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

前列腺癌细胞中骨诱导的 c-kit：对骨转移的影响

• 批准号: 8309967
• 负责人: RICARDO DANIEL BONFIL
• 金额: $ 16.53万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8309967
• 关键词: Accounting; Address; Affect; American; Antibodies; Area; Biological; Biological Assay; Blood Circulation; Body part; Bone Growth; Bone Marrow; Bone neoplasms; Cancer Patient; Cells; Clinical; Cloning; Coculture Techniques; Code; Complication; Data; Development; Disease; Distant; Dominant-Negative Mutation; Event; Experimental Models; Expression Library; Foundations; Gene Expression Microarray Analysis; Generations; Genes; Genetic; Genetic Engineering; Growth; Human; Implant; In Vitro; Ions; Knowledge; Laboratories; Lasers; Light; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of prostate; Messenger RNA; Metastatic Neoplasm to the Bone; Modeling; Molecular; Molecular Biology Techniques; Morbidity - disease rate; Mus; Neoplasm Metastasis; Obstruction; Osteoblasts; Osteoclasts; PC3 cell line; Patients; Phosphotransferases; Plasmids; Production; Prostate; Prostate Cancer therapy; Prostatic Neoplasms; Proteins; Proto-Oncogene Protein c-kit; RNA; RNA Interference; Receptor Cell; Receptor Protein-Tyrosine Kinases; Resistance; Role; SCID Mice; Sampling; Screening procedure; Signal Transduction; System; Testing; Time; bone; bone xenograft; cDNA Expression; cDNA Library; cancer cell; cell growth; chemotherapy; design; docetaxel; high risk; in vivo; killings; laser capture microdissection; lentiviral-mediated; matrigel; men; mortality; mutant; novel; overexpression; particle; research study; response; small hairpin RNA; subcutaneous; tumor; tumor growth; vector

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.

描述（由申请人提供）：骨转移是前列腺癌（PCa）患者发病和死亡的主要原因。目前，患有骨转移性疾病的PCa患者对化疗反应较差。在癌症中，受体酪氨酸激酶c-kit的失调是常见的事件。然而，其在前列腺癌中的确切作用，更具体地说，在前列腺癌骨转移中的作用，在很大程度上仍然不确定。在使用临床样本的研究中，我们已经表明，前列腺细胞的c-kit表达在恶性进展过程中逐渐增加，在PCa骨转移中观察到最高表达。我们发现c-kit在体外PCa细胞系中不表达，也不在它们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骨内注射时，我们发现在所有情况下c-kit的从头表达。重要的是，c-kit表达和化疗耐药性之间的相关性已在前列腺以外的肿瘤系统中建立，我们的初步数据表明，这可能也适用于PCa，这表明c-kit在PCa骨转移患者对化疗反应不良中的作用。综上所述，我们假设，从头表达的c-kit诱导骨源性因子是一个先决条件的PCa细胞在骨内的扩张，并负责收购化疗耐药性。为了验证我们的假设，具体的目的是：（1）确定骨诱导的PCa细胞中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c-kit表达对获得化疗耐药性的影响，和（2）鉴定参与PCa细胞中c-kit诱导的致病性骨源性因子。为了提供c-kit在骨内PCa生长中作用的因果证据，将使用慢病毒介导的RNA干扰或用含有显性阴性（激酶死亡）c-kit突变体构建体的质粒进行的干扰，以阻止骨微环境内PCa细胞中的从头c-kit诱导。PCa相关的c-kit的PCa细胞的骨内扩张的贡献将使用在我们的实验室开发的SCID-hu骨转移模型，以及其对成骨细胞和破骨细胞分化的影响进行研究。将在体内和体外使用c-kit诱导型和非诱导型c-kit PCa细胞研究PCa细胞从头c-kit表达的影响及其对多西他赛应答的后果，并研究涉及的分子机制。我们将使用激光捕获显微切割从实验性PCa骨肿瘤中提取RNA，然后使用a）从逆转录病毒cDNA表达文库中的功能性克隆和B）通过基因微阵列进行差异表达分析，以鉴定诱导PCa细胞中c-kit表达的骨源性因子（目的2）。预计这一高风险、高收益的提议将最终导致设计新的定制疗法，旨在填补伴有骨转移的PCa患者治疗的差距。