Mechanism of Met-Induced Hepatocyte Survival

Met诱导肝细胞存活的机制

• 批准号: 9078713
• 负责人: Reza Zarnegar
• 金额: $ 35.23万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-15 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9078713
• 关键词: Animal Model; Apoptosis; Breast; Cancer Biology; Cancer Etiology; Caspase; Cell Culture System; Cell Death; Cell Line; Cell Survival; Cell membrane; Cells; Cessation of life; Cisplatin; Clinical; Clinical Trials; Colon; Colon Carcinoma; Complex; Cues; Cultured Cells; Development; Disease; Drug Design; Goals; Growth; Growth Factor Receptors; HGF gene; Hepatocarcinogenesis; Hepatocyte; Human; Internet; Knock-out; Knockout Mice; Knowledge; Lead; Ligands; Liver; Liver neoplasms; MET gene; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Modification; Molecular; Monitor; Mutation; Necrosis; Patients; Pharmaceutical Preparations; Polyubiquitination; Primary carcinoma of the liver cells; Protein Kinase; RIPK1 gene; Receptor Signaling; Regulation; Resistance; Signal Pathway; Signal Transduction; Site; System; Testing; Tissues; Transgenic Mice; Tumor Cell Line; Tyrosine; Tyrosine Phosphorylation; Ubiquitination; Up-Regulation; cancer cell; cell growth; cell growth regulation; cell killing; chemotherapy; curative treatments; gain of function; genetic approach; inhibitor/antagonist; killings; kinase inhibitor; malignant breast neoplasm; meetings; mortality; mouse model; novel; overexpression; prevent; programs; public health relevance; receptor; tumor

 DESCRIPTION (provided by applicant): Liver cancer (Hepatocellular Carcinoma, HCC) is one of the leading causes of cancer mortality worldwide and unfortunately no curative therapy exists for most patients with this devastating disease because HCC is notoriously resistant to conventional chemotherapy. Thus understanding how cell growth regulation is controlled is paramount to cancer biology and the knowledge gained will facilitate rational drug design to treat HCC. Escape from cell death is a cardinal feature of the cancer cell and aversion from cell death and growth control is achieved by aberrant expression of growth and survival factors like Hepatocyte Growth Factor (HGF)-MET system. In fact overexpression or activating mutations of HGF-MET occur in a variety of human cancers including breast, colon and liver. The survival or death of cells is normally controlled by an intricate web of regulated signaling pathways intimately governed by pro-survival and pro-death ligand receptor systems. Recently it has become apparent that cell death can occur by two major disticnt programs - one called `apoptosis' which is caspase-dependent and one that is caspase-independent dubbed `programmed necrosis' or `necroptosis' (hereafter referred to as `necrosis'). RIPK1 (commonly known as Receptor Interacting Protein Kinase 1) has emerged as an important activator and executioner of necrosis. Dysregulation of cell death has dire consequences ranging from tissue degeneration to cancer. While molecular regulation of apoptosis is fairly well known, the molecular mechanisms that govern necrosis are not understood. We have recently made the novel discovery that activation of MET (a.k.a. HGFR) by its ligand HGF results in rapid recruitment of RIPK1 to the plasma membrane, RIPK1 tyrosine phosphorylation and polyubiquitination leading to inhibition of RIPK1 enzymatic activity and its degradation culminating in promotion of cell survival against necrosis. We have also discovered that RIPK1 is down regulated in human cancers including breast, colon and liver cancer (HCC). Conversely, we have discovered that blocking HGF/MET by MET inhibitors in HCC tu mor cell lines results in massive upregulation of RIPK1 and cell death. Thus, the overall goal of this proposal is to test the hypotheses that, in HCC, MET directly tyrosine phosphorylates RIPK1 inhibiting RIPK1 enzymatic activity (which is required for necrosis) and marking it for degradation thus inhibiting RIPK1- dependent HCC cell death, and that blocking the HGF-MET axis (for example, by administration of HGF-MET inhibitors) will lead to an increase in RIPK1 thus sensitizing liver cancer cells to death- inducing drugs like cisplatin. In Aim 1, we will utilize a hepatocytic cell culture system and manipulate MET and RIPK1 by genetic approaches to test our hypothesis that HGF-MET axis promotes cell survival by inhibiting RIPK1- mediated necrosis. We are the first to show that activation of MET by HGF results in rapid tyrosine phosphorylation and ubiquitination of RIPK1 at the plasma membrane via MET-RIPK1 complex formation. We have discovered that MET can directly tyrosine phosphorylate RIPK1. Using Tandem Mass Spec, we have identified this site in RIPK1 to be the Tyr384 residue. Thus we intend to investigate the importance of MET- mediated RIPK1 modifications on RIPK1 signaling and cell survival in hepatocytic cells. In Aim 2, we will directly test our hypothesis that escape from RIPK1-mediated cell death instigated by the HGF-MET axis contributes to hepatocarcinogenesis and that blocking HGF-MET causes RIPK1 upregulation sensitizing cancer cells to chemotherapeutic drugs which kill by necrosis. To accomplish this aim we will use liver-specific loss- and gain-of-function mouse models of RIPK1 and HGF, respectively, and liver tumorigenesis studies. It is well-known that HGF and MET are overexpressed in human HCC; interestingly, we have found that RIPK1 is downregulated in human cancers such as liver. Using relevant mouse models available to us, we will treat liver-specific RIPK1 knock out [LRIPKO] mice and compound LRIPKO/AlbHGF-transgenic mice and controls with DEN to induce HCC and will monitor them for liver tumor development to test our hypothesis. We anticipate that liver carcinogenesis will be dramatically enhanced and accelerated in the compound transgenic mice as compared to LRIPKO, AlbHGF-TG or wildtype controls. We anticipate that inhibiting MET with MET kinase inhibitors which are in clinical trials will induce RIPK1 upregulation in the tumors rendering them susceptible to death by chemotherapeutic drugs like cisplatin Collectively, the proposed studies will establish a new paradigm in liver tumorigenesis in which a growth factor- receptor signaling system (namely, the HGF-MET axis) promotes cancer cell survival by directly inhibiting the pro-necrosis factor, RIPK1, thereby preventing RIPK1-induced necrosis. Our studies will provide rationale for targeting HGF-MET signaling in the clinical setting of HCC to restore RIPK1 expression hence sensitizing the cancer cells to death, especially in combination with conventional chemotherapy drugs like cisplatin which kill cells by necrosis.

