Abstract 2015: Novel oncogenic roles of EVI1 in breast carcinoma
Authors: Wang Hui , Schaefer Thorsten , Konantz Martina , Reich Selina , Braun Martin , Perner Sven , Varga Zsuszanna , Moch Holger , Fehm Tanja , Kanz Lothar , Schulze-Osthoff Klaus , Lengerke Claudia 
Published: July 2016

Ecotropic viral integration site1 (EVI1) is a transcriptional regulator that is essential for the maintenance of hematopoietic stem cells and, when overexpressed in leukemia, associates with particularly aggressive disease. EVI1 expression has also been detected in a number of solid tumors including breast carcinoma (BC) where it was recently shown to predict poor survival in estrogen receptor negative (ER-) but not positive (ER+) tumors. The functional roles and molecular regulation of EVI1 in human BC are largely unexplored.

Here we confirm that EVI1 is widely expressed in both ER- and ER+ BC in a tissue microarray containing 373 individual patient samples. Unlike in leukemia, overexpression of EVI1 in BC rarely resulted from gene amplification or translocation events at its chromosomal locus 3q26. Functionally, lentiviral knockdown of EVI1 reduced proliferation, motility and in vivo tumorigenicity of human BC cells while enhancing their apoptosis sensitivity. Interestingly, estrogen supplementation rescued growth and tumorigenicity in EVI1-knockdown ER+ (but not ER-) cells. Since microarray analyses of EVI1 knockdown cells revealed alterations of several regulators of cell cycle progression (e.g. CDKN1A, CDKN1C, CDK1), apoptosis (e.g. BIK, BMF, BBC3) and growth factor signaling (e.g. NRG2, EREG, PTK2B), we further investigated the canonical MAPK pathway as a major effector axis of EVI1 in BC. Indeed, knockdown of EVI1 in BC cells effectively impaired ERK1/2 phosphorylation (i.e. MAP kinase activation), which was phenotypically reflected by impaired in vitro proliferation and in vivo tumorigenicity rates. In ER+ BC cells, these effects were functionally restored by exogenous addition of estradiol, explaining why EVI1 gains particular clinical significance in the absence of estrogen signaling (i.e. in ER- BC). Supporting this notion, ectopic expression of EVI1 and estradiol treatment co-induced p-ERK1/2. Our microarray analysis further revealed GPCR signaling molecules (e.g. CCR1, GCGR, PTGFR) as a fourth category of genes significantly modulated by EVI1 in BC. Among these, the GPR54-ligand KISS1, previously implicated in cell migration, was most strongly modulated by EVI1 knockdown and also overexpression. A corresponding promoter analysis indicated several potential EVI1-binding sites within KISS1 regulatory elements, of which two showed robust recovery rates in chromatin immunoprecipitation (ChIP) assays. Functionally, EVI1 knockdown impaired BC cell migration, which was restored by addition of Kisspeptin, a soluble gene product of KISS1.

Overall, our data characterize EVI1 as a novel oncogene in BC that impacts growth and migration via pERK and the GPR54-ligand KISS1, which we identified as a novel direct transcriptional target of EVI1-driven oncogenesis.

Citation Format: Hui Wang, Thorsten Schaefer, Martina Konantz, Selina Reich, Martin Braun, Sven Perner, Zsuszanna Varga, Holger Moch, Tanja Fehm, Lothar Kanz, Klaus Schulze-Osthoff, Claudia Lengerke. Novel oncogenic roles of EVI1 in breast carcinoma. [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 2015.