Oliver Bögler

Human Bone Marrow–Derived Mesenchymal Stem Cells for Intravascular Delivery of Oncolytic Adenovirus Δ24-RGD to Human Gliomas

Delta24-RGD is an infectivity-augmented, conditionally replicative oncolytic adenovirus with significant antiglioma effects. Although intratumoral delivery of Delta24-RGD may be effective, intravascular delivery would improve successful application in humans. Due to their tumor tropic properties, we hypothesized that human mesenchymal stem cells (hMSC) could be harnessed as intravascular delivery vehicles of Delta24-RGD to human gliomas. To assess cellular events, green fluorescent protein-labeled hMSCs carrying Delta24-RGD (hMSC-Delta24) were injected into the carotid artery of mice harboring orthotopic U87MG or U251-V121 xenografts and brain sections were analyzed by immunofluorescence for green fluorescent protein and viral proteins (E1A and hexon) at increasing times. hMSC-Delta24 selectively localized to glioma xenografts and released Delta24-RGD, which subsequently infected glioma cells. To determine efficacy, mice were implanted with luciferase- labeled glioma xenografts, treated with hMSC-Delta24 or controls, and imaged weekly by bioluminescence imaging. Analysis of tumor size by bioluminescence imaging showed inhibition of glioma growth and eradication of tumors in hMSC-Delta24-treated animals compared with controls (P < 0.0001). There was an increase in median survival from 42 days in controls to 75.5 days in hMSC-Delta24-treated animals (P < 0.0001) and an increase in survival beyond 80 days from 0% to 37.5%, respectively. We conclude that intra-arterially delivered hMSC-Delta24 selectively localize to human gliomas and are capable of delivering and releasing Delta24-RGD into the tumor, resulting in improved survival and tumor eradication in subsets of mice.

Inhibition of autophagy at a late stage enhances imatinib‐induced cytotoxicity in human malignant glioma cells

Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Imatinib mesylate, a tyrosine kinase inhibitor, is effective in the therapy of tumors including leukemias but not as a monotherapy for malignant glioma. Recently, it is thought that the adequate modulation of autophagy can enhance efficacy of anticancer therapy. The outcome of autophagy manipulation, however, seems to depend on the autophagy initiator, the combined stimuli, the extent of cellular damage and the type of cells, and it is not yet fully understood how we should modulate autophagy to augment efficacy of each anticancer therapy. In this study, we examined the effect of imatinib with or without different types of autophagy inhibitors on human malignant glioma cells. Imatinib inhibited the viability of U87‐MG and U373‐MG cells in a dose dependent manner and caused nonapoptotic autophagic cell death. Suppression of imatinib‐induced autophagy by 3‐methyladenine or small interfering RNA against Atg5, which inhibit autophagy at an early stage, attenuated the imatinib‐induced cytotoxicity. In contrast, inhibition of autophagy at a late stage by bafilomycin A1 or RTA 203 enhanced imatinib‐induced cytotoxicity through the induction of apoptosis following mitochondrial disruption. Our findings suggest that therapeutic efficiency of imatinib for malignant glioma may be augmented by inhibition of autophagy at a late stage, and that appropriate modulation of autophagy may sensitize tumor cells to anticancer therapy.

Epidermal growth factor receptor signaling intensity determines intracellular protein interactions, ubiquitination, and internalization.

Ligand activation of the epidermal growth factor receptor (EGFR) causes the binding of Cbls, which leads to EGFR polyubiquitination and internalization through endophilin complexes that contain the adaptor protein SH3-domain encoding, expressed in tumorigenic astrocytes/Cbl-interacting protein of 85 kDa/regulator of ubiquitous kinase (SETA/CIN85/Ruk). In cells grown at high density, high levels of SETA interfered in the recruitment of Casitas B-lineage (Cbl) proteins to the EGFR and reduced its polyubiquitination, suggesting that SETA has a regulatory function in the formation of the EGFR–Cbl–endophilin complex and in EGFR down-regulation. In a situation where there is EGFR signaling but no internalization or down-regulation, as is the case with the EGFR with exons 2–7 deleted (ΔEGFR) oncogene, these proteins were absent altogether. By using mAb 806, which recognizes an EGFR-activation state and preferentially immunoprecipitates ΔEGFR, we show that ΔEGFR did not interact with Cbls, SETA, or endophilin A1, providing a mechanistic explanation for its lack of internalization. As would be expected by the absence of Cbl proteins in the ΔEGFR complex, the mutant receptor was also not polyubiquitinated. The intracellular C terminus and tyrosine autophosphorylation pattern of ΔEGFR are similar to wild-type EGFR, but it signals at a lower intensity as determined by levels of EGFR phosphotyrosine. To test the implication that the lack of interaction with the Cbl–SETA–endophilin complex is because of differences in signal intensity, EGFR-expressing cells were treated with tyrphostin AG1478 EGFR inhibitor. Attenuation of wild-type EGFR signal to levels similar to that found in ΔEGFR resulted in the dissociation of SETA and Cbl proteins and a concomitant attenuation of receptor internalization.

