Association of single nucleotide polymorphisms in angiogenesis genes with expression of VEGF and VEGFR-2 in breast carcinoma.

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Background: There is vast heterogeneity in the tumor expression of angiogenic proteins, namely Vascular Endothelial Growth Factor (VEGF) and its cognate receptor VEGF Receptor-2 (VEGFR-2). Single nucleotide polymorphisms (SNPs) are widely known to be a causative factor in the regulation of the expression of various genes. In this study, we sought to associate whether or not SNPs in the Hypoxia Inducible Factor-1α (HIF-1α), VEGF, VEGFR-1, VEGFR-2, endothelial Nitric Oxide Synthase (eNOS), Neuropilin-1 (NRP-1), or NRP-2 genes influence expression of angiogenic proteins in breast cancer tissues.
 Methods: Forty-four cases of breast tumor tissues were obtained from the pathology archives at Indiana University. ER/PR, HER2 status as well as histologic grade were obtained for all specimens. DNA was extracted from tumor blocks, and genotyping was performed for the following SNPs: HIF-1α (P582S, A588T), VEGF (-2578C/A, -1498C/T, -1154 G/A, -634G/C, & 936C/T), VEGFR-1 (-962C/T), VEGFR-2 (V297I & Q472H), eNOS(-786T/C, E298D), NRP-1(F561L, V733I), NRP-2 (K123R). Subsequently, immunohistochemistry (IHC) for VEGF & VEGFR-2 was performed and an H-Score for each sample was determined. T-test and Fisher’s exact test were used to assess associations between polymorphisms and protein expression. IHC for microvessel density as well as mRNA quantification will be assessed.
 Results: The VEGF-2578 A/-1498 C/-1154 G/-634 G/+936 C haplotype (freq.= 14%, H-Score=52) resulted in a significantly lower mean VEGFR-2 IHC H-Score when compared to the other two common haplotypes CTGGC (freq.= 28%, H-Score=124, p=.026) and CTGCC (freq.= 21%, H-Score=141, p=.012). No other associations between SNPs and protein expression of VEGF & VEGFR-2 were observed. Associations between genotype, protein expression, & microvessel density with known clinical variables are planned.
 Conclusion: Our data support a novel association between the VEGF-2578 A/-1498 C/-1154 G/-634 G/+936 C haplotype and decreased VEGFR-2 expression in breast cancer. Previous reports of these SNPs have not shown association between polymorphic variability in the promoter of the VEGF ligand and its expression of its cognate receptor VEGFR-2. Elucidation of the mechanisms of polymorphic induced cross-talk between VEGF & VEGFR-2 is ongoing.