Ulrich M. Lauer

Natural Killer Cell–Mediated Lysis of Hepatoma Cells via Specific Induction of NKG2D Ligands by the Histone Deacetylase Inhibitor Sodium Valproate

Natural killer (NK) cells as components of the innate immunity substantially contribute to antitumor immune responses. However, the tumor-associated ligands engaging activating NK cell receptors are largely unknown. An exception are the MHC class I chain-related molecules MICA and MICB and the UL16-binding proteins (ULBP) which bind to the activating immunoreceptor NKG2D expressed on cytotoxic lymphocytes. A therapeutic induction of NKG2D ligands that primes cancer cells for NK cell lysis has not yet been achieved. By microarray studies, we found evidence that treatment of human hepatocellular carcinoma cells with the histone deacetylase inhibitor (HDAC-I) sodium valproate (VPA) mediates recognition of cancer cells by cytotoxic lymphocytes via NKG2D. VPA induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface, soluble and total MIC protein expression. No significant changes in the expression of the NKG2D ligands ULBP1-3 were observed. The induction of MIC molecules increased lysis of hepatocellular carcinoma cells by NK cells which was abolished by addition of a blocking NKG2D antibody. Importantly, in primary human hepatocytes, VPA treatment did not induce MIC protein expression. Taken together, our data show that the HDAC-I VPA mediates specific priming of malignant cells for innate immune effector mechanisms. These results suggest the clinical evaluation of HDAC-I in solid tumors such as hepatocellular carcinoma, especially in combination with immunotherapy approaches employing adoptive NK cell transfer.

HDAC inhibitor treatment of hepatoma cells induces both TRAIL‐independent apoptosis and restoration of sensitivity to TRAIL

Hepatocellular carcinoma (HCC) displays a striking resistance to chemotherapeutic drugs or innovative tumor cell apoptosis–inducing agents such as tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL). Recently, we found 2 histone deacetylase inhibitors (HDAC‐I), valproic acid and ITF2357, exhibiting inherent therapeutic activity against HCC. In TRAIL‐sensitive cancer cells, the mechanism of HDAC‐I–induced cell death has been identified to be TRAIL‐dependent by inducing apoptosis in an autocrine fashion. In contrast, in HCC‐derived cells, a prototype of TRAIL‐resistant tumor cells, we found a HDAC‐I‐mediated apoptosis that works independently of TRAIL and upregulation of death receptors or their cognate ligands. Interestingly, TRAIL resistance could be overcome by a combinatorial application of HDAC‐I and TRAIL, increasing the fraction of apoptotic cells two‐ to threefold compared with HDAC‐I treatment alone, whereas any premature HDAC‐I withdrawal rapidly restored TRAIL resistance. Furthermore, a tumor cell–specific downregulation of the FLICE inhibitory protein (FLIP) was observed, constituting a new mechanism of TRAIL sensitivity restoration by HDAC‐I. In contrast, FLIP levels in primary human hepatocytes (PHH) from different donors were upregulated by HDAC‐I. Importantly, combination HDAC‐I/TRAIL treatment did not induce any cytotoxicity in nonmalignant PHH. In conclusion, HDAC‐I compounds, exhibiting a favorable in vivo profile and inherent activity against HCC cells, are able to selectively overcome the resistance of HCC cells toward TRAIL. Specific upregulation of intracellular FLIP protein levels in nonmalignant hepatocytes could enhance the therapeutic window for clinical applications of TRAIL, opening up a highly specific new treatment option for advanced HCC. (HEPATOLOGY 2006;43:425–434.)

Epigenetic combination therapy as a tumor-selective treatment approach for hepatocellular carcinoma.

Innovative epigenetic therapeutics comprise histone deacetylase inhibitors (HDAC‐I) and demethylating agents (DA). It was recently found that HDAC‐I compounds exhibit profound therapeutic activities against hepatocellular carcinoma (HCC). A comprehensive preclinical investigation was performed on the potential of a combined HDAC‐I/DA epigenetic regimen for the highly chemotherapy‐resistant HCC entity.