Alix/AIP1 Antagonizes Epidermal Growth Factor Receptor Downregulation by the Cbl-SETA/CIN85 Complex

ABSTRACT The assembly of the Cbl-SETA/CIN85-endophilin complex at the C terminus of the epidermal growth factor receptor (EGFR) following ligand activation mediates its internalization and ubiquitination. We found that the SETA/CIN85-interacting protein Alix/AIP1, which also binds endophilins, modulates this complex. Alix was found to associate indirectly with EGFR, regardless of its activation state, and with ΔEGFR, which signals at low intensity and does not bind Cbls or SETA/CIN85. In agreement with this, Alix interaction did not occur via SETA/CIN85. However, SETA/CIN85 and Alix were capable of mutually promoting their interaction with the EGFR. Increasing the level of Alix weakened the interaction between SETA/CIN85 and Cbl and reduced the tyrosine phosphorylation of c-Cbl and the level of ubiquitination of EGFR, SETA/CIN85, and Cbls. This antagonism of the Cbl-SETA/CIN85 complex by Alix was reflected in its diminution of EGFR internalization. In agreement with this, small interfering RNA-mediated knockdown of Alix promoted EGFR internalization and downregulation. It has been suggested that SETA/CIN85 promotes receptor internalization by recruiting endophilins. However, Alix was also capable of increasing the level of endophilin associated with EGFR, implying that this is not sufficient to promote receptor internalization. We propose that Alix inhibits EGFR internalization by attenuating the interaction between Cbl and SETA/CIN85 and by inhibiting Cbl-mediated ubiquitination of the EGFR.

Constitutive EGFR signaling confers a motile phenotype to neural stem cells

The epidermal growth factor receptor (EGFR) has been shown to play an important role in brain development, including stem and precursor cell survival, proliferation, differentiation, and migration. To further examine the temporal and spatial requirements of erbB signals in uncommitted neural stem cells (NSCs), we expressed the ligand-independent EGF receptor, EGFRvIII, in C17.2 NSCs. These NSCs are known to migrate and to evince a tropic response to neurodegenerative environments in vivo but for which an underlying mechanism remains unclear. We show that enhanced erbB signaling via constitutive kinase activity of EGFRvIII in NSCs sustains an immature phenotype and enhances NSC migration.

SETA/CIN85/Ruk and its binding partner AIP1 associate with diverse cytoskeletal elements, including FAKs, and modulate cell adhesion

The adaptor protein SETA/CIN85/Ruk is involved in regulating diverse signal transduction pathways, including the internalization of tyrosine kinase receptors via the Cbl ubiquitin ligases, and attenuating PI3K activity by interaction with its regulatory subunit. Here we present evidence for a new aspect of SETA function, based on the initial observation that it co-localizes with actin in microfilaments and at focal adhesions, and with microtubules. Although there was no evidence for direct molecular interactions between SETA and cytoskeletal proteins, the SETA-interacting protein AIP1, which is a rat ortholog of the Xenopus src substrate Xp95, strongly interacted with structural proteins of the cytoskeleton, including actin and tubulins. Both SETA and AIP1 interacted with focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (PYK-2), and c-Cbl interacted with PYK-2. AIP1, which interacted more strongly than either SETA or c-Cbl, required an intact consensus tyrosine kinase phosphorylation sequence at Y319 to bind to focal adhesion kinases, which suggests that phosphorylation is an important mediator of this complex. SETA, which interacted as a dimer with focal adhesion kinases, promoted the interaction between PYK-2 and AIP1. Direct analysis of the impact of these proteins on cell adhesion, by use of an electrical cell-substrate impedance sensor (ECIS), showed that SETA promoted cell adhesion while AIP1 and c-Cbl reduced it. Furthermore, the ability of AIP1 and AIP1 mutants to decrease cell adhesion in ECIS analysis correlated with their presence in PYK-2 complexes, providing a direct link between AIP1-mediated molecular interactions and cellular behavior. Transfection of AIP1 also reduced the level of phosphorylation of endogenous PYK-2 and FAK, suggesting that this protein may directly regulate focal adhesion kinases, and thereby cell adhesion. These data are the first to implicate the adaptor protein SETA and its binding partner AIP1 as being involved with the cytoskeleton and in the regulation of cell adhesion, and suggest that they may be part of the focal adhesion kinase regulatory complex.

