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。它最常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是重要的，了解它在正常发展中的作用是必不可少的。通过各种研究，发起人实验室发现，pRb对于成骨细胞的谱系承诺是必需的，并且在缺乏pRb的动物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，印度刺猬（Ihh）在Rb1-/-颅细胞分化过程中下调，暗示该因子是pRb在骨发育和癌症中功能的潜在中介。该项目的目的是确定Ihh在成骨细胞谱系承诺中的作用，以及Ihh在骨肉瘤中作为pRb功能中介的可能作用。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是否对成骨细胞的承诺是必要的。这将通过首先将细胞分化为脂肪细胞和成骨细胞来确定Ihh-/-颅细胞是否具有双能性来完成。通过连续分化这些细胞来评估干细胞的干性，也就是说，用成骨培养基分化，直到完全分化，然后再次复制和分化。除了这些研究之外，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向pRb缺失的颅细胞添加Ihh的效果。预计Ihh的缺失会增加颅骨中双能祖细胞的数量（类似于pRb的缺失），而将rIhh添加到Rb1-/-培养物中会损害它们的双能能力。还将评估Ihh在骨肉瘤模型中丢失的影响，以确定Ihh在骨肿瘤发生中的作用。为此，将动物配对为携带Trp53和Ihh的条件等位基因，然后通过将Osterix-Cre等位基因引入该群体，在成骨细胞中删除这两个等位基因。将监测动物的骨肉瘤形成情况，分离肿瘤细胞并对其进行鉴定，以评估其脂肪细胞和成骨细胞分化能力。此外，将使用E18.5胚胎的颅骨细胞并将这些细胞与成骨培养基进行分化，以评估p53和Ihh在成骨细胞的承诺和分化中的缺失。预计Ihh的缺失将有助于成骨细胞的转化和

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.