| Part?Association between genetic polymorphisms within chemokine decoy receptor genes and regional lymph node metastasis in patients with breast cancerThe inhibitory effect of two chemokine decoy receptors (CDRs), DARC and D6, on breast cancer metastasis is mainly due to their capability in sequestrating angiogenic and inflammatory chemokines. DARC is widely expressed on erythrocytes and vascular endothelial cells, and D6 is mainly expressed on lymphatic endothelial cells. Lymphatic and hematogenous dissemination are two common ways for metastasis of breast cancer. Thus, DARC and D6 serve as systemic barrier to metastasis. We hypothesized that the genetic polymorphisms within CDR genes may influence the gene expression and protein activity of CDRs, thus leading to different metastasis potential of breast cancer. To test our hypothesis, we investigated the relationship between regional lymph node metastasis (RLNM) and ten genetic variations in DARC and CCBP2 (encoding D6) in 785 breast cancer patients with negative RLNM and 678 patients with positive RLNM. There were significant association between RLNM with four studied single nucleotide polymorphisms (SNPs), i.e., rs3027012 and rs 12075 in DARC, and rs2228468 and rs 1366046 in CCBP2. After correction of multiple comparisons by false discovery rate, SNP rs 12075 continued to be significantly associated with RLNM with its A-allele significantly decreasing the risk of RLNM (OR=0.5,95% CI,0.35-0.69, P=4.0×10-4), and SNP rs2228468 tended to be significantly associated with RLNM (P=0.052). The data suggest that genetic polymorphisms within CDR genes may affect the risk of RLNM in patients with breast cancer.Part IIThe functional role of genetic polymorphisms within chemokine decoy receptor genes in metastasis potential of breast cancer The data in Part I have shown the association between RLNM in patients with breast cancer and the four genetic variants in DARC and CCBP2. rs 12075 (G42A) and rs2228468 (S373Y) located in the exons are non-synonymous SNPs, respectively. We generated'pcDNA3.1-DARC-42G','pcDNA3.1-DARC-42A','pcDNA3.1-D6-373S' and'pcDNA3.1-D6-373Y'plasmid constructs. Stable transfectants of 231-DARC-42G,231-DARC-42A,231-D6-373S,231-D6-373Y,231-DARC-42A-D6-373Y, 231-DARC-42G-D6-373S and 231-vector were generated from MDA-MB-231 cells. Significant SNPs in DARC and CCBP2 had no differential effects on gene expression in breast cancer cells in vitro, respectively. No interaction effect of these two SNPs on gene expression was observed, either. The effects of the two SNPs on their capability in sequestrating chemokines and in inducing senescence were further investigated both in vitro and in vivo. Regarding the effect of rs 12075 (G42A), the levels of CXCL8, CCL2, and CCL5 in the supernatant of 231-DARC-42A transfectants were about 30-35% lower than those of 231-DARC-42G transfectants (P=0.031, P=0.028, and P=0.027, respectively). Regarding the effect of rs2228468 (S373Y), the level of CCL2 in the supernatant of 231-D6-373Y transfectants was 16.4% lower than that of 231-D6-373S transfectants (a borderline P-value of 0.069). DARC-rs 12075 has no differential effect on senescence in vitro. The mean tumor volumes were 2.97cm3 for 231-DARC-42G xenografts and 1.80cm3 for 231-DARC-42A xenografts in five-weeks, respectively. But the difference in tumor volume for 231-D6-373Y xenografts and 231-D6-373S xenografts was not obvious. The levels of mouse CCL2 and CCL5 in 231-DARC-42A xenografts were significantly lower than those in 231-DARC-42G xenografts (P=0.034 and P=0.038, respectively). A trend was noted that the level of mouse CCL2 in 231-D6-373Y xenografts was about 11.4% lower than that in 231-D6-373S xenografts (P=0.075). The synthetic effects on chemokine sequestration were also observed in 231-DARC-42A-D6-373Y cells and xenografts expressing high levels of both DARC and D6. Furthermore, erythrocytes of 42GA heterozygote sequestrated significantly more chemokines than those of 42GG homozygote ex vivo. Taken together, our data suggest that genetic polymorphisms in DARC and CCBP2 might affect metastasis potential of breast cancer by altering the chemokine sequestrating capability of their proteins. |
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