Invasion as target for therapy of glioblastoma multiforme

The survival of cancer patients suffering from glioblastoma multiforme is limited to just a few months even after treatment with the most advanced techniques. The indefinable borders of glioblastoma cell infiltration into the surrounding healthy tissue prevent complete surgical removal. In addition, genetic mutations, epigenetic modifications and microenvironmental heterogeneity cause resistance to radio- and chemotherapy altogether resulting in a hardly to overcome therapeutic scenario. Therefore, the development of efficient therapeutic strategies to combat these tumors requires a better knowledge of genetic and proteomic alterations as well as the infiltrative behavior of glioblastoma cells and how this can be targeted. Among many cell surface receptors, members of the integrin family are known to regulate glioblastoma cell invasion in concert with extracellular matrix degrading proteases. While preclinical and early clinical trials suggested specific integrin targeting as a promising therapeutic approach, clinical trials failed to deliver improved cure rates up to now. Little is known about glioblastoma cell motility, but switches in invasion modes and adaption to specific microenvironmental cues as a consequence of treatment may maintain tumor cell resistance to therapy. Thus, understanding the molecular basis of integrin and protease function for glioblastoma cell invasion in the context of radiochemotherapy is a pressing issue and may be beneficial for the design of efficient therapeutic approaches. This review article summarizes the latest findings on integrins and extracellular matrix in glioblastoma and adds some perspective thoughts on how this knowledge might be exploited for optimized multimodal therapy approaches.     

EphA2 as a novel molecular marker and target in glioblastoma multiforme

We investigated the presence of EphA2, and its ligand, ephrinA1, in glioblastoma multiforme (GBM), a malignant neoplasm of glial cells, and normal brain. We also initially examined the functional importance of the interaction between EphA2 and ephrinA1 in glioma cells. Expression and localization of EphA2 and ephrinA1 in human GBM and normal brain were examined using Western blotting, immunofluorescence, and immunohistochemistry. A functional role for EphA2 was investigated by assessing the activation status of the receptor and the effect of ephrinA1 on the anchorage-independent growth and invasiveness of GBM cells. We found EphA2 to be elevated in approximately 90% of GBM specimens and cell lines but not in normal brain, whereas ephrinA1 was present at consistently low levels in both GBM and normal brain. EphA2 was activated and phosphorylated by ephrinA1 in GBM cells. Furthermore, ephrinA1 induced a prominent, dose-dependent inhibitory effect on the anchorage-independent growth and invasiveness of GBM cells highly overexpressing EphA2, which was not seen in cells expressing low levels of the receptor. Thus, EphA2 is both specifically overexpressed in GBM and expressed differentially with respect to its ligand, ephrinA1, which may reflect on the oncogenic processes of malignant glioma cells. EphA2 seems to be functionally important in GBM cells and thus may play an important role in GBM pathogenesis. Hence, EphA2 represents a new marker and novel target for the development of molecular therapeutics against GBM.     

Dynamic inhibition of ATM kinase provides a strategy for glioblastoma multiforme radiosensitization and growth control

Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.Key words: AKT, DNA repair, KU-60019, temozolomide     

Dynamic inhibition of ATM kinase provides a strategy for glioblastoma multiforme radiosensitization and growth control

Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.     

mTOR controls FLIPS translation and TRAIL sensitivity in glioblastoma multiforme cells

The tumor-selective, proapoptotic, death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a mediator of antitumor drug activity and in itself is a promising agent for the treatment of human malignancies. Like many tumors, however, glioblastoma multiforme (GBM), the most fatal form of glioma, exhibits a range of TRAIL sensitivity, and only a small percentage of GBM tumors undergo TRAIL-induced apoptosis. We here show that TRAIL resistance in GBM is a consequence of overexpression of the short isoform of the caspase-8 inhibitor, c-FLICE inhibitory protein (FLIP(S)), and that FLIP(S) expression is in turn translationally enhanced by activation of the Akt-mammalian target of rapamycin (mTOR)-p70 S6 kinase 1 (S6K1) pathway. Conversely, pharmacologic or genetic inhibition of mTOR, or the mTOR target S6K1, suppresses polyribosomal accumulation of FLIP(S) mRNA, FLIP(S) protein expression, and TRAIL resistance. In archived material from 12 human GBM tumors, PTEN status was a predictor of activation of the Akt-mTOR-S6K1 pathway and of FLIP(S) levels, while in xenografted human GBM, activation status of the PTEN-Akt-mTOR pathway distinguished the tumors inherently sensitive to TRAIL from those which could be sensitized by the mTOR inhibitor rapamycin. These results define the mTOR pathway as a key limiter of tumor elimination by TRAIL-mediated mechanisms, provide a means by which the TRAIL-sensitive subset of GBM can be identified, and provide rationale for the combined use of TRAIL with mTOR inhibitors in the treatment of human cancers.     

