PTEN TUMOR SUPPRESSOR AND SIGNAL TRANSDUCTION

描述：（改编自研究者摘要）PTEN/MMAC 1是一种肿瘤 在多种癌症中突变的抑制基因。PTEN编码磷酸酶 认识到重要的第二信使， 磷脂酰肌醇-3，4，5-三磷酸（PIP 3，4，5），并去除 肌醇环上的3 '-磷酸。因此，PTEN拮抗 磷脂酰肌醇（PI）-3激酶，其在磷酸化肌醇环时， 同样的位置。尽管PI-3激酶和PTEN可能影响许多 信号通路，也许是最好理解的通路种类，PTEN作用于 胰岛素受体和PI-3激酶的下游和AKT的上游。通过 底物的磷酸化，AKT调节许多参与的靶点， 转录、翻译、细胞周期和凋亡。测试 假设PTEN调节基因表达，他们开发了一种腺病毒 系统在感染后两小时内表达PTEN-/-肿瘤 细胞PTEN表达与AKT的快速抑制相关。标记 将不同感染时间的RNA与寡核苷酸芯片杂交 代表了超过四万个基因通过比较 未感染的细胞、腺-β-半乳糖苷酶和腺-PTEN感染的细胞，它们 确定了32名候选人，这些候选人在3小时内至少被诱导了三次。 感染他们为24名候选人准备了探针，其中8名 在北方印迹上证实了PTEN特异性诱导。最易诱导的基因 胰岛素受体底物（IRS）-2。IRS-2的诱导作用在 蛋白质水平。IRS-2是IRS基因家族的一员，其功能是 传递胰岛素、胰岛素样生长因子和细胞因子受体的衔接蛋白 信号. IRS-2的主要作用是将活化的受体与PI-3连接 激酶。在PTEN感染的细胞中，诱导的IRS-2与 PI-3激酶。因此，他们的初步数据表明， 诱导反馈回路激活PI-3激酶。本补助金申请将 本研究旨在探讨PTEN对IRS-2的调控机制，并对PTEN在IRS-2中的作用机制进行初步探讨。 IRS-2在肿瘤发生中的作用。他们将使用重组腺病毒， 药理学试剂，以确定细胞能够通过其 诱导IRS 2应答PTEN。这将包括使用腺病毒， 表达PTEN突变、显性阴性AKT和显性活性FKHR、GSK-3， 和RAF。还将测试PI-3激酶、MEK和mTOR的抑制剂。他们 他们不仅会分析这些药物诱导IRS-2的能力， 还比较了它们与PTEN对AKT、MAPK、GSK-3和S6的影响 激酶活性PI-3激酶和AKT的p85和p110亚基是有效的 致癌基因为了确定IRS-2在癌症中是否发生了基因改变， 将在细胞系和原发性肿瘤中筛选突变和影响， 含有野生型PTEN。因为IRS-2在细胞内起着衔接蛋白的作用， 他们将研究PTEN对胰岛素信号转导的影响， 小鼠的胰岛素信号传导。最后，PTEN杂合子小鼠在 多器官系统他们将用IRS-2+/-小鼠饲养PTEN+/-小鼠， IRS-2缺失是否能够减弱肿瘤表型。

DESCRIPTION: (Adapted from the investigator's abstract) PTEN/MMAC1 is a tumor suppressor that is mutated in many forms of cancer. PTEN encodes a phosphatase that recognizes the important second messenger, phosphatidylinositol-3,4,5-triphosphate (PIP3,4,5), and removes the 3'-phosphate from the inositiol ring. PTEN therefore antagonizes phosphatidylinositol (PI)-3 kinase, which phosphorylates the inositol ring at the same position. Although PI-3 kinase and PTEN potentially influence many signaling pathways, perhaps the best understood pathway species, PTEN acts downstream of the insulin receptor and PI-3 kinase and upstream of AKT. Through the phosphorylation of substrates, AKT regulates many targets involved in transcription, translation, the cell cycle, and apoptosis. To test the hypothesis that PTEN regulates gene expression, they developed an adenoviral system that expressed PTEN within two hours of infection in PTEN-/- tumor cells. PTEN expression was associated with a rapid inhibition of AKT. Labeled RNAs from different times of infection were hybridized to oligonucleotide chips representing over 40,000 genes. By comparing the level of expression between uninfected, Adeno-beta-galactosidase, and Adeno-PTEN infected cells, they identified 32 candidates that were induced at least threefold within 3 hours of infection. They prepared probes for 24 of the candidates, of which 8 demonstrated PTEN-specific induction on Northern blots. The most induced gene was the insulin receptor substrate (IRS)-2. Induction of IRS-2 was confirmed at the protein level. IRS-2 is a member of a family of IRS genes that function as adaptor proteins for the transmission of insulin, IGF, and cytokine receptor signals. The major role of IRS-2 is to link an activated receptor to PI-3 kinase. In PTEN infected cells, the induced IRS-2 coimmunoprecipitated with PI-3 kinase. Thus, their preliminary data suggests that the expression of PTEN induced a feedback-loop to activated PI-3 kinase. This grant application will attempt to explore the mechanism through which PTEN regulates IRS-2 and define the role of IRS-2 in tumorigenesis. They will use recombinant adenoviruses and pharmacological agents to identify the mode through which cells are able to induce IRS2 in response to PTEN. This will include the use of adenoviruses that express PTEN mutations, dominant-negative AKT and dominant active FKHR, GSK-3, and RAF. Inhibitors of PI-3 kinase, MEK, and mTOR will also be tested. They will not only analyze these agents for their ability to induce IRS-2, they will also compare them to PTEN regarding their effect on AKT, MAPK, GSK-3, and S6 kinase activity. The p85 and p110 subunits of PI-3 kinase and AKT are potent oncogenes. To determine whether IRS-2 is altered genetically in cancer they will screen for mutations and implications in cells lines and primary tumors containing wild type PTEN. Because IRS-2 functions as an adapter protein in the transduction of insulin signals, they will investigate the effect of PTEN on insulin signaling in mice. Finally, PTEN heterozygous mice have tumors in multiple organ systems. They will breed PTEN+/- mice with IRS-2+/- mice to see if IRS-2 loss is able to attenuate the tumor phenotype.