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.
Liver cancer (Hepatocellular Carcinoma, HCC) is one of the leading causes of cancer death in the world; unfortunately, no cure exists for most patients because the tumor is unresponsive to current chemotherapy drugs. Hepatocyte Growth Factor (HGF) and its receptor MET are known to stimulate liver cancer growth. We have found that they do so by decreasing the abundance of death-inducing molecules such as RIPK1 which Met tags for destruction. A reduction in death-inducing molecules like RIPK1 results in chemotherapy resistance and an inability for chemotherapeutic drugs to kill cancer cells. The goal of this proposal is to investigate how exactly the HGF-MET axis down-regulates RIPK1 in liver cancer using state-of-the art approaches in cultured cells and animal models and then to determine strategies that prevent the HGF-MET system from down-modulating RIPK1. We will explore one potential strategy: we hypothesize that, by inhibiting the HGF-Met axis with specific anti-HGF/MET drugs in cancer cells, HGF and MET can no longer send pro-growth and pro-survival signals inside the cells. In turn, inhibiting HGF-MET signaling will stop cells from growing and will prevent MET from tagging RIPK1 for destruction. RIPK1 accumulation plus reduced growth cues will make cancer cells less likely to survive, especially when treated with standard chemotherapy drugs which induced cell death. Our ideas are very novel since our discovery that HGF-MET down-regulates RIPK1 has never been described before. Plus, we are the first to try to inhibit liver cancer growth by our planned approach.
