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（由申请人提供）：肝癌（肝细胞癌，HCC）是全世界癌症死亡的主要原因之一，不幸的是，对于大多数患有这种毁灭性疾病的患者来说，没有治疗方法，因为 HCC 对传统化疗具有众所周知的耐药性。细胞生长调节的控制对于癌症生物学至关重要，所获得的知识将有助于合理设计治疗 HCC 的药物。逃避细胞死亡是癌细胞的一个主要特征，通过避免细胞死亡和生长控制可以实现。生长和存活因子如肝细胞生长因子 (HGF)-MET 系统的异常表达事实上，HGF-MET 的过度表达或激活突变发生在多种人类癌症中，包括乳腺癌、结肠癌和肝癌。细胞死亡是由一个复杂的调节信号通路网络控制的，而这些信号通路又受到促生存和促死亡配体受体系统的密切控制。依赖于 caspase 的细胞和不依赖于 caspase 的细胞被称为“程序性坏死”或“坏死性凋亡”（以下简称“坏死”），RIPK1（通常称为受体相互作用蛋白激酶 1）已成为坏死的重要激活剂和执行者。细胞死亡的失调会产生从组织退化到癌症的可怕后果。我们最近发现，配体 HGF 激活 MET（又名 HGFR）会导致 RIPK1 快速募集到质膜，RIPK1 酪氨酸磷酸化和多泛素化导致 RIPK1 酶活性受到抑制。我们还发现，RIPK1 在人类癌症中表达下调，其中包括促进细胞存活、抵抗坏死。在乳腺癌、结肠癌和肝癌 (HCC) 中，我们发现在 HCC 肿瘤细胞系中通过 MET 抑制剂阻断 HGF/MET 会导致 RIPK1 的大量上调和细胞死亡。因此，该提案的总体目标是进行测试。假设在 HCC 中，MET 直接酪氨酸磷酸化，抑制 RIPK1 酶活性（坏死所需）并标记其降解，从而抑制 RIPK1 依赖性HCC 细胞死亡，并且阻断 HGF-MET 轴（例如，通过施用 HGF-MET 抑制剂）将导致 RIPK1 增加，从而使肝癌细胞对顺铂等死亡诱导药物敏感。利用肝细胞培养系统并通过遗传方法操纵 MET 和 RIPK1 来检验我们的假设，即 HGF-MET 轴通过抑制 RIPK1 介导的坏死来促进细胞存活。我们首次证明，HGF 激活 MET 会通过 MET-RIPK1 复合物的形成，导致膜质上的 RIPK1 快速酪氨酸磷酸化和泛素化。我们使用串联质谱发现 MET 可以直接磷酸化 RIPK1。确定 RIPK1 中的该位点为 Tyr384 残基，因此我们打算研究 MET 介导的 RIPK1 修饰对的重要性。肝细胞中的 RIPK1 信号传导和细胞存活 在目标 2 中，我们将直接检验我们的假设，即逃避 HGF-MET 轴引发的 RIPK1 介导的细胞死亡有助于肝癌发生，并且阻断 HGF-MET 会导致 RIPK1 上调，从而使癌细胞对肝癌敏感。为了实现这一目标，我们将使用肝脏特异性功能丧失和功能获得的小鼠模型。 RIPK1 和 HGF 分别与肝脏肿瘤发生研究 众所周知，HGF 和 MET 在人类 HCC 中过度表达；有趣的是，我们使用现有的相关小鼠模型发现 RIPK1 在人类癌症中表达下调。我们将用 DEN 治疗肝脏特异性 RIPK1 敲除 [LRIPKO] 小鼠和复合 LRIPKO/AlbHGF 转基因小鼠和对照小鼠以诱导 HCC，并监测它们的肝脏肿瘤发展，以检验我们的假设。我们预计，与 LRIPKO、AlbHGF-TG 或野生型对照相比，复合转基因小鼠的肝癌发生将显着增强和加速。我们预计，使用临床试验中的 MET 激酶抑制剂抑制 MET 将诱导 RIPK1 上调。在肿瘤中使它们 总的来说，拟议的研究将建立肝脏肿瘤发生的新范例，其中生长因子受体信号系统（即 HGF-MET 轴）通过直接抑制促坏死来促进癌细胞存活RIPK1，防止 RIPK1 诱导的坏死，我们的研究将为在 HCC 临床环境中靶向 HGF-MET 信号传导以恢复 RIPK1 表达提供理论基础。使癌细胞对死亡敏感，特别是与顺铂等传统化疗药物联合使用，通过坏死杀死细胞。