Tom M. Ganten

Yes and PI3K bind CD95 to signal invasion of glioblastoma.

Invasion of surrounding brain tissue by isolated tumor cells represents one of the main obstacles to a curative therapy of glioblastoma multiforme. Here we unravel a mechanism regulating glioma infiltration. Tumor interaction with the surrounding brain tissue induces CD95 Ligand expression. Binding of CD95 Ligand to CD95 on glioblastoma cells recruits the Src family member Yes and the p85 subunit of phosphatidylinositol 3-kinase to CD95, which signal invasion via the glycogen synthase kinase 3-beta pathway and subsequent expression of matrix metalloproteinases. In a murine syngeneic model of intracranial GBM, neutralization of CD95 activity dramatically reduced the number of invading cells. Our results uncover CD95 as an activator of PI3K and, most importantly, as a crucial trigger of basal invasion of glioblastoma in vivo.

Enhanced caspase-8 recruitment to and activation at the DISC is critical for sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by chemotherapeutic drugs

AbstractTumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumour activity upon systemic administration in mice without showing the deleterious side effects observed with other apoptosis-inducing members of the TNF family such as TNF and CD95L. TRAIL may, thus, have great potential in the treatment of human cancer. However, about 60% of tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of tumour resistance to TRAIL, we investigated hepatocellular carcinoma (HCC) cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs, for example, 5-fluorouracil (5-FU), rendered the TRAIL-resistant HCC cell lines sensitive to TRAIL-induced apoptosis. Analysis of the TRAIL death-inducing signalling complex (DISC) revealed upregulation of TRAIL-R2. Caspase-8 recruitment to and its activation at the DISC were substantially increased after 5-FU sensitisation, while FADD recruitment remained essentially unchanged. 5-FU pretreatment downregulated cellular FLICE-inhibitory protein (cFLIP) and specific cFLIP downregulation by small interfering RNA was sufficient to sensitise TRAIL-resistant HCC cell lines for TRAIL-induced apoptosis. Thus, a potential mechanism for TRAIL sensitisation by 5-FU is the increased effectiveness of caspase-8 recruitment to and activation at the DISC facilitated by the downregulation of cFLIP and the consequent shift in the ratio of caspase-8 to cFLIP at the DISC.

Preclinical Differentiation between Apparently Safe and Potentially Hepatotoxic Applications of TRAIL Either Alone or in Combination with Chemotherapeutic Drugs

Purpose: Tumor necrosis factor-related apoptosis–inducing ligand (TRAIL/Apo2L) exhibits potent antitumor activity on systemic administration in nonhuman primates without deleterious side effects for normal tissue. However, there is a controversy about the potential toxicity of TRAIL on human hepatocytes. The use of different recombinant TRAIL forms only partially explains the contradicting reports on TRAIL sensitivity in primary human hepatocytes (PHH). Experimental Design: To clarify this issue, we comprehensively tested four different recombinant forms of TRAIL for their apoptosis-inducing capacity on PHH obtained from a total of 55 human livers between day 1 and day 8 of in vitro culture. Results: One day after single-cell isolation, all but one recombinant form of TRAIL [i.e., an untagged form of TRAIL (TRAIL.0)] induced apoptosis in PHH. Apoptosis induction by TRAIL in these cells could only be fully inhibited by concomitant blockade of TRAIL receptor 1 and TRAIL receptor 2. At day 4 of in vitro culture, when surrogate markers indicated optimal hepatocyte in vitro function, only high doses of cross-linked FLAG-TRAIL killed PHH whereas the other three recombinant TRAIL forms did not. Strikingly, cotreatment of day 4 PHH with cisplatin sensitized for TRAIL-induced apoptosis whereas 5-fluorouracil, etoposide, gemcitabine, irinotecan, or oxaliplatin, which are commonly used in the treatment of gastrointestinal cancers, did not. Conclusion: Our data show that whereas TRAIL alone or together with selected chemotherapeutic drugs seems to be safe, the combination of TRAIL with cisplatin is toxic to PHH.

