Dominant-negative CREB inhibits tumor growth and metastasis of human melanoma cells

Xie, Shanhai; Price, Janet E.; Luca, Mario; Jean, Didier; Ronai, Ze’ev; Bar‐Eli, Menashe

doi:10.1038/sj.onc.1201358

Cited by 121 publications

(128 citation statements)

References 18 publications

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“…CREB, a critical regulator of immediate early gene transcription, has been shown to be activated by growth factors (33) and play an important role in the acquisition of the metastatic phenotype of human melanoma cells (21). Although the exact mechanism of action remains to be elucidated, our results suggest that the CRE-decoy oligonucleotides may provide a powerful means of combating cancers by regulating the expression of cAMP-sensitive genes.…”

Section: Fig 1 Cre-decoy Oligonucleotide Competes With Cre Enhancermentioning

confidence: 77%

Dual Blockade of Cyclic AMP Response Element- (CRE) and AP-1-directed Transcription by CRE-transcription Factor Decoy Oligonucleotide

Park

Nesterova

Agrawal³

et al. 1999

Journal of Biological Chemistry

119

116

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Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins has important therapeutic potential. Synthetic double-stranded phosphorothioate oligonucleotides with high affinity for a target transcription factor can be introduced into cells as decoy cis-elements to bind the factors and alter gene expression. The CRE (cyclic AMP response element)-transcription factor complex is a pleiotropic activator that participates in the induction of a wide variety of cellular and viral genes. Because the CRE cis-element, TGACGTCA, is palindromic, a synthetic single-stranded oligonucleotide composed of the CRE sequence self-hybridizes to form a duplex/hairpin. Herein we report that the CRE-palindromic oligonucleotide can penetrate into cells, compete with CRE enhancers for binding transcription factors, and specifically interfere with CREand AP-1-directed transcription in vivo. These oligonucleotides restrained tumor cell proliferation, without affecting the growth of noncancerous cells. This decoy oligonucleotide approach offers great promise as a tool for defining cellular regulatory processes and treating cancer and other diseases.Eukaryotic transcription is regulated by the interplay of various protein factors at promoters (1, 2). It has been shown that prokaryotic repressors can function as negative regulators of eukaryotic promoters (3,4). This observation suggests that displacement of activating proteins might provide a general strategy for gene-specific repression in eukaryotes. Several approaches have been undertaken to control eukaryotic gene expression through such displacement.In one approach, trans-dominant mutants are generated that interfere with the function of transactivators. Mutants are generated that retain the ability to bind to cis-regulatory DNA sequences but that have dysfunctional transcriptional activation domains. These mutant transcription factors compete with their functional, wild-type counterparts for binding to the enhancer sequences and prevent the activation or repression of the target gene. Although this strategy has been successful, in vitro (5-8), the generation of such mutants is not always possible. The transcription factor must be well characterized such that the activation domain(s) is identified and can be mutated. Also, even with sufficient knowledge to generate such mutants, difficult gene therapy procedures would be required to express these proteins in vivo.Promoter competition strategy has also been utilized whereby plasmids containing cis-acting elements in common with the targeted gene are introduced in high copy number into cells (9). At high copy number, a majority of the transcription factors can be competitively bound away from the native enhancer sequences with gene expression accordingly regulated. Because these plasmids must be stably maintained at high copy number in target cells, a requirement that is difficult to achieve in vivo, this approach has also been limiting.Another alternative is to employ oligonucleotides to form triple heli...

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Section: Fig 1 Cre-decoy Oligonucleotide Competes With Cre Enhancermentioning

confidence: 77%

Dual Blockade of Cyclic AMP Response Element- (CRE) and AP-1-directed Transcription by CRE-transcription Factor Decoy Oligonucleotide

Park

Nesterova

Agrawal³

et al. 1999

Journal of Biological Chemistry

119

116

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“…On the other hand, CREB, and additional family members such as ATF1, may contribute to melanoma cell viability (111). Interference with CREB function by ectopic expression of a dominant negative variant, capable of sequestering endogenous CREB as well as other associated proteins away from DNA sites, decreased radiation resistance of the human melanoma cell line MeWo by 3-10-fold (111), as well as their tumorigenic and metastatic potential in nude mice compared with parental controls (112). However, growth properties in vitro were not altered.…”

