[Cancer Research 62, 5962-5966, October 15, 2002]
© 2002 American Association for Cancer Research
Down-Regulation of COOH-Terminal Binding Protein Expression in Malignant Melanomas Leads to Induction of MIA Expression1
Ina Poser,
Michaela Golob,
Michael Weidner,
Reinhard Buettner and
Anja K. Bosserhoff
Institute of Pathology, RWTH Aachen, D-52074 Aachen [I. P.]; Institute of Pathology, University of Bonn, D-53127 Bonn [R. B.]; Institute of Pathology, University of Regensburg, D-93053 Regensburg [I. P., M. G., A. K. B.]; and Roche Diagnostics, D-82377 Penzberg [M. W.], Germany
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ABSTRACT
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Malignant transformation of melanocytes to melanoma cells closely parallels activation of MIA expression and involves a promoter region that we referred to previously as a HCR (highly conserved region). The HCR element interacts with the melanoma-associated transcription factor and confers strong activation of the promoter. Furthermore, mutation and deletion studies described in this study revealed that the permissive site for cell-specific promoter activity was located directly 5' to the HCR region. Changes in the DNA sequence 5' adjacent to the melanoma-associated transcription factor binding site led to an MIA promoter activity in benign melanocytes and nonmelanocytic cells that usually do not express MIA. Detailed analysis revealed binding of T-cell factor family transcription factors to the repressor element. Because this family is known to interact with COOH-terminal binding protein, we explored the role of COOH-terminal binding protein 1(CtBP1) in silencing MIA gene expression. By reporter gene analysis, we determined a strict negative regulation of MIA promoter activity in melanoma cells by CtBP1. Furthermore, we observed strong expression of CtBP1 in primary melanocytes but a loss of wild-type CtBP1 expression in malignant melanoma in vitro and in vivo. Therefore, we speculate that CtBP1 has an important negative role in MIA regulation, and loss of CtBP1 is implicated in melanoma progression.
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INTRODUCTION
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Previously, we described the isolation of MIA, an Mr 11,000 protein secreted from malignant melanoma cells into the tissue culture supernatant (1, 2, 3)
. Purified MIA induces detachment of malignant melanoma cells in vitro and causes significant alteration of cell morphology as melanoma cells round up (3
, 4) . MIA expression in vivo correlates with progressive malignancy of melanocytic tumors (5, 6, 7)
. In recent studies, we measured enhanced MIA protein levels specifically in the serum of patients with metastatic melanomas (8
, 9)
. Analyzing normal skin and skin-derived melanocytic tumors by semiquantitative RT-PCR3
revealed absence of significant MIA mRNA levels in normal skin and melanocytes, very low levels in some benign melanocytic nevi, and high levels in all primary and metastatic malignant melanomas (6
, 7)
. Absence of MIA mRNA expression in benign melanocytes cultured from normal skin biopsies has also been reported by a different study (10)
.
By analyzing transcriptional mechanisms involved in the regulation of MIA expression, we have provided previously an initial characterization of the promoter and shown that it is strongly activated in melanoma cells (7
, 11)
. We identified a 30-bp region in the MIA promoter essentially required for strong and specific activity in melanoma cells, which is subject to binding by the protein MATF. In this study, we concentrated on a second cis-regulatory element in the 30-bp region located 5' adjacent to the MATF binding site, which functions as a silencer element in benign skin melanocytes and is depressed in malignant melanoma cells.
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MATERIALS AND METHODS
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Cell Lines and Cell Culture Conditions.
The melanoma cell lines Mel Im and Mel Ju have been described in detail previously (1
, 12)
. For tissue culture, the cells were maintained in DMEM supplemented with 400 units/ml penicillin, 50 µg/ml streptomycin, 300 µg/ml L-glutamine, and 10% FCS (Sigma, Deisenhofen, Germany) and split 1:3 every third day.
Human primary melanocytes derived from normal skin were cultivated in melanocyte medium MGM-3 (Life Technologies, Inc., Eggenstein, Germany) under a humidified atmosphere of 5% CO2 at 37°C. Cells were used in passages 2–4 and not later than 3 days after trypsinization. Cells were detached for subcultivation or assay with 0.05% trypsin, 0.04% EDTA in PBS.
Transfection Experiments.
