Robert M. Pitti

Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer

Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apo1/CD95 (ref. 1). One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are potentially harmful to the organism, such as virus-infected or tumour cells. Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was amplified in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL.

A novel receptor for Apo2L/TRAIL contains a truncated death domain

Apo2 ligand (Apo2L [1], also called TRAIL for tumor necrosis factor (TNF)-related apoptosis-inducing ligand [2]) belongs to the TNF family and activates apoptosis in tumor cells. Three closely related receptors bind Apo2L: DR4 and DR5, which contain cytoplasmic death domains and signal apoptosis, and DcR1, a decoy receptor that lacks a cytoplasmic tail and inhibits Apo2L function [3-5]. By cross-hybridization with DcR1, we have identified a fourth Apo2L receptor, which contains a cytoplasmic region with a truncated death domain. We subsequently named this protein decoy receptor 2 (DcR2). The DcR2 gene mapped to human chromosome 8p21, as did the genes encoding DR4, DR5 and DcR1. A single DcR2 mRNA transcript showed a unique expression pattern in human tissues and was particularly abundant in fetal liver and adult testis. Upon overexpression, DcR2 did not activate apoptosis or nuclear factor-kappaB; however, it substantially reduced cellular sensitivity to Apo2L-induced apoptosis. These results suggest that DcR2 functions as an inhibitory Apo2L receptor.

Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL

Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non–small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine–galactose–sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.

Cullin3-Based Polyubiquitination and p62-Dependent Aggregation of Caspase-8 Mediate Extrinsic Apoptosis Signaling

Cell-surface death receptors such as DR4 and DR5 trigger apoptosis through a death-inducing signaling complex (DISC) that recruits the apical protease caspase-8. Apoptosis commitment requires efficient activation and autocatalytic release of caspase-8 into the cytoplasm to engage executioner caspases. While DISC recruitment initiates caspase-8 stimulation, full activation of the protease depends on further molecular aggregation events that are not fully understood. Here, we show that death receptor ligation induces polyubiquitination of caspase-8, through a previously unknown interaction of the DISC with a cullin3 (CUL3)-based E3 ligase. CUL3-mediated caspase-8 polyubiquitination required the RING box protein RBX1, whereas the deubiquitinase A20 reversed this modification. The ubiquitin-binding protein p62/sequestosome-1 promoted aggregation of CUL3-modified caspase-8 within p62-dependent foci, leading to full activation and processing of the enzyme and driving commitment to cell death. These results identify a mechanism that positively controls apoptosis signaling by polyubiquitination and aggregation of a key initiator caspase.

Interaction of the TNF homologues BLyS and APRIL with the TNF receptor homologues BCMA and TACI.

BLyS (also called TALL-1, THANK, or BAFF) [1] [2] [3] [4] is a member of the tumor necrosis factor (TNF) gene family that stimulates proliferation and immunoglobulin production by B cells. BLyS interacts with the TNF receptor (TNFR) homologue TACI (transmembrane activator and CAML-interactor) [5], and treatment of mice with a TACI-Fc fusion protein abolishes germinal center formation after antigenic challenge [6]. Here we report a novel interaction between BLyS and another TNFR homologue, BCMA (B cell maturation antigen) [7] [8]. Further, the TNF homologue APRIL [9], a close relative of BLyS, also bound to BCMA and TACI. BLyS or APRIL activated nuclear factor-kappaB (NF-kappaB) through TACI and BCMA, and each ligand stimulated immunoglobulin M (IgM) production by peripheral blood B cells. These results define a dual ligand-receptor system that may play an important role in humoral immunity.

Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA

A new member of the tumor necrosis factor (TNF) cytokine family, designated Apo-2 ligand (Apo-21) [1] or TRAIL [2], has been shown recently to induce apoptosis in various tumor cell lines; however, its biological role is unknown. Here, we show that Apo-21, activated apoptosis in T-cell-enriched cultures of peripheral blood lymphocytes stimulated by interleukin-2 (IL-2), but not in unstimulated cells. This finding suggests that, like Fas/Apo-1 ligand and TNF [3-5], Apo-2L may play a role in regulating post-stimulation apoptosis of mature lymphocytes. Studies on the mechanism of Apo-2L action demonstrated marked membrane blebbing, a hallmark of apoptosis, within a few minutes of the addition of Apo-2L to tumor cells. Ectopic expression of a dominant negative mutant of FADD, a cytoplasmic protein that mediates death signalling by Fas/Apo-1 and by TNF receptor type 1 (TNFR1) [6-9], inhibited the induction of apoptosis by anti-Fas/Apo-1 antibody, but had little effect on Apo-2L function. In contrast, expression of CrmA, a cowpox virus-derived inhibitor of the Ced-2-like proteases ICE [10] and CPP32/Yama [11,12], blocked the induction of apoptosis by either Apo-2L or anti-Fas/Apo-1 antibody. These results suggest that Apo-2L activates a rapid, FADD-independent pathway to trigger a cell-death programme that requires the function of cysteine proteases such as ICE or CPP32/Yama.

