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Stabilization of β-Catenin by Genetic Defects in Melanoma Cell Lines

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Science  21 Mar 1997:
Vol. 275, Issue 5307, pp. 1790-1792
DOI: 10.1126/science.275.5307.1790

Abstract

Signal transduction by β-catenin involves its posttranslational stabilization and downstream coupling to the Lef and Tcf transcription factors. Abnormally high amounts of β-catenin were detected in 7 of 26 human melanoma cell lines. Unusual messenger RNA splicing and missense mutations in the β-catenin gene (CTNNB1) that result in stabilization of the protein were identified in six of the lines, and the adenomatous polyposis coli tumor suppressor protein (APC) was altered or missing in two others. In the APC-deficient cells, ectopic expression of wild-type APC eliminated the excess β-catenin. Cells with stabilized β-catenin contained a constitutive β-catenin-Lef-1 complex. Thus, genetic defects that result in up-regulation of β-catenin may play a role in melanoma progression.

The protein β-catenin is an important signaling protein in both Xenopus laevis and Drosophila melanogaster development (1). The proposed pathway, which is initiated by the wnt-1/wingless receptors, involves the posttranslational stabilization of β-catenin, leading to its accumulation in the cytoplasm and nucleus. In the nucleus, β-catenin is thought to interact with the Lef and Tcf families of transcription factors and thus directly regulates expression of target genes (2). The wnt-1 proto-oncogene also stabilizes β-catenin in mammalian cell culture and promotes tumor formation when expressed in mouse mammary tissue (3). The potential role of β-catenin signaling in cancer is supported by the observation that the APC tumor suppressor protein down-regulates excess intracellular β-catenin when it is ectopically expressed in colon cancer cells containing defective APC (4). The regulatory mechanism for β-catenin turnover requires the NH2-terminal region of the protein. Deletion of this sequence, or mutation of four serine or threonine residues therein, result in the accumulation of β-catenin and thus activate its role in signaling (57). Conceivably then, mutations that stabilize β-catenin may contribute to loss of cell growth control in tumorigenesis.

Previously, a mutant form of β-catenin, containing a Ser37 → Phe37 (S37F) substitution, was identified in the 888 mel cell line as a melanoma-specific antigen recognized by tumor-infiltrating lymphocytes (8). Because it was possible that this mutation increased the stability of β-catenin, we determined β-catenin concentrations in these cells and in 25 other melanoma cell lines. Seven of the lines, including the 888 mel cell, contained elevated amounts of β-catenin relative to normal human neonatal melanocytes (NHEM) (Fig. 1A). Two of the seven appeared to have APC alterations as well: the 1335 mel cells contained a truncated APC and the 928 mel cells had no detectable APC. The truncated APC was not immunoprecipitated by antibody specific to the COOH-terminal sequence of APC, suggesting it was a COOH-terminal truncation similar to that observed in colon cancers (Fig. 1B).

Fig. 1.

Analysis of β-catenin and APC in melanoma cell lines. (A) Protein-equivalent amounts of total cell lysate from the indicated cell lines were subjected to SDS-PAGE and immunoblotting (13). The blot was cut horizontally and developed with anti-APC2 (APC; top) or anti-β-catenin (β-cat; bottom). The β-catenin blot was developed with 125I-labeled protein A, and the counts per minute (CPM) for each β-catenin band is indicated below each lane. For (A) and (B), values at left indicate positions and molecular masses in kilodaltons of protein standards, and NHEM indicates a normal neonatal human melanocyte. All other cell lines were derived from human melanomas (16). (B) APC was immunoprecipitated from protein-equivalent amounts of the cell lysates, and the precipitates were analyzed for APC and β-catenin with SDS-PAGE and immunoblotting (13). (C) Size-exclusion chromatography was performed on approximately 800 μg of total protein from each lysate, and fractions were analyzed for β-catenin with SDS-PAGE and immunoblotting. Total lysate (L) and column fraction (Fract.) numbers are shown at top, and arrows indicate the elution positions of protein standards. Longer exposures are presented for cell lines with lower concentrations of total β-catenin.

