Modeling human prostate cancer by cellular reprogramming

 描述（由申请人提供）：虽然细胞重编程通常用于干细胞生物学和再生医学中的应用，但该方法也可用作人类疾病（包括癌症）的基因工程模型的基础。特别是，我们提出，定向谱系转换/转分化可以与基因靶向方法结合使用，用于创建新的人类癌症模型。在本申请中，我们将使用成纤维细胞的重编程以及组织重组和CRISPR介导的基因靶向来生成小鼠和人类前列腺组织以用于前列腺癌的建模。在我们的初步研究中，我们已经表明，小鼠成纤维细胞可以重新编程为前列腺组织使用三步的过程，涉及多能性因子的瞬时诱导，前列腺上皮细胞的转录调节因子的表达，和组织重组与泌尿生殖间充质随后肾移植。基于我们的初步发现，我们假设这种重编程方法可以与CRISPR介导的肿瘤抑制基因的基因靶向结合使用，以产生新型的前列腺癌基因工程人类模型。我们现在将使用两个特定目的研究前列腺癌的人模型的产生：（1）研究人成纤维细胞重编程为前列腺上皮以鉴定用于前列腺上皮分化的特化的最佳条件，并通过重编程的前列腺组织的分子分析;和（2）通过使用CRISPR-PCR的基因靶向和重编程来建模人前列腺癌。介导的基因靶向，用于人前列腺癌中突变的肿瘤抑制基因的特异性改变，随后产生已经历致癌转化的重编程的人前列腺组织。结合起来，这些研究将为人类癌症建模的创新方法提供基础，这将对人类前列腺癌的发生和发展的分子机制产生重要的新见解。

 DESCRIPTION (provided by applicant): While cellular reprogramming is generally utilized for applications in stem cell biology and regenerative medicine, this approach can also be used as the basis for genetically-engineered models of human disease, including cancer. In particular, we propose that directed lineage conversion/transdifferentiation can be used in combination with gene targeting methods for the creation of novel models of human cancer. In this application, we will use reprogramming of fibroblasts together with tissue recombination and CRISPR-mediated gene targeting to generate mouse and human prostate tissue for modeling of prostate cancer. In our preliminary studies, we have shown that mouse fibroblasts can be reprogrammed to prostate tissue using a three-step process involving transient induction of pluripotency factors, expression of transcriptional regulators of prostate epithelium, and tissue recombination with urogenital mesenchyme followed by renal grafting. Based on our preliminary findings, we hypothesize that this reprogramming approach can be used in combination with CRISPR-mediated gene targeting of tumor suppressor genes to generate novel genetically-engineered human models of prostate cancer. We will now investigate the generation of human models of prostate cancer using two specific aims: (1) Investigation of reprogramming of human fibroblasts into prostate epithelium to identify optimal conditions for specification of prostate epithelial differentiation and by molecular analyses of reprogrammed prostate tissue; and (2) Modeling of human prostate cancer by gene targeting and reprogramming using CRISPR-mediated gene targeting for the specific alteration of tumor suppressor genes that are mutated in human prostate cancer, followed by generation of reprogrammed human prostate tissue that has undergone oncogenic transformation. In combination, these studies will provide the basis for an innovative approach for human cancer modeling, which should yield important new insights into the molecular mechanisms of human prostate cancer initiation and progression.