For transient transfections, 3 x 105 cells/well were seeded into 6-well plates and transiently transfected with 0.5 µg of reporter plasmids using the Lipofectamine plus method (Life Technologies, Inc.), according to the manufacturer’s instructions. The following plasmids were transfected: 1386-pGL2 [1386 bp of the human MIA promoter region in pGL2-basic (Ref. 7
)], trimer c-pGL2 [trimer of oligo c (see Fig. 1A
) in pGL2-promoter], mut Y-pGL2 (mutated TCF4 binding site in 1386-pGL2, see Fig. 1A
), del Y-pGL2 (deleted TCF4 binding site in 1386-pGL2, see Fig. 1A
), pBATßCat [expression vector for ß-catenin (Ref. 13
)], pCMX-PL1-CtBP1 [expression vector for CtBP1 (Ref. 14
)], and pMH TCF4 (expression vector for hTCF4). Twenty-four h after transfection, the cells were lysed, and the luciferase activity in the lysate was measured. To normalize transfection efficiency, 0.2 µg of an pRL-TK plasmid (Promega Corp., Madison, WI) was cotransfected into each well, and Renilla luciferase activity measured by a luminometric assay (Promega). The amount of measured Renilla luciferase activity was used to normalize efficiency. To ensure equal amounts of transfected DNA in each experiment, the plasmid pBS (Stratagene, Heidelberg, Germany) was cotransfected. All transfections experiments were repeated at least three times.
RNA Isolation and RT-PCR.
Expression of TCF4 and CTPB1 mRNA was measured by RT-PCR. For RT-PCR, total cellular RNA was isolated from cultured cells and malignant melanomas using the RNeasy kit (Qiagen, Hilden, Germany). The integrity of the RNA preparations was controlled on an 1% agarose/formaldehyde gel. First-strand cDNA was synthesized using 1 µg of total cellular RNA as template, 1 µg of random primer (Pharmacia, Freiburg, Germany), 4 µl of 5x First Strand Buffer (Life Technologies, Inc.), 2 µl of 10 mM DTT, 1 µl of 10 mM deoxynucleotide triphosphates, and 1 µl of Superscript II (Life Technologies, Inc.) in a volume of 20 µl. For PCR amplification, 2 µl of the cDNA preparation were used as a template, and the following program was applied: 32 cycles of 45 s at 94°C, 30 s at 60°C, and 1 min at 72°C, with a final extension of 5 min at 72°C. The following primers were used: hCtBP1for94, CGA CCT CCG ATC ATG AAC; hCtBP1rev947, GCT AAA GCT GAA GGG TTC C; TCF4 for205, TCA CCA ACA GCG AAT GGC; and TCF4 rev809, AGG AAG GAT AGC CTG GCG. PCR products were separated on a 1.8% agarose gel and stained with ethidium bromide. Furthermore, quantitative real time-PCR was performed on a Lightcycler (Roche, Mannheim, Germany). Two µl of cDNA template, 2.4 µl of 25 mM MgCl2, 0.5 µM forward and reverse primer (hCtBP1 LC for, CAT CAT CGT CCG GAT TG; hCtBP1 LC rev, CCA TCC GAC AAG TAA GGG), and 2 µl of SybrGreen LightCycler Mix in a total of 20 µl were applied to the following PCR program: 30 s at 95°C (initial denaturation); 20°C/s temperature transition rate up to 95°C for 20 s, 10 s at 58°C, 20 s at 72°C, and 10 s in single acquisition mode at 87°C, repeated for 40 times (amplification). The PCR reaction was evaluated by melting curve analysis following the manufacturer’s instructions and checking the PCR products on 1.8% agarose gels. To verify amplification of the correct template, PCR products were sequenced.
Gel Shift Experiments.
The double-stranded oligomeric binding sites oligo c (5'-Ggc tcg agt agg cat ttt ctt tgg ccc ata-3'), hum Y (5'-GTG AGC TGC TTT GGA CCT TAT C-3'), mut Y (5'-GTG AGC GTC GAC TCA CCT TAT C-3'), and wt-TCF (5'-GAT CTG AAT TGC TTT GGG CTC GA-3') were phospholabeled and used for gel mobility-shift assays. Nuclear extracts were prepared from primary melanocytes, Mel Im and Mel Ju cells and gel shifts were performed as described previously (7)
. Competition experiments were performed using a 50-fold excess of the wild-type or mutated binding sites. For supershifting experiments, an antibody directed against TCF4 was used. The antibody was generated in rabbits using GST-TCF4aa1-52.
MIA-ELISA.
