A new TRAIL to therapy of hepatocellular carcinoma: Blocking the proteasome
Authors: Akira Anan, Gregory J. Gores
Journal: Hepatology (2005)

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer mortality worldwide, largely because therapies for advanced HCC are limited and often ineffective. Most currently available systemic chemotherapeutic agents are cytotoxic agents with a narrow therapeutic window (often a peephole for the HCC patient). Not only are most HCC intrinsically resistant to available therapies, but, HCC usually arises in a liver with cirrhosis. The cirrhosis alters drug distribution and decreases hepatic drug metabolism complicating chemotherapy administration. Portal hypertension with splenic sequestration of platelets and white blood cells also accentuates the leucopenia and thrombocytopenia of many chemotherapeutic agents necessitating dose reduction, thereby, compromising therapy. Thus, additional chemotherapeutic agents are necessary for the treatment of advanced HCC. The ideal agent would not cause bone marrow suppression, would not be hepatotoxic, and would target specific molecular pathways uniquely amplified in transformed hepatocytes.1 The development of targeted therapies is currently an area of broad scientific and clinical interest in oncology. HCC, hepatocellular carcinoma; cFLIP, cellular FLICE inhibitory protein; DISC, death inducing signaling complex; DR, death receptor; FADD, Fas-associated death domain protein; I-κB, inhibitor of kappa B; NF-κB, nuclear factor kappa B; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TRAIL-R, TRAIL-receptor. The proteasome is a large barrel-like, multiprotein complex located within the cytoplasm of cells. It is responsible for proteolytic cleavage of proteins, so marked for degradation by the conjugation of ubiquitins to lysine residues. Ubiquitination is executed by three enzymatic steps; the last one is performed by an E3 ligase which confers specificity to the ubiquitination process. Previous studies have revealed that the proteasome system regulates cell cycle proteins, tumor suppressor molecules, oncogenes, transcriptional factors, and pro- and anti-apoptotic proteins.2-4 Transformed cells are more susceptible to apoptosis following proteasome inhibition than are normal cells.5-7 Although multiple mechanisms have been proposed to explain this observation, the exact mechanisms by which proteasome inhibitors exert their anticancer effects are still being elucidated. Proteasome inhibition, therefore, is a novel therapeutic strategy for several human cancers.8, 9 Indeed, bortezomib (known as PS-341, Velcade) is the first proteasome inhibitor to enter clinical trials and was recently approved by the US Food and Drug Administration and European Medicine Evaluation Agency for the treatment of refractory multiple myeloma.4 Unfortunately, bortezomib as monotherapy has proven disappointing for the treatment of solid malignancies including HCC.9, 10 These observations do not, however, exclude a role for bortezomib and other proteasome inhibitors as therapeutic agents in combination therapy. TRAIL/Apo2 ligand is another promising, novel anticancer agent. TRAIL selectively induces apoptosis in transformed cells but not in normal cells.11, 12 TRAIL and TRAIL agonistic monoclonal antibodies are now in phase I and II clinical trials. Emerging information from these trials indicate anti-cancer activity without evidence for hepatotoxicity, the latter was a potential and initial concern.13 TRAIL induces cell death by binding to its cognate death receptors, TRAIL-R1 and TRAIL-R2 (also known as DR4 and DR5). Ligand binding-induced conformation changes in the receptors initiates receptor aggregation, recruitment of the adaptor molecule Fas-associated death domain (FADD), and recruitment plus activation of the proteases caspase-8 and 10.14 Active caspases 8/10 then initiate a cascade of events culminating in cell death. The multimeric protein complex consisting of the receptor, FADD and caspase 8/10 is referred to as the death inducing signaling complex (DISC). Previous studies suggested that HCC cells may be resistant to TRAIL-mediated apoptosis, despite expression of TRAIL receptors.15, 16 The molecular mechanisms mediating TRAIL resistance are complex, have recently been reviewed,17 and will not be further discussed. Strategies to overcome TRAIL resistance are likely to prove useful in cancer therapy. Recent studies have demonstrated that inhibition of proteasome function effectively sensitizes cells to TRAIL by regulating several factors, normally reduced in neoplastic cells through enhanced proteasome degradation.18-20 Ganten et al. in this issue of