Susan Finniss

GRP78 is overexpressed in glioblastomas and regulates glioma cell growth and apoptosis

We characterized the expression and function of the endoplasmic reticulum protein GRP78 in glial tumors. GRP78 is highly expressed in glioblastomas but not in oligodendrogliomas, and its expression is inversely correlated with median patient survival. Overexpression of GRP78 in glioma cells decreases caspase 7 activation and renders the cells resistant to etoposide- and cisplatin-induced apoptosis, whereas silencing of GRP78 decreases cell growth and sensitizes glioma cells to etoposide, cisplatin, and gamma-radiation. Thus, GRP78 contributes to the increased apoptosis resistance and growth of glioma cells and may provide a target for enhancing the therapeutic responsiveness of these tumors.

Polynuclear platinum anticancer drugs are more potent than cisplatin and induce cell cycle arrest in glioma

We have evaluated the efficacy of the multinuclear platinum chemotherapeutics BBR3464, BBR3571, and BBR3610 against glioma cells in culture and animal models and investigated their mechanism of action at the cellular level. In a clonogenic assay, BBR3610, the most potent compound, had an IC90 dose (achieving 90% colony formation inhibition) that was 250 times lower than that of cisplatin for both LNZ308 and LN443 glioma cells. In subcutaneous xenografts of U87MG glioma cells, BBR3610 approximately doubled the time it took for a tumor to reach a predetermined size and significantly extended survival when these cells were implanted intracranially. Analysis of apoptosis and cell cycle distribution showed that BBR compounds induced G2/M arrest in the absence of cell death, while cisplatin predominantly induced apoptosis. Interestingly, the BBR compounds and cisplatin both induced extracellular signal-regulated kinase 1/2 phosphorylation, and inhibition of this pathway at the level of MEK antagonized the induction of G2/M arrest or apoptosis, respectively. Analysis of Chk1 and Chk2 status did not show any differential effects of the drugs, and it is thus unlikely to underlie the difference in response. Similarly, the drugs did not differentially modulate survivin levels, and knockdown of survivin did not convert the response to BBR3610 to apoptosis. Together, these findings support continued development of BBR3610 for clinical use against glioma and provide a framework for future investigation of mechanism of action.

The Localization of Protein Kinase Cδ in Different Subcellular Sites Affects Its Proapoptotic and Antiapoptotic Functions and the Activation of Distinct Downstream Signaling Pathways

Protein kinase Cδ (PKCδ) regulates cell apoptosis and survival in diverse cellular systems. PKCδ translocates to different subcellular sites in response to apoptotic stimuli; however, the role of its subcellular localization in its proapoptotic and antiapoptotic functions is just beginning to be understood. Here, we used a PKCδ constitutively active mutant targeted to the cytosol, nucleus, mitochondria, and endoplasmic reticulum (ER) and examined whether the subcellular localization of PKCδ affects its apoptotic and survival functions. PKCδ-Cyto, PKCδ-Mito, and PKCδ-Nuc induced cell apoptosis, whereas no apoptosis was observed with the PKCδ-ER. PKCδ-Cyto and PKCδ-Mito underwent cleavage, whereas no cleavage was observed in the PKCδ-Nuc and PKCδ-ER. Similarly, caspase-3 activity was increased in cells overexpressing PKCδ-Cyto and PKCδ-Mito. In contrast to the apoptotic effects of the PKCδ-Cyto, PKCδ-Mito, and PKCδ-Nuc, the PKCδ-ER protected the cells from tumor necrosis factor–related apoptosis-inducing ligand–induced and etoposide-induced apoptosis. Moreover, overexpression of a PKCδ kinase-dead mutant targeted to the ER abrogated the protective effect of the endogenous PKCδ and increased tumor necrosis factor–related apoptosis-inducing ligand–induced apoptosis. The localization of PKCδ differentially affected the activation of downstream signaling pathways. PKCδ-Cyto increased the phosphorylation of p38 and decreased the phosphorylation of AKT and the expression of X-linked inhibitor of apoptosis protein, whereas PKCδ-Nuc increased c-Jun NH2-terminal kinase phosphorylation. Moreover, p38 phosphorylation and the decrease in X-linked inhibitor of apoptosis protein expression played a role in the apoptotic effect of PKCδ-Cyto, whereas c-Jun NH2-terminal kinase activation mediated the apoptotic effect of PKCδ-Nuc. Our results indicate that the subcellular localization of PKCδ plays important roles in its proapoptotic and antiapoptotic functions and in the activation of downstream signaling pathways. (Mol Cancer Res 2007;5(6):627–39)