 描述（由适用提供）：肝癌（肝细胞癌，HCC）是全球癌症死亡率的主要原因之一，不幸的是，对于大多数患有这种毁灭性疾病的患者，由于HCC对常规化疗具有抗药性。了解细胞生长调节的控制方式对癌症生物学的控制至关重要，而获得的知识将有助于理性的药物设计以治疗HCC。从细胞死亡中逃脱是癌细胞的基本特征，细胞死亡和生长控制的厌恶是通过异常表达生长和生存因子（如肝细胞生长因子（HGF）-MET系统）实现的。实际上，HGF-MET的过表达或激活突变发生在包括乳腺癌，结肠和肝脏在内的各种人类癌症中。细胞的存活或死亡通常受到由促生物和死亡配体受体系统密切控制的调节信号通路的复杂网络控制。最近，很明显，两个主要的脱发程序可能会发生细胞死亡 - 一种称为caspase依赖性的“凋亡”，另一种称为caspase独立的，被称为“编程坏死”或“坏死”（以下称为“坏死”）。 RIPK1（通常称为受体相互作用蛋白激酶1）已成为重要的激活剂和坏死的执行器。细胞死亡的失调具有从组织变性到癌症的结果。虽然凋亡的分子调节是众所周知的，但尚不清楚控制坏死的分子机制。 We have recently made the novel discovery that activation of MET (a.k.a. HGFR) by its ligand HGF results in rapid recruitment of RIPK1 to the plasma membrane, RIPK1 tyrosine phosphorylation and polyubiquitination leading to inhibition of RIPK1 enzymatic activity and its degradation culminating in promotion of cell survival against necrosis.我们还发现，RIPK1在包括乳腺癌，结肠癌和肝癌（HCC）在内的人类癌症中受到调节。相反，我们发现在HCC TU MOR细胞系中通过MET抑制剂阻断HGF/MET会导致RIPK1和细胞死亡的大规模上调。 That, the overall goal of this proposal is to test the hypotheses that, in HCC, MET directly tyrosine phosphorylates RIPK1 inhibiting RIPK1 enzymatic activity (which is required for necrosis) and marking it for degradation thus inhibiting RIPK1- dependent HCC cell death, and that blocking the HGF-MET axis (for example, by administration of HGF-MET inhibitors) will lead to an increase在RIPK1中，从而使肝癌细胞对死亡 - 诱导诸如顺铂等药物的敏感性。在AIM 1中，我们是第一个证明HGF激活HGF激活的人会导致RIPK1通过MET-RIPK1络合物形成在质膜上快速酪氨酸磷酸化和RI​​PK1的泛素化。我们发现MET可以直接酪氨酸磷酸化RIPK1。使用串联质量规格，我们在RIPK1中确定了该站点为Tyr384居住。我们打算研究Met介导的RIPK1修饰对肝细胞细胞中RIPK1信号传导和细胞存活的重要性。在AIM 2中，我们将直接检验我们的假设，即HGF-MET轴刺激了RIPK1介导的细胞死亡，这有助于肝癌发生，并且阻断HGF-MET会导致RIPK1导致RIPK1上调使癌细胞敏感到因坏死而杀死的化学治疗药物。为了实现这一目标，我们将分别使用RIPK1和HGF的肝特异性损失和功能性小鼠模型，以及肝肿瘤发生研究。众所周知，HGF和MET在人类HCC中过表达。有趣的是，我们发现RIPK1在肝脏等人类癌症中被下调。使用相关的小鼠模型，我们将处理肝脏特异性RIPK1敲除[Lripko]小鼠和复合Lripko/albhGF-TENSGENIC小鼠和DEN的对照来诱导HCC，并将监测它们以测试我们的假设。我们预计，与LRIPKO，AlbHGF-TG或WildType对照相比，在化合物转基因小鼠中，肝癌发生将在化合物转基因小鼠中动态增强和加速。预计抑制临床试验中的MET激酶抑制剂将诱导RIPK1上调。 拟议的研究易受化学治疗药物（如顺铂）的死亡，将在肝肿瘤发生中建立一个新的范式，其中生长因子受体受体信号系统（即HGF-MET Axis）可通过直接抑制促疾病因素来促进癌细胞的存活。我们的研究将为靶向HGF-MET信号在HCC的临床环境中靶向HGF-MET信号传导的基本原理，从而恢复RIPK1表达，从而使癌细胞致死，尤其是与常规化学疗法药物（如顺铂）结合使用，例如顺铂，这些药物通过坏死而杀死细胞。