Racial disparity in breast cancer and functional germ line mutation in galectin-3 (rs4644): a pilot study

Abstract

For reasons largely unknown, Caucasian women are at a significantly higher risk of developing breast cancer than Asian women. Over a decade ago, mutations in BRCA1/2 were identified as genetic risk factors; however, the discovery of additional breast cancer genes and genes contributing to racial disparities are lacking. We report a functional germline mutation (polymorphism) in the galectin-3 gene at position 191 (rs4644) substituting proline with histidine (P64H), which results in susceptibility to matrix metalloproteinase cleavage and acquisition of resistance to drug-induced apoptosis. This substitution correlates with incidence of breast cancer and racial disparity. Genotype analysis of 338 Caucasian (194 disease free and 144 breast cancer patients) and 140 Asian (79 disease free and 61 breast cancer patients) women showed that the allele homozygous for H64 exists in disease free Caucasian and Asian women at a frequency of 12% and 5%, respectively, versus 37% and 82% in breast cancer patients. The data indicate that H/H allele is associated with increased breast cancer risk in both races. The data implicate galectin-3 H(64) in breast cancer and explain, in part, the noted racial disparity, thus providing a novel target for diagnosis and treatment.

Figure 1

A. Distribution of nsSNP rs4644 among cell lines and their tumorigenicity. B. Multiple sequence alignment for galectin-3 of chicken and a few mammalian species. The dots in alignment represent gaps. Grey boxes show the identity between the proteins. X represents either H or P. The numbers represent the position of amino acid in human galectin-3.

Figure 2

Genotype distribution of nsSNP rs4644 in normal population by race. A. Genotype distribution of rs4644 from 144 cancer-free Caucasian and 20 Asian volunteer women. B. Data from 50 Caucasian and 59 Asian unrelated women were retrieved from Ensembl release 46 (from the database we have excluded family related members). (http://www.ensembl.org/Homo_sapiens/snpview?snp=rs4644). Black bar: H- homozygous alleles; lined bar: H/P- heterozygous alleles; Open bar: P- homozygous alleles. Inset: Breast cancer incidence by race. Rates are expressed as cases per 100,000. Statistics were generated from malignant cases only. Surveillance, Epidemiology, and End Results (SEER) Program (www.seer.cancer.gov) SEER Stat Database: Incidence - SEER 17 Regs Limited-Use, Nov 2006 Sub (1995-2004), National Cancer Institute, DCCPS, Surveillance Research Program, Cancer Statistics Branch, released April 2007, based on the November 2006 submission.

Figure 3

Genotype distribution of nsSNP rs4644 in breast cancer patients by race. A. Genotype distribution of rs4644 from 141 Caucasian and 61 Asian breast cancer patients was determined using independent sequencing and RFLP analyses. B. RFLP and sequence analysis of the C191A site of the galectin-3 gene. 324-bp amplified DNA was digested with NcoI and the resulting fragments were separated by electrophoresis on a 2% agarose gel at 100 V for 30 min. PCR product containing the H allele can be digested by NcoI creating two fragments of 171 and 153 bp length (lanes H/H and H/P). Sequencing of the galectin-3 gene around the C191A site was performed in Applied Genomics Technology Center using the sense primer (inset: DNA sequence chromatograms indicating allelic variation). The odds ratio of breast cancer for the H/H genotype compared to the P/P genotype for Caucasian and Asian patients were 2.7; 95% CI- 1.4-4.9, P=0.001 and 94.6; 95% CI, 10.0-892.4, P<0.001 respectively.

Figure 4

Cisplatin induced apoptosis of BT-549 cells and cloned variants. I: Percent apoptosis in cells treated with 25uM cisplatin for 72 hr at 37°C and analyzed following MTT assay. ODs in the untreated control cells were given a value of 100 and relative percent apoptosis was calculated as 100%, and the values are the average of triplicate experiments. Bars represent ±SD. * P>0.001 II: Western blot analysis of the expression of PARP in cells treated with 50 μM cisplatin after 24, 48, and 72 hr. A: BT-549; B: BT-549 Gal-3 H⁶⁴; C: BT-549 Gal-3 P⁶⁴. β-Actin was used as loading control. 1- Untreated; 2- 24 hr; 3- 48 hr; 4- 72 hr. Data shown is representative of three clones for each variant. III: Immunohistochemical analysis of intact and cleaved galectin-3 in breast cancer progression. A-C: Intact protein was visualized using monoclonal anti-galectin-3 antibody TIB166; A’-C’: full length plus cleaved galectin-3 using polyclonal galectin-3 antibody hL31. For detailed description see(10). A-A’: Normal breast tissue; B-B’: Ductal hyperplasia; C-C’: Infiltrating lobular carcinoma. Arrows in B’ and C’ indicate the cleaved protein. Representative pictures are presented from a study of 5 normal breast tissues, 10 lobular hyperplasia and 8 infiltrating lobular carcinoma cases. x200