Michael T. Eby

Modulation of the NF-κB pathway by virally encoded Death Effector Domains-containing proteins

Death Effector Domains (DEDs) have been known to mediate the recruitment of Caspase 8 and its homologs to the aggregated death-inducing signaling complex (DISC), consisting of the death domain (DD)-containing receptors and various signaling proteins. In addition, several viruses were recently shown to encode proteins with DEDs (also called FLICE inhibitory proteins or vFLIPs) which have the ability of blocking cell death induced by DD-containing receptors. We provide evidence that vFLIPs can also modulate the NF-κB pathway and physically interact with several signaling proteins, such as the TRAFs, RIP, NIK and the IKKs. Modulation of the NF-κB pathway may play a role in the natural history of infection by these viruses.

Activation of the NF-κB pathway by Caspase 8 and its homologs

Caspase 8 is the most proximal caspase in the caspase cascade and has been known for its role in the mediation of cell death by various death receptors belonging to the TNFR family. We have discovered that Caspase 8 can activate the NF-κB pathway independent of its activity as a pro-apoptotic protease. This property is localized to its N-terminal prodomain, which contains two homologous death effector domains (DEDs). Caspase 10 and MRIT, two DEDs-containing homologs of Caspase 8, can similarly activate the NF-κB pathway. Dominant-negative mutants of the Caspase 8 prodomain can block NF-κB induced by Caspase 8, FADD and several death receptors belonging to the TNFR family. Caspase 8 can interact with multiple proteins known to be involved in the activation of the NF-κB pathway, including the serine-threonine kinases RIP, NIK, IKK1 and IKK2. Thus, DEDs-containing caspases and caspase homolog(s) may have functions beyond their known role in the mediation of cell death.

Eiger and its receptor, Wengen, comprise a TNF-like system in Drosophila

In mammals, members of the tumor necrosis factor (TNF) family play an important role in the regulation of cellular proliferation, differentiation and programmed cell death. We describe isolation and characterization of an orthologous ligand/receptor axis in Drosophila. The ligand, designated Eiger, is a type II membrane glycosylated protein, which can be cleaved at residue 145 and released from the cell surface as a soluble factor, thereby representing the first potential cytokine to be described in Drosophila. Eiger exists in two alternatively spliced isoforms, Eiger long (Eiger-L) and Eiger short (Eiger-s), both of which are expressed throughout development and in the adult. We also describe the isolation and characterization of a novel Drosophila member of the TNF receptor family, designated Wengen, which is a type I membrane protein that can physically interact with the recently described TRAF2 homolog dTRAF2. Both Eiger and Wengen are expressed in distinctive patterns during embryogenesis and Eiger is responsive to genotoxic stress. Forced expression of Eiger-L, Eiger-s or Wengen, caused apoptotic cell death which could be rescued by caspase inhibitors or the JNK phosphatase Puckered. In addition, Eiger-induced cell killing was attenuated by RNAi-mediated suppression of Wengen. Our results illustrate that Eiger and Wengen represent proximal components of an evolutionarily conserved TNF-like signaling pathway in Drosophila.

Activation of the c-Jun N-terminal Kinase/Stress-activated Protein Kinase Pathway by Overexpression of Caspase-8 and Its Homologs

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.

Role of MRIT/cFLIP in Protection Against Chemotherapy-Induced Apoptosis

MRIT (Mach-related inducer of toxicity)/cFLIP (cellular FADD like interlukin1-β converting enzyme inhibitory protein) is a proteolytically inactive structural homologue of caspase-8, which is known to protect against death receptors-induced apoptosis by blocking the activation of caspase-8. We have observed that exogenous expression of MRITα1/cFLIPL isoform also protects against cell death induced by a diverse group of chemotherapeutic drugs with different mechanisms of action, including doxorubicin, etoposide, cytosine arabinoside, daunorubicin, chlorambucil and cisplatin. However, MRITα1/cFLIPL failed to protect against apoptosis induced by paclitaxel and vincristine, two microtubule-damaging agents. Although MRITα1/cFLIPL protects against chemotherapy-induced apoptosis in both solid tumor and hematopoietic cell lines, this effect was more pronounced in the former. MRITα1/cFLIPL expression is decreased during drug-induced apoptosis and exogenous expression of MRITα1/cFLIPL delays the activation of caspase-8 and -3 during drug- induced apoptosis. These results suggest that MRITα1/cFLIPL may be an important determinant of both death receptor- and chemotherapy-induced apoptosis and strategies aimed at downregulating its expression deserve further study as a way to overcome multidrug resistance to cancer therapy.

Cytotoxicity of Tumor Necrosis Factor related apoptosis-inducing ligand towards Ewing's sarcoma cell lines

Death ligands of the Tumor Necrosis Factor (TNF) family are known to induce apoptosis upon binding to their cognate receptors. However, the clinical utility of these cytokines as anticancer agents has been limited due to unacceptable toxicity. TRAIL is a recently isolated death ligand that possesses selective anti-tumor activity against a number of cancer cell lines without significant systemic toxicity. In this report we present evidence that cell lines derived from Ewings Sarcoma (ES) are uniformly sensitive to TRAIL-mediated apoptosis. Furthermore, unlike TNF-α, treatment with TRAIL fails to induce the anti-apoptotic and pro-inflammatory NF-κB pathway in the ES cell lines. Our results suggest that TRAIL may prove to be a useful agent for the treatment of Ewings sarcoma and related peripheral neuroectodermal tumors.

Differential inactivation of caspase-8 in lung cancers (Cancer Biology and Therapy (2002) 1, (65-69) DOI: 10.4161/cbt.1.1.45)

1074 Cancer Biology & Therapy Volume 14 issue 11 The Editors were recently provided information regarding evidence of image manipulation in a research paper published in Cancer Biology & Therapy in 2002. Uncertainty exists as to the impropriety of the manipulation. The publisher was notified by the senior author at the request of the Dean of Southwestern Medical School to publish an Expression of Concern regarding “Differential Inactivation of Caspase-8 in Lung Cancers” (http://dx.doi.org/10.4161/cbt.1.1.45), a research paper by Narayan Shivapurkar, Shinichi Toyooka, Michael T Eby, Chun Xian Huang, Ubaradka G Sathyanarayana, H Thomas Cunningham, Jyotsna L Reddy, Elizabeth Brambilla, Takashi Takahashi, John D Minna, Preet M Chaudhary, and Adi F Gazdar. The allegations concern Figure 3.