Katrin E. Tagscherer

Differentiation therapy exerts antitumor effects on stem-like glioma cells.

Purpose: Stem-like tumor cells comprise a highly tumorigenic and therapy-resistant tumor subpopulation, which is believed to substantially influence tumor initiation and therapy resistance in glioma. Currently, therapeutic, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population; retinoic acid is well known as a potent modulator of differentiation and proliferation in normal stem cells. In glioma, knowledge about the efficacy of retinoic acid–induced differentiation to target the stem-like tumor cell pool could have therapeutic implications. Experimental Design: Stem-like glioma cells (SLGC) were differentiated with all-trans retinoic acid–containing medium to study the effect of differentiation on angiogenesis, invasive growth, as well as radioresistance and chemoresistance of SLGCs. In vivo effects were studied using live microscopy in a cranial window model. Results: Our data suggest that in vitro differentiation of SLGCs induces therapy-sensitizing effects, impairs the secretion of angiogenic cytokines, and disrupts SLGCs motility. Further, ex vivo differentiation reduces tumorigenicity of SLGCs. Finally, we show that all-trans retinoic acid treatment alone can induce antitumor effects in vivo. Conclusions: Altogether, these results highlight the potential of differentiation treatment to target the stem-like cell population in glioblastoma. Clin Cancer Res; 16(10); 2715–28. ©2010 AACR.

Apoptosis-based treatment of glioblastomas with ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins

Defects in the apoptotic signaling cascades contribute to the poor therapeutic response of malignant gliomas. As glioblastomas are characterized by high expression levels of anti-apoptotic Bcl-2 family proteins, we studied the effects of the novel Bcl-2 inhibitor, ABT-737, on malignant glioma cells. ABT-737 treatment released the pro-apoptotic Bax protein from its binding partner Bcl-2 and potently induced apoptotic cell death in glioblastoma cells in vitro and in vivo. The local administration of ABT-737 prolonged the survival in an intracranial glioma xenograft model. Downregulation of Mcl-1 and overexpression of Bcl-2 sensitized the cells to ABT-737-mediated apoptosis. Moreover, ABT-737 potentiated the cytotoxicity of the chemotherapeutic drugs vincristine and etoposide, and of the death ligand TRAIL. As glioma stem cells may play a crucial role for the tumor progression and the resistance to treatment in glioblastomas, we investigated the effects of ABT-737 on the subpopulation of glioma cells exhibiting stem cell characteristics. Inhibition of proliferation and induction of apoptosis by ABT-737 were less efficient in glioma stem cells than in non-stem cell-like glioma cells. As the resistance of glioma stem cells was associated with high Mcl-1 expression levels, ABT-737 treatment combined with downregulation of Mcl-1 could represent a promising novel approach in glioblastoma treatment.

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.

Notch1 signaling promotes survival of glioblastoma cells via EGFR-mediated induction of anti-apoptotic Mcl-1

The Notch1-mediated signaling pathway has a central role in the maintenance of neural stem cells and contributes to growth and progression of glioblastomas, the most frequent malignant brain tumors in adults. Here, we demonstrate that the Notch1 receptor promotes survival of glioblastoma cells by regulation of the anti-apoptotic Mcl-1 protein. Notch1-dependent regulation of Mcl-1 occurs cell type dependent at a transcriptional or post-translational level and is mediated by the induction of epidermal growth factor receptor (EGFR). Inhibition of the Notch1 pathway overcomes apoptosis resistance and sensitizes glioblastoma cells to apoptosis induced by ionizing radiation, the death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or the Bcl-2/Bcl-XL inhibitor ABT-737. In conclusion, targeting Notch1 might represent a promising novel strategy in the treatment of glioblastomas.

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 15 kDa) 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.

