Abstract 2270: Metformin and the thioredoxin system influence the radiosensitivity of luminal versus basal/triple-negative phenotype breast cancer.
Authors: Zhang Yimin , Woolston Caroline M. , Storr Sarah J. , Patel Poulam M. , Morgan David AL , Martin Stewart G. 
Published: August 2014

Abstract
Patients with basal/triple-negative breast cancer have a worse prognosis to radiotherapy (RT) than those with luminal tumours. Deregulated redox proteins, such as the thioredoxin (Trx) system, often provide protection from increased oxidative stress, such as that induced by ionising radiation. We sought to determine if altered redox homeostasis, with a focus on the Trx system, could explain such differences between breast cancer phenotypes. In vitro clonogenic assays were performed to compare inherent radiosensitivity of basal (MDA-MB-231) and luminal (MCF-7) breast cancer cell lines. When irradiated in 2D conditions both lines exhibited similar response to single and fractionated irradiation. As tissue architecture can influence phenotypic behaviour, radiosensitivity was also examined when cells were irradiated in 3D (matrigel). In the 3D system, the surviving fraction (SF) of MCF-7 was significantly lower than that of MDA-MB-231 (SF 6Gy: MCF-7 0.009% v MDA-MB-231 0.15%), which is consistent with the clinical response. Metformin (used for type2 diabetes and which affects Trx expression/activity) was used to examine whether modulating Trx family expression alters radiosensitivity and whether any difference is observed between basal v luminal breast cancer cells. Western blot analysis showed that Trx and TxNIP (the endogenous inhibitor of Trx) levels were lower in MCF-7 than MDA-MB-231’s and that metformin decreased Trx expression in MCF-7 with no effect on MDA-MB-231’s. Expression of TxNIP was dramatically attenuated, in both cell lines, by metformin. In 2D assays of radiosensitivity 10mM metformin did not alter MDA-MB-231 response but substantially increased MCF-7’s (sensitizer enhancement ratio =1.5). To elucidate potential mechanisms Human Phospho-Kinase Arrays (R&D systems) were used, ± metformin, and showed that MDA-MB-231 had higher endogenous P53 activity than MCF-7, and that AMPKα1 activity (a target of metformin), was elevated in MCF-7 but inhibited in MDA-MB-231 following treatment. The expression of total and phospho-AMPKα (pAMPK) was subsequently assessed, using standard immunohistochemistry, on a tissue microarray of 166 early stage breast cancers from patients treated by breast conserving surgery and RT. Althougth pAMPK results did not reach significance high levels of total AMPKα was significantly associated with ER positive tumours (P&lt;0.0000001). Furthermore, high total-AMPKα was also associated with progression-free (P=0.021) and overall survival (P=0.000018). Results suggest that luminal breast cancer has higher inherent radiosensitivity than basal/triple-negative phenotype, that metformin radiosensitizes luminal but has no effect on basal phenotype, which may relate to the ER state, Trx system and AMPKα expression and activities of P53 and AMPKα1.
Citation Format: Yimin Zhang, Caroline M. Woolston, Sarah J. Storr, Poulam M. Patel, David AL Morgan, Stewart G. Martin. Metformin and the thioredoxin system influence the radiosensitivity of luminal versus basal/triple-negative phenotype breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2270. doi:10.1158/1538-7445.AM2013-2270