A substantial amount of β-catenin was coimmunoprecipitated with wild-type (WT) APC from five other lines with high levels of β-catenin. The accumulation of β-catenin on WT APC is characteristic of β-catenin stabilization, as has been observed in particular with NH2-terminal deletion mutants of β-catenin (5). The 1088 mel cell appeared to contain a truncated β-catenin that accumulated on the APC protein. Another characteristic of stabilized β-catenin is its migration in a monomeric pool upon size fractionation chromatography (5, 9, 10). All of the melanoma cells with elevated amounts of β-catenin exhibited a substantial pool of monomeric β-catenin (Fig. 1C). In addition, two of the cell lines with normal amounts of β-catenin, the 1280 and 1300 mel, also contained some monomeric β-catenin.

Up-regulation of β-catenin in the 928 and 1335 mel cell lines may have resulted from loss of WT APC, as has been proposed for colon cancer cells (4). To test this hypothesis, we transiently expressed WT APC in the 928 mel cells and costained them with antibodies specific to APC and β-catenin. The 928 mel cells that were positive for ectopically expressed APC contained low concentrations of β-catenin relative to nontransfected cells, which exhibited excessive nuclear and cytoplasmic staining (Fig. 2). The ability of APC to down-regulate β-catenin in the 928 mel cells suggests that they contain WT β-catenin. In contrast, ectopic expression of WT APC in the 888 mel cells did not down-regulate the endogenous mutant β-catenin, but instead resulted in its accumulation on the WT APC.

Fig. 2.

Down-regulation of β-catenin by ectopic expression of WT APC. The 928 mel and 888 mel cells were transiently transfected with a plasmid encoding human WT APC, and 48 hours later, cells were fixed and costained with anti-APC (left) and anti-β-catenin (right) (18).

The wnt-1 proto-oncogene activates β-catenin signaling by reducing the rate of β-catenin degradation (3), whereas the APC tumor suppressor enhances this rate (4). To examine whether the high steady-state amount of β-catenin in the melanoma cells was due to a reduced rate of turnover, we performed pulse-chase analysis of β-catenin on representative cell lines. The β-catenin in the SK23 mel cell line, which contains WT APC and normal amounts of β-catenin, had a half-life (T1/2) of less than 30 min (Fig. 3A). In contrast, the β-catenin in the 888 mel cells, which contained the S37F mutation, had a T1/2 of >4.5 hours. The β-catenin in the 928 mel cells, which lack WT APC, and in the 624 mel cells, which contain a mutant β-catenin (Table 1), also had an extended T1/2. The 888 mel cells contain mRNAs for both WT and mutant β-catenins (8), but the relative contribution of their products to the half-life analysis is unknown. The results suggest that the WT β-catenin is a minor fraction of the total or that the mutant form dominantly interferes with the turnover of the WT protein. The 1088 mel cells contain both a full-length β-catenin with an intermediate T1/2 of ∼2 hours and a truncated β-catenin with an extended T1/2 of >4.5 hours.

Fig. 3.

Pulse-chase analysis of β-catenin. (A) Melanoma cells were pulse-labeled with [35S]methionine, chased with cold methionine for the indicated times, and then lysed (20). β-Catenin was immunoprecipitated and then analyzed with SDS-PAGE and fluorography. The cell lines are indicated to the left of each panel at the position of the β-catenin band. DN indicates the position of the NH2-terminal truncated form of β-catenin in the 1088 mel cells. (B) ATT20 cell lines stably expressing either WT β-catenin or the S37A mutant were subjected to pulse-chase analysis (20). (C) SW480 cells were transiently cotransfected with plasmids encoding a COOH-terminal (APC3) or central (APC25) fragment of APC and either the WT or S37A mutant of β-catenin (20). APC25 down-regulates β-catenin but APC3 does not (4).

Table 1.

Mutations in melanoma cell lines with accumulated β-catenin. AA, amino acid.

