Oncogenic K-Ras turns death receptors into metastasis-promoting receptors in human and mouse colorectal cancer cells.

BACKGROUND & AIMS: Death receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential. METHODS: Isogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor-related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein- or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach. RESULTS: CD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras-dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras-driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras-proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras-deficient cells against apoptosis. CONCLUSIONS: Oncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/LIM kinase pathway, and this is essential for K-Ras/Raf1-driven metastasis formation.

TRAIL and other TRAIL receptor agonists as novel cancer therapeutics.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo2L, is a member of the TNF superfamily (TNFSF) of cytokines. TRAIL gained much attention during the past decade due to the demonstration of its therapeutic potential as a tumor-specific apoptosis inducer. TRAIL was identified as a protein with high homology to other members of the TNF cytokine family, especially to the ligand of Fas/Apo-1 (CD95), CD95L (FasL/APO-1L). TRAIL has been shown to induce apoptosis selectively in many tumor cell lines without affecting normal cells and tissues, making TRAIL itself as well as agonists of the two human receptors of TRAIL which can submit an apoptotic signal, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), promising novel biotherapeutics for cancer therapy. An increasing number of publications now shows that TRAIL resistance in primary human tumor cells will have to be overcome and that sensitization to TRAIL-induced apoptosis will be required in many cases. Therefore, it will also be instrumental to develop suitable diagnostic tests to identify patients who will benefit from TRAIL-based novel anticancer therapeutics and those who will not. Interestingly, the first clinical results even in monotherapy with TRAIL as well as various agonistic TRAIL receptor-specific antibodies have shown encouraging results. This chapter provides a compact overview on the biochemistry of the TRAIL/TRAIL-R system, the physiological role of TRAIL and its receptors and the results of clinical trials with TRAIL and various TRAIL-R agonistic antibodies.

Following TRAIL's path in the immune system.

The members of the tumour necrosis factor (TNF) superfamily of cytokines play important roles in the regulation of various immune-cell functions. Likewise, induction of cell death by apoptosis is indispensable for the normal functioning of the immune system. There are two major pathways of apoptosis induction. The intrinsic, or mitochondrial, pathway is regulated by the activation and interaction of members of the Bcl-2 family. The extrinsic, or death receptor, pathway is triggered by certain TNF family members when they engage their respective cognate receptors on the surface of the target cell. Hence, cell-to-cell-mediated death signals are induced by activation of these death receptor-ligand systems. Besides TNF itself and the CD95 (Fas/APO-1) ligand (FasL/Apo1L), the TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) belongs to the subfamily of ligands that is responsible for extrinsic induction of cell death. Depending on their status of stimulation, TRAIL can be expressed by various cells of the immune system, amongst them natural killer (NK) cells, T cells, natural killer T cells (NKT cells), dendritic cells and macrophages. TRAIL has been implicated in immunosuppressive, immunoregulatory and immune-effector functions. With respect to pathological challenges, TRAIL and its receptors have been shown to play important roles in the immune response to viral infections and in immune surveillance of tumours and metastases. In this review we summarize the current knowledge on the role of TRAIL and its receptors in the immune system and, based on this, we discuss future directions of research into the diverse functions of this fascinating receptor-ligand system.

TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development.

TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in established tumor cell lines but not nontransformed cells. Herein, we demonstrate a role for the apoptosis-inducing TRAIL receptor (TRAIL-R) as a metastasis suppressor. Although mouse models employing tumor transplantation have shown that TRAIL can reduce tumor growth, autochthonous tumor models have generated conflicting results with respect to the physiological role of the TRAIL system during tumorigenesis. We used a multistage model of squamous cell carcinoma to examine the role of TRAIL-R throughout all steps of tumor development. DMBA/TPA-treated TRAIL-R-deficient mice showed neither an increase in number or growth rate of benign papillomas nor an increase in the rate of progression to squamous cell carcinoma. However, metastasis to lymph nodes was significantly enhanced, indicating a role for TRAIL-R specifically in the suppression of metastasis. We also found that adherent TRAIL-R-expressing skin carcinoma cells were TRAIL resistant in vitro but were sensitized to TRAIL upon detachment by inactivation of the ERK signaling pathway. As detachment from the primary tumor is an obligatory step in metastasis, this provides a possible mechanism by which TRAIL-R could inhibit metastasis. Hence, treatment of cancer patients with agonists of the apoptosis-inducing receptors for TRAIL may prove useful in reducing the incidence of metastasis.

TRAIL: a multifunctional cytokine.

The Tumor necrosis factor (TNF)-Related Apoptosis Inducing Ligand, TRAIL, has gained much attention due to its specific anti-tumor potential without toxic side effects. TRAIL binds to a complex receptor system. In humans there are two death-inducing receptors for TRAIL while only one is present in mice. The signaling induced by these receptors leads to apoptosis but might also result in activation of survival signals. To assess the safety and possible side effects of TRAIL-based cancer therapy it is necessary to understand the physiological role of the TRAIL/TRAIL-R system. This has been addressed in mice deficient either for TRAIL or for its only murine apoptosis-inducing receptor, TRAIL-R (MK/mDR5). In this review we will discuss their phenotypes and the results of recent studies on the role of TRAIL in the homeostasis of the immune system, the influence of the TRAIL/TRAIL-R system on infection and autoimmune diseases and the still controversial role of TRAIL in tumorigenesis. Clinical trials with TRAIL and other TRAIL receptor agonists are now under way. It will be exciting to determine which TRAIL-R agonists, either alone or in combination with other anti-cancer therapeutics, will result in better outcome of cancer treatment in the future.

TRAIL enhances efficacy of radiotherapy in a p53 mutant, Bcl-2 overexpressing lymphoid malignancy.

BACKGROUND AND PURPOSE: Resistance to apoptosis is a contributing factor in the response to radiotherapy. Aim of this study was to evaluate whether TRAIL--in a soluble isoleucine zippered form--enhances the cytotoxic effect of irradiation on tumour cells with a blockade in the mitochondrial apoptosis route and/or a dysfunctional p53 pathway. MATERIALS AND METHODS: The p53 mutant human T acute lymphoblastic leukemia line Jurkat transduced with the Bcl-2 gene was used as model system in vitro and in a subcutaneous transplant setting in immunodeficient mice. Sensitivity to single and combined treatment was read out by apoptosis hallmarks and clonogenic survival in vitro, and by bioluminescence and palpation in vivo. RESULTS: Jurkat cells overexpressing Bcl-2 did not undergo apoptosis after irradiation, but the combination with TRAIL synergistically induced apoptosis without breaking mitochondrial resistance. TRAIL also reduced clonogenic survival after irradiation. In vivo, radiotherapy or TRAIL alone delayed tumour outgrowth, but combination treatment had the most profound effect. CONCLUSIONS: Isoleucine zippered TRAIL can strongly enhance the efficacy of tumour therapy with ionising radiation in an unfavourable setting of p53 mutation and Bcl-2 overexpression.