Modeling human prostate cancer by cellular reprogramming

 描述(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的应用，但这种方法也可以用作包括癌症在内的人类疾病基因工程模型的基础。特别是，我们建议定向谱系转换/转分化可以与基因打靶方法相结合来创建新的人类癌症模型。在这项应用中，我们将使用成纤维细胞的重编程、组织重组和CRISPR介导的基因打靶来产生小鼠和人的前列腺组织，用于前列腺癌的建模。在我们的初步研究中，我们已经证明，小鼠成纤维细胞可以通过三个步骤重新编程为前列腺组织，包括瞬时诱导多能性因子，表达前列腺上皮转录调节因子，以及在肾移植后与泌尿生殖间充质组织重组。根据我们的初步发现，我们假设这种重编程方法可以与CRISPR介导的肿瘤抑制基因靶向相结合，产生新的前列腺癌基因工程人类模型。我们现在将使用两个特定的目标来研究人类前列腺癌模型的产生：(1)研究人成纤维细胞向前列腺上皮细胞的重编程，以确定规范前列腺上皮分化的最佳条件和重编程的前列腺组织；以及(2)通过基因打靶和使用CRISPR介导的基因打靶进行重编程来建立人前列腺癌的模型，以特异地改变人前列腺癌中突变的肿瘤抑制基因，然后产生经过癌基因转化的重编程的人前列腺组织。总而言之，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.