Mechanism of Met-Induced Hepatocyte Survival

 描述(申请人提供)：肝癌(肝细胞癌)是全球癌症死亡的主要原因之一，不幸的是，由于肝癌对传统化疗具有众所周知的抗药性，目前还没有针对这种毁灭性疾病的大多数患者的根治疗法。因此，了解细胞生长调控对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肝癌的药物。逃脱细胞死亡是癌细胞的一个基本特征，而细胞死亡和生长控制的厌恶是通过生长和生存因子的异常表达实现的，如肝细胞生长因子(HGF)-MET系统。事实上，HGF-MET的过度表达或激活突变在包括乳腺癌、结肠癌和肝癌在内的各种人类癌症中都存在。细胞的存活或死亡通常由一个错综复杂的信号通路网络控制，这些信号通路由支持生存和支持死亡的配体受体系统密切控制。近来发现，细胞死亡可以通过两种主要的程序发生--一种是caspase依赖的“细胞凋亡”，另一种是caspase非依赖性的“程序性坏死”或“坏死性下垂”(以下简称“坏死”)。RIPK1(俗称受体相互作用蛋白激酶1)是一种重要的坏死激活剂和执行者。细胞死亡的失调会造成从组织退化到癌症的可怕后果。虽然细胞凋亡的分子调控是众所周知的，但管理坏死的分子机制还不清楚。我们最近有了一个新的发现，激活MET(又名。HGFR)通过其配体HGF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酶活性的抑制和它的降解，最终促进细胞抗坏死存活。我们还发现RIPK1在包括乳腺癌、结肠癌和肝癌(HCC)在内的人类癌症中表达下调。相反，我们发现在肝癌细胞系中用MET抑制剂阻断HGF/MET会导致RIPK1的大量上调和细胞死亡。因此，这项建议的总体目标是测试以下假设，即在肝癌中，MET直接使RIPK1酪氨酸磷酸化，抑制RIPK1的酶活性(这是坏死所必需的)并将其标记为降解，从而抑制依赖RIPK1的肝癌细胞死亡，以及阻断HGF-MET轴(例如，通过给予HGF-MET抑制剂)将导致RIPK1的增加，从而使肝癌细胞对顺铂等致死药物敏感。在目标1中，我们将利用肝细胞培养系统，通过遗传学方法操纵MET和RIPK1，以验证我们的假设，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后，通过MET-RIPK1复合体的形成，导致质膜上RIPK1的快速酪氨酸磷酸化和泛素化。我们发现MET可以直接使RIPK1酪氨酸磷酸化。利用串联质谱仪，我们已经确定RIPK1中的这个位点是Tyr384残基。因此，我们打算研究MET介导的RIPK1修饰对RIPK1信号转导和肝细胞存活的重要性。在目标2中，我们将直接测试我们的假设，即逃离HGF-MET轴引发的RIPK1介导的细胞死亡有助于肝癌的发生，并且阻断HGF-MET会导致RIPK1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，我们将分别使用肝脏特异性的RIPK1和HGF功能丧失和功能获得的小鼠模型，以及肝脏肿瘤发生研究。众所周知，HGF和MET在人类肝细胞癌中高表达；有趣的是，我们发现RIPK1在人类癌症如肝癌中表达下调。利用我们现有的相关小鼠模型，我们将处理肝脏特异的RIPK1基因敲除[LRIPKO]小鼠、复合LRIPKO/AlbHGF转基因小鼠和DEN对照小鼠来诱导肝癌，并将监测它们的肝脏肿瘤发展情况，以验证我们的假设。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著增强和加速。我们预计，用处于临床试验中的MET激酶抑制剂抑制MET会诱导肿瘤中RIPK1的上调，从而使它们成为可能 这些研究将在肝脏肿瘤发生中建立一种新的范式，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因子RIPK1来促进癌细胞存活，从而防止RIPK1诱导的坏死。我们的研究将为在肝癌的临床环境中靶向HGF-MET信号转导以恢复RIPK1表达从而使癌细胞致死提供理论依据，特别是与传统的化疗药物如顺铂联合使用时，顺铂通过坏死杀死细胞。

 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.