Telaprevir‐based triple therapy in liver transplant patients with hepatitis C virus: A 12‐week pilot study providing safety and efficacy data

After liver transplantation (LT), the management of recurrent hepatitis C virus (HCV) infections still remains a major challenge. In HCV genotype 1 patients not undergoing transplantation, the introduction of protease inhibitor (PI)–based regimens has increased the sustained virological response rate significantly. This pilot study investigated both the safety and efficacy of telaprevir (TVR)‐based triple therapy in HCV‐infected LT patients with a special emphasis on drug‐drug interactions between immunosuppressants and PIs. Safety and efficacy data were gathered for 12 weeks for 9 HCV‐infected LT patients who were treated with a combination of TVR, pegylated interferon, and ribavirin (RBV) in parallel with immunosuppressive drugs such as tacrolimus (TAC; n = 4), cyclosporine A (CSA; n = 4), and sirolimus (SIR; n = 1). Seven of the transplant patients completed the 12 weeks of triple therapy. At week 4, 4 of the patients were found to be HCV RNA–negative, and importantly, 8 were found to be negative at week 12. During the 12‐week course of triple therapy, short‐term measurements of immunosuppressant trough levels required individual dose reductions in all patients (CSA, 2.5‐fold; SIR, 7‐fold; and TAC, 22‐fold). Furthermore, two‐thirds of the patients exhibited hematological side effects requiring RBV dose reductions, the administration of erythropoietin, or even blood transfusions. In conclusion, this pilot study provides evidence showing that TVR‐based triple therapy is effective within the first 4 to 12 weeks in LT patients suffering from HCV genotype 1 recurrence, and it also provides evidence showing that drug‐drug interactions between TVR and immunosuppressants can be handled appropriately through the close monitoring of trough levels and adequate dosage adjustments. Liver Transpl, 2012.

Diffusion-Weighted MRI of Advanced Hepatocellular Carcinoma During Sorafenib Treatment: Initial Results

OBJECTIVE The objective of our study was to evaluate signal changes of advanced hepatocellular carcinoma in diffusion-weighted MRI in the early-response monitoring of oral therapy with the multikinase inhibitor sorafenib. CONCLUSION Hepatocellular carcinoma lesions exhibit characteristic but unusual apparent diffusion coefficient (ADC) changes during sorafenib therapy, consisting of early decrease in ADC after therapy onset followed by a reincrease. The ADC changes seem to reflect the underlying pathophysiologic mechanisms in tumor necrosis (most probably hemorrhagic) induced by this novel targeted agent early after therapy onset and may indicate tumor reactivation in the later follow-up period.

Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.

Early MRI response monitoring of patients with advanced hepatocellular carcinoma under treatment with the multikinase inhibitor sorafenib

BackgroundNew therapeutic principles in clinical oncology require the adjustment of response criteria to govern therapy decisions. For advanced hepatocellular carcinoma (HCC) a new era has recently begun by the approval of the multikinase inhibitor sorafenib. As a unique feature, HCC usually develops in a diseased liver and current imaging technologies employing classical response criteria have not been prospectively evaluated for this new treatment.MethodsMRI signal patterns were assessed in 21 advanced HCC patients receiving sorafenib. MRI was performed at baseline and in short-term intervals thereafter. Signal changes under therapy on T1WI, T2WI and post-gadolinium images including necrosis volume and its ratio to the entire tumor volume were compared to baseline imaging. To assess the association between the categorical variables, Fishers exact tests were applied for a statistical analysis. Survey time ranged from 2–65 weeks, and a total of 39 target lesions were evaluated.ResultsSignal abnormalities during sorafenib therapy were disclosed by T1WI and T2WI in 15/21 patients. The predominant tumor signal change was hyperintensity on both T1WI and T2WI. Interestingly, most patients developed MRI signal changes within 4 weeks of therapy; in contrast, two non-responders did not show any signal alteration at follow-up. Under therapy, 16/21 patients presented with new or progressive necrosis, whereas 7 patients achieved temporarily >75% tumor necrosis under sorafenib. Significantly associated MRI variables were increase in T1WI signal and tumor necrosis (p = 0.017) as well as increase of tumor necrosis with an elevated ratio of necrotic to vital tumor areas (p = 0.002). Remarkably, some (3/13) of the patients developing necrotic tumor areas showed a relevant (>20%) increase in tumor volume, which should be considered in the assessment of imaging studies.ConclusionAs sorafenib induces early intralesional necrosis with profound changes in T1WI/T2WI MRI signal intensities and measurable necrotic tumor areas in most HCC patients, early MRI-based evaluation could pave the way for its rationale and cost-effective application.

