Regulation of Tumor Cell Dormancy in Human Prostate Cancer Bone Metastasis

这个项目的总体目标是提高我们对调控PCa骨转移瘤生长或休眠的机制的理解。这是一个主要的临床问题，因为它解释了在不可预测的微小残留病期后晚期转移复发的原因。我们的初步数据，主要基于我们新建立的人自发性骨转移异种移植模型，以及文献共同表明，PCa骨转移的生长可能受到可溶性因子Sparc和Dkk1的旁分泌和/或自分泌相互作用的抑制，这种相互作用明显地通过促进转移细胞的EMT表型(EMT=上皮向间充质转化)来抑制肿瘤的定植。因此，该项目的第一个目标将是首次获得直接的功能证据，证明在我们的自发转移的PCa异种移植模型(WP1)中，Sparc或Dkk1的基因修饰(例如，可诱导的敲除)是否会改变骨髓(BM)的定植。我们的初步工作表明，我们的模型紧密地概括了EMT的过程，它们具有临床和功能相关性，并且我们能够在体内监测从休眠到定植的转换。在WP2中，我们将确定Dkk1是否不仅通过直接靶向肿瘤细胞，而且还通过靶向骨髓基质细胞来影响肿瘤细胞的休眠。为此，我们将使用主要受体LRP5的Dkk1结合位点突变(LRP5-G170V)或辅助受体Krm1和/或Krm2敲除的免疫缺陷小鼠进行基于细胞系的PCA异种移植实验。此外，我们将把我们新建立的患者来源的异种移植(PDX)模型‘C5’移植到这些小鼠身上，该模型会自发转移到骨中，并表达Dkk1和SPARC。WP1和WP2被嵌入到申请人和英国克伦坡大学骨学和生物力学研究所(Thorsten Schinke教授)之间的长期密切合作中。Schinke教授将贡献他在静态、动态和细胞组织形态计量学方面的专业知识，并提供LRP5-G170V和KRM1/2 k.O.WP2的小鼠。WP1和WP2可能表明，Sparc和Dkk1以及Dkk1受体激活水平的升高抑制了从休眠到定植的转换。预期的结果将为未来如何在微小残留癌症阶段保持Sparc和Dkk1的表达以防止转移复发(可能是在第二个资助期)的实验提供理论基础。此外，为了通过一种无偏见的方法确定前列腺癌转移生长的新标记物，我们将使用最近建立的生物发光成像方法-异种移植原发瘤和相应的骨转移瘤的原位激光消融和随后的蛋白质组分析(WP3，与H.Schlüter教授，UKE合作)。由此产生的候选人将在第二个资助期进行功能验证。

The general aim of this project is to improve our understanding of the mechanisms that regulate outgrowth or otherwise dormancy of PCa bone metastases. This is a major clinical problem as it accounts for late metastatic relapse after unpredictable periods of minimal residual disease. Our preliminary data, which are mainly based on our newly established spontaneous bone metastasis xenograft models of human PCa, and the literature collectively suggest that the outgrowth of PCa bone metastases might be suppressed by a paracrine and/or autocrine interplay of the soluble factors Sparc and Dkk1 that apparently inhibits tumor colonization by promoting the EMT-phenotype of metastatic cells (EMT=epithelial-to-mesenchymal transition). The first aim of this project will therefore be to obtain for the first time direct functional evidence whether the bone marrow (BM) colonization changes upon genetic modification (e.g., inducible knockdown) of Sparc or Dkk1 in our spontaneous metastasis PCa xenograft models (WP1). Our preliminary work demonstrates that our models closely recapitulate the process of EMT, that they have clinical and functional relevance, and that we are able to monitor the switch from dormancy to colonization in vivo. In WP2 we will determine whether Dkk1 exerts its effects on tumor cell dormancy not only by directly targeting the tumor cells, but also the BM stromal cells. For this purpose, we will use immunodeficient mice with mutated Dkk1-binding site of the main receptor Lrp5 (Lrp5-G170V) or with knockout of the co-receptors Krm1 and/or Krm2 for cell line-based PCa xenograft experiments. In addition, we will engraft these mice with our newly established patient-derived xenograft (PDX) model 'C5' that spontaneously metastasizes to the bone and expresses Dkk1 and Sparc. WP1 and WP2 are embedded into a long-standing, close collaboration between the applicant and the Institute of Osteology and Biomechanics at UKE (Prof. Thorsten Schinke). Prof. Schinke will contribute his expertise in static, dynamic and cellular histomorphometry and provide the Lrp5-G170V and Krm1/2 k.o. mice for WP2. WP1 and WP2 might demonstrate that elevated levels of Sparc and Dkk1 and Dkk1 receptor activation suppress the switch from dormancy to colonization. The expected results would provide a rationale for future experiments on how to maintain Sparc and Dkk1 expression in the phase of minimal residual cancer to prevent metastatic relapse (presumably in a second funding period). Moreover, in order to identify novel markers of metastatic outgrowth in PCa by an unbiased approach, we will use a recently established method of bioluminescence imaging-guided in situ laser ablation of xenograft primary tumors and corresponding bone metastases and subsequent proteome analysis (WP3, cooperation with Prof. H. Schlüter, UKE). The resulting candidates will be functionally validated in a second funding period.