Abstract A18: Inflaming breast cancer development: Expression of aromatase is increased by TNFα via the early growth response genes

Introduction: Breast cancer remains the leading cause of cancer-related death in Western women. In postmenopausal cases, up to 70% of tumors are classed as estrogen receptor positive (ER+), dependent on estrogen for continued growth and proliferative advantage. Adjuvant antiestrogen therapies are considered the cornerstone approach to the treatment of such tumors, and as such research is ongoing to maximize the effectiveness of drug treatments. The major source of estrogens for growth of ER+ breast cancers is local conversion of androgen precursors by the enzyme P450 aromatase. Inflammatory factors such as tumor necrosis factor-α (TNFα) stimulate transcription of the CYP19A1 gene that encodes aromatase, via its adipose-specific promoter I.4 (PI.4). The pathways by which this is achieved are not fully understood. Increased concentrations of TNFα are frequently detected within the stromal and epithelial compartments of an ER+ breast tumor microenvironment, contributing to a more invasive tumor fed by the increases in local estrogen production which are, in part, being driven by this critical cytokine. This study aims to identify the mechanisms underlying TNFα-dependent aromatase induction in the context of estrogen-dependent breast cancer. Methods: A custom gene array was performed to assess mRNA changes in primary human breast adipose fibroblasts (BAFs) that had been treated for 1 hr with 5ng/ml TNFα. This data was validated by qRT-PCR. Luciferase reporter assays were performed in C0S7 cells and EMSA assays were carried out using γP-32 detection. Results: A custom array showed that mRNA expression of the early growth response gene Egr1 was increased in BAFs that had been treated for 1 hr with TNFα. The family of early growth response genes Egr1, Egr2, Egr3 and Egr4 were all found to be significantly upregulated in a time-dependent manner in response to TNFα treatment, as validated by qRT-PCR. This occurs via induction of the NFkB pathway, as Egr mRNA induction was reduced in a dose-dependent manner upon pathway inhibition with the IKK-inhibiting compound BAY-11-7082. Egr2, Egr3 and Egr4 were able to induce Pl.4-driven luciferase reporter activity, with Egr2 proving to be the most potent activator. Serial 5′ deletion analysis of the 1 kb PI.4 promoter region revealed that the Egr2-induced promoter activity was mediated by a 41 bp sequence at the 3′ end, a region that did not contain any putative Egr binding sites. EMSA confirmed that there was no binding of any of the four Egr transcription factors to the 41 bp region. Mutagenesis of the 41 bp fragment is being carried out to narrow down the specific region of interest, and novel transcription factor binding sites will be confirmed with further luciferase assays and EMSA. Conclusion: We have elucidated a novel pathway by which TNFα is able to induce aromatase expression and thus estrogen biosynthesis in the breast via induction of the NFkB pathway and early growth response genes. Understanding TNFα signalling and the transcription factors it activates to turn on PI.4 CYP19A1 expression in BAFs is vital to the understanding of breast cancer pathology. These insights may help in the development of diagnostic tools or novel therapeutics in order to tackle the growing instances of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A18.