Sendai virus vectors as an emerging negative‐strand RNA viral vector system

The power to manipulate the genome of negative‐strand RNA viruses, including the insertion of additional non‐viral genes, has led to the development of a new class of viral vectors for gene transfer approaches. The murine parainfluenza virus type I, or Sendai virus (SeV), has emerged as a prototype virus of this vector group, being employed in numerous in vitro as well as animal studies over the last few years. Extraordinary features of SeV are the remarkably brief contact time that is necessary for cellular uptake, a strong but adjustable expression of foreign genes, efficient infection in the respiratory tract despite a mucus layer, transduction of target cells being independent of the cell cycle, and an exclusively cytoplasmic replication cycle without any risk of chromosomal integration.

Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene

The low transduction efficiency of viral and nonviral vectors is a major limitation in tumour gene therapy. The HSV-1 tegument protein VP22 has been shown to exhibit a novel intercellular transport property. VP22 wild-type as well as VP22 fusion proteins efficiently spread from the original expressing cell to numerous neighbouring cells, so that protein transport by VP22 chimaeric polypeptides into the surrounding cells offers a possible compensation for the inadequate gene transfer efficiencies. To improve the therapeutic efficacy of the E. coli cytosine deaminase (CD) suicide gene we made use of the VP22 transport property in CD transducing adenoviral (Ad) vectors. C- and N-terminal fusions of CD linked in-frame with VP22 were generated and cloned into recombinant adenoviral vectors. Following in vitro transduction immunofluorescence analysis of Ad-transduced producer cells coplated with naive cells confirmed that the characteristic foci pattern of central producer and adjoining neighbour cells displaying nuclear staining was retained. After transduction of rat hepatoma cells with adenoviral vectors and subsequent incubation with the prodrug 5-FC, we observed enhanced cell cytotoxicity when comparing the CD-VP22 fusion (Ad-CD-VP22) with Ad-vectors expressing the CD gene only (Ad-CD). Thereby employment of Ad-vectors encoding VP22 fusion proteins opens up new possibilities to potentiate the efficiency of suicide gene therapy for the treatment of solid tumours.

Direct and Natural Killer Cell-Mediated Antitumor Effects of Low-Dose Bortezomib in Hepatocellular Carcinoma

Purpose: Hepatocellular carcinoma (HCC) displays particular resistance to conventional cytostatic agents. Alternative treatment strategies focus on novel substances exhibiting antineoplastic and/or immunomodulatory activity enhancing for example natural killer (NK) cell antitumor reactivity. However, tumor-associated ligands engaging activating NK cell receptors are largely unknown. Exceptions are NKG2D ligands (NKG2DL) of the MHC class I-related chain and UL16-binding protein families, which potently stimulate NK cell responses. We studied the consequences of proteasome inhibition with regard to direct and NK cell–mediated effects against HCC. Experimental Design: Primary human hepatocytes (PHH) from different donors, hepatoma cell lines, and NK cells were exposed to Bortezomib. Growth and viability of the different cells, and immunomodulatory effects including alterations of NKG2DL expression on hepatoma cells, specific induction of NK cell cytotoxicity and IFN-γ production were investigated. Results: Bortezomib treatment inhibited hepatoma cell growth with IC50 values between 2.4 and 7.7 nmol/L. These low doses increased MICA/B mRNA levels, resulting in an increase of total and cell surface protein expression in hepatoma cells, thus stimulating cytotoxicity and IFN-γ production of cocultured NK cells. Importantly, although NK cell IFN-γ production was concentration-dependently reduced, low-dose Bortezomib neither induced NKG2DL expression or cell death in PHH nor altered NK cell cytotoxicity. Conclusions: Low-dose Bortezomib mediates a specific dual antitumor effect in HCC by inhibiting tumor cell proliferation and priming hepatoma cells for NK cell antitumor reactivity. Our data suggest that patients with HCC may benefit from Bortezomib treatment combined with immunotherapeutic approaches such as adoptive NK cell transfer taking advantage of enhanced NKG2D-mediated antitumor immunity.