MIA protein expression was measured using an ELISA system (Roche), following the manufacturer’s instructions. Briefly, monoclonal antibodies coupled with biotin or peroxidase, respectively, were used to quantify MIA in a 96-well plate coated with streptavidin. ABTS was used as substrate and quantified at an absorbance of 405 nm.
Invasion Assay.
Invasion assays were performed in Boyden chambers containing polycarbonate filters with 8-µm pore size (Costar, Bodenheim, Germany). Filters were coated with a commercially available reconstituted basement membrane (Matrigel, diluted 1:3 in H2O; Becton Dickinson, Heidelberg, Germany). The lower compartment was filled with fibroblast-conditioned medium as a chemoattractant. Melanoma cells were harvested by trypsinization for 2 min, resuspended in DMEM without FCS at a density 2 x 105 cells/ml, and placed in the upper compartment of the chamber. After incubation at 37°C for 4 h, filters were removed. Cells adhering to the lower surface were fixed, stained, and counted.
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RESULTS
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Previous studies revealed a 30-bp region in the MIA promoter, which is responsible for melanoma-specific regulation and strong MIA expression (11)
. We were able to show that a region highly conserved between the human and murine promoter (HCR; Fig. 1A
) interacts with the transcription factor MATF and is essential for strong MIA promoter activity in melanoma cells.
In this study, we further investigated the function of the 30-bp region in cell-specific regulation of MIA expression. Therefore, we concentrated on a second conserved binding site located in the 30-bp region 5' of the HCR region. Deletion of 9 bp or insertion of mutations within this site led to complete loss of melanoma-specific activity of the promoter (Fig. 1B)
. In reporter gene assays, no activity of the wild-type promoter was found in primary melanocytes or HeLa cells, but strong and specific activity was found in the melanoma cell lines. In contrast, a promoter LUC reporter mutated in the conserved site 5' adjacent of the HCR region conferred strong activity in HeLa cells and primary melanocytes. From these results, we concluded that the element upstream of the HCR site recruits a negative transcriptional regulator.
Detailed analysis of the repressor element showed that it conforms to a TCF binding site (Fig. 1C)
. Indeed, gel shift analysis proved binding of TCF4 (Fig. 2A)
, and TCF4 binding was verified using an anti-TCF4 antibody in supershift experiments (Fig. 2B)
. Furthermore, competition with a consensus TCF binding site disrupted the gel shift complex (Fig. 2A
, Lane 4), and also a mutated form of the region did not form a shifted complex with TCF4 (Fig. 2A
, Lane 2). Surprisingly, expression of TCF4 in melanocytes and melanoma cells measured by RT-PCR did not differ in expression level (data not shown), and furthermore, TCF4 binding to the MIA promoter region was equally observed in both melanocytes and melanoma cells (Fig. 2B)
.
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Fig. 2. Gel shift assays identify TCF/LEF binding to the MIA promoter. A, TCF4 binding to the promoter site was verified by cold competition. Mutation of the binding site (mut Y, Lane 2) led to a loss of specific binding compared with oligo c (Lane 1). Furthermore, competition with oligo c and a TCF consensus site (wt-TCF) was possible (Lanes 3 and 4). Gel shifts were performed using nuclear extracts of the cell line Mel Im. B, TCF4 binding to hum Y was detected in primary human melanocytes (PHM) as well as in melanoma cells (Mel Im and Mel Ju). Bandshifts from nuclear extracts were supershifted by the anti-TCF4 antibody.
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It is known that TCF transcription factors are coactivated by nuclear ß-catenin. We therefore performed cotransfection experiments to analyze the effect of ß-catenin on MIA promoter activity. Neither a positive nor a negative effect on MIA promoter activity was observed after cotransfecting a series of different amounts of ß-catenin expression plasmid (Fig. 3)
.
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Fig. 3. Effect of cotransfected ß-catenin on MIA promoter activity. An expression plasmid for ß-catenin was cotransfected with the MIA promoter (1386-pGL2) in a series of different concentrations (0.5, 0.25, and 0.125 µg/transfection). No significant changes in MIA promoter activity were detected. Bars, SD.
