Functional Dissection of Essential Wnt Signaling Receptors in Breast Stem Cells

项目摘要 Wnt信号传导用于维持体细胞和胚胎干细胞的再生潜力。然而，在这方面， 由于异位Wnt信号传导对于上皮组织是高度致癌的，所以这种激活是有风险的;因此， 在异位Wnt信号传导存在下维持和发育的组织可能具有过度的肿瘤风险， 在生命后期的发展。我们已经发现Wnt信号受体之一Lrp 5的辅助功能， 我们提出可能是负责Wnt信号的致癌活性;如果我们是正确的，Lrp 5 活性可以在未来基于干细胞的治疗应用中进行操纵。 乳腺上皮干细胞的研究为研究体干细胞的活性提供了一个很好的机会。 只要一个乳腺干细胞移植到一个乳房，就可以再生一个完整的乳腺导管树。 新的宿主脂肪垫我们以前的工作表明：1）乳腺干细胞功能与 Wnt信号传导活性，以及2）Wnt信号传导受体Lrp 5是维持乳腺干细胞所需的 活性，尽管事实上，更有效的Wnt信号传导受体，Lrp 6，共表达。这反映 维持乳腺干细胞的Wnt配体需要Lrp 5和Lrp 6两者存在 以产生Wnt配体。因此，我们称Lrp 5为乳腺干细胞功能的“看门人”。 然而，最近我们发现1）Lrp 5对乳腺上皮细胞生长具有深远的影响， 不依赖于经典的Wnt信号通路（β连环蛋白/TCF依赖性反式激活）。LRP5 通过一种全新的机制促进葡萄糖摄取，葡萄糖剥夺足以复制表型 Lrp 5的功能丧失; 2）不寻常的可溶性Wnt配体Wnt 3a可以促进生长， 乳腺上皮细胞在体内的发育，并且不需要Lrp 5存在。 我们的假设是Lrp 5促进肿瘤的发展是对异位Wnt信号的反应，但不是 Wnt诱导的干细胞积累所需的，伴随着乳腺再生激活 潜力在本申请中，我们的目标是发现1）Lrp 5如何调节葡萄糖摄取和乳腺癌的发生。 上皮细胞生长，和2）Lrp 5介导的葡萄糖摄取是否需要大量增加， 伴随细胞转化的葡萄糖摄取。因此，需要增加的葡萄糖摄取来提供能量， 肿瘤细胞的代谢重编程特征（“瓦尔堡”效应），使肿瘤细胞 特别易受葡萄糖摄取不足的影响。 该项目的目的是1）定义Lrp 5的新活性，Lrp 5是一种细胞表面受体，以前被描述为 典型的Wnt信号受体; 2）提出一种新的范例，即Wnt信号的致癌性可能 通过降低与Lrp 5相关的代谢活性来改善; 3）应用独特的技术（IFAST 免疫沉淀）和来自骨中Lrp 5研究的知识资源（其中Lrp 5支配骨 密度和人类患者中Lrp 5的突变产生成骨细胞功能的获得和丧失 活动）。

PROJECT SUMMARY Wnt signaling is used to maintain the regenerative potential of somatic and embryonic stem cells. However, this activation comes at a risk, since ectopic Wnt signaling is highly oncogenic for epithelial tissues; thus, tissues maintained and developed in the presence of ectopic Wnt signaling may have an excess risk of tumor development later in life. We have found an auxiliary function for one of the Wnt signaling receptors, Lrp5, that we propose could be responsible for the oncogenic activity of Wnt signaling; if we are correct, Lrp5 activity could be manipulated in future applications of stem cell-based cures. Study of mammary epithelial stem cells offers a remarkable opportunity to study somatic stem cell activity. Just a single mammary stem cell can regenerate a complete mammary ductal tree upon transplantation to a new host fat pad. Our previous work has shown that 1) mammary stem cell function is directly related to Wnt signaling activity, and 2) the Wnt signaling receptor Lrp5 is required to maintain mammary stem cell activity, despite the fact that the more potent Wnt signaling receptor, Lrp6, is co-expressed. This reflects the fact that the Wnt ligand(s) that maintain mammary stem cells require both Lrp5 and Lrp6 to be present to generate a Wnt ligand. We therefore call Lrp5 a “gatekeeper” of mammary stem cell function. However, recently we have found 1) that Lrp5 has a profound effect on mammary epithelial cell growth that is independent of the canonical Wnt signaling pathway (βcatenin/TCF-dependent transactivation). Lrp5 promotes glucose uptake by a totally novel mechanism, and glucose deprivation is sufficient to phenocopy loss of function of Lrp5; 2) that the unusual soluble Wnt ligand, Wnt3a, can promote growth and development of mammary epithelial cells in vivo, and does not require Lrp5 to be present. Our hypothesis is that Lrp5 promotes tumor development in response to ectopic Wnt signaling, but is not required for the Wnt-induced stem cell accumulation that accompanies the activation of breast regenerative potential. In this application we aim to find out 1) how Lrp5 regulates glucose uptake and mammary epithelial cell growth, and 2) whether Lrp5-mediated glucose uptake is required for the massive increase in glucose uptake that accompanies cell transformation. Thus, increased glucose uptake is required to fuel the metabolic reprogramming characteristic of tumor cells (the “Warburg” effect), making tumor cells particularly susceptible to deficiencies of glucose uptake. This project aims to 1) define a novel activity for Lrp5, a cell surface receptor previously described as a canonical Wnt signaling receptor; 2) address a novel paradigm, that the oncogenicity of Wnt signaling may be ameliorated by reducing the metabolic activity associated with Lrp5; 3) apply unique technical (IFAST immunoprecipitation) and knowledge resources from the study of Lrp5 in bone (where Lrp5 governs bone density, and mutations of Lrp5 in human patients generate gain- and loss-of-function for osteoblast activity).