Understanding glioblastoma tumor biology: the potential to improve current diagnosis and treatments

Glioblastoma (GBM) is a highly malignant brain cancer characterized by uncontrolled cellular proliferation, diffuse infiltration, a tendency for necrosis, significant angiogenesis, intense resistance to apoptosis, and widespread genomic aberrations. Prognosis is poor and treatments are largely palliative, although there are subsets of patients that have prolonged survival. Greater understanding of the tumor biology of GBM has been achieved in the past decade, leading to the prospect of novel targeted therapies and biomarker-based individualization of therapy. The goal of this review is to describe the tumor biology and pathologic features of GBM, guidelines for classification and diagnosis, the current status of prognostic and predictive biomarkers, and the role of the blood-brain barrier in delivering therapy for GBM.     

Insulin-like growth factor receptor I mediates resistance to anti-epidermal growth factor receptor therapy in primary human glioblastoma cells through continued activation of phosphoinositide 3-kinase signaling.

Overexpression of the epidermal growth factor receptor (EGFR) has been shown previously to correlate with enhanced malignant potential of many human tumor types, including glioblastoma multiforme (GBM). Anti-EGFR targeting has been demonstrated to enhance apoptosis and reduce both cellular invasion and angiogenic potential. It remains unclear whether absolute EGFR expression levels are sufficient to predict which tumors will respond best to anti-EGFR therapy. We have identified two primary GBM cell lines with equivalent EGFR expression levels with very different sensitivities to the EGFR receptor tyrosine kinase inhibitor, AG1478. This was apparent despite similar reductions in EGFR signaling in both cell lines, as measured by phospho-EGFR levels. AG1478 enhanced both spontaneous and radiation-induced apoptosis and reduced invasive potential in the GBM(S), but not in the GBM(R), cell line. The resistant GBM(R) cell line demonstrated an up-regulation of insulin-like growth factor receptor I (IGFR-I) levels on AG1478 administration. This resulted in sustained signaling through the phosphoinositide 3-kinase pathway, resulting in potent antiapoptotic and proinvasion effects. Cotargeting IGFR-I with EGFR greatly enhanced both spontaneous and radiation-induced apoptosis of the GBM(R) cells and reduced their invasive potential. Akt1 and p70(s6k) appeared to be important downstream targets of IGFR-I-mediated resistance to anti-EGFR targeting. These findings suggest that IGFR-I signaling through phosphoinositide 3-kinase may represent a novel and potentially important mechanism of resistance to anti-EGFR therapy.     

Inhibition of cell death by ribosomal protein L35a

In order to better understand how tumor cells develop resistance to chemotherapy drugs, we screened a human cDNA expression library in Jurkat cells for cDNA's that conferred resistance to doxorubicin-induced cell death. One of the cDNA's isolated in the screen codes for ribosomal protein L35a, a component of the large subunit of the ribosome. Jurkat cells engineered to overexpress L35a protein were more resistant not only to doxorubicin but also to UV-irradiation, anti-Fas antibody, and serum starvation compared to Jurkat cells expressing endogenous levels of L35a. Jurkat cells overexpressing L35a did not have increased levels of the anti-apoptotic proteins Bcl-2 or Bcl-xL, the drug efflux pump P-glycoprotein, nor altered cellular growth kinetics or total protein synthesis. Our results provide new insight into L35a function and suggest that it may have a role in the cellular response to cytotoxic damage. Since L35a RNA is overexpressed in a significant number of glioblastoma multiforme (GBM) brain tumors, our results may stimulate further investigation into the possible role of L35a in the resistance of GBM to cytotoxic therapy.     

Molecular mechanisms of temozolomide resistance in glioblastoma multiforme

Glioblastoma multiforme (GBM; WHO astrocytoma grade IV) is considered incurable owing to its inherently profound resistance towards current standards of therapy. Considerable effort is being devoted to identifying the molecular basis of temozolomide resistance in GBMs and exploring novel therapeutic regimens that may improve overall survival. Several independent DNA repair mechanisms that normally safeguard genome integrity can facilitate drug resistance and cancer cell survival by removing chemotherapy-induced DNA adducts. Furthermore, subpopulations of cancer stem-like cells have been implicated in the treatment resistance of several malignancies including GBMs. Thus, a growing number of molecular mechanisms contributing to temozolomide resistance are being uncovered in preclinical studies and, consequently, we are being presented with a broad range of potentially novel targets for therapy. A substantial future challenge is to successfully exploit the increasing molecular knowledge contributing to temozolomide resistance in robust clinical trials and to ultimately improve overall survival for GBM patients.     

