cells

“…We have excluded SNAIL and ETS-1 as responsible factors for regulating CDH13 expression (data not shown). This study focused on whether CDH13 promoter activity might be under the control of transcription factor BRN2, based on the in silico detection of consensus binding sequences of BRN2 in the proximal promoter region of CDH13, on observations of increased levels of BRN2 [12][13][14][15][16] and decreased levels of T-cadherin 9 expression in melanomas, and on observations of opposing effects of BRN2 [12][13][14][15][16] and T-cadherin 9 on melanoma cell behavior.…”

“…10,11 BRN2 expression is upregulated in melanoma cell lines and in melanoma lesions where cells expressing high levels of BRN2 show enhanced invasive and metastatic capacity. [12][13][14][15][16] Silencing of BRN2 in melanoma cell lines reduces proliferation, invasion and tumorigenesis. [14][15][16][17] In contrast, silencing of T-cadherin in melanocytes increases invasive capacity, whereas ectopic re-expression in several T-cadherin-deficient melanoma cell lines reduces attachment-independent growth, migration and invasion in vitro and also their tumorigenicity in vivo.…”