Cancer and Gene Regulation by the pRB/E2F Pathway

项目总结（见说明）： 癌症发展需要改变控制细胞增殖、凋亡和终末分化的调节机制。该项目的中心是视网膜母细胞瘤蛋白（人类的pRB，小鼠的pRb）肿瘤抑制剂。pRB通过直接调节其潜在的转录程序在这些生物过程中的每一个中起关键作用。已经鉴定了许多pRB响应性mRNA，并且最近的研究显示pRB在miRNA的调节中的直接作用。然而，目前还不清楚pRB的各种功能如何有助于其肿瘤抑制作用。本项目（项目3）将研究两个关键pRb功能的机制。目的1和2中的实验将确定mlRNA调节如何有助于pRb在间充质分化和肿瘤发生中的作用。这些研究的理由有两个方面。首先，我们已经表明，pRb是间充质特化的一个关键决定因素，pRb缺失促进去分化和细胞可塑性。第二，在与项目1的合作中，我们已经证明Rb家族的缺失会导致 包括间充质可塑性的候选调节物的充分研究（例如miR-17~92簇）和不充分表征的miRNA的去调节。在目标1中，我们将使用基于细胞的测定来确定这些pRb调节的miRNA的功能，包括建立它们由pRb调节的机制、它们的靶标的身份以及它们在间充质分化和pRb调节的可塑性中的作用。在目标2中，我们将直接测试miR-17~92簇以及更广泛的miRNA如何影响Rb突变型骨肉瘤小鼠模型中产生的肿瘤的生长和可塑性。在这些间充质研究的同时，我们将研究pRb的促凋亡作用的机制基础。我们已经表明，化疗治疗促进转录活性pRb-E2 F1复合物的形成，选择性诱导凋亡基因。我们的数据表明，翻译后修饰的E2 F 1和pRB控制这种复合物的形成。目标3中的实验将使用功能获得和功能丧失突变体来确定这些翻译后修饰如何影响pRB和E2 f1调节mRNA和miRNA程序的能力，这些mRNA和miRNA程序是这些蛋白在细胞凋亡与细胞增殖中的作用的基础。

PROJECT SUMMARY (See instructions): Cancer development requires alterations in the regulatory mechanisms that control cell proliferation, apoptosis and terminal differentiation. This project is centered on the retinoblastoma protein (pRB in humans, pRb in mouse) tumor suppressor. pRB plays a key role in each of these biological processes by directly regulating their underlying transcriptional programs. Numerous pRB-responsive mRNAs have been identified and more recent studies show a direct role for pRB in the regulation of miRNAs. However, it remains unclear how the various functions of pRB contribute to its tumor suppressive roles. This project (Project 3) will investigate the mechanisms that underlie two key pRb functions. Experiments in Aims 1 and 2 will determine how mlRNA regulation contributes to pRb's role in mesenchymal differentiation and tumorigenesis. The rationale for these studies is twofold. First, we have shown that pRb is a key determinant of mesenchymal specification, and that pRb loss promotes dedifferentiation and cellular plasticity. Second, in collaboration with Project 1, we have shown that Rb family loss causes the deregulation of both well-studied (e.g. the miR-17~92 cluster) and poorly characterized miRNAs that include candidate regulators of mesenchymal plasticity. In Aim 1, we will use cell-based assays to determine the function of these pRb-regulated miRNAs including establishing the mechanism of their regulation by pRb, the identity of their targets and their roles in mesenchymal differentiation and pRb regulated plasticity. In Aim 2, we will directly test how the miR-17~92 cluster, and miRNAs more broadly, influence the growth and plasticity of tumors arising in a mouse model of Rb mutant osteosarcoma. In parallel with these mesenchymal studies, we will investigate the mechanistic basis for pRb's pro-apoptotic role. We have shown that chemotherapeutic treatment promotes formation of a transcriptionally active pRb-E2F1 complex that selectively induces apoptotic genes. Our data suggest that post-translational modifications of both E2f 1 and pRB control the formation of this complex. Experiments in Aim 3 will use gain-of-function and loss-of-function mutants to establish how these post-translational modifications affect pRB and E2f1 's ability to regulate the mRNA and miRNA programs that underlie the roles of these proteins in apoptosis versus cell proliferation.