Monitoring autophagy in glioblastoma with antibody against isoform B of human microtubule-associated protein 1 light chain 3

Autophagy, an evolutionarily conserved response to stress, has recently been implicated in cancer initiation and progression, but the detailed mechanisms and functions have not yet been fully elucidated. One major obstacle to our understanding is lack of an efficient and robust method to specifically monitor autophagic cells in cancer specimens. To identify molecular events associated with autophagy, we performed cDNA microarray analysis of autophagic glioblastoma cell lines. Based on the analysis, we raised a polyclonal antibody against isoform B of human microtubule-associated protein 1 light chain 3 (LC3B). Application of the anti-LC3B antibody revealed the presence of autophagic cells in both in vitro and in vivo settings. Of the 65 glioblastoma tissues, 31 had highly positive cytoplasmic staining of LC3B. The statistical interaction between cytoplasmic staining of LC3B and Karnofsky Performance Scale score was significant. High expression of LC3B was associated with an improved outcome for patients with poorer performance, whereas, for patients with normal performance, survival was better for patients with low staining than with high staining of LC3B. Anti-LC3B antibody provides a useful tool for monitoring the induction of autophagy in cancer cells and tissues.

m6A Demethylase ALKBH5 Maintains Tumorigenicity of Glioblastoma Stem-like Cells by Sustaining FOXM1 Expression and Cell Proliferation Program.

The dynamic and reversible N6-methyladenosine (m6A) RNA modification installed and erased by N6-methyltransferases and demethylases regulates gene expression and cell fate. We show that the m6A demethylase ALKBH5 is highly expressed in glioblastoma stem-like cells (GSCs). Silencing ALKBH5 suppresses the proliferation of patient-derived GSCs. Integrated transcriptome and m6A-seq analyses revealed altered expression of certain ALKBH5 target genes, including the transcription factor FOXM1. ALKBH5 demethylates FOXM1 nascent transcripts, leading to enhanced FOXM1 expression. Furthermore, a long non-coding RNA antisense to FOXM1 (FOXM1-AS) promotes the interaction of ALKBH5 with FOXM1 nascent transcripts. Depleting ALKBH5 and FOXM1-AS disrupted GSC tumorigenesis through the FOXM1 axis. Our work uncovers a critical function for ALKBH5 and provides insight into critical roles of m6A methylation in glioblastoma.

Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors.

Mutations within the kinase domain and truncations of the epidermal growth factor receptor are rare events in bladder cancer : Implications for therapy

Purpose: It has previously been reported that the patient response to gefitinib depends on the presence of mutations within the kinase domain of epidermal growth factor receptor (EGFR) or the expression of its truncated form, EGFR variant III (EGFRvIII). The focus of this study was to determine if these alterations are present within the tyrosine kinase and ligand-binding domain of EGFR in urothelial carcinoma. Experimental Design: The kinase domain found within exons 18 to 21 of the EGFR from 11 bladder cancer cell lines and 75 patient tumors were subjected to automated sequencing. EGFRvIII expression was determined by immunohistochemistry using a urothelial carcinoma tissue microarray, and its expression was subsequently verified by reverse transcription PCR, real-time PCR, and Western blot analysis, using an EGFRvIII-transfected glioblastoma cell line and glioblastoma tumors as positive controls. Results: Our analysis failed to detect mutations within the tyrosine kinase domain of EGFR in the 11 cell lines and 75 patients tested. The initial analysis of EGFRvIII expression by immunohistochemistry revealed that at least 50% of the patient tumors expressed EGFRvIII in a urothelial carcinoma tissue microarray. Conflicting reports exist, however, regarding the extent of EGFRvIII expression in tissues owing to the specificity of the antibodies and the methodologies used. Therefore, we sought to validate this observation by reverse transcription PCR, real-time PCR, and Western blot analysis. In these assays, none of the samples were positive for EGFRvIII except for control transfectants and glioblastomas. Conclusions: When our results are taken together, we conclude that alterations within the tyrosine kinase domain and expression of EGFRvIII are rare events in bladder cancer. The present study has clinical implications in selecting tyrosine kinase inhibitors for the therapy of urothelial carcinoma.