The role of pRb-dependent Ihh in osteogenesis and osteosarcoma

描述(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调控失调。在视网膜母细胞瘤和骨肉瘤中最常受到直接影响。要了解为什么pRb的直接突变在某些组织的癌症发展中是重要的，了解它在正常发育中的作用是至关重要的。通过各种研究，赞助商的实验室发现，pRb是成骨细胞向世系承诺所必需的，缺乏pRb的动物头盖骨中有更多的双能祖细胞，这可能导致恶性表型。该实验室最近还发现，在Rb1-/-头盖骨细胞分化过程中，印度刺猬(IHH)的表达下调，这意味着该因子可能是pRb在骨骼发育和癌症中的功能调节因子。本项目的目的是确定IHH在成骨细胞的谱系承诺中的作用，以及IHH作为骨肉瘤中PRB功能的中介的可能作用。使用体外和体内工具，将评估缺乏IHH的头盖骨细胞的双能性/干性，以确定IHH是否对成骨细胞的定位是必要的。这将通过首先将细胞分化为脂肪细胞和成骨细胞来完成，以确定IHH-/-颅骨细胞是否具有双能性。干细胞将通过连续分化这些细胞来评估，也就是说，用成骨介质分化，直到完全分化，然后复制和再次分化。除了这些研究外，还将利用重组IHH(RIHh)和脂肪细胞/成骨细胞分化方案来评估在分化过程中加入IHH对PRB缺陷的头盖骨细胞的影响。预计失去IHH将增加头盖骨中的双能祖细胞池(类似于PRB的丧失)，而在Rb1-/-培养中加入rIhh将削弱其双能能力。还将评估骨肉瘤模型中IHH丢失的影响，以确定IHH在骨肿瘤发生中的作用。为此，动物将交配携带条件等位基因Trp53和IHH，之后将通过将Osterix-Cre等位基因引入成骨细胞来删除这两个等位基因。动物将被监测骨肉瘤的形成，肿瘤细胞将被分离并鉴定以评估其脂肪细胞和成骨细胞的分化能力。此外，将使用来自E18.5胚胎的头盖骨细胞并用成骨培养液分化这些细胞来评估成骨细胞承诺和分化过程中P53和IHH的丢失。预计IHH的丢失将有助于成骨细胞的转化和 促进肿瘤发生。

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.