Functional characterization of bone metastasis-initiating and radioresistant prostate tumor cells

一旦前列腺肿瘤扩散到骨骼，它通常无法治愈。骨中的支持位点通过特殊的分子成分维持癌细胞的存活和增殖，如C-X-C基序趋化因子配体12 (CXCL12)，它也介导肿瘤细胞归巢到骨。反过来，骨转移性前列腺癌细胞表达不同的蛋白质，调节骨重塑和肿瘤发生，包括Endo 180胶原受体。前列腺肿瘤有一个分层组织，其中癌干细胞（CSC）维持和增殖肿瘤肿块。许多研究表明，与肿瘤体相比，CSC具有较高的放射抗性，并且最近的许多临床研究使用CSC相关的生物标志物来预测放疗结果。肿瘤转移是由进化的CSC群体在最坏的情况下驱动的。为了支持这一点，代表不同水平转移过程的细胞，包括癌症患者血液中的循环肿瘤细胞（CTC）和骨髓中的弥散性肿瘤细胞（DTC），表达CSC标记物，转移启动细胞共享CSC的关键特征，包括形成肿瘤和激活CSC特异性信号机制的能力。我们的研究揭示了一些蛋白参与转移性和放射耐药前列腺肿瘤细胞的调节。其中包括醛脱氢酶蛋白1A1 (ALDH1A1)，它是放射耐药CSC的标记物，在PCa转移中有高表达。另一个例子是CXCL12受体CXCR4，它是CSC的标志物，在前列腺癌放疗后CTC上调，与放疗结果相关。最后，在高致瘤性放射耐药PCa细胞中，作为PCa转移关键调控因子之一的胶原受体Endo180/MRC2的表达上调。我们假设，在原发性肿瘤的骨髓DTC、CTC或CSC中，在基因组和转录组水平上检测到的某些信号通路的激活可以预测骨转移的发展，另一方面，可能参与骨重塑和转移对放疗的反应。本研究的第一个目的是研究由CXCR4、ALDH1A1和Endo180介导的信号通路在骨转移启动和放射抵抗前列腺肿瘤细胞和骨重塑中的调节作用，以及这些机制在转移发展过程中可能的相互作用。本研究的第二个目标是对代表不同转移过程水平的前列腺肿瘤细胞（原发肿瘤、CSC、CTC、DTC、耐辐射PCa细胞）的基因组和转录组学分析进行公正和系统的分析，以验证CSC相关信号通路的激活。这些机制和表型将通过CRISPR/Cas9介导的候选基因敲除、转移性小鼠模型、体外和体内骨重塑监测以及候选基因与临床数据和患者对治疗反应的相关性来验证。

Once prostate tumor spreads to the bone, it typically cannot be cured. The supportive sites in bone maintain survival and proliferation of cancer cells by special molecular components such as C-X-C motif chemokine ligand 12 (CXCL12) which also mediates tumor cell homing to bone. In turn, metastatic PCa cells in bone express different proteins that regulate both, bone remodeling and tumorigenesis including Endo 180 collagen receptor. Prostate tumors have a hierarchical organization where cancer stem cells (CSC) maintain and propagate the tumor mass. A number of studies showed a high radioresistance of CSCs compared to the tumor bulk, and many recent clinical studies use CSC related biomarkers for prediction of radiotherapy outcome. Tumor metastases are driven by the evolved populations of CSC at their worst. In support of this, cells representing different levels of metastatic processes including circulating tumor cells (CTC) in the blood of cancer patients and disseminated tumor cells (DTC) in their bone marrow express CSC markers, and metastasis initiating cells are sharing key CSC features including ability to form tumors and to activate CSC specific signaling mechanisms. Our studies revealed a few proteins that are involved in regulation of metastatic and radioresistant prostate tumor cells. That includes aldehyde dehydrogenase protein 1A1 (ALDH1A1) which is a marker of radioresistant CSC and has a high expression in PCa metastases. Another example is CXCL12 receptor, CXCR4, which is a marker of CSC, upregulates in CTC after radiotherapy and correlates with radiotherapy outcome in PCa. Finally, expression of collagen receptor Endo180/MRC2 which is one of the key regulators of PCa metastases is upregulated in highly tumorigenic radioresistant PCa cells. We assume that activation of certain signaling pathways detected on the genomic and transcriptomic levels in bone-marrow DTC, CTC or CSC in primary tumors can be prognostic for bone metastasis development and, on other hand, can be involved in the bone remodeling and response of metastases to radiotherapy. The first objective of this study is investigation of the role of signaling pathways mediated by CXCR4, ALDH1A1 and Endo180 for regulation of bone metastasis-initiating and radioresistant prostate tumor cells and bone remodeling, and possible interaction of these mechanisms during metastatic development. The second objective of this study is an unbiased and systematic analysis of genomic and transcriptomic profiling of prostate tumor cells representing different levels of metastatic processes (primary tumors, CSC, CTC, DTC, radioresistant PCa cells) to validate activation of CSC related signaling pathways. These mechanisms and phenotypes will be validated by using CRISPR/Cas9 mediated knockout of candidate genes, metastatic mice models, monitoring of bone remodeling in vitro and in vivo, and correlation of candidate genes to clinical data and patients’ response to therapy.