The promise of TRAIL—potential and risks of a novel anticancer therapy

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising new anticancer biotherapeutic. As shown by many preclinical studies, TRAIL efficiently induces apoptosis in numerous tumor cell lines but not in the majority of normal cells. However, an increasing number of publications report on a predominance of TRAIL resistance in primary human tumor cells, which require sensitization for TRAIL-induced apoptosis. Sensitization of cancer cells by treatment with chemotherapeutic drugs and irradiation has been shown to restore TRAIL sensitivity in many TRAIL-resistant tumor cells. Accordingly TRAIL treatment has been successfully used in different in vivo models for the treatment of tumors also in combination with chemotherapeutics without significant toxicity. However, some reports demonstrated toxicity of TRAIL alone or in combination with chemotherapeutic drugs in normal cells. This review summarizes data concerning the apoptosis-inducing pathways and efficacy of TRAIL, alone or in combination with chemotherapeutic drugs, in primary cancer cells compared to the unwanted effects of TRAIL treatment on normal tissue. We discuss the different in vitro tumor cell models and the potential of different recombinant forms of TRAIL or agonistic antibodies to TRAIL death receptors. Most preclinical studies show a high efficiency of a combinatorial TRAIL-based therapy in animal models and in primary human ex vivo tumor cells with a low toxicity in normal cells. Accordingly clinical phase I/II studies have begun and will be developed further with caution.

Proteasome inhibition sensitizes hepatocellular carcinoma cells, but not human hepatocytes, to TRAIL.

TRAIL exhibits potent anti‐tumor activity on systemic administration in mice. Because of its proven in vivo efficacy, TRAIL may serve as a novel anti‐neoplastic drug. However, approximately half of the tumor cell lines tested so far are TRAIL resistant, and potential toxic side effects of certain recombinant forms of TRAIL on human hepatocytes have been described. Pretreatment with the proteasome inhibitor MG132 and PS‐341 rendered TRAIL‐resistant hepatocellular carcinoma (HCC) cell lines but not primary human hepatocytes sensitive for TRAIL‐induced apoptosis. We investigated the different levels of possible MG132‐induced interference with resistance to apoptotic signal transduction. Although proteasome inhibition efficiently suppressed nuclear factor‐kappaB (NF‐κB) activity, specific suppression of NF‐κB by mutIκBα failed to sensitize TRAIL‐resistant cell lines for TRAIL‐induced apoptosis. In contrast to the previously reported mechanism of sensitization by 5‐fluorouracil (5‐FU), cellular FLICE‐inhibitory protein (cFLIP)L and cFLIPS were markedly upregulated in the TRAIL death inducing signaling complex (DISC) by proteasome inhibitor pretreatment. Compared with 5‐FU pretreatment, caspase‐8 was more efficiently recruited to the DISC in MG132 pretreated cells despite the presence of fewer death receptors and more cFLIP in the DISC. But downregulation of cFLIP by short interference RNA (siRNA) further sensitized the HCC cell lines. In conclusion, these results show that otherwise chemotherapy‐resistant tumor cells can be sensitized for TRAIL‐induced apoptosis at the DISC level in the presence of high levels of cFLIP, which suggests the existence of an additional factor that modulates the interaction of FADD and the TRAIL death receptors. Of clinical relevance, proteasome inhibitors sensitize HCC cells but not primary human hepatocytes for TRAIL‐induced apoptosis. (HEPATOLOGY 2005.)

Bortezomib Sensitizes Primary Human Astrocytoma Cells of WHO Grades I to IV for Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis

Purpose: Malignant gliomas are the most aggressive human brain tumors without any curative treatment. The antitumor effect of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in gliomas has thus far only been thoroughly established in tumor cell lines. In the present study, we investigated the therapeutic potential of TRAIL in primary human glioma cells. Experimental Design: We isolated primary tumor cells from 13 astrocytoma and oligoastrocytoma patients of all four WHO grades of malignancy and compared the levels of TRAIL-induced apoptosis induction, long-term tumor cell survival, caspase, and caspase target cleavage. Results: We established a stable culture model for isolated primary human glioma cells. In contrast to cell lines, isolated primary tumor cells from all investigated glioma patients were highly TRAIL resistant. Regardless of the tumor heterogeneity, cotreatment with the proteasome inhibitor bortezomib efficiently sensitized all primary glioma samples for TRAIL-induced apoptosis and tremendously reduced their clonogenic survival. Due to the pleiotropic effect of bortezomibenhanced TRAIL DISC formation upon TRAIL triggering, down-regulation of cFLIPL and activation of the intrinsic apoptosis pathway seem to cooperatively contribute to the antitumor effect of bortezomib/TRAIL cotreatment. Conclusion: TRAIL sensitivity of tumor cell lines is not a reliable predictor for the behavior of primary tumor cells. The widespread TRAIL resistance in primary glioma cells described here questions the therapeutic clinical benefit of TRAIL as a monotherapeutic agent. Overcoming TRAIL resistance by bortezomib cotreatment might, however, provide a powerful therapeutic option for glioma patients.

