Regulation of the PCa Metastatic Phenotype by the HSC Niche

计划2 项目摘要 概述：前列腺癌（PCa）具有惊人的扩散，侵袭和生存能力， 骨髓一旦到达那里，播散性肿瘤细胞（DTC）可能会休眠多年。之前我们展示了， 在转移过程中，循环PCa细胞靶向容纳造血干细胞（HSC）的“小生境”。的 niche调节HSC静止。 继续这项工作，我们发现从小生境中回收的DTC在CD 133 +/CD 44+细胞中高度富集。 群体并表达与“茎样”表型相关的基因。表型从CD 133- 在从其他组织回收的细胞中未观察到CD 133 +/CD 44-至CD 133 +/CD 44+群体。进一步的研究表明 HSC小生境本身是DTC表型向不太成熟、更易癌变转变的关键， 干细胞样细胞-一种对化疗有抗性表型。我们的目标是更全面地了解 CD 133 +/CD 44+群体的生物学。 假设：DTC一旦进入HSC生态位，就会获得癌干细胞样状态 代表促进休眠和对治疗的抗性的分子途径。 目的1：从骨髓中分离CD 133 +/CD 44 + DTC细胞，并对其进行生物学特性鉴定。 我们将确定从骨髓中分离的CD 133 +/CD 44+是否产生（i）体外球和（ii）皮下球。 体内有限稀释的肿瘤，（iii）与非小生境相比，表达更高水平的干细胞标志物 （iv）使用双标记，我们将鉴定哪种DTC群体（CD 133 +/CD 44+或 CD 133-/CD 44-）导致骨转移。 目的2：阐明CD 133-/CD 44-细胞向CD 133 +/CD 44+细胞转化的调控机制。 DTC表型在进入生态位后的变化依赖于生长停滞特异性-6。以限定 涉及受体的shRNA将靶向三种受体酪氨酸激酶（Tyro 3、Ax 1、Mer 受体）结合GAS 6。(ii)由GAS 6信号传导激活的细胞内信号传导途径将被激活。 定义为鉴定阻断CD 133-/CD 44-转化为CD 133 +/CD 44+细胞的机制。 目的3：确定人类DTC表达CD 133、CD 44和GAS 6受体的程度。 我们知道0.5-8%的原发性肿瘤细胞表达干细胞样表型，但在我们的骨髓模型中，20- 30%的DTC表达CD 133 +/CD 44+。根据这些数据集，我们预测20%或更多的 从男性回收的DTC将表达干细胞样表型。为了验证我们的假设， 以下每个患者人群的样本，男性前列腺切除术前（原发性、CTC、DTC），患者 初次治疗后未接受过激素治疗的前列腺癌，PSA值升高（DTC和CTC），去势抵抗 将评价具有临床明显转移性疾病（CTC和DTC）的患者的以下表达： CD 133、CD 44和GAS 6受体。

Project 2 Project Summary Overview: Prostate cancers (PCa) have an astonishing ability to disseminate, invade and survive in the marrow. Once there, disseminated tumor cells (DTCs) may lie dormant for years. Previously we showed that during metastasis circulating PCa cells target the „niche‟ that houses hematopoietic stem cells (HSC). The niche regulates HSC quiescence. Continuing this work, we show that DTCs recovered from the niche are highly enriched in the CD133+/CD44+ population and express genes associated with a “stem-like” phenotype. The shift in phenotype from CD133- /CD44- to CD133+/CD44+ populations is not seen in cells recovered from other tissues. Further studies show that the HSC niche itself is central to the shift in the DTC phenotype towards a less mature, more cancer stem-like cell–a phenotype which is resistant to chemotherapy. The goal is to more fully understand the biology of the CD133+/CD44+ population. Hypothesis: The acquisition of a cancer stem cell-like state by DTCs once they engage the HSC niche represents a molecular pathway which facilitates dormancy and resistance to therapy. Aim 1: Characterize the biology of CD133+/CD44+ DTC cells isolated from the marrow. We will determine if CD133+/CD44+ isolated from marrow generate (i) spheres in vitro and (ii) generate s.c. tumors in limiting dilution in vivo, (iii) express higher levels of stem cell markers compared to non-niche engaged DTCs, and (iv) using double labeling we will identify which population of DTCs (CD133+/CD44+ or CD133-/CD44-) leads to bone metastases. Aim 2: Elucidate the mechanisms regulating the conversion of CD133-/CD44- into CD133+/CD44+ cells. The change in DTC phenotype upon entering the niche is dependent on growth arrest specific-6. To define the receptors involved shRNA will target each of the three receptor tyrosine kinases (Tyro3, Axl, Mer receptors) that bind to GAS6. (ii) The intracellular signaling pathways activated by GAS6 signaling will be defined to identify mechanisms to block the conversion of CD133-/CD44- into CD133+/CD44+ cells. Aim 3: Identify the extent to which DTCs in humans express CD133, CD44 and GAS6 receptors. We know that 0.5-8% of primary tumor cells express a stem-like phenotype, yet in our models in marrow, 20- 30% of the DTCs express CD133+/CD44+. Based on these data sets, we predict that 20% or more of the DTCs recovered from men will express the stem-like phenotype. To test our hypothesis, 20 sets of paired samples in each of the following patient populations, men pre-prostatectomy (primary, CTCs, DTCs), patients with hormone naive PCa after primary therapy with rising PSA values (DTCs & CTCs), and castrate resistant patients with clinically evident metastatic disease (CTC and DTCs) will be evaluated for the expression of CD133, CD44 and GAS6 receptors.