The PEA-15 Protein Regulates Autophagy via Activation of JNK

PEA-15/PED (phosphoprotein enriched in astrocytes 15 kDa/phosphoprotein enriched in diabetes) is a death effector domain-containing protein which is known to modulate apoptotic cell death. The mechanism by which PEA-15 inhibits caspase activation and increases ERK (extracellular-regulated kinase) activity is well characterized. Here, we demonstrate that PEA-15 is not only pivotal in the activation of the ERK pathway but also modulates JNK (c-Jun N-terminal kinase) signaling. Upon overexpression of PEA-15 in malignant glioma cells, JNK is potently activated. The PEA-15-induced JNK activation depends on the phosphorylation of PEA-15 at both phosphorylation sites (serine 104 and serine 116). The activation of JNK is substantially inhibited by siRNA-mediated down-regulation of endogenous PEA-15. Moreover, we demonstrate that glioma cells overexpressing PEA-15 show increased signs of autophagy in response to classical autophagic stimuli such as ionizing irradiation, serum deprivation, or rapamycin treatment. In contrast, the non-phosphorylatable mutants of PEA-15 are not capable of promoting autophagy. The inhibition of JNK abrogates the PEA-15-mediated increase in autophagy. In conclusion, our data show that PEA-15 promotes autophagy in glioma cells in a JNK-dependent manner. This might render glioma cells more resistant to adverse stimuli such as starvation or ionizing irradiation.

Inhibition of caspases primes colon cancer cells for 5-fluorouracil-induced TNF-α-dependent necroptosis driven by RIP1 kinase and NF-κB.

Resistance towards the drug 5-fluorouracil (5-FU) is a key challenge in the adjuvant chemotherapy of colorectal cancer (CRC), and novel targeted approaches are required to improve the therapeutic outcome. Necroptosis is a recently discovered form of programmed cell death, which depends on receptor interacting protein 1 (RIP1) and particularly occurs under caspase-deficient conditions. The targeted induction of necroptosis represents a promising strategy to overcome apoptosis resistance in cancer. The aim of this study was to systematically explore the usage of pan-caspase inhibitors to sensitize resistant CRC cells for 5-FU. We found that pan-caspase inhibitors facilitated 5-FU-induced necroptosis, which was mediated by autocrine secretion of tumor necrosis factor α (TNF-α). TNF-α production was driven by nuclear factor κB (NF-κB) and required RIP1 kinase. In vivo xenograft experiments showed that the novel pan-caspase inhibitor IDN-7314 in combination with 5-FU synergistically blocked tumor growth. Ex vivo experiments with fresh human CRC tissue specimens further indicated that a subgroup of patients could benefit from combinatory treatment. Thereby, elevated levels of secreted TNF-α and expression of components of the necroptotic pathway might help to predict the sensitivity to pro-necroptotic therapies. Together, our results shed new light on the molecular regulation of necroptosis by NF-κB and RIP1. Moreover, we identify necroptotic cell death as an important effector mechanism of 5-FU-mediated anti-tumoral activity. On the basis of this study, we propose pan-caspase inhibitors as a novel approach in the adjuvant chemotherapy of CRC.

The soluble Decoy Receptor 3 is regulated by a PI3K-dependent mechanism and promotes migration and invasion in renal cell carcinoma

BackgroundOverexpression of Decoy Receptor 3 (DcR3), a soluble member of the tumor necrosis factor receptor superfamily, is a common event in several types of cancer. In renal cell carcinoma (RCC), DcR3 overexpression is associated with lymph node and distant metastasis as well as a poor prognosis. However, the functional role and regulation of DcR3 expression in RCC is so far unknown.MethodsModulation of DcR3 expression by siRNA and ectopic gene expression, respectively, was performed in ACHN and 769-P RCC cell lines. Functional effects of a modulated DcR3 expression were analyzed with regard to migration, invasion, adhesion, clonogenicity, and proliferation. Furthermore, quantitative RT-PCR and immunoblot analyses were performed to evaluate the expression of downstream mediators of DcR3. In further experiments, luciferase assays, quantitative RT-PCR and immunoblot analyses were applied to study the regulation of DcR3 expression in RCC. Additionally, an ex vivo tissue slice culture technique combined with immunohistochemistry was used to study the regulation of DcR3 expression in human RCC specimens.ResultsHere, we show that DcR3 promotes adhesion, migration and invasiveness of RCC cells. The DcR3-dependent increase in cellular invasiveness is accompanied with an up-regulation of integrin alpha 4, matrixmetalloproteinase 7 and urokinase plasminogen activator (uPA). Further, we identified a signaling pathway regulating DcR3 expression in RCC. Using in vitro experiments as well as an ex vivo RCC tissue slice culture model, we demonstrate that expression of DcR3 is regulated in a PI3K/AKT-dependent manner involving the transcription factor nuclear factor of activated T-cells (NFAT).ConclusionsTaken together, our results identify DcR3 as a key driver of tumor cell dissemination and suggest DcR3 as a promising target for rational therapy of RCC.