Cilengitide induces autophagy-mediated cell death in glioma cells.

We studied the effect of the integrin inhibitor cilengitide in glioma cells. Cilengitide induced cell detachment and decreased cell viability, and induction of autophagy followed by cell apoptosis. In addition, cilengitide decreased the cell renewal of glioma stem-like cells (GSCs). Inhibition of autophagy decreased the cytotoxic effect of cilengitide. Pretreatment of glioma cells and GSCs with cilengitide prior to γ-irradiation resulted in a larger increase in autophagy and a more significant decrease in cell survival. We found that cilengitide induced autophagy collectively in glioma cells, xenografts, and GSCs, which contributed to its cytotoxic effects and sensitized these cells to γ-radiation.

TRAIL conjugated to nanoparticles exhibits increased anti-tumor activities in glioma cells and glioma stem cells in vitro and in vivo

Glioblastomas (GBM) are characterized by resistance to chemotherapy and radiotherapy, and therefore, alternative therapeutic approaches are needed. TRAIL induces apoptosis in cancer but not in normal cells and is considered to be a promising anti-tumor agent. However, its short in vivo half-life and lack of efficient administration modes are serious impediments to its therapeutic efficacy. Nanoparticles (NP) have been used as effective delivery tools for various anticancer drugs. TRAIL was conjugated to magnetic ferric oxide NP by binding the TRAIL primary amino groups to activated double bonds on the surface of the NP. The effect of NP-TRAIL was examined on the apoptosis of glioma cells and self-renewal of glioma stem cells (GSCs). In addition, the ability of the NP-TRAIL to track U251 cell-derived glioma xenografts and to affect cell apoptosis, tumor volume, and survival among xenografted rats was also examined. Conjugation of TRAIL to NP increased its apoptotic activity against different human glioma cells and GSCs, as compared with free recombinant TRAIL. Combined treatment with NP-TRAIL and γ-radiation or bortezomib sensitized TRAIL-resistant GSCs to NP-TRAIL. Using rhodamine-labeled NP and U251 glioma cell-derived xenografts, we demonstrated that the NP-TRAIL were found in the tumor site and induced a significant increase in glioma cell apoptosis, a decrease in tumor volume, and increased animal survival. In summary, conjugation of TRAIL to NP increased its apoptotic activity both in vitro and in vivo. Therefore, NP-TRAIL represents a targeted anticancer agent with more efficient action for the treatment of GBM and the eradication of GSCs.

Phosphorylation of Protein Kinase Cδ on Distinct Tyrosine Residues Induces Sustained Activation of Erk1/2 via Down-regulation of MKP-1 ROLE IN THE APOPTOTIC EFFECT OF ETOPOSIDE

The mechanism underlying the important role of protein kinase Cδ (PKCδ) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKCδ in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKCδ since the phosphorylation of Erk1/2 was inhibited by a PKCδ-KD mutant and PKCδ small interfering RNA. We recently reported that phosphorylation of PKCδ on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKCδtyrosine mutants, we found that the phosphorylation of PKCδon these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKCδ was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKCδ. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKCδ and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKCδon tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis.

