Silencing cAMP-response Element-binding Protein (CREB) Identifies CYR61 as a Tumor Suppressor Gene in Melanoma

Abstract

Metastatic progression of melanoma is associated with overexpression and activity of cAMP-response element-binding protein (CREB). However, the mechanism by which CREB contributes to tumor progression and metastasis remains unclear. Here, we demonstrate that stably silencing CREB expression in two human metastatic melanoma cell lines, A375SM and C8161-c9, suppresses tumor growth and experimental metastasis. Analysis of cDNA microarrays revealed that CREB silencing leads to increased expression of cysteine-rich protein 61 (CCN1/ CYR61) known to mediate adhesion, chemostasis, survival, and angiogenesis. Promoter analysis and chromatin immunoprecipitation assays demonstrated that CREB acts as a negative regulator of CCN1/CYR61 transcription by directly binding to its promoter. Re-expression of CREB in CREB-silenced cells rescued the low CCN1/CYR61 expression phenotype. CCN1/ CYR61 overexpression resulted in reduced tumor growth and metastasis and inhibited the activity of matrix metalloproteinase-2. Furthermore, its overexpression decreased melanoma cell motility and invasion through Matrigel, which was abrogated by silencing CCN1/CYR61 in low metastatic melanoma cells. Moreover, a significant decrease in angiogenesis as well as an increase in apoptosis was seen in tumors overexpressing CCN1/CYR61. Our results demonstrate that CREB promotes melanoma growth and metastasis by down-regulating CCN1/ CYR61 expression, which acts as a suppressor of melanoma cell motility, invasion and angiogenesis.Cutaneous melanoma is the most aggressive type of skin cancer, and it can metastasize very rapidly (1). An estimated 62,480 new cases of melanoma were diagnosed in the United States during 2008, 8,420 of which resulted in death (2). The transition of melanoma from the radial growth phase to the vertical growth phase to metastasis is accompanied by multiple molecular changes (3-8). We and others have shown that two transcription factors, activating transcription factor-1 (ATF-1) 2 and cAMP-response element-binding protein (CREB), are activated and overexpressed in melanoma during its progression toward the malignant phenotype (9 -13).CREB and ATF-1 belong to the leucine zipper class of transcription factors. Stimuli such as growth factors, neurotransmitters, inflammatory biolipids, stress signals, or other factors that elevate intracellular cAMP or Ca 2ϩ levels can activate CREB/ATF-1 through phosphorylation at Ser 133 by protein kinase A or mitogen-activated protein kinases (MAPK) (14 -17). Following activation, CREB/ATF-1 regulates the expression of genes that suppress apoptosis, induce cell proliferation, and mediate inflammation and tumor metastasis by binding to cAMP-response elements (CREs) within the promoter and enhancer regions of these genes (15, 18 -20).A number of reports have suggested that CREB is involved in melanoma progression We have demonstrated previously that quenching CREB activity in metastatic melanoma cells by means of a dominant-negative form of CREB (KCREB) leads to a decrease in their tumorigenicity a...

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