HEPATOLOGY report that proteasome inhibitors can also sensitize hepatocellular carcinoma cells, but not primary human hepatocytes, to TRAIL-induced apoptosis.21 This article provides three very intriguing new insights regarding how proteasome inhibition sensitizes HCC cells to TRAIL-mediated apoptosis: (1) upregulation of TRAIL receptor 1 and 2 by proteasome inhibition was not sufficient to explain the sensitization of HCC cells to TRAIL cytotoxicity; (2) proteasome inhibition sensitized HCC cells to TRAIL-induced apoptosis by greatly enhancing recruitment of FADD and caspase-8 to the TRAIL receptor complex without altering FADD and caspase-8 cellular protein levels; and (3) proteasome inhibitors do not sensitize primary human hepatocytes to TRAIL-mediated apoptosis. Each of these will be discussed briefly. Previous reports suggested that upregulation of TRAIL receptor expression may be a mechanism to explain augmentation of TRAIL cytotoxicity, not only by conventional chemotherapeutic drugs or irradiation,22 but also by proteasome inhibition.23-25 Furthermore, a recent report suggests that MG132, an experimental proteasome inhibitor, enhances TRAIL-R2 expression at both protein and mRNA levels via upregulation of the transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP).25 In the study by Ganten et al., however, proteasome inhibition did not require upregulation of TRAIL receptors 1 or 2 in order to sensitize the cells to TRAIL cytotoxicity. Given this observation, the authors next focused on TRAIL DISC formation as an alternative mechanism explaining the phenomenon of TRAIL sensitization. Their data suggest that proteasome inhibition markedly enhances FADD and caspase-8 recruitment to the TRAIL DISC. The enhanced recruitment of FADD and caspase 8/10 to the DISC was also accompanied by enhanced recruitment of the anti-apoptotic protein cFLIP. Although usually thought of as an anti-apoptotic protein, cFLIP can promote caspase-8 activation,26 and in this regard, could potentially explain enhanced DISC conversion of procaspase 8 to its active form. However, we do not believe that alterations in cFLIP cellular protein levels explain the observations of Ganten et al. because recent data by Ashkenazi and coworkers and observations in the current study by Ganten et al. clearly demonstrated that cFLIP is anti-apoptotic in TRAIL signaling.27 Rather, we invoke a potential new mechanism for their observations, a putative protein which promotes death receptor signaling (Fig. 1). In our putative model, the expression of this protein would be enhanced by oncogenes but would be rapidly degraded in cancer cells via the proteasome. However, when the proteasome is inhibited, its cellular levels would increase, thereby, sensitizing the cells to TRAIL-mediated cytotoxicity. In this putative model, we postulate that because expression of the putative protein is oncogene driven, proteasome inhibition would not sensitize normal cells to TRAIL killing. This interpretation of the Ganten observations can be tested experimentally, especially given the advances in proteomics. Proteasome inhibition sensitizes HCC cells to TRAIL-mediated apoptosis. We postulate the existence of a protein which enhances TRAIL DISC formation. Expression of this protein is oncogene driven, and thus, it would have low-level expression in normal hepatocytes. In cancer cells, this protein is rapidly degraded by ubiquitin-proteasome pathway. However, when the proteasome is inhibited, its cellular levels increase sensitizing the cells to TRAIL-mediated apoptosis. The current study by Ganten et al. also provides a new therapeutic strategy for the treatment of HCC. The combination of proteasome inhibition plus a TRAIL agonist may well be clinically useful for the treatment of this disease. Such a strategy is potentially attractive because it is mechanism based and targeted. Although bortezomib has mild hepatotoxicity in clinical trials,28 it should prove safe and, aside from a neuropathy, is well tolerated. To date, the initial concerns regarding TRAIL hepatotoxicity have not been observed in human trials. This combination therapy, therefore, approaches the ideal therapy described previously. Preclinical and clinical studies are ongoing in several hematological malignancies and solid tumors using bortezomib not only alone but also in combination with other chemotherapeutic agents.9 Hopefully, as TRAIL agonists become more widely available such studies will also be conducted in HCC. Only evidence-based results in human trials will ultimately prove the usefulness of this strategy. We wait for these trials. The authors thank Erin Bungum for superb secretarial assistance.