Equine herpesvirus type 1-mediated oncolysis of human glioblastoma multiforme cells

The cytolytic animal virus equine herpesvirus type 1 (EHV-1) was evaluated for its oncolytic potential against five human glioblastoma cell lines. EHV-1 productively infected four of these cell lines, and the degree of infection was positively correlated with glioma cell death. No human major histocompatibility complex class 1 (MHC-I) was detected in the resistant glioma line, while infection of the susceptible glioma cell lines, which expressed human MHC-I, were blocked with antibody to MHC-I, indicating that human MHC-I acts as an EHV-1 entry receptor on glioma cells.     

Secretome signature of invasive glioblastoma multiforme

The incurability of malignant glioblastomas is mainly attributed to their highly invasive nature coupled with resistance to chemo- and radiation therapy. Because invasiveness is partially dictated by the proteins these tumors secrete we used SILAC to characterize the secretomes of four glioblastoma cell lines (LN18, T98, U118 and U87). Although U87 and U118 cells both secreted high levels of well-known invasion promoting proteins, a Matrigel invasion assay showed U87 cells to be eight times more invasive than U118 cells, suggesting that additional proteins secreted by U87 cells may contribute to the highly invasive phenotype. Indeed, we identified a number of proteins highly or exclusively expressed by U87 cells as compared to the less invasive cell lines. The most striking of these include ADAM9, ADAM10, cathepsin B, cathepsin L1, osteopontin, neuropilin-1, semaphorin-7A, suprabasin, and chitinase-3-like protein 1. U87 cells also expressed significantly low levels of some cell adhesion proteins such as periostin and EMILIN-1. Correlation of secretome profiles with relative levels of invasiveness using Pavlidis template matching further indicated potential roles for these proteins in U87 glioblastoma invasion. Antibody inhibition of CH3L1 reduced U87 cell invasiveness by 30%.     

Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion.     

Growth inhibition and modulation of antigenic phenotype in human melanoma and glioblastoma multiforme cells by caffeic acid phenethyl ester (CAPE)

The active component of the honeybee hive product propolis, caffeic acid phenethyl ester (CAPE), has been shown to display increased toxicity toward various oncogene-transformed cell lines in comparison with their untransformed counterparts (Su et al., 4: 231-242, 1991). This observation provides support for the concept that it is the transformed phenotype which is specifically sensitive to CAPE. In the present study, we have determined the effect of CAPE on the growth and antigenic phenotype of a human melanoma cell line, HO-1, and a human glioblastoma multiforme cell line, GBM-18. For comparison, we have also tested the effects of mezerein (MEZ), mycophenolic acid (MPA) and retinoic acid (RA), which can differentially modulate growth, differentiation and the antigenic phenotype in these human tumor cell lines. Growth of both cell lines was suppressed by CAPE in a dose-dependent fashion, with HO-1 cells being more sensitive than GBM-18 cells. The antiproliferative effect of CAPE was enhanced in both cell types if CAPE and MEZ were used in combination. Growth suppression was associated with morphological changes in H0-1 cells, suggesting induction of a more differentiated phenotype. CAPE also differentially modulated the expression of several antigens on the surface of the two tumor cell lines. These results suggest a potential role for CAPE as an antitumor agent, an antigenic modulating agent and possibly a differentiation inducing agent.     

CDK1 inhibition sensitizes normal cells to DNA damage in a cell cycle dependent manner

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner.

Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.     

Rapamycin-mediated mTOR inhibition attenuates survivin and sensitizes glioblastoma cells to radiation therapy

Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma cells to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma cells followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (γ-H2AX) was increased. p21 protein was also induced on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.     

CTL recognition of a novel HLA-A*0201-binding peptide derived from glioblastoma multiforme tumor cells

Genetic instability of tumor cells can result in translation of proteins that are out of frame, resulting in expression of neopeptides. These neopeptides are not self-proteins and therefore should be immunogenic. By eluting peptides from human glioblastoma multiforme (GBM) tumor cell surfaces and subjecting them to tandem mass spectrometry, we identified a novel peptide (KLWGLTPKVTPS) corresponding to a frameshift in the 3′ beta-hydroxysteroid dehydrogenase type 7 (HSD3B7) gene. HLA-binding algorithms predicted that a 9-amino acid sequence embedded in this peptide would bind to HLA-A*0201. We confirmed this prediction using an HLA-A*0201 refolding assay followed by live cell relative affinity assays, but also showed that the 12-mer binds to HLA-A*0201. Based on the 9-mer sequence, optimized peptide ligands (OPL) were designed and tested for their affinities to HLA-A*0201 and their abilities to elicit anti-peptide and CTL capable of killing GBM in vitro. Wild-type peptides as well as OPL induced anti-peptide CTL as measured by IFN-γ ELISPOTS. These CTL also killed GBM tumor cells in chromium-51 release assays. This study reports a new CTL target in GBM and further substantiates the concept that rational design and testing of multiple peptides for the same T-cell epitope elicits a broader response among different individuals than single peptide immunization.     

