Increased Apoptosis Induction in Hepatocellular Carcinoma by a Novel Tumor-Targeted TRAIL Fusion Protein Combined With Bortezomib

Abstract

As the result of an increasing incidence and a prevalent therapy resistance of hepatocellular carcinoma (HCC), there is a strong need for novel strategies to enhance treatment responses in HCC. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed as a promising anticancer drug because it can selectively induce apoptosis in cancer cells, but not in healthy cells. Nevertheless, most tumor cells show TRAIL resistance, emphasizing the requirement for apoptosis-sensitizing agents and TRAIL molecules with improved tumor specificity. In this study, we employed a recombinant TRAIL molecule, in which three TRAIL protomers were expressed as a single polypeptide chain (scTRAIL), and a novel TRAIL variant, in which scTRAIL was additionally fused to an antibody fragment recognizing epidermal growth factor receptor (EGFR) to improve its HCC-targeting properties. We analyzed the proapoptotic effects of both TRAIL versions in combination with the proteasome inhibitor bortezomib (BZB) in hepatoma cells and primary human hepatocytes as well as in intact explants from HCC and healthy liver tissue. We demonstrate that EGFR-targeted TRAIL in combination with BZB induced significantly higher caspase activation and cell death in hepatoma cells, but not in primary hepatocytes. Importantly, when incubated with fresh liver explants, the combination of EGFR-targeted TRAIL and BZB displayed selective cytotoxicity for HCC, but not for tumor-free liver tissue, which could even be verified in liver explants from the same individuals. Unlike nontargeted TRAIL, EGFR-targeted TRAIL combined with BZB exerted no toxicity in liver tissues from nonalcoholic fatty liver disease patients. Conclusion: EGFRtargeted TRAIL reveals increased antitumor activity toward HCC without inducing toxicity to tumor-free liver tissue and might therefore represent a promising novel strategy for HCC treatment. (HEPATOLOGY 2013;57:625-636) H epatocellular carcinoma (HCC) is a global health problem with increasing incidence. 1 In western countries, less than 50% of patients are eligible for potential curative treatment, including resection, transplantation, or local ablation. The limited therapeutic options and its resistance to systemic chemotherapy have triggered the search for moleculartargeted therapies for liver cancer. There is evidence of aberrant activation of several signaling cascades, such as the epidermal growth factor receptor (EGFR)/

Hepatitis B Virus X Protein (HBx) Is Responsible for Resistance to Targeted Therapies in Hepatocellular Carcinoma: Ex Vivo Culture Evidence

“…Bortezomib is clinically effective in hematologic malignancies (14,36). Recently, PIs exhibited antitumor effects against diverse solid neoplasms, including tumors of the breast, lung, prostate, and liver; additionally, laboratory findings have revealed that combining bortezomib with other drugs may enhance the treatment of HCC (17,(23)(24)(25)(26). HBV infection is a major etiologic cause of HCC, with more than 60% of cases in Asia and Africa and at least 20% of HCC cases in Europe, Japan, and the United States associated with chronic infection with HBV (5).…”

“…Bortezomib was also shown to differentially affect the expression of E2F1, p21, and p27 (21) and to mediate a specific dual antitumor effect via natural killer (NK) cell antitumor reactivity (22). An international, multicenter phase II trial (23) and several preclinical studies (24)(25)(26) of bortezomib in HCC patients have been reported. However, to our knowledge, no studies have focused on the relationship between bortezomib and HBV-related HCC (HBV-HCC).…”

BRaf and MEK Inhibitors Differentially Regulate Cell Fate and Microenvironment in Human Hepatocellular Carcinoma

“…The study was performed according the Ethics committees of Hannover Medical School and the University of Heidelberg, Germany. Culturing of ex vivo liver of patients with hepatocellular carcinoma was described (22). Hepatocellular carcinoma or healthy liver tissue was cut into 125 mm 3 pieces under sterile conditions and incubated in 24-well plates with medium control or 7.5 mg/mL sorafenib.…”

“…Because chemokine and growth factor secretion by hepatocellular carcinoma cells was modulated by MAPK inhibitors in vitro, we analyzed the effects of sorafenib treatment on the microenvironment on ex vivo liver tissue in a standardized fashion (22,27). Fresh hepatocellular carcinoma liver tissue (n ¼ 6) was obtained from patients with hepatocellular carcinoma and healthy liver tissue (n ¼ 38) from patients with liver metastases of nonhepatic origin.…”

“…Molecular analyses of mutations in codon 600 of BRaf, codons 12 and 13 of NRas revealed that all hepatocellular carcinoma samples carried wild-type sequences of these kinases (data not shown). Defined tissue pieces were treated with inhibitors as described (22,27). Treatment with sorafenib reduced secretion of CXCL8, CXCL10, CCL5, and IL-1RA in both supernatant and tissue of most patients (Figs.…”