Jaromir Sykora

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.

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.

Prognostic significance of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression in patients with breast cancer

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis upon binding to TRAIL receptors 1 and 2 (TRAIL-R1/DR4 and TRAIL-R2/DR5). TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) have no or only a truncated cytoplasmic death domain. Consequently, they cannot induce apoptosis and instead have been proposed to inhibit apoptosis induction by TRAIL. Agonists for the apoptosis-inducing TRAIL-R1 and TRAIL-R2 are currently tested in clinical trials. To determine the expression pattern of all surface-bound TRAIL receptors and their prognostic clinical value, we investigated tumour samples of 311 patients with breast cancer by immunohistochemistry. TRAIL receptor expression profiles were correlated with clinico-pathological data, disease-free survival and overall survival. TRAIL-R1 was more strongly expressed in better differentiated tumours, and correlated positively with surrogate markers of a better prognosis (hormone receptor status, Bcl-2, negative nodal status), but negatively with the expression of Her2/neu and the proliferation marker Ki67. In contrast, TRAIL-R2 and TRAIL-R4 expression correlated with higher tumour grades, higher Ki67 index, higher Her2/neu expression and a positive nodal status at the time of diagnosis, but with lower expression of Bcl-2. Thus, the TRAIL receptor expression pattern was predictive of nodal status. Patients with grade 1 and 2 tumours, who had TRAIL-R2 but no TRAIL-R1, showed a positive lymph node status in 47% of the cases. Vice versa, only 19% had a positive nodal status with high TRAIL-R1 but low TRAIL-R2. Most strikingly, TRAIL-R4 and -R2 expression negatively correlated with overall survival of breast cancer patients. Although TRAIL-R2 correlated with more aggressive tumour behaviour, mammary carcinoma could be sensitised to TRAIL-R2-induced apoptosis, suggesting that TRAIL-R2 might therefore be used to therapeutically target such tumours. Hence, determination of the TRAIL receptor expression profile may aid in defining which breast cancer patients have a higher risk of lymph node metastasis and worse overall survival and on the other hand will help to guide TRAIL-based tumour therapy.

Prognostic Value of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (TRAIL) and TRAIL Receptors in Renal Cell Cancer

Purpose: The death ligand tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R) are involved in immune surveillance and tumor development. Here, we studied a possible association between the expression of TRAIL/TRAIL-Rs and the prognosis in patients with renal cell carcinomas (RCC). Experimental Design: A tissue microarray containing RCC tumor tissue samples and corresponding normal tissue samples from 838 patients was generated. Expression of TRAIL and TRAIL-Rs was examined by immunohistochemistry and the effect of TRAIL and TRAIL-R expression on disease-specific survival was assessed. Results: High TRAIL-R2 expression levels were associated with high-grade RCCs (P < 0.001) and correlated negatively with disease-specific survival (P = 0.01). Similarly, high TRAIL expression was associated with a shorter disease-specific survival (P = 0.01). In contrast, low TRAIL-R4 expression was associated with high-stage RCCs (P < 0.001) as well as with the incidence of distant metastasis (P = 0.03) and correlated negatively with disease-specific survival (P = 0.02). In patients without distant metastasis, multivariate Cox regression analyses revealed that TRAIL-R2 and TRAIL are independent prognostic factors for cancer-specific survival (in addition to tumor extent, regional lymph node metastasis, grade of malignancy, and type of surgery). Conclusion: High TRAIL-R2, high TRAIL, and low TRAIL-R4 expression levels are associated with a worse disease-specific survival in patients with RCCs. Therefore, the assessment of TRAIL/TRAIL-R expression offers valuable prognostic information that could be used to select patients for adjuvant therapy studies. Moreover, our findings are of relevance for a potential experimental therapeutic administration of TRAIL-R agonists in patients with RCCs.

