Bone-induced c-kit in Prostate Cancer Cells: Implications for Bone Metastasis

描述(申请人提供)：骨转移是前列腺癌(PCa)患者发病率和死亡率的主要原因。目前，患有骨转移疾病的前列腺癌患者对化疗的反应很差。在癌症中，受体酪氨酸激酶c-kit的解除调控是一个常见的事件。然而，它在PCa中的确切作用，更具体地说，在PCa骨转移中的作用，在很大程度上仍不确定。在使用临床样本的研究中，我们已经表明，在恶性进展过程中，前列腺细胞c-kit的表达逐渐增加，在前列腺癌骨转移中的表达最高。我们发现，c-kit既不在体外培养的前列腺癌细胞系中表达，也不在由其形成的皮下肿瘤中表达。然而，当这些相同的细胞与骨髓来源的细胞共培养或使用不同的实验模型植入骨内时，我们发现所有病例中都有c-kit的从头表达。重要的是，在前列腺癌以外的肿瘤系统中，c-kit的表达与化疗耐药之间已经建立了相关性，我们的初步数据表明，这可能也适用于前列腺癌，这表明c-kit在前列腺癌骨转移患者对化疗的不良反应中发挥了作用。综上所述，我们假设由骨源性因子诱导的c-kit从头表达是PCA细胞在骨内扩张的先决条件，并对获得化疗耐药负责。为了验证我们的假设，具体目的是：(1)确定骨诱导的c-kit表达在骨内肿瘤生长和骨反应中的功能意义，以及pca细胞从头表达c-kit对获得化疗耐药的影响，以及(2)确定参与诱导c-kit的骨源性因素。为了提供c-kit在骨内PCa生长中作用的因果证据，将慢病毒介导的RNA干扰或与含有显性阴性(激酶死亡)c-kit突变结构的质粒一起转染，以阻止c-kit在骨微环境中的从头诱导。我们将利用我们实验室建立的骨转移SCID-Hu模型来研究PCA相关的c-kit在PCA细胞骨内扩张中的作用，以及它对成骨细胞和破骨细胞分化的影响。我们将在体内和体外用c-kit诱导和非诱导的c-kit PCa细胞研究c-kit的从头表达对PCa细胞表达的影响及其对多西紫杉醇反应的影响，并对相关的分子机制进行探讨。我们将利用激光捕获显微切割技术从实验性PCa骨肿瘤中提取RNA，然后使用a)从逆转录病毒cDNA表达文库中进行功能克隆和b)基因芯片差异表达分析来鉴定骨源性因子诱导PCa细胞中c-kit的表达(目标2)。预计这项高风险、高收益的提案将最终设计出新的量身定做的疗法，旨在填补前列腺癌患者骨转移治疗的空白。

DESCRIPTION (provided by applicant): Bone metastasis is the main cause of morbidity and mortality in prostate cancer (PCa) patients. Currently, PCa patients with bone metastatic disease respond poorly to chemotherapy. In cancer, deregulation of the receptor tyrosine kinase c-kit is a frequent event. However, its precise role in PCa and, more specifically in PCa bone metastasis, remains largely undetermined. In studies using clinical samples, we have shown that c-kit expression by prostate cells is gradually increased during malignant progression, with the highest expression seen in PCa bone metastases. We found that c-kit is neither expressed by PCa cell lines in vitro, nor by subcutaneous tumors formed by them. However, when these same cells are either co-cultured with bone marrow-derived cells or injected intraosseously using different experimental models, we found a de novo expression of c-kit in all the cases. Importantly, a correlation between c-kit expression and chemoresistance has been established in tumor systems other than prostate, and our preliminary data indicate that this might be also true for PCa, suggesting a role for c-kit in the poor response of patients with PCa bone metastasis to chemotherapy. Taken together, we hypothesize that de novo expression of c-kit induced by bone-derived factors is a prerequisite for the expansion of PCa cells within the bone, and is responsible for the acquisition of chemotherapy-resistance. To test our hypothesis, the specific aims are: (1) Determine the functional significance of bone-induced c-kit expression in PCa cells in intraosseous tumor growth and bone response, and the effect of de novo c-kit expression by PCa cells on the acquisition of chemoresistance, and (2) Identify causative bone-derived factors involved in the induction of c-kit in PCa cells. To provide causal evidence for the role of c-kit in intraosseous PCa growth, lentiviral-mediated RNA interference or transfect ion with a plasmid containing a dominant-negative (kinase-dead) c-kit mutant construct will be used to impede de novo c-kit induction in PCa cells within the bone microenvironment. The contribution of PCa-associated c-kit to intraosseous expansion of PCa cells will be studied using the SCID-hu model of bone metastasis developed in our laboratory, as well as its effect on osteoblast and osteoclast differentiations. The effect of de novo c-kit expression by PCa cells and its consequence to response to docetaxel will be studied in vivo and in vitro with c-kit-inducible and non-inducible c-kit PCa cells, and molecular mechanisms involved will be investigated. Using laser capture microdisection we will extract RNA from experimental PCa bone tumors and then use a) functional cloning from a retroviral cDNA expression library and b) differential expression analysis by gene microarray, to identify bone-derived factors inducing c-kit expression in PCa cells (Aim 2). It is expected that this high-risk, high-gain proposal will culminate in the design of new tailored therapies aimed at filling the gap in the treatment of PCa patients with bone metastasis.