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More recently, CtBP1 was identified to negatively regulate TCF transcription factors. We therefore tested whether CtBP1 is involved in regulating the MIA TCF4 element. We analyzed the expression pattern of CtBP1 and measured down-regulation of wild-type CtBP1 expression in melanoma cells compared with primary melanocytes (Fig. 4A)
. To rule out cell culture artifacts, further measurements were performed with RNA extracted from malignant melanomas (Fig. 4, A–I)
compared with normal skin (Fig. 4B, J–N)
. To quantify mRNA expression in melanocytes, melanoma cell lines and tissue real-time RT-PCR was performed. The results show clearly the marked down-regulation of CTBP1 in melanoma. Amplification of CtBP1 on genomic DNA isolated from melanoma cell lines was possible, proving that the gene is not deleted in human melanoma (data not shown). Taken together, our results clearly indicate down-regulation of CtBP1 in parallel with up-regulation of MIA in malignant melanoma cells both in vitro and in vivo.
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Fig. 4. Expression of CtBP in melanocytes and malignant melanoma. A, RT-PCR detection of CtBP1 expression in primary human melanocytes (PHM) and malignant melanoma cell lines. PHMs were shown to express CtBP1 strongly in contrast to all 11 melanoma cell lines, which showed complete loss or strongly reduced expression of wild-type CtBP1 expression. Bottom, results of quantitative RT-PCR (PHMs were set as 100%). B, RT-PCR detection of CtBP1 expression in malignant melanoma revealed loss of expression in all analyzed tumor tissues (Lanes A–I). In normal skin biopsies (Lanes J–N), strong expression of wild-type CtBP1 was detected. Bottom, results of quantitative RT-PCR [normal skin (N) was set as 100%].
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Reporter gene assays were performed to analyze the functional consequences of the loss of CtBP1 expression. The activity of the full-length MIA promoter (1386 bp) was strongly repressed after cotransfection of CtBP1 in melanoma cells (Fig. 5A)
. Testing a minimal promoter construct under control of the trimeric MIA-promoter TCF site revealed that the strong repressor function of the MIA promoter elicited by CtBP1 is located at this site (Fig. 5B)
. The CtBP1 repressor function on both the full-length promoter and the trimeric MIA-TCF element was dose dependent. Also, no effect on MIA promoter activity by CtBP1 was observed on the mut Y construct in which the TCF binding site was mutated (Fig. 5C
; for sequence of mut Y, see Fig. 1A
). The repressor function elicited by CtBP1 was not influenced by cotransfecting ß-catenin (Fig. 5D)
. In contrast, simultaneous transfection of TCF4 and CtBP1 expression plasmids into melanoma cells inhibited the CtBP1 repressor function (Fig. 6)
. MIA promoter activity (trimer c construct) could significantly be restored by TCF4 compared with its repressed activity after sole CtBP1 transfection.
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Fig. 5. Role of CtBP1 in MIA promoter activity. CtBP1 expression plasmids were cotransfected in several doses (0.5, 0.25, and 0.125 µg/transfection) with MIA promoter reporter plasmids. Either the full-length MIA promoter (1386-pGL2; A) or a pGL2-promoter construct carrying a trimer of oligo c region (trimer c; B) was used. Experiments were performed in the melanoma cell lines Mel Im and Mel Ju. CtBP1 repressed the MIA promoter activity in a dose-dependent fashion. Using the mutated MIA promoter mut Y, no regulation by CtBP1 (0.5 µg/transfection) was found (C). Repression of the MIA promoter activity by CtBP1 was unaffected by cotransfection of ß-catenin (D). Bars, SD.
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Fig. 6. TCF4 counteracts CtBP1 repression activity. TCF4 and CtBP1 expression plasmids were cotransfected into Mel Im and Mel Ju, respectively. To confirm interactions of TCF4 and CtBP1, the pGL2-promoter construct carrying a trimer of oligo c region (trimer c-pGL2) was used. TCF4 was able to reduce the inhibitory effect of CtBP1 on the MIA promoter. Bars, SD.
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Reexpression of CtBP1 also had a negative influence on endogenous MIA protein expression measured by MIA ELISA. A strong down-regulation by 50.23% (Mel Im) and 59.2% (Mel Ju) was found in the cells transfected with 0.5 µg of CtBP1 expression plasmid compared with the mock-transfected controls.
To analyze the effect of loss of CtBP1 expression on melanoma progression, we transfected the melanoma cell line Mel Im with CtBP1 and compared its invasive potential using invasion assays to the mock-transfected cells. The CtBP1-transfected cells showed a marked reduction of invasion by 60% (±7.9%; P < 0.001, Student’s t test).