The PEA-15/PED protein protects glioblastoma cells from glucose deprivation-induced apoptosis via the ERK/MAP kinase pathway

PEA-15 (phosphoprotein enriched in astrocytes 15u2009kDa) is a death effector domain-containing protein, which is involved in the regulation of apoptotic cell death. Since PEA-15 is highly expressed in cells of glial origin, we studied the role of PEA-15 in human malignant brain tumors. Immunohistochemical analysis of PEA-15 expression shows strong immunoreactivity in astrocytomas and glioblastomas. Phosphorylation of PEA-15 at Ser116 is found in vivo in perinecrotic areas in glioblastomas and in vitro after glucose deprivation of glioblastoma cells. Overexpression of PEA-15 induces a marked resistance against glucose deprivation-induced apoptosis, whereas small interfering RNA (siRNA)-mediated downregulation of endogenous PEA-15 results in the sensitization to glucose withdrawal-mediated cell death. This antiapoptotic activity of PEA-15 under low glucose conditions depends on its phosphorylation at Ser116. Moreover, siRNA-mediated knockdown of PEA-15 abolishes the tumorigenicity of U87MG glioblastoma cells in vivo. PEA-15 regulates the level of phosphorylated extracellular-regulated kinase (ERK)1/2 in glioblastoma cells and the PEA-15-dependent protection from glucose deprivation-induced cell death requires ERK1/2 signaling. PEA-15 transcriptionally upregulates the Glucose Transporter 3, which is abrogated by the inhibition of ERK1/2 phosphorylation. Taken together, our findings suggest that Ser116-phosphorylated PEA-15 renders glioma cells resistant to glucose deprivation-mediated cell death as encountered in poor microenvironments, for example in perinecrotic areas of glioblastomas.

Differential expression of the TRAIL/TRAIL-receptor system in patients with inflammatory bowel disease.

TNF-related apoptosis inducing-ligand (TRAIL) is a potent inducer of apoptosis and plays an important role in immune regulation. To explore the role of TRAIL in inflammatory bowel disease (IBD), we examined the expression of the TRAIL/TRAIL-receptor system in colonic resections from patients with ulcerative colitis and Crohns disease in comparison to normal colon and appendicitis. TRAIL and TRAIL-receptor (TRAIL-R) expression was assessed in resections of normal colon, colon of IBD patients, and appendicitis by immunohistochemistry. TRAIL was downregulated in enterocytes of patients with IBD, but was upregulated in mononuclear cells in areas of active mucosal inflammation. For TRAIL-R1, we detected a strong downregulation in the surface epithelium in IBD but not in appendicitis. TRAIL-R2 and TRAIL-R4 were strongly downregulated in the surface epithelium in any kind of mucosal inflammation. TRAIL and TRAIL-R1 are downregulated in enterocytes, and TRAIL is upregulated in mononuclear cells only in IBD but not in normal colon or appendicitis. This may point to a pathophysiologic role of the TRAIL system in inflammatory bowel disease.

Troglitazone-mediated sensitization to TRAIL-induced apoptosis is regulated by proteasome-dependent degradation of FLIP and ERK1/2-dependent phosphorylation of BAD

Resistance to apoptosis is one reason for the poor response of malignant brain tumors to therapy. The PPARγ-modulating drug Troglitazone down-regulates the anti-apoptotic FLIP protein and sensitizes glioblastoma cells to apoptosis induced by the death ligand TRAIL. To investigate the molecular basis of an experimental combination therapy for malignant gliomas with TRAIL and Troglitazone, we investigated the Troglitazone-induced signaling cascades and the expression of TRAIL receptors and FLIP in malignant gliomas. Troglitazone down-regulated the FLIP protein through accelerated ubiquitin/proteasome-dependent degradation, which might be mediated by a Troglitazone-induced increase in reactive oxygen species. Moreover, Troglitazone induced the phosphorylation of the MAP kinase ERK1/2 as well as of the BAD protein. Inhibition of either PPARγ or MEK1/2 blocked the Troglitazone-mediated phosphorylation of BAD and further increased the synergistic induction of glioma cell death by TRAIL and Troglitazone. Immunohistochemical analysis demonstrated that FLIP and TRAIL-R2 were significantly higher expressed in anaplastic (WHO grade III) than in diffuse (WHO grade II) gliomas. High FLIP and low TRAIL-R2 expression levels were associated with a poor prognosis of patients. Our findings warrant a further pre-clinical evaluation of an experimental anti-glioma therapy with TRAIL and Troglitazone, potentially in conjunction with a MAP kinase inhibitor.

Bortezomib-Mediated Up-Regulation of TRAIL-R1 and TRAIL-R2 Is Not Necessary for but Contributes to Sensitization of Primary Human Glioma Cells to TRAIL

In Response: In their Letter, Kyritsis et al. discussed the interesting question of the contribution of up-regulation of the apoptosis-inducing tumor necrosis factor–related apoptosis inducing ligand (TRAIL) receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), to the sensitization of tumor cells to TRAIL