Dysregulated metabolism and the regulation of aromatase in breast adipose stromal cells in obesity and cancer

Abstract

Background The risk of breast cancer in postmenopausal women is increased two-fold with obesity, and the majority of breast tumours that arise are oestrogen-dependent. After menopause, when the ovaries cease to produce oestrogens, it is the local production of oestrogens within the breast adipose stromal cells (ASCs) which promotes and sustains tumour growth. This is largely due to the increased expression of aromatase, responsible for the conversion of androgens to oestrogens. Aromatase expression in breast cancer is known to be under the control of a proximal promoter, promoter PII, which is maximally activated by cAMP-dependent mechanisms. We have previously demonstrated that the LKB1/AMPK pathway is a key negative regulator of aromatase expression within the breast by inhibiting the nuclear translocation of the CREB co-activator CRTC2 [1]. We have also demonstrated that the tumour-derived factor prostaglandin E2 (PGE2) and the obesity-associated factor leptin stimulate aromatase expression by inhibiting LKB1 and AMPK expression and activity. Hypoxia inducible factor-1a (HIF1a) is emerging as a potent regulator of glycolysis in tumour cells and we have identified a putative hypoxia response element in aromatase promoter Pll immediately adjacent the cAMP response element, known to be bound by the CREBCRTC2 complex in ASCs in breast cancer. We therefore hypothesise that HIF1a may be involved in regulating aromatase in breast cancer. Materials and methods Primary human breast ASCs were isolated from tissue after breast reduction surgery. Real-time PCR, Western blotting, immunofluorescence and high content screening were used to assess HIF1a expression and localisation after PGE2 treatment. Chromatin immunoprecipitation (ChIP) was performed to examine the interaction of HIF1a with aromatase promoter PII and reporter assays were performed to assess the effect of HIF1a on PII activity. Double immunohistochemistry for HIF1a and aromatase was also performed on sections of formalin-fixed paraffin-embedded breast tissue from breast cancer patients and cancer-free patients.