Functional Dissection of Essential Wnt Signaling Receptors in Breast Stem Cells

项目总结 WNT信号被用来维持体细胞和胚胎干细胞的再生能力。然而， 这种激活是有风险的，因为异位Wnt信号对上皮组织具有高度的致癌性；因此， 在存在异位Wnt信号的情况下维持和发育的组织可能有额外的肿瘤风险 在生命的后期发展。我们发现了Wnt信号受体之一Lrp5的辅助功能， 我们提出的可能对Wnt信号的致癌活性负责；如果我们是正确的，LRP5 在以干细胞为基础的治疗方法的未来应用中，可以对活性进行操纵。 乳腺上皮干细胞的研究为体细胞活性的研究提供了极好的机会。 仅一个乳腺干细胞就可以在移植到人乳腺导管后再生完整的乳腺导管树 新主人肥胖垫。我们之前的工作表明：1)乳腺干细胞的功能与 Wnt信号活性，以及2)Wnt信号受体LRP5是维持乳腺干细胞所必需的 活性，尽管更强大的Wnt信号受体LRP6是共表达的。这反映了 维持乳腺干细胞的Wnt配体(S)需要同时存在LRP5和LRP6 以产生Wnt配体。因此，我们将LRP5称为乳腺干细胞功能的“守门人”。 然而，最近我们发现1)LRP5对乳腺上皮细胞的生长有深远的影响 不依赖于典型的Wnt信号通路(β连环蛋白/Tcf依赖的反式激活)。LRP5 通过一种全新的机制促进葡萄糖摄取，而剥夺葡萄糖就足以使表观复制 LRP5功能的丧失；2)异常的可溶性Wnt配体Wnt3a可以促进生长和 乳腺上皮细胞在体内的发育，不需要LRP5的存在。 我们的假设是，LRP5促进肿瘤的发展是对异位Wnt信号的反应，但不是 Wnt诱导的干细胞积累需要伴随乳房再生的激活 潜力。在这个应用中，我们的目标是找出1)LRP5是如何调节葡萄糖摄取和乳房的 上皮细胞生长，以及2)LRP5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，需要增加葡萄糖摄取量来为 肿瘤细胞的代谢重编程特性(“Warburg”效应)，使肿瘤细胞 特别容易受到葡萄糖摄取不足的影响。 该项目旨在1)定义LRP5的新活性，LRP5是一种细胞表面受体，以前被描述为 规范的Wnt信号受体；2)提出了一种新的范例，即Wnt信号的致瘤性可能 通过减少与LRP5相关的代谢活动来改善；3)应用独特的技术(IFAST 免疫沉淀)和来自骨中LRP5研究的知识资源(其中LRP5主宰骨骼 人类患者LRP5的密度和突变导致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).