Section: Signal Transduction: Immediate Eventsmentioning

confidence: 99%

The Regulation of Normal Melanocyte Proliferation

Halaban

2000

Pigment Cell Research

169

124

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“…Recent evidence supports the belief that a variety of transcription factors are important in the pathogenesis of melanoma, including activating transcription factor-1 (ATF-1) (Bohm et al, 1995;, cAMP response elementbinding protein (CREB) Jean et al, 1998b;Rutberg et al, 1994;Xie et al, 1997b), transcription factor AP-2 (Huang et al, 1998;Jean et al, 1998a), micropthalmia transcription factor (Mitf) (Goding, 2000;King et al, 1999;Fang and Setaluri, 1999), and nuclear factor-kappa B (NF-kB) . In particular, we have previously demonstrated that the transition of melanoma to the metastatic phenotype is associated with a loss of expression of the transcription factor AP-2 (Bar- Eli, 1997Eli, , 1999).…”

Section: Introductionmentioning

confidence: 99%

“…After cell debris was removed by centrifugation, cell extracts were assayed for Renilla luciferase activity using the dual luciferase reporter assay system (Promega, Madison) or b-gal activity. Samples were normalized to the same relative luciferase or b-gal activity and assayed for CAT activity (Xie et al, 1997b). The cell extracts were incubated with 0.025 mCi of [14-C]chloramphenicol and 0.35 mM acetylcoenzyme A (Sigma, St. Louis, MO, USA) in 25 mM TrisHCl, pH 7.9, for 2 h at 378C.…”

Section: Transient Transfection and Cat Assaysmentioning

confidence: 99%

Dominant-negative transcription factor AP-2 augments SB-2 melanoma tumor growth in vivo

et al. 2001

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We have previously demonstrated that the transition of melanoma to the metastatic phenotype is associated with a loss of expression of the transcription factor AP-2. To further investigate the role of AP-2 in the progression of human melanoma, we attempted to inactivate AP-2 in primary cutaneous SB-2 melanoma cells by using a dominant-negative AP-2, or AP-2B, gene. AP-2B is an alternatively spliced AP-2 variant capable of inhibiting AP-2 trans-activator function. Stable transfection of primary cutaneous melanoma SB-2 cells with the dominant-negative AP-2B gene was con®rmed by RT ± PCR and Northern blot analyses. Electromobility shift assay using nuclear extracts from these cell lines demonstrated decreased functional binding of AP-2B-transfected cells to the AP-2 consensus binding sequence compared with neo-transfected controls. In addition, CAT activity driven by a construct containing the AP-2 consensus binding sequence was downregulated in the AP-2B transfected cells, indicating AP-2 activity was quenched in the transfected cells. Orthotopic (subcutaneous) injection of the dominant-negative (AP-2B)-transfected cell lines into nude mice increased their tumorigenicity compared to control neo-transfected cells. The AP-2B-transfected cells displayed an increase in MMP-2 expression (by Northern blot) and MMP-2 activity (by zymography), which resulted in an increase in invasiveness through Matrigel-coated ®lters. The AP-2B-transfected tumors also displayed an increase in MMP-2 expression, microvessel density, and angiogenesis in vivo. These results demonstrate that inactivation of AP-2 contributes to the progression of melanoma, at least partially via deregulation of the MMP-2 gene.

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Dominant-negative CREB inhibits tumor growth and metastasis of human melanoma cells

Cited by 121 publications

References 18 publications

Dual Blockade of Cyclic AMP Response Element- (CRE) and AP-1-directed Transcription by CRE-transcription Factor Decoy Oligonucleotide

Dual Blockade of Cyclic AMP Response Element- (CRE) and AP-1-directed Transcription by CRE-transcription Factor Decoy Oligonucleotide

The Regulation of Normal Melanocyte Proliferation

Dominant-negative transcription factor AP-2 augments SB-2 melanoma tumor growth in vivo

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