A galectin-3 sequence polymorphism confers TRAIL sensitivity to human breast cancer cells

Abstract

Background: A common polymorphism, rs4644, coding for Pro64 or His64 of the carbohydrate-binding protein galectin-3, influences the susceptibility of galectin-3 to cleavage by matrix metalloproteinases and is associated with breast cancer incidence. Because forced expression of galectin-3 in a galectin-3 null breast cancer cell line confers sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the authors sought to determine whether the His64/Pro64 polymorphism of galectin-3 affects the sensitivity to TRAIL.

Methods: Genomic DNA of breast cell lines was analyzed for the single nucleotide polymorphism rs4644, and cytotoxicity was determined with the MTT assay.

Results: When a collection of 9 breast cancer cell lines that express galectin-3 was examined for lectin, galactoside-binding, soluble, 3 (LGALS3) genotype and sensitivity to doxorubicin and TRAIL, doxorubicin sensitivity was not found to be related to LGALS3 genotype. In contrast, none of the 5 cell lines that were homozygous for Pro64 galectin-3 were found to be sensitive to TRAIL, but 2 of 2 homozygous His64 cell lines and 1 of 2 heterozygous His64 cell lines were sensitive to TRAIL. Forced expression of galectin-3 of defined genotype in galectin-3 null cells was used to more directly test the effect of the Pro64His mutation on TRAIL sensitivity. High levels of expression of His64 galectin-3 rendered BT549 cells sensitive to TRAIL and resistant to doxorubicin, but cells expressing Pro64 galectin-3 remained resistant to TRAIL and sensitive to doxorubicin.

Conclusions: The results of the current study indicate that the naturally occurring Pro64His mutation in galectin-3 increases sensitivity to death receptor-mediated apoptosis. This finding could be relevant to disparities in breast cancer outcomes across population groups, and could guide the design of future clinical trials of TRAIL-based therapies.

Figure 1

Genotype and expression levels of galectin-3 in breast cancer cell lines. LGALS3 genotype at the rs4644 locus was determined by sequence analysis. PP denotes homozygous Pro64; HH, homozygous His64; and HP, heterozygous. Expression of the galectin-3 protein was determined by Western analysis.

Figure 2

Sensitivity of breast cancer cells to TRAIL and doxorubicin. Cells of homozygous His64 genotype (open bars), heterozygous (hashed bars) or homozygous Pro64 (solid bars) were treated 4h with 100 ng/ml TRAIL (A) or overnight with 5 ug/ml doxorubicin (B) and cell viability was determined with the MTT assay.

Figure 3

Forced expression of galectin-3 of defined genotype in BT549 galectin-3 null breast cancer cells. Western blot detection of galectin-3, PTEN, Akt, and phospho-Akt in total cell lysates of stable transfectants. BT, BT549 parental cells; VC, control containing vehicle plasmid; gal25A and gal25B, His64 galectin-3-expressing clones; 25C, B3, and C2 Pro64 galectin-3-expressing clones. Beta-actin expression is shown as loading control.

Figure 4

Sensitivity to TRAIL and resistance to doxorubicin conferred by expression of His64 galectin-3 but not Pro64 galectin-3. Parental BT549 cells and BT549 transfectants expressing vehicle plasmid (BT and VC, open bars) or transfectants expressing His64 galectin-3 (25A and 25B, hashed bars) or Pro64 galectin-3 (25C, B3, and C2, solid bars) were treated 4h with TRAIL (A) or overnight with doxorubicin (B) and cell viability was measured by the MTT assay. Stippled bars represent dead cell in the absence of TRAIL or doxorubicin.