TRAIL/bortezomib cotreatment is potentially hepatotoxic but induces cancer-specific apoptosis within a therapeutic window

Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) represents a novel promising anticancer biotherapeutic. However, TRAIL‐resistant tumor cells require combinatorial regimens to sensitize tumor but not normal cells for TRAIL‐induced apoptosis. Here, we investigated the mechanism of the synergistic antitumor effect of bortezomib in combination with TRAIL in hepatoma, colon, and pancreatic cancer cells in comparison to the toxicity in primary human hepatocytes (PHH). TRAIL cotreatment at high but clinically relevant concentrations of bortezomib caused toxicity in PHH which potentially limits the clinical applicability of bortezomib/TRAIL cotreatment. However, at low concentrations of bortezomib TRAIL‐resistant hepatoma, colon and pancreatic cancer cell lines but not PHH were efficiently sensitized for TRAIL‐induced apoptosis. RNA interference and TRAIL receptor blockage experiments revealed that in bortezomib‐treated hepatoma cells TRAIL‐R1/TRAIL‐R2 up‐regulation, enhanced TRAIL DISC formation and cFLIPL down‐regulation in addition to accumulation of Bak cooperatively sensitized for TRAIL. Bim, although accumulated upon bortezomib treatment, did not play a causal role for TRAIL sensitization in Hep3b cells. Combined treatment with bortezomib and TRAIL massively reduced the clonogenic capacity of hepatoma cells in vitro. Surviving clones could be resensitized for repeated TRAIL treatment. Conclusion: Bortezomib/TRAIL cotreatment bears the risk of severe hepatotoxicity at high but clinically relevant concentrations of bortezomib. However, within a wide therapeutic window bortezomib sensitized different cancer cells but not PHH for TRAIL‐induced apoptosis. (HEPATOLOGY 2007;45:649–658.)

Sirolimus Use in Liver Transplant Recipients With Hepatocellular Carcinoma: A Randomized, Multicenter, Open-Label Phase 3 Trial.

Background We investigated whether sirolimus-based immunosuppression improves outcomes in liver transplantation (LTx) candidates with hepatocellular carcinoma (HCC). Methods In a prospective-randomized open-label international trial, 525 LTx recipients with HCC initially receiving mammalian target of rapamycin inhibitor–free immunosuppression were randomized 4 to 6 weeks after transplantation into a group on mammalian target of rapamycin inhibitor–free immunosuppression (group A: 264 patients) or a group incorporating sirolimus (group B: 261). The primary endpoint was recurrence-free survival (RFS); intention-to-treat (ITT) analysis was conducted after 8 years. Overall survival (OS) was a secondary endpoint. Results Recurrence-free survival was 64.5% in group A and 70.2% in group B at study end, this difference was not significant (P = 0.28; hazard ratio [HR], 0.84; 95% confidence interval [95% CI], 0.62; 1.15). In a planned analysis of RFS rates at yearly intervals, group B showed better outcomes 3 years after transplantation (HR, 0.7; 95% CI, 0.48-1.00). Similarly, OS (P = 0.21; HR, 0.81; 95% CI, 0.58-1.13) was not statistically better in group B at study end, but yearly analyses showed improvement out to 5 years (HR, 0.7; 95% CI, 0.49-1.00). Interestingly, subgroup (Milan Criteria-based) analyses revealed that low-risk, rather than high-risk, patients benefited most from sirolimus; furthermore, younger recipients (age ⩽60) also benefited, as well sirolimus monotherapy patients. Serious adverse event numbers were alike in groups A (860) and B (874). Conclusions Sirolimus in LTx recipients with HCC does not improve long-term RFS beyond 5 years. However, a RFS and OS benefit is evident in the first 3 to 5 years, especially in low-risk patients. This trial provides the first high-level evidence base for selecting immunosuppression in LTx recipients with HCC.

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.

Human and Mouse VEGFA-Amplified Hepatocellular Carcinomas Are Highly Sensitive to Sorafenib Treatment

UNLABELLED Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet therapeutic options for advanced HCC are limited. We identify a subset of mouse and human HCCs harboring VEGFA genomic amplification, displaying distinct biologic characteristics. Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions; stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor (HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib-the first-line drug in advanced HCC-targets multiple kinases, including VEGFRs, but has only an overall mild beneficial effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensitive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival of sorafenib-treated patients with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A-blocking drugs. SIGNIFICANCE Using a mouse model of inflammation-driven cancer, we identified a subclass of HCC carrying VEGFA amplification, which is particularly sensitive to VEGF-A inhibition. We found that a similar amplification in human HCC identifies patients who favorably responded to sorafenib-the first-line treatment of advanced HCC-which has an overall moderate therapeutic efficacy.