Normal stem cells and their transition to disease in the skin

 描述(申请人提供)：干细胞及其环境，即所谓的生态位，不仅是维持适当的组织动态平衡的关键组件，也是维持疾病状态(如癌症)的关键组件。在一个完整的动物中，随着时间的推移无法跟踪相同的细胞是一个双重挑战，这极大地限制了我们对正常组织动态平衡期间出现问题的原因以及肿瘤生长初期表现出的细胞行为的理解。具体地说，这个障碍阻碍了我们理解特定细胞的作用 以及它们的地理位置如何促进它们的生长，无论是正常的还是癌症的。我们现在第一次有能力解决这些问题，因为我的实验室率先使用了活成像技术来再生皮肤干细胞，并建立了一种方法，允许我们在一只完整的活小鼠身上随着时间的推移跟踪相同的细胞。通过这些方法，我们了解到，首先，干细胞所在的位置影响它们促进生长的能力。其次，作为一种扰动的结果，例如干细胞库的丧失，其他细胞可以获得一种以前没有的促进生长的新能力。第三，在克隆中诱导致癌突变，例如稳定Wnt效应器β-catenin的突变，将推动异常生长，最终通过招募非细胞自主的野生型细胞导致肿瘤形成。然而，目前尚不清楚1)什么信号机制调节最终导致肿瘤发展的异常细胞行为，2)细胞在组织中的位置是否影响不同的肿瘤结果，3)干细胞和其他类型的细胞采用哪些早期细胞行为导致肿瘤发展。为了识别导致癌症的异常干细胞/祖细胞活动，我们使用了两种截然不同的人类皮肤肿瘤；一种是良性的，一种是恶性的，我们可以在小鼠模型中忠实地概括出来。我们计划1)利用尖端的外显子组和RNA测序方法来识别与肿瘤上皮细胞和间质细胞群体相关的靶基因突变；2)通过宫内注射和随后的体内成像方法来评估这些基因在小鼠体内的过表达和shRNA敲除在癌症中的作用。总之，这些方法将使我们能够了解新的和已建立的靶基因如何改变干细胞的行为，并确定导致恶性肿瘤生长的最初连续步骤。了解干细胞在癌症中作用的机制和信号通路对改变当前的治疗策略具有很大的希望。我们的计划旨在确定导致皮肤肿瘤启动的相关细胞和基因，这可能被证明与扩展应用于其他类型的癌症有关。

 DESCRIPTION (provided by applicant): Stem cells and their environment, the so-called niche, are critical components that sustain not only proper tissue homeostasis, but also diseased states such as cancer. The inability to follow the same cells over time in an intact animal is a bi challenge that has greatly limited our understanding of what goes awry during normal tissue homeostasis and the cellular behaviors exhibited in the initial stages of tumor growth. Specifically, this roadblock has hindered our ability to understand both the role of specific cells and how their location contributes to their growth, whether it be normal or cancerous. For the first time, we are now capable of addressing these questions since my lab has pioneered the use of live imaging in skin stem cell regeneration and established an approach that allows us to follow the same cells over time in an intact, live mouse. By these approaches we have learned that first, the location where stem cells reside influences their ability to contribute to growth. Second, as a consequence of a perturbation, such as loss of a stem cell pool, other cells can acquire a new ability to contribute to growth that they did not possess previously. Third, the induction of oncogenic mutations in clones, such as mutations that stabilize the Wnt effector β-catenin, will drive aberrant growths ultimately leading to tumor formation through the recruitment of non cell-autonomous wild-type cells. However, it is still unclear 1) what signaling mechanisms regulate the aberrant cellular behaviors that will eventually cause tumor development, 2) whether a cell's position within a tissue influences different tumoral outcomes, and 3) what early cell behaviors are adopted by stem and other cell types that lead to tumor development. To identify the aberrant stem cell/progenitor activities that cause cancer, we are using two contrasting human skin tumors; a benign and a malignant form, which we can faithfully recapitulate in a mouse model. We plan to 1) utilize cutting-edge exome and RNA sequencing approaches to identify target gene mutations associated with tumoral epithelial and stromal cell populations and 2) to assess the role of these genes towards cancer by in vivo overexpression and shRNA knockdown in the mouse via in utero injection and subsequent functional validation using our in vivo imaging approach. Together, these approaches will allow us to understand how novel and established target genes alter stem cell behaviors and identify the initial sequential steps that lead to malignant tumor growth. Understanding the mechanisms and signaling pathways underlying the role of stem cells in cancer holds great promise to transform current therapeutic strategies. Our plan aims to identify the relevant cells and genes that are responsible for skin tumor initiation, which may prove relevant for an extended application to other types of cancer.