Deregulation of MSI RNA-binding proteins promotes intestinal tumorigenesis

描述（由申请人提供）：结直肠癌（CRC）是全球癌症相关死亡的主要原因。CRC包含一个明确的癌症干细胞群，能够自我更新和分化为子细胞，子细胞构成了肿瘤的主体。最近的研究表明，正常的肠干细胞是CRC的起源细胞，因为激活Wnt途径的致癌突变（主要通过APC肿瘤抑制基因的基因失活）仅在肠干细胞中发生时才导致肿瘤形成，而不是其分化的后代。因此，了解正常肠道干细胞中干细胞命运决定的机制如何被解除管制并有助于CRC的个体发生是一个优先事项。成体干细胞通常经历不对称的细胞命运决定，以确保在稳态期间维持干细胞与分化细胞的适当比例。在黑腹果蝇的成神经细胞中，这一过程由Musashi RNA结合蛋白的活性控制，其哺乳动物直系同源物Msi 1和Msi 2在哺乳动物肠干细胞和人CRC中高度表达。我们提出，Msi蛋白的失调改变了CRC中的细胞命运决定，使得干细胞以其分化后代为代价进行扩增，从而导致促进侵袭性未分化肿瘤。这一假设将使用基因改变的小鼠来解决，其中Msi蛋白水平可以在肠道干细胞中调节。最初，我们将确定Msi功能的获得是否足以诱导干细胞驱动的肿瘤发生。为此，已经产生了药物诱导型Msi 1和Msi 2转基因小鼠，使得能够在精确的时空控制下有效诱导Msi活性。确定是否需要Msi活动 对于干细胞驱动的肠癌的APCmin/+模型中肿瘤发生的发生和进展，产生Msi 1和Msi 2的条件性缺失等位基因。Msi基因在肿瘤进展的开始或过程中的缺失将明确决定其在疾病个体发生中的功能作用。最后，使用体内CLIP-Seq方法，我们最近鉴定了许多与Wnt通路调控相关的Msi 1 RNA结合靶点，并且还观察到Msi基因在体内响应Wnt信号传导而被激活。因此，我们将解决的假设，Msi是一个重要的效应Wnt介导的肠道转化和Msi驻留在一个负反馈回路与Wnt信号通路，以管理正常的平衡之间的干细胞和它们的分化后代在肠道。这项研究将为干细胞驱动的癌症启动和发展的基本机制提供新的见解。此外，由于癌症干细胞通常对常规疗法如放疗或化疗具有抗性，并且它们的存活可以促进肿瘤复发，我们预测我们的研究将确定用于治疗CRC和其他干细胞驱动的肿瘤的新的治疗靶点。

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally. CRC contains a well-defined cancer stem cell population capable of both self-renewal and differentiation into daughter cells that comprise the bulk of the tumor. It has recently been shown that normal intestinal stem cells are the cell of origin in CRC, as oncogenic mutations activating the Wnt pathway (primarily through genetic inactivation of the APC tumor suppressor) result in tumor formation only when they occur in an intestinal stem cell, and not its differentiated progeny. It is therefore a priority to understand how mechanisms underlying stem cell fate determination in normal intestinal stem cells can become deregulated and contribute to the ontogeny of CRC. Adult stem cells often undergo asymmetric cell fate determination to insure that the proper ratio of stem cells to differentiated cells is maintained during homeostasis. In neuroblasts of Drosophila melanogaster this process is governed by the activity of the Musashi RNA binding protein, whose mammalian orthologs, Msi1 and Msi2, are highly expressed in mammalian intestinal stem cells and in human CRC. We propose that deregulation of Msi proteins alters cell fate determination in CRC such that stem cells expand at the expense of their differentiated progeny, thus resulting in the promotion of aggressive, undifferentiated tumors. This hypothesis will be addressed using genetically altered mice in which Msi protein levels can be modulated in the intestinal stem cells. Initially, we will determin whether Msi gain of function is sufficient to induce stem cell-driven tumorigenesis. To this end, drug inducible Msi1 and Msi2 transgenic mice have been generated, enabling efficient induction of Msi activity with precise spatio-temporal control. To determine whether Msi activity is required for the onset and progression tumorigenesis in the APCmin/+ model of stem cell-driven intestinal cancer, conditional deletion alleles of both Msi1 and Msi2 were generated. Deletion of Msi genes at the onset or during the course of tumor progression will unequivocally determine their functional role in disease ontogeny. Finally, using an in vivo CLIP-Seq approach, we have recently identified a number of Msi1 RNA binding targets associated with regulation of the Wnt pathway and have also observed that Msi genes are activated in response to Wnt signaling in vivo. We will therefore address the hypothesis that Msi is an important effector of Wnt-mediated intestinal transformation and that Msi resides in a negative feedback loop with the Wnt signaling pathway to govern the normal balance between stem cells and their differentiated progeny in the intestine. This study will provide novel insight into the fundamental mechanisms by which stem cell-driven cancers are initiated and progress. Moreover, as cancer stem cells are often resistant to conventional therapeutics such as radiation or chemotherapy and their survival can promote tumor recurrence, we predict that our studies will identify novel therapeutic targets for the treatment of CRC and other stem cell- driven tumors.