PP4 and IGF-1 Signaling in Breast Tumorigenesis

描述(申请人提供)：胰岛素样生长因子1(IGF-1)通过促进增殖和保护癌细胞免受凋亡，参与维持恶性表型。胰岛素受体底物1(IRS-1)是连接IGF-1受体及其下游信号通路的IGF-1途径的关键细胞内信号分子。IRS-1的结构性激活在包括乳腺癌在内的人类肿瘤中是一种常见的事件。因此，IRS-1和IRS-1介导的IGF-1信号的任何负调控因子都可能具有潜在的抗肿瘤活性。肿瘤坏死因子-α(TNF-α)通过抑制IGF-1信号转导发挥抗乳腺癌细胞增殖的作用。我们的初步研究表明，蛋白磷酸酶4(PP4)在肿瘤坏死因子-α刺激后与IRS-1和IRS-4相互作用并下调IRS-4。在这一应用中，我们将验证我们的假设，即PP4通过去磷酸化和下调IRS-1来介导肿瘤坏死因子-α对IGF-1信号的拮抗作用，从而在乳腺癌细胞中发挥其肿瘤抑制作用。具体目标是： 目的1.研究PP4对乳腺癌细胞IRS-1去磷酸化和下调的作用机制。我们将研究IRS1-PP4相互作用在MCF-7乳腺癌细胞中的功能意义。我们将通过二维磷酸肽作图和微测序来定位IRS-1中的PP4去磷酸化位点(S)。我们将通过研究IRS-1在乳腺癌细胞IGF-1信号中的磷酸化缺陷和磷酸化模拟突变体来确定PP4介导的去磷酸化的功能意义。 目的2.研究PP4在IGF-1信号转导和乳腺肿瘤发生中的作用。我们将使用基因敲除、siRNA和显性-负性突变的方法来检验PP4介导的肿瘤坏死因子-α对IGF-1刺激的乳腺癌细胞生长、存活或运动的拮抗作用，从而发挥肿瘤抑制作用。这些研究将为PP4在乳腺癌中对IGF-1信号的新调控提供新的见解。此外，这项研究可能有助于将PP4确定为乳腺癌治疗的新靶点。

DESCRIPTION (provided by applicant): Insulin-like growth factor 1 (IGF-1) participates in the maintenance of the malignant phenotype by enhancing proliferation and protecting cancer cells from apoptosis. Insulin receptor substrate 1 (IRS-1) is a key intracellular signaling molecule of the IGF-1 pathway that connects the IGF-1 receptor to its downstream signaling pathways. Constitutive activation of IRS-1 is a frequent event in human tumors including breast cancer. Thus, any negative regulator of IRS-1 and IRS-1-mediated IGF-1 signaling may have potential antitumor activity. Tumor necrosis factor alpha (TNF-alpha) exerts its anti-proliferative effect on breast cancer cells by impairing IGF-1 signaling. Our preliminary studies suggest that protein phosphatase 4 (PP4) interacts with and down-regulates IRS-1 and IRS-4 following TNF-alpha stimulation. In this application, we will test our hypothesis that PP4 plays a role in mediating the antagonistic effect of TNF-alpha on IGF-1 signaling by dephosphorylating and down-regulating IRS-1, and thus exerts its tumor suppressive function in breast cancer cells. The specific aims are: Aim 1. Study the mechanism of IRS-1 dephosphorylation and down-regulation by PP4 in breast cancer cells. We will study the functional significance of IRS1-PP4 interaction in MCF-7 breast cancer cells. We will map the PP4 dephosphorylation site(s) within IRS-1 by 2-D phosphopeptide mapping and microsequencing. We will determine the functional significance of PP4-mediated dephosphorylation by studying phosphorylation-deficient and phosphorylation-mimetic mutants of IRS-1 in IGF-1 signaling in breast cancer cells. Aim 2. Study the role of PP4 in IGF-1 signaling and breast tumorigenesis. We will test the hypothesis that PP4 mediates the antagonistic effect of TNF-alpha on IGF-1-stimulated breast cancer cell growth, survival or motility, and thus, acts as a tumor suppressor using gene knockout, siRNA, and dominant-negative mutant approaches. These studies will provide new insight into the novel regulation of IGF-1 signaling by PP4 in breast cancer. Furthermore, this study may lead to the identification of PP4 as a novel target for breast cancer therapeutics.