The Biological and Clinical Relevance of EVI1 Expression in Prostate Carcinogenesis

肿瘤干细胞（CSCs）是癌症患者肿瘤发生、进展和治疗耐药的重要调节因子。我们和其他研究小组的前期工作表明，胚胎干细胞蛋白SOX2是卵巢癌、乳腺癌和前列腺癌（PCa）的CSC标志物，并且在肺癌和不同类型的鳞状癌中起致癌作用。在这篇论文中，我们建议通过研究干细胞转录调控因子Ecotropic viral integration site 1 （EVI1）来扩展我们对PCa干细胞分子调控的知识。EVI1主要在健康和恶性造血干细胞中被研究。在急性髓性白血病（AML）中，高EVI1表达与特别不良的临床结果相关。尽管EVI1在包括PCa在内的多种实体肿瘤中表达，但关于EVI1在实体肿瘤中的报道很少，在PCa中几乎没有报道。我们实验室的初步数据表明，在人类PCa中，EVI1是一种干细胞标记物，也增强了疾病的侵袭性。因此，我们建议分析EVI1在PCa中的表达、作用和分子靶点，并特别关注其与PCa csc的关系。我们将分析多达四个独立的PCa患者队列中的EVI1蛋白表达，这些患者包括原发肿瘤、局部淋巴结和激素难治性远处转移或仅原发肿瘤，并研究高EVI1表达是否可能代表PCa进展和预后的新生物标志物。接下来，我们将探讨扩增、易位或激活突变是否会导致EVI1蛋白过表达。利用我们实验室现有的SOX2调控区1和2的慢病毒报告系统、先前发表的表型标记和醛脱氢酶（ALDH）活性测定，我们将探索EVI1表达与已建立的PCa干细胞标记之间的关系。我们将利用慢病毒构建的EVI1表达修饰的人PCa细胞，探讨EVI1表达在PCa细胞中介导的潜在功能（包括维持PCa干细胞的特性、生长、增殖、抗凋亡、迁移、侵袭和体内致瘤性）。体内致瘤性实验将在已建立的NSG小鼠异种移植实验中进行，并分别使用新开发的斑马鱼异种移植模型进行。通过RNA-Seq和ChIP分析，在PCa细胞中探索EVI1的分子靶基因，以确定可药物靶点。先前报道的相关靶点通路（如Smad3/ tgf - β， BCL蛋白）和三氧化二砷（ATO）最近被报道降低白血病细胞中EVI1蛋白的稳定性，将在体外和最终在体内探索其在PCa细胞中抑制EVI1介导的on- genic效应的功效。该项目的数据将提高我们对前列腺癌分子发病机制的理解，并有助于个性化治疗的发展。

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.