Proliferation marker pKi-67 occurs in different isoforms with various cellular effects

The Ki‐67 antigen, pKi‐67, is a commonly used proliferation marker in research and pathology. It has been recognized that the protein exists in two different splice variants that differ in one exon. In the current work, we present three new splice variants of human pKi‐67 consisting of two naturally occurring isoforms and one atypical version. Additionally, data is presented indicating that alternative splicing of the pKi‐67 N‐terminus is common in tumor cell lines. Analyzing 93 tissues mainly consisting of brain tumor specimens, we found evidence that long and short isoform can be expressed independently of each other. Induction of mitosis in human peripheral blood mononuclear cells revealed that short pKi‐67 appears earlier in the cell cycle than the long isoform and reaches its expression maximum when transcription of the latter sets in. Finally, transfection of mammalian culture cells with exon 7 (specific for the long pKi‐67 isoform and not present in the short isoform) in a tetracycline regulated expression system decreased the rate of cell proliferation without affecting the cell cycle. In summary, we present evidence that the pKi‐67 N‐terminus is differentially spliced resulting in at least five different isoforms with different functions.

Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma

Glioblastoma (GBM) is the most aggressive primary brain tumor with poor prognosis. Here, we studied the effects of phenformin, a mitochondrial complex I inhibitor and more potent chemical analog of the diabetes drug metformin on the inhibition of cell growth and induction of apoptosis of glioma stem cells (GSCs) using both in vitro and in vivo models. Phenformin inhibited the self-renewal of GSCs, decreased the expression of stemness and mesenchymal markers and increased the expression of miR-124, 137 and let-7. Silencing of let-7 abrogated phenformin effects on the self-renewal of GSCs via a pathway associated with inhibition of H19 and HMGA2 expression. Moreover, we demonstrate that phenformin inhibited tumor growth and prolonged the overall survival of mice orthotopically transplanted with GSCs. Combined treatments of phenformin and temozolomide exerted an increased antitumor effect on GSCs in vitro and in vivo. In addition, dichloroacetate, an inhibitor of the glycolysis enzyme pyruvate dehydrogenase kinase, that decreases lactic acidosis induced by biguanides, enhanced phenformin effects on the induction of cell death in GSCs and prolonged the survival of xenograft-bearing mice. Our results demonstrate for the first time that phenformin targets GSCs and can be efficiently combined with current therapies for GBM treatment and GSC eradication.

Preferential expression of functional IL-17R in glioma stem cells: potential role in self-renewal

Gliomas are the most common primary brain tumor and one of the most lethal solid tumors. Mechanistic studies into identification of novel biomarkers are needed to develop new therapeutic strategies for this deadly disease. The objective for this study was to explore the potential direct impact of IL-17−IL-17R interaction in gliomas. Immunohistochemistry and flow cytometry analysis of 12 tumor samples obtained from patients with high grade gliomas revealed that a considerable population (2–19%) of cells in all malignant gliomas expressed IL-17RA, with remarkable co-expression of the glioma stem cell (GSC) markers CD133, Nestin, and Sox2. IL-17 enhanced the self-renewal of GSCs as determined by proliferation and Matrigel® colony assays. IL-17 also induced cytokine/chemokine (IL-6, IL-8, interferon-γ-inducible protein [IP-10], and monocyte chemoattractant protein-1 [MCP-1]) secretion in GSCs, which were differentially blocked by antibodies against IL-17R and IL-6R. Western blot analysis showed that IL-17 modulated the activity of signal transducer and activator of transcription 3 (STAT3), nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), glycogen synthase kinase-3β (GSK-3β) and β-catenin in GSCs. While IL-17R-mediated secretion of IL-6 and IL-8 were significantly blocked by inhibitors of NF-κB and STAT3; NF-κB inhibitor was more potent than STAT3 inhibitor in blocking IL-17-induced MCP-1 secretion. Overall, our results suggest that IL-17–IL-17R interaction in GSCs induces an autocrine/paracrine cytokine feedback loop, which may provide an important signaling component for maintenance/self-renewal of GSCs via constitutive activation of both NF-κB and STAT3. The results also strongly implicate IL-17R as an important functional biomarker for therapeutic targeting of GSCs.