The Biological and Clinical Relevance of EVI1 Expression in Prostate Carcinogenesis

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Cancer stem cells (CSCs) are essential regulators of tumor initiation, progression and therapy resistance in cancer patients. Previous work from our and other groups indicate that the embryonic stem cell protein SOX2 is a CSC marker in ovarian, breast and prostate carcinoma (PCa), and furthermore plays oncogenic roles in lung and different types of squamous carcinomas. In this proposal, we propose to expand our knowledge on the molecular regulation of PCa stem cells by investigating the transcriptional stem cell regulator Ecotropic viral integration site 1 (EVI1). EVI1 has been mainly studied in healthy and malignant blood stem cells. In acute myeloid leukemia (AML), high EVI1 expression associates with particularly adverse clinical outcome. Even though EVI1 is expressed in several solid tumors including PCa, only few reports are available on EVI1 in solid tumors, and almost none in PCa. Preliminary data from our labs suggest that, in human PCa, EVI1 is a stem cell marker and enhances disease ag-gressiveness as well. We therefore propose to analyze expression, roles and molecular targets of EVI1 in PCa with particular focus on its relationship to PCa CSCs. We will analyze EVI1 protein expression in up to four independent PCa patient cohorts consisting either of primary tumors, localized lymph node and hormone-refractory distant metastases or only of primary tumors, and investigate whether high EVI1 ex-pression might represent a novel biomarker for PCa progression and prognosis. Next, we will explore whether amplification, translocation or activating mutations result in EVI1 protein overexpression. Using a lentiviral reporter system for the SOX2 regulatory regions 1 and 2 available in our lab, previously pub-lished phenotypic markers and aldehyde dehydrogenase (ALDH) activity assays, we will explore the as-sociation between EVI1 expression and established PCa stem cell markers. Potential functions mediated by EVI1 expression in PCa cells (including maintenance of PCa stem cell identity, growth, proliferation, apoptosis resistance, migration, invasion and in vivo tumorigenicity) will be explored using human PCa cells with modified EVI1 expression generated using lentiviral constructs. In vivo tumorigenicity assays will be performed in established NSG mouse xenograft assays, and respectively using a newly developed zebrafish xenograft model. Molecular target genes of EVI1 will be explored in PCa cells via RNA-Seq and ChIP analyses to identify drugable targets. Previously reported relevant target pathways (e.g. Smad3/TGF-beta, BCL proteins) and arsenic trioxide (ATO), which has recently been reported to reduce EVI1 protein stability in leukemic cells, will be explored for their efficacy to inhibit EVI1-mediated on-cogenic effects in PCa cells in vitro and eventually also in vivo.The data emerging from this project will improve our understanding of the molecular pathogenesis in PCa and contribute to the development of personalized treatment.