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）是世界范围内癌症死亡的主要原因之一，不幸的是，大多数患有这种毁灭性疾病的患者没有治愈性治疗，因为HCC对常规化疗具有众所周知的抗性。因此，了解细胞生长调节是如何控制的是至关重要的癌症生物学和知识获得将促进合理的药物设计，以治疗肝癌。逃避细胞死亡是癌细胞的主要特征，并且通过生长和存活因子如肝细胞生长因子（HGF）-MET系统的异常表达来实现对细胞死亡和生长控制的厌恶。事实上，HGF-MET的过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。细胞的存活或死亡通常由受促存活和促死亡配体受体系统密切控制的受调节信号通路的复杂网络控制。最近，已经变得明显的是，细胞死亡可以通过两种主要的不同程序发生-一种称为“凋亡”，其是半胱天冬酶依赖性的，另一种称为“程序性坏死”或“坏死性凋亡”（下文称为“坏死”），其是半胱天冬酶非依赖性的。RIPK 1（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刽子手。细胞死亡的失调具有从组织变性到癌症的可怕后果。虽然细胞凋亡的分子调控是相当众所周知的，但支配坏死的分子机制尚不清楚。我们最近有了新的发现，MET（又名MET）的激活可能是一个新的发现。通过其配体HGF对RIPK 1（HGFR）的作用导致RIPK 1快速募集至质膜，RIPK 1酪氨酸磷酸化和多聚泛素化导致RIPK 1酶活性的抑制及其降解，最终促进细胞存活对抗坏死。我们还发现RIPK 1在人类癌症中下调，包括乳腺癌，结肠癌和肝癌（HCC）。相反，我们发现在HCC莫尔细胞系中通过MET抑制剂阻断HGF/MET导致RIPK 1的大量上调和细胞死亡。因此，本提案的总体目标是检验以下假设：在HCC中，MET直接酪氨酸磷酸化RIPK 1，抑制RIPK 1酶活性（坏死所需）并标记其降解，从而抑制RIPK 1依赖性HCC细胞死亡，并阻断HGF-MET轴例如，通过施用HGF-MET抑制剂）将导致RIPK 1的增加，从而使肝癌细胞对死亡诱导药物如顺铂敏感。在目的1中，我们将利用肝细胞培养系统，并通过遗传方法操纵MET和RIPK 1来验证我们的假设，即HGF-MET轴通过抑制RIPK 1介导的坏死来促进细胞存活。我们是第一个发现MET被HGF激活后，通过MET-RIPK 1复合物的形成，导致RIPK 1在质膜上快速的酪氨酸磷酸化和泛素化。我们已经发现MET可以直接使RIPK 1酪氨酸磷酸化。使用串联质谱，我们已经确定RIPK 1中的该位点为Tyr 384残基。因此，我们打算研究MET介导的RIPK 1修饰对肝细胞中RIPK 1信号传导和细胞存活的重要性。在目的2中，我们将直接检验我们的假设，即逃避由HGF-MET轴引发的RIPK 1介导的细胞死亡有助于肝癌发生，并且阻断HGF-MET导致RIPK 1上调，使癌细胞对通过坏死杀死的化疗药物敏感。为了实现这一目标，我们将分别使用RIPK 1和HGF的肝脏特异性功能丧失和获得小鼠模型，以及肝脏肿瘤发生研究。众所周知，HGF和MET在人类HCC中过表达;有趣的是，我们发现RIPK 1在人类癌症如肝癌中下调。使用我们可用的相关小鼠模型，我们将用DEN治疗肝脏特异性RIPK 1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。我们预期，与LRIPKO、AlbHGF-TG或野生型对照相比，在复合转基因小鼠中，肝癌发生将显著增强和加速。我们预期，用临床试验中的MET激酶抑制剂抑制MET将诱导肿瘤中的RIPK 1上调，从而使它们成为肿瘤。 易受化疗药物如顺铂的影响而死亡总的来说，所提出的研究将建立肝肿瘤发生的新范例，其中生长因子-受体信号传导系统（即，HGF-MET轴）通过直接抑制促坏死因子RIPK 1，从而防止RIPK 1诱导的坏死来促进癌细胞存活。我们的研究将为在HCC的临床环境中靶向HGF-MET信号传导以恢复RIPK 1表达从而使癌细胞对死亡敏感提供理论基础，特别是与通过坏死杀死细胞的常规化疗药物如顺铂组合。