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To ensure that substitution of Ser37 was responsible for the reduced rate of protein turnover, we transfected murine pituitary ATT20 cells, which exhibit rapid turnover of endogenous β-catenin (5), with plasmids encoding epitope-tagged Ser37 → Ala37 (S37A) or WT β-catenin. The exogenous WT β-catenin had a T1/2 of ∼40 min, whereas the S37A β-catenin had a T1/2 of >4 hours (Fig. 3B). To determine if the S37A β-catenin was responsive to APC-dependent turnover, we coexpressed it with an APC25 cDNA in SW480 human colon cancer cells that contain only truncated APC. The APC25 fragment down-regulates β-catenin, whereas the control APC3 fragment does not (4). Recovery of the epitope-tagged β-catenins revealed that WT, but not the S37A β-catenin, was degraded in response to the coexpressed APC25 fragment (Fig. 3C). These results demonstrate that a single point mutation has a marked effect on the T1/2 of β-catenin.

Sequencing of β-catenin cDNAs from the other melanoma lines with β-catenin accumulation revealed three additional point mutations affecting serine residues (Table 1). As with the 888 mel cells, the mutations identified in the 501 and 1241 mel cells were C to T transitions that produced an S37F substitution. Interestingly, C-to-T transitions are also common in the p53 gene in melanomas, and may be an effect of ultraviolet radiation (11). The mutation in 624 mel predicts Ser45 → Tyr45 (S45Y) substitution, and pulse-chase analysis of this cell suggests that it may prolong the T1/2 of β-catenin (Fig. 3). Moreover, coexpression of an S45Y β-catenin with APC25 indicated it was refractory to APC-dependent turnover in SW480 cells (12). The serines at position 37 and 45 are likely important phosphorylation sites, as the quadruple substitution of Ser33, Ser37, Thr41, and Ser45 markedly reduced the phosphorylation of β-catenin in Xenopus (7). Two novel β-catenin mRNAs, one lacking exons 2 and 3, and the other lacking exons 2, 3, and 4, were identified in the 1088 mel cells. Initiation normally occurs at codon 1 in exon 2; however, initiation at codon 88, the first ATG in exon 4, would account for a truncated β-catenin approximately the size of that detected in the 1088 mel cells (Fig. 1A). A more severely truncated β-catenin, predicted from initiation at codon 174 in exon 5 of the other alternative mRNA, has not been detected. Whether the β-catenin mRNA isoforms in this cell are due to a mutation or to unusual mRNA processing is unclear. None of the other melanoma cells contained these mRNAs. Sequencing of β-catenin cDNAs from the APC-deficient 1335 and 928 mel cells identified only wild-type sequence, as did sequencing of the 1280 mel, 1300 mel, SK23 mel, and NHEM lines.

Recently, β-catenin has been shown to functionally interact with Lef-Tcf transcription factors when overexpressed in Xenopus oocytes (2). To determine if this interaction occurs in the melanoma cell lines, we immunoprecipitated β-catenin from some of the lines and examined the precipitates for Lef-1. Lef-1 was preferentially coimmunoprecipitated by anti-β-catenin from the cells containing stabilized β-catenin (Fig. 4). This raises the possibility that in these cells a constitutive β-catenin-Lef-Tcf complex may result in persistent transactivation of as yet unidentified target genes.

Fig. 4.

Coimmunoprecipitation of Lef-1 with β-catenin. β-Catenin was immunoprecipitated from ∼600 μg total protein from the indicated cell lysates, and the precipitates were analyzed with SDS-PAGE and immunoblotting for β-catenin and Lef-1 (13).

Of the 26 melanoma cell lines we examined, 8 are defective in β-catenin regulation because of β-catenin mutations, unusual β-catenin mRNA splicing, or inactivation of APC. We hypothesize that these mutations are selected in tumor progression. The mutation in the 888 mel line was unlikely to be generated by in vitro culture, as it was also present in the 1290 mel line, which was derived from a new tumor from the same patient after a 3-year remission (8). Moreover, the mutation was also identified in the uncultured tumor material from which the 1290 mel was derived. The stabilizing mutations in β-catenin are also consistent with a proposed function for APC in colon cancer. The ability of WT, but not mutant APC to down-regulate β-catenin in colon cancer cells led us to propose that up-regulation of β-catenin may contribute to cancer progression (4). In the melanoma cells, β-catenin mutations were identified in cells that appeared to express WT APC, whereas high amounts of WT β-catenin were found in cells expressing mutant APC. Thus, up-regulation of β-catenin may be a common feature of tumorigenesis that can be effected through mutations in the APC or β-catenin genes or other genes that function in this pathway.

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