The role of pRb-dependent Ihh in osteogenesis and osteosarcoma

描述（由申请人提供）：视网膜母细胞瘤蛋白（pRb）是一种肿瘤抑制因子，在大多数人类癌症中发现突变或失调。最常见的是直接影响视网膜母细胞瘤和骨肉瘤。为了理解为什么pRb的直接突变在某些组织的癌症发展中很重要，理解它在正常发展中的作用是必不可少的。通过各种研究，申办方实验室发现pRb对于成骨细胞的谱系定型是必需的，并且在缺乏pRb的动物的颅骨中存在更多的双能祖细胞，这可能导致恶性表型。该实验室最近还发现，在分化过程中，印度刺猬（Ihh）在Rb 1-/-颅骨细胞中下调，这意味着该因子是pRb在骨发育和癌症中功能的潜在介质。本研究的目的是探讨Ihh在成骨细胞谱系定型中的作用，以及Ihh在骨肉瘤中作为pRb功能介导者的可能作用。使用体外和体内工具，将评估缺乏Ihh的颅骨细胞的双能性/干性，以确定Ihh是否是成骨细胞定型所必需的。这将通过首先将细胞分化成脂肪细胞和成骨细胞以确定Ihh-/-颅骨细胞是否是双能的来实现。干细胞将通过连续分化这些细胞来评估，即用成骨培养基分化直至实现完全分化，然后重新铺板并再次分化。除了这些研究之外，还将利用重组Ihh（rIhh）和成脂细胞/成骨细胞分化方案来评估在分化过程中向pRb缺陷的头盖骨细胞中加入Ihh的效果.预计Ihh的丢失将增加颅骨中双能祖细胞的池（类似于pRb丢失），并且将rIhh添加到Rb 1-/-培养物中将损害其双能能力。还将评估Ihh损失在骨肉瘤模型中的作用以确定Ihh在骨肿瘤发生中的作用。为此，将使动物交配以携带Trp 53和Ihh的条件性等位基因，之后通过将Osterix-Cre等位基因引入该群体，将两者在成骨细胞中缺失。将监测动物的骨肉瘤形成，并分离和表征肿瘤细胞，以评估其脂肪细胞和成骨细胞分化能力。此外，将使用来自E18.5胚胎的颅骨细胞并用成骨培养基分化这些细胞来评估成骨细胞定型和分化中p53和Ihh两者的损失。预计Ihh的丢失将有助于成骨细胞的转化， 促进肿瘤发生。 公共卫生相关性：本项目的目的是了解骨骼发育中的一个关键因素如何促进这一过程以及肿瘤形成。随着癌症发病率在全球范围内的上升，确定各种蛋白质相互作用以帮助肿瘤发生的方式对于治疗和治愈疾病是必要的。虽然这个项目的重点是骨肉瘤，但它也将有助于更广泛地了解发育蛋白质如何影响癌症的发生和生长。

DESCRIPTION (provided by applicant): The retinoblastoma protein (pRb) is a tumor suppressor that is found mutated or dysregulated in most human cancers. It is most commonly directly affected in retinoblastoma and osteosarcoma. To appreciate why direct mutation of pRb is important in cancer development in certain tissues, understanding its role in normal development is essential. Through various studies, the sponsor's lab found that pRb is necessary for lineage commitment of osteoblasts and that there are more bipotent progenitors in the calvarium of animals lacking pRb, which could contribute to malignant phenotypes. The lab also found more recently that Indian hedgehog (Ihh) is downregulated in Rb1-/- calvarial cells during differentiation, implicating this factor as a potential mediator of pRb's function in bone development and cancer. The purpose of this project is to determine the role of Ihh in lineage commitment of osteoblasts as well as the possible roles for Ihh as a mediator of pRb function in osteosarcoma. Using both in vitro and in vivo tools, the bipotency/stemness of calvarial cells lacking Ihh will be assessed to determine if Ihh is necessary for osteoblast commitment. This will be accomplished by first differentiating the cells into adipocytes and osteoblasts to determine if Ihh-/- calvarial cells are bipotent. Stemness will be assessed by serially differentiating these cells, that is, differentiating with osteogenic media until full differentiation has been achieved, then replating and differentiating again. In addition to these studies, the effect of adding Ihh to pRb- deficient calvarial cells during differentiation will be assessed utilizing recombinant Ihh (rIhh) and the adipoctye/osteoblast differentiation protocols. It is expected that loss of Ihh will increase the pool of bipotent progenitor cells in the calvariu (similar to pRb loss) and that adding rIhh to Rb1-/- cultures will impair their bipotent capabilitis. The effect of Ihh loss in an osteosarcoma model will also be assessed to determine a role for Ihh in bone tumorigenesis. To this end, animals will be mated to carry conditional alleles of Trp53 and Ihh, after which both will be deleted in osteoblasts by introducing an Osterix-Cre allele into this population. Animals will be monitored for osteosarcoma formation and tumor cells will be isolated and characterized to assess their adipocyte and osteoblast differentiation abilities. In addition, the loss of both p53 and Ihh in osteoblast commitment and differentiation will be assessed using calvarial cells from E18.5 embryos and differentiating these cells with osteogenic media. It is expected that Ihh loss will contribute to transformation of osteoblasts and promote tumorigenesis. PUBLIC HEALTH RELEVANCE: This purpose of this project is to understand how a key factor in bone development contributes to this process as well as tumor formation. As the incidence of cancer rises worldwide, determining ways in which various proteins interact to aid in tumorigenesis is necessary to work toward treating and curing the disease. Although this project focuses on osteosarcoma, it will also contribute to the broader understanding of how developmental proteins affect cancer initiation and growth.