p53-dependent regulation of Mcl-1 contributes to synergistic cell death by ionizing radiation and the Bcl-2/Bcl-XL inhibitor ABT-737

Treatment with the Bcl-2/Bcl-XL inhibitor ABT-737 is a promising novel strategy to therapeutically induce apoptotic cell death in malignant tumors such as glioblastomas. Although many studies have demonstrated that ABT-737 acts synergistically with chemotherapeutic drugs, the possibility of a combined treatment with ionizing radiation (IR) and ABT-737 has not yet been thoroughly investigated. Similarly, the relationship between p53 function and the pro-apoptotic effects of ABT-737 are still obscure. Here, we demonstrate that IR and ABT-737 synergistically induce apoptosis in glioblastoma cells. The sensitivity to ABT-737-mediated cell death is significantly increased by the IR-dependent accumulation of cells in the G2/M cell cycle phase. Wild type p53 function inhibits the efficacy of a combined IR and ABT-737 treatment via a p21-dependent G1 cell cycle arrest. Moreover, mutant as well as wild type p53 counteract the pro-apoptotic activity of ABT-737 by maintaining the expression levels of the Mcl-1 protein. Thus, p53 regulates the sensitivity to ABT-737 of glioblastoma cells. Our results warrant a further evaluation of a novel combination therapy using IR and ABT-737. The efficacy of such a therapy could be substantially enhanced by Mcl-1-lowering strategies.

BLOC1S2 interacts with the HIPPI protein and sensitizes NCH89 glioblastoma cells to apoptosis

The HIPPI (HIP-1 protein interactor) protein is a multifunctional protein that is involved in the regulation of apoptosis. The interaction partners of HIPPI include HIP-1 (Huntingtin-interacting protein-1), Apoptin, Homer1c, Rybp/DEDAF, and BAR (bifunctional apoptosis regulator). In search for other binding partners of HIPPI, we performed a yeast two hybrid screen and identified BLOC1S2 (Biogenesis of lysosome-related organelles complex-1 subunit 2) as a novel HIPPI-interacting protein. In co-immunoprecipitation assays, BLOC1S2 specifically associates with HIPPI, but not with HIP-1. To study the expression of BLOC1S2 on the protein level, we generated a mouse monoclonal antibody specific for BLOC1S2 and a multiple tissue array comprising 70 normal and cancer tissue samples of diverse origin. BLOC1S2 protein is widely expressed in normal tissue as well as in malignant tumors with a tendency towards lower expression levels in certain subtypes of tumors. On the subcellular level, BLOC1S2 is expressed in an organellar-like pattern and co-localizes with mitochondria. Over-expression of BLOC1S2 in the presence or absence of HIPPI does not induce apoptosis. However, BLOC1S2 and HIPPI sensitize NCH89 glioblastoma cells to the pro-apoptotic actions of staurosporine and the death ligand TRAIL by enhancing caspase activation, cytochrome c release, and disruption of the mitochondrial membrane potential. Given its interaction with HIPPI and its pro-apoptotic activity, BLOC1S2 might play an important functional role in cancer and neurodegenerative diseases.