Trafficking of the EGF receptor to the nucleus: Mechanisms and Effects

描述(申请人提供)：肝脏显示出独特的生长和再生能力。例如，在三分之二的肝脏被切除后，完全的肝脏再生在几天到几周内发生。慢性肝细胞损伤可导致肝再生调节受损，从而导致肝细胞癌，这是世界上最常见的恶性肿瘤之一。父母赠款的假设是，HGF通过c-met，通过在肝细胞核内诱导InsP3介导的钙信号来调节肝脏的生长。这个FIRCA应用程序将调查这是否是受体酪氨酸激酶(RTK)在一系列组织中更普遍的作用机制。具体地说，FIRCA的应用假设是，表皮生长因子受体(EGFR)与c-Met一样，通过在细胞核内诱导InsP3介导的钙信号来调节细胞生长，并且EGFR的这一作用介导了常见恶性肿瘤的细胞增殖。这一假说将通过以下具体目标来验证：1.将确定在常见恶性肿瘤中EGFR是否以及如何到达细胞核。我们将测试小窝中的一个EGFR亚群是否向细胞核流动。受体在细胞内的运动将通过以及通过细胞分级研究来监测。在肝细胞中确定的通路将在乳腺癌、肺癌、前列腺癌和结肠癌的细胞中进行测试。2.EGF是否以及如何增加胞核内的钙离子，有待进一步研究。靶向InsP3缓冲区将被用来确定EGF是否像HGF一样，特异性地在细胞核内诱导InsP3的形成。RNA干扰技术将用于比较由EGF和HGF激活的PLC亚型，并确定这些PLC亚型是否因细胞类型而异。3.核钙信号在EGF诱导的细胞生长中的作用将被确定。我们将确定EGF诱导的细胞增殖是否通过阻断(A)EGFR向细胞核移动，(B)EGF诱导细胞核内形成钙信号，或(C)激活细胞核内钙依赖蛋白，如CaMKII来中断。这些研究将揭示生长因子及其相应的受体酪氨酸激酶如何控制完整细胞中的核钙，并确定这可能在调节肿瘤生长中发挥的独特作用。这项研究将主要在巴西的UFMG与达维森·戈麦斯合作进行，作为NIH P01 DK57751项目1的延伸。 与公共健康相关：肝脏和其他器官内细胞的生长受生长因子及其受体(称为受体酪氨酸激酶)的调节。这些研究将揭示生长因子及其相应的受体酪氨酸激酶如何控制细胞核内的信号，并确定这一信号通路在各种肿瘤中异常生长中可能发挥的独特作用。

DESCRIPTION (provided by applicant): The liver displays a unique ability to grow and regenerate. For example, complete hepatic regeneration occurs within days to weeks after two-thirds of the liver has been resected. Chronic hepatocellular damage can lead to impaired regulation of liver regeneration, which results in hepatocellular carcinoma, one of the most common malignancies in the world. The hypothesis of the parent grant is that HGF, via c-met, regulates growth in the liver by inducing InsP3-mediated Ca2+ signals within the nucleus of hepatocytes. This FIRCA application would investigate whether this is a more general mechanism of action of receptor tyrosine kinases (RTKs) across a range of tissues. Specifically, the hypothesis of this FIRCA application is that the Epidermal Growth Factor receptor (EGFR), like c-met, regulates cell growth by inducing InsP3-mediated Ca2+ signals within the nucleus, and that this action of EGFR mediates cell proliferation in common malignancies. This hypothesis will be tested through the following specific aims: 1. whether and how the EGFR reaches the nucleus in common malignancies will be determined. We will test whether a sub-population of EGFRs in caveolae traffic to the nucleus. Intracellular movement of the receptor will be monitored by as well as by cell fractionation studies. Pathways identified in liver cells will be tested in cells derived from breast, lung, prostate, and colon cancers. 2. Whether and how EGF increases Ca2+ in the nucleus will be determined. Targeted InsP3 buffers will be used to determine whether EGF, like HGF, specifically induces InsP3 formation within the nucleus. RNA interference techniques will be used to compare PLC isoforms activated by EGF and HGF, and to determine whether these PLC isoforms vary among cell types. 3. The role of nuclear Ca2+ signals in EGF-induced cell growth will be determined. We will determine whether EGF-induced cell proliferation is disrupted by blocking either (a) movement of EGFR to the nucleus, (b) EGF-induced formation Ca2+ signals in the nucleus, or (c) activation of Ca2+-dependent proteins within the nucleus, such as CaMKII. These studies will reveal how growth factors and their corresponding receptor tyrosine kinases control nuclear Ca2+ in intact cells, and identify the distinct role this may play in regulating tumor growth. This research will be performed primarily at UFMG in Brazil in collaboration with Dawidson Gomes as an extension of Project 1 of NIH P01 DK57751. PUBLIC HEALTH RELEVANCE: Growth of cells within the liver and other organs is regulated by growth factors and their receptors, known as receptor tyrosine kinases. These studies will reveal how growth factors and their corresponding receptor tyrosine kinases control signaling within the cell nucleus, and identify the distinct role this signaling pathway may play in the abnormal growth that occurs in a wide variety of tumors.