Identification of a ligand for the death-domain-containing receptor Apo3

The tumor necrosis factor (TNF) cytokine family regulates development and function of the immune system [1]. TNF is expressed primarily by activated lymphocytes and macrophages and induces gene transcription or apoptosis in target cells [2,3]. We have identified a novel relative of TNF that binds to the recently discovered, death-domain-containing receptor called Apo3 [4] (also known as DR3, WSL-1, TRAMP or LARD [5-9]). The Apo3 ligand (Apo3L) is a 249 amino-acid, type II transmembrane protein. The extracellular sequence of Apo3L shows highest identity to that of TNF. We detected Apo3L mRNA in many human tissues and mapped its encoding gene to chromosome 17p13, near the p53 tumor-suppressor gene. Soluble Apo3L induced apoptosis and nuclear factor kappaB (NF-kappaB) activation in human cell lines. Caspase inhibitors blocked apoptosis induction by Apo3L, as did a dominant-negative mutant of the cell death adaptor protein Fas-associated death domain protein (FADD/MORT1), which is critical for apoptosis induction by TNF [3]. Dominant-negative mutants of several factors that play a key role in NF-kappaB induction by TNF [10] inhibited NF-kappaB activation by Apo3L. Thus, Apo3L has overlapping signaling functions with TNF, but displays a much wider tissue distribution.

Identification of a new member of the tumor necrosis factor family and its receptor, a human ortholog of mouse GITR

The tumor necrosis factor (TNF) and TNF receptor (TNFR) gene superfamilies regulate diverse biological functions, including cell proliferation, differentiation, and survival [1] [2] [3]. We have identified a new TNF-related ligand, designated human GITR ligand (hGITRL), and its human receptor (hGITR), an ortholog of the recently discovered murine glucocorticoid-induced TNFR-related (mGITR) protein [4]. The hGITRL gene mapped to chromosome 1q23, near the gene for the TNF homolog Fas/CD95 ligand [5]. The hGITR gene mapped to chromosome 1p36, near a cluster of five genes encoding TNFR homologs [1] [6]. We found hGITRL mRNA in several peripheral tissues, and detected hGITRL protein on cultured vascular endothelial cells. The levels of hGITR mRNA in tissues were generally low; in peripheral blood T cells, however, antigen-receptor stimulation led to a substantial induction of hGITR transcripts. Cotransfection of hGITRL and hGITR in embryonic kidney 293 cells activated the anti-apoptotic transcription factor NF-kappaB, via a pathway that appeared to involve TNFR-associated factor 2 (TRAF2) [7] and NF-kappaB-inducing kinase (NIK) [8]. Cotransfection of hGITRL and hGITR in Jurkat T leukemia cells inhibited antigen-receptor-induced cell death. Thus, hGITRL and hGITR may modulate T lymphocyte survival in peripheral tissues.

An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

Proapoptotic Activation of Death Receptor 5 on Tumor Endothelial Cells Disrupts the Vasculature and Reduces Tumor Growth

The proapoptotic death receptor DR5 has been studied extensively in cancer cells, but its action in the tumor microenvironment is not well defined. Here, we uncover a role for DR5 signaling in tumor endothelial cells (ECs). We detected DR5 expression in ECs within tumors but not normal tissues. Treatment of tumor-bearing mice with an oligomeric form of the DR5 ligand Apo2L/TRAIL induced apoptosis in tumor ECs, collapsing blood vessels and reducing tumor growth: Vascular disruption and antitumor activity required DR5 expression on tumor ECs but not malignant cells. These results establish a therapeutic paradigm for proapoptotic receptor agonists as selective tumor vascular disruption agents, providing an alternative, perhaps complementary, strategy to their use as activators of apoptosis in malignant cells.