Elevated Aromatase (CYP19A1) Expression Is Associated with a Poor Survival of Patients with Estrogen Receptor Positive Breast Cancer

Abstract

Genetic variants in CYP19A1, the gene encoding aromatase, have been reported to be associated with circulating estrogen concentrations, a key risk factor for breast cancer. The mechanism underlying this association is still unclear; it has been suggested that some of these variants may alter the expression and/or activity of aromatase. Here we analyzed the expression of intra-tumoral CYP19A1 messenger RNA (mRNA) and the genotypes of rs10046, a well-characterized single nucleotide polymorphism in CYP19A1, in 138 breast cancer patients and 15 breast cancer cell lines. The genotype TT was detected in 36 patients and six cell lines, genotype CT in 55 patients and five cell lines, and genotype CC in 28 patients and four cell lines. We found no evidence for a significant association of CYP19A1 levels with rs10046 genotypes, although expression tended to be higher in tumors and cell lines with the homozygous risk genotype TT. We also found no evidence for a significant association of rs10046 genotypes with breast cancer prognosis. In contrast, high CYP19A1 expression was highly significantly associated with a poor overall, disease-free, and metastasis-free survival in estrogen receptor-positive but not negative breast cancer patients. Moreover, CYP19A1 mRNA was significantly elevated in postmenopausal patients and in patients older than 50 years, and a trend towards a positive correlation with ER status and ESR1 mRNA expression was observed. These findings highlight the key role of aromatase in estrogen receptor-positive breast cancer biology.

Fig. 1

Association of CYP19A1 mRNA expression with a, b rs10046 SNP genotype, c, d ER (estrogen receptor) status, and e, f ESR1 (estrogen receptor 1) mRNA expression. Analyses are in human primary breast tumors (a, c, e) and in human breast cancer cell lines (b, d, f). CC, CT, and TT are the genotypes of SNP rs10046. The numbers of patients and cell lines in each group (n) are shown in parentheses. The y-axes in a–f show normalized relative CYP19A1 mRNA levels (log(2) values). P values (P, in parentheses above each panel) were determined by a, b ANOVA, c, d unpaired, two-sided t tests, and e, f Spearman’s rank correlation. r, Spearman’s rho (rank correlation coefficient)

Fig. 2

CYP19A1 mRNA expression in human untransformed breast epithelial and breast cancer cell lines. The y-axis shows mean relative CYP19A1 mRNA levels (log(2) values) of duplicate measurements. Expression levels of the indicated individual untransformed cells (Normal, black bars), breast cancer cell lines with an epithelial morphology (Epithelial, white bars), and breast cancer cell lines with a fibroblastoid morphology in culture (Fib, hatched bars) are shown. For normalization, the mean expression level of the four untransformed cell lines was set to unity (i.e., 2⁰ in graph), and the levels of individual cell lines are presented relative to those. Boxes underneath the bar graph indicate a positive (black) or negative (white) status of ER, PR, and CDH1 (E-cadherin). ER and PR status are based on [45, 46], and CDH1 status is based on determination of mRNA levels by qRT-PCR. Gray, status not available

Fig. 3

Association of CYP19A1 mRNA expression with established clinical and histopathological parameters. Boxplots according to a age at breast cancer onset in years, b menopausal status, c tumor type, d tumor size according to TNM classification, e tumor stage, f tumor grade, g lymph node status, h in paired primary tumors and lymph node metastases (LN) of 17 patients, i p53 status, j progesterone receptor (PR) status, k HER2-status, and l molecular subtype, i.e., HER2-type (HER), Luminal A (Lu.A), Luminal B (Lu.B), and triple negative (TN). neg, negative; pos, positive. The numbers of patients in each group (n) are shown in parentheses. The y-axes show normalized relative CYP19A1 mRNA levels (log(2) values). P values (P, in parentheses above each panel) were determined by e, l ANOVA; h paired, two-sided t tests; and unpaired, two-sided t tests in all other panels

Fig. 4

Association of CYP19A1 mRNA expression with the survival of human breast cancer patients. Kaplan-Meier analyses of the overall survival (a, d, g), disease-free survival (b, e, h) and metastasis-free survival (c, f, i) in unselected patients (a–c; n = 100), ER-positive patients (d–f; n = 40), and ER-negative patients (g–i; n = 56) are shown. Patient subgroups with high and low CYP19A1 expression and their numbers (n) are indicated in each panel. ER, estrogen receptor