Cancer heterogeneity and “The Struggle for Existence”: Diagnostic and analytical challenges

The notions of inter- and intra-tumour heterogeneity (ITH) have been recognised for many years but recent advances in sequencing technology are allowing the true extent of both forms of heterogeneity to be revealed in detail for the first time. In this review we examine the current evidence for ITH, the possibility of cancers following a branched rather than linear evolutionary path and the potential implications both of these may have for the mechanisms of drug resistance acquisition. We also note that although clearly present in many cases, heterogeneity and branched evolution are not universal, with cases of tumour homogeneity and linear evolution still detected relatively frequently.     

SapC-DOPS-induced lysosomal cell death synergizes with TMZ in glioblastoma

SapC-DOPS is a novel nanotherapeutic that has been shown to target and induce cell death in a variety of cancers, including glioblastoma (GBM). GBM is a primary brain tumor known to frequently demonstrate resistance to apoptosis-inducing therapeutics. Here we explore the mode of action for SapC-DOPS in GBM, a treatment being developed by Bexion Pharmaceuticals for clinical testing in patients. SapC-DOPS treatment was observed to induce lysosomal dysfunction of GBM cells characterized by decreased glycosylation of LAMP1 and altered proteolytic processing of cathepsin D independent of apoptosis and autophagic cell death. We observed that SapC-DOPS induced lysosomal membrane permeability (LMP) as shown by LysoTracker Red and Acridine Orange staining along with an increase of sphingosine, a known inducer of LMP. Additionally, SapC-DOPS displayed strong synergistic interactions with the apoptosis-inducing agent TMZ. Collectively our data suggest that SapC-DOPS induces lysosomal cell death in GBM cells, providing a new approach for treating tumors resistant to traditional apoptosis-inducing agents.     

Activation of Multiple ERBB Family Receptors Mediates Glioblastoma Cancer Stem-like Cell Resistance to EGFR-Targeted Inhibition

Epidermal growth factor receptor (EGFR) signaling is strongly implicated in glioblastoma (GBM) tumorigenesis. However, molecular agents targeting EGFR have demonstrated minimal efficacy in clinical trials, suggesting the existence of GBM resistance mechanisms. GBM cells with stem-like properties (CSCs) are highly efficient at tumor initiation and exhibit therapeutic resistance. In this study, GBMCSC lines showed sphere-forming and tumor initiation capacity after EGF withdrawal from cell culture media, compared with normal neural stem cells that rapidly perished after EGF withdrawal. Compensatory activation of related ERBB family receptors (ERBB2 and ERBB3) was observed in GBM CSCs deprived of EGFR signal (EGF deprivation or cetuximab inhibition), suggesting an intrinsic GBM resistance mechanism for EGFR-targeted therapy. Dual inhibition of EGFR and ERBB2 with lapatinib significantly reduced GBM proliferation in colony formation assays compared to cetuximab-mediated EGFR-specific inhibition. Phosphorylation of downstream ERBB signaling components (AKT, ERK1/2) and GBM CSC proliferation were inhibited by lapatinib. Collectively, these findings show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members (ERBB2, ERBB3) enabling GBM CSC proliferation, and therefore simultaneous blockade of multiple ERBB family members may be required for more efficacious GBM therapy.     