Interfering with mitochondrial dynamics sensitizes glioblastoma multiforme to temozolomide chemotherapy

Glioblastoma multiforme (GBM) is a primary tumour of the central nervous system (CNS) that exhibits the highest degree of malignancy. Radiotherapy and chemotherapy are essential to prolong the survival time of patients. However, clinical work has demonstrated that sensitivity of GBM to chemotherapy decreases with time. The phenomenon of multi‐drug resistance (MDR) reminds us that there may exist some fundamental mechanisms in the process of chemo‐resistance. We tried to explore the mechanism of GBM chemo‐resistance from the perspective of energy metabolism. First, we found that the oxidative phosphorylation (OXPHOS) level of SHG44 and U87 cells increased under TMZ treatment. In further studies, it was found that the expression of PINK1 and mitophagy flux downstream was downregulated in GBM cells, which were secondary to the upregulation of TP53 in tumour cells under TMZ treatment. At the same time, we examined the mitochondrial morphology in tumour cells and found that the size of mitochondria in tumour cells increased under the treatment of TMZ, which originated from the regulation of AMPK on the subcellular localization of Drp1 under the condition of unbalanced energy supply and demand in tumour cells. The accumulation of mitochondrial mass and the optimization of mitochondrial quality accounted for the increased oxidative phosphorylation, and interruption of the mitochondrial fusion process downregulated the efficiency of oxidative phosphorylation and sensitized GBM cells to TMZ, which was also confirmed in the in vivo experiment. What is more, interfering with this process is an innovative strategy to overcome the chemo‐resistance of GBM cells.     

Towards developing biomarkers for glioblastoma multiforme: a proteomics view

Glioblastoma multiforme (GBM) is one of the most aggressive and lethal forms of the primary brain tumors. With predominance of tumor heterogeneity and emergence of new subtypes, new approaches are needed to develop tissue-based markers for tumor typing or circulatory markers to serve as blood-based assays. Multi-omics data integration for GBM tissues would offer new insights on the molecular view of GBM pathogenesis useful to identify biomarker panels. On the other hand, mapping differentially expressed tissue proteins for their secretory potential through bioinformatics analysis or analysis of the tumor cell secretome or tumor exosomes would enhance our understanding of the tumor microenvironment and prospects for targeting circulatory biomarkers. In this review, the authors first present potential biomarker candidates for GBM that have been reported and then focus on plausible pipelines for multi-omic data integration to identify additional, high-confidence molecular panels for clinical applications in GBM.     

Autotaxin is overexpressed in glioblastoma multiforme and contributes to cell motility of glioblastoma by converting lysophosphatidylcholine to lysophosphatidic acid

Autotaxin (ATX) is a multifunctional phosphodiesterase originally isolated from melanoma cells as a potent cell motility-stimulating factor. ATX is identical to lysophospholipase D, which produces a bioactive phospholipid, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC). Although enhanced expression of ATX in various tumor tissues has been repeatedly demonstrated, and thus, ATX is implicated in progression of tumor, the precise role of ATX expressed by tumor cells was unclear. In this study, we found that ATX is highly expressed in glioblastoma multiforme (GBM), the most malignant glioma due to its high infiltration into the normal brain parenchyma, but not in tissues from other brain tumors. In addition, LPA1, an LPA receptor responsible for LPA-driven cell motility, is predominantly expressed in GBM. One of the glioblastomas that showed the highest ATX expression (SNB-78), as well as ATX-stable transfectants, showed LPA1-dependent cell migration in response to LPA in both Boyden chamber and wound healing assays. Interestingly these ATX-expressing cells also showed chemotactic response to LPC. In addition, knockdown of the ATX level using small interfering RNA technique in SNB-78 cells suppressed their migratory response to LPC. These results suggest that the autocrine production of LPA by cancer cell-derived ATX and exogenously supplied LPC contribute to the invasiveness of cancer cells and that LPA1, ATX, and LPC-producing enzymes are potential targets for cancer therapy, including GBM.