Cells can undergo a morphologically distinct form of death, termed apoptosis, when they are damaged or no longer needed. Apoptosis is of central importance for development and tissue homeostasis. On the other hand, aberrant regulation of apoptosis is associated with a variety of human diseases, including cancer, autoimmunity, neurodegenerative disorders, liver diseases, and ischemic stroke. Since the basic cell death program is constitutively expressed in virtually all animal cells, precise regulatory mechanisms must exist that restrict the activation of apoptosis to unwanted cells.
Regulation of Programmed Cell Death in Drosophila
My laboratory pioneered Drosophila as a model system for cell death research. We described and characterized the first cell death genes in the fly, Reaper and Hid, and the first Drosophila caspase. Reaper, Hid, and Grim activate apoptosis by binding to and inactivating inhibitor of apoptosis proteins (IAPs), which in turn directly inhibit caspases, the key executioners of apoptosis. By antagonizing IAPs, Reaper-family proteins remove powerful "brakes on death." Because Reaper stimulates the auto-ubiquitination and degradation of IAPs, it irreversibly destroys key protection against cell death. Significantly, reaper, hid, and grim are transcriptionally regulated by many different death-inducing stimuli and act as integrators for relaying different apoptotic signals to the core death program. Collectively, our work defined how different signaling pathways are integrated by a transcriptional mechanism to induce cell death and how apoptosis is initiated through the release of caspase inhibition. Because IAPs are commonly overexpressed in human tumors and promote cancer cell survival, they are promising targets for cancer therapy. A conserved IAP-binding motif found in Reaper, Hid, Grim, and mammalian Smac/Diablo provided the basis for generating small-molecule IAP antagonists for the clinic. However, Reaper only kills well upon recruitment into a complex with Hid at the mitochondrial outer membrane (MOM). We exploited these findings to generate bivalent small molecules that contain both an IAP-binding motif and a mitochondrial targeting sequence. These compounds provide improved targeting of the X-linked IAP (XIAP) at the MOM and selectively kill certain types of cancer cells.
Death by Degradation: IAPs, Stem Cells, Apoptosis, and Tumor Suppression in the Mouse
We are using mouse genetics to investigate whether concepts originally developed in Drosophila apply to the regulation of mammalian apoptosis. This work illustrated the conserved and central role of the ubiquitin-proteasome system in the regulation of apoptosis in both Drosophila and mammals. Moreover, it revealed the importance of XIAP and its antagonist ARTS (apoptosis-related protein in the TGFβ signaling pathway for the regulation of stem cell apoptosis, wound healing, and tumorigenesis. We found that the mammalian XIAP acts as an E3-ubiquitin ligase to inhibit caspases and that inactivation of this protein protects mice against tumor growth. Conversely, the pro-apoptotic XIAP antagonist ARTS induces stem cell apoptosis and is a tumor suppressor. This indicates that apoptosis plays an important role as a frontline defense against cancer by restricting the number of normal stem cells. On the other hand, Sept4/ARTS-/- mice display dramatic improvement in wound healing and remarkable regeneration of hair follicles (HFs), and inactivation of XIAP abrogates these phenotypes and impairs wound healing. These results suggest that apoptosis plays an important role in regulating stem cell–dependent regeneration and indicate that this pathway may be a target for regenerative medicine.
Despite the long-standing interest in developing XIAP inhibitors as cancer therapeutics, virtually all compounds developed so far primarily antagonize cIAP1,2 and are ineffective against XIAP. To obtain potent and specific small-molecule inhibitors of XIAP, we conducted a cell-based high-throughput screen for compounds that modulate degradation of a fluorescent XIAP substrate. We screened 100,000 molecules and identified two specific inhibitors of XIAP. These novel compounds promote effective killing of certain melanoma cells, and they sensitize glioma cells to apoptosis mediated by TRAIL, a death receptor ligand. Our results provide proof of concept for the discovery of effective inhibitors of XIAP and their potential utility in the clinic.
Signaling by Apoptotic Cells
Developing tissues can often compensate for the massive loss of cells in response to damage, such as radiation. We found that Drosophila cells that undergo apoptosis upon stress or injury can stimulate tissue repair by secreting mitogens that induce proliferation of adjacent progenitor cells. These secreted mitogens include Wingless and BMP/TGFβ-family proteins. Similar phenomena occur in vertebrates and appear to contribute to both wound healing and tumorigenesis. On the other hand, we also observed that strong induction of apoptosis in the Drosophila wing disc can cause apoptosis of other cells at some distance, indicating that dying cells can release long-range death factors. We subsequently identified Eiger, the Drosophila tumor necrosis factor (TNF) homolog, as the signal responsible for this apoptosis-induced apoptosis (AiA). Eiger is produced in apoptotic cells and, via the activation of the JNK pathway, propagates the initial apoptotic stimulus throughout the tissue. The mechanism of AiA is conserved in evolution and contributes to the coordinated cell death of HF cells in mice. During the regressive phase of the HF cycle, TNFα is produced by apoptotic cells and is required for normal cell death in the HF. AiA provides a mechanism to explain the coordinated behavior of dying cells in normal development and under pathological conditions.
Nonlethal Use of Apoptotic Proteins and the Ubiquitin-Proteasome System for Cellular Remodeling
Apoptotic proteins can serve nonlethal functions for cellular remodeling in both Drosophila and mammals. We originally defined the role of ubiquitin-pathway enzymes for caspase activation and protein degradation during sperm differentiation, but similar mechanisms operate in mammals and in other cells, including neurons. More recently, we discovered a new mechanism for regulating the activity of the 26S proteasome, the proteolytic complex responsible for the degradation of most intracellular proteins. We first showed that the basal activity of proteasomes can be dynamically increased by the conserved proteasome regulator PI31. Subsequently, we found that ADP ribosylation of PI31 by Tankyrase (TNKS) promotes 26S assembly and stimulates 26S activity in both Drosophila and human cells. Moreover, inhibition of TNKS by either RNA interference or small-molecule inhibitors reduces protein breakdown. These results reveal a novel mechanism of proteasome regulation that can be targeted with existing compounds. Since NAD+ is the source of the ADP-ribose group, proteasome regulation by TNKS provides a potential new link between cell metabolism and the regulation of protein degradation. Our work also indicates that TNKS inhibitors may be useful for the treatment of multiple myeloma and other cancers sensitive to proteasome inhibition.
Part of this work was supported by a grant from the National Institutes of Health.
As of June 3, 2013