Abstract 4561: Dissecting mechanisms of CART signalling through the estrogen receptor in ER+ breast cancer

Abstract

Abstract The cocaine- and amphetamine-regulated transcript (CART) was first discovered as a peptide up-regulated by the administration of cocaine and amphetamine to rats. CART peptides are involved in regulating physiological processes, such as feeding, reward and neuro-protection. However, recent studies have associated high CART expression with worse overall survival in patients with small-bowel carcinoid tumours [1] and estrogen receptor-positive (ER+), lymph node-negative breast cancer [2]. Interestingly, in ER+ breast cancer, CART was also shown to be associated with poor patient response to tamoxifen, suggesting CART may play a role in conferring tamoxifen resistance [2]. The aim of this study was to elucidate the mechanism(s) by which CART signals in ER+ breast cancer. In order to test whether CART could mediate the ligand-independent activation of ER-alpha, MAP-Kinase pathway activation and levels of downstream gene-targets of ER-alpha were assessed post CART stimulation. Additionally, the ability of CART to activate ER-alpha using three LXD motifs (nuclear receptor co-activator recognition motifs) present within the CART sequence was also assessed. This was achieved through selectively mutating these motifs and testing whether CART still possessed the ability to activate ER-alpha using an ERE-Dual luciferase reporter assay. Treatment of cells with CART demonstrated an increase in MAP-Kinase activity through the detection of increasing phosphorylated ERK levels. An increase in the phosphorylation of ER-alpha at serine 118 (a phosphorylation site thought to be involved in tamoxifen resistance) was also evident following CART stimulation. CART stimulation also resulted in an increase in levels of the progesterone receptor, a known ER-alpha gene target. Mutagenesis of each LXD motif within CART resulted in significant decreases in ER-alpha activity, suggesting a potential structure-function relationship between CART and ER-alpha. In conclusion, we suggest that CART can activate ER-alpha in a ligand-independent manner through the MAP-Kinase pathway, and also potentially through specific LXD motifs within its sequence. Further investigation into the relationship between CART and ER-alpha will help us gain a better understanding of, not only the potential structure-function relationship between CART and ER-alpha, but also the role CART plays with regards to tamoxifen resistance in ER+ breast cancer. [1] Landerholm K et al., Expression of cocaine- and amphetamine-regulated transcript is associated with worse survival in small bowel carcinoid tumors. Clin Cancer Res. 2012 Jul 1;18(13):3668-76 [2] DJ Brennan, DP O’Connor et al., The cocaine- and amphetamine-regulated transcript mediates ligand-independent activation of ERα, and is an independent prognostic factor in node-negative breast cancer. Oncogene 2011 Dec 5. Doi: 10.1038/onc.2011.519 Citation Format: Brian Mooney, Sudipto Das, Rut Klinger, William Gallagher, Darran O’Connor. Dissecting mechanisms of CART signalling through the estrogen receptor in ER+ breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4561.