Beta-catenin mutations in cell lines established from human colorectal cancers

Abstract

beta-catenin has functions as both an adhesion and a signaling molecule. Disruption of these functions through mutations of the beta-catenin gene (CTNNB1) may be important in the development of colorectal tumors. We examined the entire coding sequence of beta-catenin by reverse transcriptase-PCR (RT-PCR) and direct sequencing of 23 human colorectal cancer cell lines from 21 patients. In two cell lines, there was apparent instability of the beta-catenin mRNA. Five different mutations (26%) were found in the remaining 21cell lines (from 19 patients). A three-base deletion (codon 45) was identified in the cell line HCT 116, whereas cell lines SW 48, HCA 46, CACO 2, and Colo 201 each contained single-base missense mutations (codons 33, 183, 245, and 287, respectively). All 23 cell lines had full-length beta-catenin protein that was detectable by Western blotting and that coprecipitated with E-cadherin. In three of the cell lines with CTNNB1 mutations, complexes of beta-catenin with alpha-catenin and APC were detectable. In SW48 and HCA 46, however, we did not detect complexes of beta-catenin protein with alpha-catenin and APC, respectively. These results show that selection of CTNNB1 mutations occurs in up to 26% of colorectal cancers from which cell lines are derived. In these cases, mutation selection is probably for altered beta-catenin function, which may significantly alter intracellular signaling and intercellular adhesion and may serve as a complement to APC mutations in the early stages of tumorigenesis.

Figure 1

(a) Sequence analysis for the cell lines Colo 201 and 205 with the corresponding wild-type sequence. These cell lines are derived from the primary and metastatic tumor in the same patient and show a missense mutation in codon 287 (indicated by arrow), which results in an A-to-G substitution. The mutation is homozygous and this case demonstrates that CTNNB1 mutation and allelic loss occurred before tumor metastasis. (b) The effects of the mutations on the primary structure of β-catenin and the level of evolutionary conservation of the domains in which the mutations are occurring. In SW 48 the missense mutation results in substitution of a highly conserved serine residue (from Drosophila to man) to a tyrosine residue. In HCT 116, a three-base deletion results in loss of a similarly conserved serine residue. HCA 46 shows substitution of a highly conserved alanine residue (from Drosophila to man) by a serine residue as a result of a missense mutation. In CACO 2 and Colo 201/205, a missense mutation results in substitution of alanine for glycine and serine for asparagine, respectively, in residues that are conserved from Xenopus to man.

Figure 2

(a–c) Coprecipitation of β-catenin in the cell lines with CTNNB1 mutations. β-catenin was precipitated and blotted respectively with antibodies to (a) E-cadherin, (b) α-catenin, and (c) APC. All cell lines showed normal coprecipitation with E-cadherin (a). SW 48 failed to coprecipitate with α-catenin (b), and HCA 46 failed to coprecipitate with APC (c). In all other cases coprecipitation was observed as expected. In two of the cell lines, CCO7 and SW1417, there was no evidence of β-catenin mRNA by RT-PCR. Normal-sized protein was demonstrated by Western blot analysis (d), thus suggesting that there was loss of message stability in these cell lines.