Abstract A45: <i>EVI1</i> – a novel oncogene in breast carcinoma
Authors: Hui Wang, Thorsten Schäfer, Martina Konantz, Selina Reich, Martin Braun, Sven Perner, Zsuzsanna Varga, Holger Moch, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke
Journal: Cancer Research (2016)

Abstract Ecotropic viral integration site 1 (EVI1) is a nuclear transcription factor essential for the maintenance of hematopoietic stem cells. Aberrant expression of EVI1 is found in ~10% of acute myeloid leukemia, where it associates with particularly aggressive disease. EVI1 is also expressed in solid tumors, but remains largely unstudied in this setting. A recent analysis of primary breast carcinoma (BC) samples suggests that enhanced EVI1 expression indicates poor outcome specifically in estrogen receptor (ER) negative tumors (Patel et al., 2011). Here, we explored expression, functional roles and molecular targets of EVI1 in human BC. EVI1 expression was analyzed by qRT-PCR and immunoblot analysis in 8 human BC cell lines and by immunohistochemistry in a cohort of 373 primary BC patient samples collected at the University Hospital Zurich. EVI1 was found to be expressed in most BC samples independently of their ER status. Long-term survival was influenced by EVI1 in ER-, but not ER+ patients, confirming previous results. To investigate the function of EVI1 in BC, cell lines with high and low endogenous EVI1 expression were transduced with EVI1 shRNA knockdown or inducible EVI1-expressing lentiviral particles. Growth, proliferation and cell cycle assays demonstrated that EVI1 promoted BC cell growth by accelerating cell cycle transition. Consistently, EVI1 knockdown BC cells showed reduced in vivo tumor growth compared to control cells in two xenotransplantation models (NSG mice and zebrafish embryo). Interestingly, estrogen supplementation rescued proliferation and tumorigenicity of EVI1-knockdown ER+ but not ER- BC cells, indicating that signaling through the ER pathway might override EVI1-dependent effects on proliferation. Conversely, EVI1-induced proliferation might be particularly important for tumors that are not regulated via the ER pathway, which is in line with its documented prognostic significance in such tumor types. In addition to its effects on proliferation, EVI1 was also found to inhibit BC cell apoptosis in response to both intrinsic and extrinsic stimuli, as previously shown by our group in acute lymphoblastic leukemia cells (Konantz et al, 2013). Moreover, in vitro migration assays showed impaired migration in EVI1 knockdown MDAMB231 cells suggesting that EVI1 regulates BC biology via different mechanisms. Currently, we are exploring the molecular mechanisms by which EVI1 mediates these different effects. Microarray analysis revealed molecules of the MAPK pathways, pro-apoptotic genes and, most interestingly, genes involved in GPCR signaling pathway (e.g. KISS1) as most significantly regulated by EVI1. Analysis of the KISS1 promoter revealed several potential EVI1-binding sites, and a direct association of EVI1 protein with one of these could be confirmed by CHIP. We are currently further exploring the relevance of this binding site for EVI1-mediated activation of KISS1 using luciferase KISS1 reporter assays and site-directed-mutagenesis. In ER- BC, KISS1 expression was reported to enhance cell motility and invasiveness and to reduce cell adhesion. Interestingly, the impaired migration observed in EVI1 knockdown MDAMB231 cells could be indeed rescued by stimulation with Kisspeptin. Neither EVI1 overexpression nor stimulation with Kisspeptin could influence the poorly migrating ER+ MCF7 cells. Taken together, our data identify EVI1 as a novel oncogene particularly important for ER- BC biology. Next to stimulation of BC cell proliferation and reduction of apoptosis sensitivity, we show that EVI1 stimulates ER- BC cell migration via activation of its direct molecular target KISS1. Citation Format: Hui Wang, Thorsten Schäfer, Martina Konantz, Selina Reich, Martin Braun, Sven Perner, Zsuzsanna Varga, Holger Moch, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke. EVI1 – a novel oncogene in breast carcinoma. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A45.