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DISCUSSION
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A series of studies have described highly cell type-specific expression of MIA in malignant melanomas and even used MIA serum levels as a surrogate marker to detect melanoma metastasis. By investigating transcriptional mechanisms mediating melanoma-specific gene expression, we previously identified a region in the MIA promoter conferring strong and cell type-specific expression of MIA in malignant melanoma cells. A binding site (HCR) was characterized in this region interacting with a factor referred to as MATF, which strongly activates the MIA promoter in melanoma cells (11)
.
With our tests of small deletions and mutated constructs of the MIA promoter, we here clearly provide additional evidence that a negative cis-regulatory element is located 5' adjacent to the HCR region and is critically important in melanoma-specific expression. Defects in this promoter element lead to loss of its specific expression and to unscheduled activity of the promoter in MIA-negative cells, such as HeLa cells or primary melanocytes. Our analysis reveals that TCF4 binds to this region. Interestingly, ß-catenin does not seem to function as a coregulator of this TCF-dependent element. We therefore concentrated on the TCF corepressor CtBP1. CtBP1 was first identified as a cytoplasmatic protein binding to the COOH-terminal region of the adenoviral protein E1A and attenuating its ability to activate transcription. CtBP recognizes PXDLS motifs in DNA-binding proteins and functions as a transcriptional corepressor in Drosophila, Xenopus, and mammals. The precise mechanisms by which CtBP influences transcription are still under investigation (15)
. Meanwhile, a number of studies have provided unequivocal evidence that CtBP1 binds to and regulates HMG-box proteins including Sox6 and TCF4 (16
, 17) . As shown previously for Sox6, this study implicates that TCF4 can act as a transcriptional repressor, depending on the context of coregulatory factors. In vitro reporter gene assays revealed that CtBP1 functions as a strong repressor of MIA promoter activity, and that this repressor function requires the TCF binding element in the MIA promoter. Our expression studies further indicate that loss of TCF/CtBP1 binding, and consequently loss of suppression of MIA expression in nonmelanoma cells may be important for melanoma progression. In the context of the MIA promoter, TCF is obviously used as a negative regulator in combination with CtBP1. This could be proven by simultaneous transfections of TCF4 and CtBP1 expression plasmids into melanoma cells. Here TCF4 negatively regulates CtBP1-induced repression of MIA promoter activity by interacting with CtBP1.
Further analysis revealed CtBP1 to be strongly expressed in primary melanocytes. In contrast, melanoma cells in vitro and in vivo were shown to have lost or strongly down-regulated wild-type CtBP1 expression. The mechanisms of loss of CtBP1 have to be evaluated in further ongoing studies. As for other proteins, e.g., p16, several mechanisms are feasible, such as mutations, promoter hypermethylation, promoter inactivation, or posttranscriptional mechanisms.
CtBP has been speculated to be involved in normal cell growth control. It was shown previously that the binding of CtBP to adenoviral E1A correlates with inhibition of E1A plus H-ras cotransformation, tumorigenesis, and metastasis (18, 19, 20)
. Furthermore, a repressor function of CtBP on E2F-medited transcription via RB was detected (21)
. In our study, invasion assays pointed to a role of CtBP1 also in cell migration because reexpression of CtBP1 in melanoma cells induced a reduction of the invasive potential. It therefore appears that loss of the CtBP1 corepressor function may be a critical event in the pathogenesis of many different malignant tumors.
In summary, we here describe for the first time an important role of CtBP1 in MIA regulation and MIA-dependent effects of melanoma progression. Additional experiments will address the question of whether CtBP1 is not only involved in up-regulation of MIA expression but also of other genes in malignant melanomas.
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ACKNOWLEDGMENTS
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We are indebted to Astrid Hamm and Claudia Abschlag for technical assistance and to Jürgen Behrens (University of Erlangen, Germany) for providing the ß-catenin construct.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from the Deutsche Forschungsemeinschaft and the Deutsche Krebshilfe (to A. B. and R. B.). 
2 To whom requests for reprints should be addressed, at Institute of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. Phone: 49-941-944-6705; Fax: 49-941-944-6602; E-mail: anja.bosserhoff{at}klinik.uni-regensburg.de
3 The abbreviations used are: RT-PCR, reverse transcription-PCR; MATF, melanoma-associated transcription factor; CtBP, COOH-terminal binding protein; HCR, highly conserved region; TCF, T-cell factor.
Received 12/17/01.
Accepted 8/16/02.
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ABSTRACT
INTRODUCTION
), primary human melanocytes (PHM, 
), and HeLa cells (
). Bars, SD. C, TCF consensus site in the murine and human MIA promoter.