Impact of pharmacogenetic CYP2B6 variants on steroid hormone metabolism in breast cancer cells

Background

Cytochrome P450 2B6 (CYP2B6) is expressed in breast cancer (BC) and the variants CYP2B6*4 (rs2279343) and CYP2B6*9 (rs3745274), in combination termed CYP2B6*6, were shown to correlate with increased risk for steroid hormone-dependent prostate and breast cancer, while the variant CYP2B6*5 (rs3211371) was associated with poor prognosis in hormone receptor positive BC. CYP2B6 is involved in the metabolism of about 8 % of common drugs and steroid hormones like testosterone (T) and estrogen (E2). E2 is associated with BC risk and has been shown to induce CYP2B6 expression via estrogen receptor in BC cells. CYP2B6 has been reported to hydroxylate E2 to 2-OH-E2, which in contrast to the CYP1B1 metabolite 4-OH-E2 seems not to affect BC carcinogenesis. CYP1B1 is also expressed in BC and is strongly associated with BC risk and poor prognosis, among others due to 4-OH-E2 formation. Metabolism of T by CYP2B6 to 16α- and 16β-OH-T has been shown, the latter not being converted to E2 by aromatase. There is lack of knowledge about the impact of pharmacogenetic CYP2B6 variants on metabolism of these steroid hormones, which might influence BC risk and progression. We aimed to reproduce reported steroid hormone metabolism of CYP2B6 compared to CYP1B1 and further examined potential differences in E2 and T metabolite formation between different CYP2B6 variants.

Methods

Isolated CYP2B6 and CYP1B1 enzymes were incubated with E2 and T to assess which metabolites are formed. Potential differences in E2 and T metabolism by CYP2B6 variants was assessed with newly established T47D cell lines stably overexpressing either GFP (control), CYP2B6*1 (wildtype), *4, *5, *6 or *9. The expression was validated using quantitative real-time PCR (qPCR) and western blot. T47D cell lines were incubated with E2 or T and metabolite formation was quantified  by LC-MS/MS. Metabolite levels were normalized to the expression levels of different CYP2B6 variants by western blot.

Results

Incubation of E2 with isolated CYP2B6 or CYP1B1 enzymes showed 4-OH-E2 to be mainly formed by CYP1B1, but not CYP2B6 (approx. 1000-fold difference). CYP2B6 formed about 5-fold more 2-OH-E2 than 4-OH-E2. Metabolism of T by CYP1B1 resulted in formation of 16α-OH-T, while CYP2B6 only formed 16β-OH-T instead. 16α-OH-T formation by CYP2B6 was also detected, but the signal was below limit of quantification. First experiments with CYP2B6 variants overexpressed in T47D cells revealed altered activities regarding E2 or T metabolism. Compared to CYP2B6 wildtype, CYP2B6*9 showed an about 2-fold increased 2-OH-E2 and about 3-fold higher 16β-OH-T formation. Also CYP2B6*5 showed an approx. 1.5-fold higher 2-OH-E2 formation than CYP2B6*1.

Discussion

Our findings indicate that CYP2B6 is involved in T and E2 metabolism mainly forming the metabolites 16β-OH-T and 2-OH-E2, while CYP1B1 forms 16α-OH-T and 4-OH-E2. Since 16α-OH-T is converted to E2 while 16β-OH-T is not, and 2-OH-E2 shows lower carcinogenicity than 4-OH-E2, CYP2B6 derived metabolites seem to be less harmful than those of CYP1B1. In our results the CYP2B6 alleles CYP2B6*5 and CYP2B6*9, which were previously associated with BC risk or poor prognosis, showed an increased steroid hormone metabolism of E2 and T. Thus, our results indicate that a misbalanced steroid hormone metabolism may influence BC risk or development. Further experiments will be conducted to systematically address K_m and V_max values for the different CYP2B6 variants.