Mer receptor tyrosine kinase inhibition impedes glioblastoma multiforme migration and alters cellular morphology

Glioblastoma multiforme (GBM) is an aggressive brain tumor, fatal within 1 year from diagnosis in most patients despite intensive multimodality therapy. The migratory and microscopically invasive nature of GBM as well as its resistance to chemotherapy renders conventional therapies inadequate in its treatment. Although Mer receptor tyrosine kinase (RTK) inhibition has been shown to decrease the long-term survival and improve the chemosensitivity of GBM in vitro, its role in malignant cellular migration has not been previously evaluated. In this study, we report for the first time a role for Mer RTK in brain tumor migration and show that Mer inhibition profoundly impedes GBM migration and alters cellular morphology. Our data demonstrate that Mer RTK inhibition results in altered signaling through focal adhesion kinase (FAK) and RhoA GTPase and a transformation of cytoskeletal organization, suggesting both molecular and structural mechanisms for the abrogation of migration. We also describe a novel and translational method of Mer RTK inhibition using a newly developed monoclonal antibody, providing proof of principle for future evaluation of Mer-targeted translational therapies in the treatment of GBM. Previous findings implicating Mer signaling in glioblastoma survival and chemotherapy resistance coupled with our discovery of the role of Mer RTK in GBM cellular migration support the development of novel Mer-targeted therapies for this devastating disease.     

Inhibition of LSD1 sensitizes glioblastoma cells to histone deacetylase inhibitors

Glioblastoma multiforme (GBM) is a particularly aggressive brain tumor and remains a clinically devastating disease. Despite innovative therapies for the treatment of GBM, there has been no significant increase in patient survival over the past decade. Enzymes that control epigenetic alterations are of considerable interest as targets for cancer therapy because of their critical roles in cellular processes that lead to oncogenesis. Several inhibitors of histone deacetylases (HDACs) have been developed and tested in GBM with moderate success. We found that treatment of GBM cells with HDAC inhibitors caused the accumulation of histone methylation, a modification removed by the lysine specific demethylase 1 (LSD1). This led us to examine the effects of simultaneously inhibiting HDACs and LSD1 as a potential combination therapy. We evaluated induction of apoptosis in GBM cell lines after combined inhibition of LSD1 and HDACs. LSD1 was inhibited by targeted short hairpin RNA or pharmacological means and inhibition of HDACs was achieved by treatment with either vorinostat or PCI-24781. Caspase-dependent apoptosis was significantly increased (>2-fold) in LSD1-knockdown GBM cells treated with HDAC inhibitors. Moreover, pharmacologically inhibiting LSD1 with the monoamine oxidase inhibitor tranylcypromine, in combination with HDAC inhibitors, led to synergistic apoptotic cell death in GBM cells; this did not occur in normal human astrocytes. Taken together, these results indicate that LSD1 and HDACs cooperate to regulate key pathways of cell death in GBM cell lines but not in normal counterparts, and they validate the combined use of LSD1 and HDAC inhibitors as a therapeutic approach for GBM.     

Activated EGFR signaling increases proliferation,survival, and migration and blocks neuronal differentiation in post-natal neural stem cells

Recent evidence supports the notion that transformation of undifferentiated neural stem cell (NSC) precursors may contribute to the development of glioblastoma multiforme (GBM). The over-expression and mutation of the epidermal growth factor receptor (EGFR), along with other cellular pathway mutations, plays a significant role in GBM maintenance progression. Though EGFR signaling is important in determining neural cell fate and conferring astrocyte differentiation, there is a limited understanding of its role in NSC and tumor stem cell (TSC) biology. We hypothesized that EGFR expression and mutation in post-natal NSCs may contribute to cellular aggressiveness including enhanced cellular proliferation, survival and migration. Stable subclones of C17.2 murine NSCs were transfected to over-express either the wild-type EGFR (wtEGFR) or its most common mutated variant EGFRvIII. Activated EGFR signaling in these cells induced behaviors characteristic of GBM TSCs, including enhanced proliferation, survival and migration, even in the absence of EGF ligand. wtEGFR activation was also found to block neuronal differentiation and was associated with a dramatic increase in chemotaxis in the presence of EGF. EGFRvIII expression lead to an increase in NSC proliferation and survival, while it simultaneously blocked neuronal differentiation and promoted glial fate. Our findings suggest that activated EGFR signaling enhances the aggressiveness of NSCs. Understanding the regulatory mechanisms of NSCs may lend insight into deregulated mechanisms of GBM TSC invasion, proliferation, survival and resistance to current treatment modalities.     

Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor-related apoptosis-inducing ligand and induces an alternative cell death pathway

Human glioblastoma (GBM) cells are notorious for their resistance to apoptosis-inducing therapeutics. We have identified lanatoside C as a sensitizer of GBM cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death partly by upregulation of the death receptor 5. We show that lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis in a GBM xenograft model in vivo. Lanatoside C on its own serves as a therapeutic agent against GBM by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential, and early intracellular ATP depletion. In conclusion, lanatoside C sensitizes GBM cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative cell death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy to counter resistance of cancer cells to apoptosis.     

A sphingosine kinase inhibitor induces cell death in temozolomide resistant glioblastoma cells

Purpose  Sphingosine kinase is an oncogene that is up-regulated in several solid tumors. The product of the sphingosine kinase activity, sphingosine-1-phosphate is a potent mitogen involved in diverse cell processes such as cell survival and migration. Current standard therapy in the treatment of glioblastoma multiforme (GBM) is a combination of surgery, radiation, and chemotherapy using the drug temozolomide (TMZ). However, virtually all tumors become resistant to TMZ. Therefore, new drug targets are necessary. In this study, we investigated whether inhibiting sphingosine kinase could induce cell death in TMZ-resistant GBM cells. Methods  To study TMZ resistance in vitro, we have generated TMZ-resistant cell lines from established GBM cells. We used a potent inhibitor of sphingosine kinase to study its effect on colony formation and cell growth in GBM cells with a limited dilution and WST assay. Moreover, cell death was determined by measuring caspase-3 activity using flow cytometry. Results  A sphingosine kinase inhibitor reduced cell colony formation and activated caspase-3 in both TMZ-sensitive and resistant GBM cells. Conclusion  Addition of a sphingosine kinase inhibitor to the standard chemotherapy regimen against GBM may be beneficial.     

脑胶质瘤中RAD18的表达与放射治疗抵抗相关性分析

目的 探讨RAD18与多形性胶质母细胞瘤(Glioblastoma multiforme,GBM)放射抵抗的相关性,以期为改善脑胶质瘤放射抵抗提供新的治疗靶点。方法 将人脑胶质瘤细胞系A172分别转染空白和装有RAD18的质粒载体,应用克隆实验检测经相同剂量射线照射后两组细胞增殖情况,最后采用qRT-PCR方法检测原发性及放射性粒子近距离治疗后复发性GBM样本中RAD18 mRNA的表达情况,进行统计学分析。结果 经转染RAD18质粒过表达的GBM细胞经放射治疗后增殖情况高于转染空白质粒的GBM细胞(P＜0.001);经近距离放射性粒子治疗后复发性GBM中RAD18 mRNA的表达水平高于原发性GBM(P＜0.01)。结论 复发性GBM放射治疗的抵抗性可能与RAD18的蛋白过表达有关。     

The efficacy of lapatinib and nilotinib in combination with radiation therapy in a model of NF2 associated peripheral schwannoma

Despite multimodal treatment that includes surgery, radiation and chemotherapy, virtually all glioblastomas (GBM) recur, indicating that these interventions are insufficient to eradicate all malignant cells. To identify potential new therapeutic targets in GBMs, we examined the expression and function of proteins that are associated with therapy resistance and cancer cell survival. We measured the expression of eight such proteins in 50 GBM samples by immunohistochemistry and analyzed patient survival. We report that GBM patients with high expression of ABCG2 (also called BCRP) or XIAP at the protein level had worse survival than those with low expression. The adjusted hazard ratio for ABCG2 was 2.35 and for XIAP was 2.65. Since glioma stem cells (GSCs) have been shown to be more resistant than bulk tumor cells to anti-cancer therapies and to express high levels of these proteins, we also sought to determine if ABCG2 and XIAP have functional roles in GSCs. We used small molecule inhibitors to treat patient-derived GBM tumorspheres in vitro and observed that inhibitors of ABCG2, Ko143 and fumitremorgin, significantly reduced self-renewal. These results suggest that ABCG2 and XIAP proteins may be useful indicators of patient survival and that inhibition of ABCG2 may be a promising therapeutic strategy in GBMs.     

Searching for a cure: gene therapy for glioblastoma

Glioblastoma multiforme (GBM) is an invariably fatal malignancy. The lethality of GBM has been linked to the highly invasive nature of GBM cells, their escape from immune cell oversight and their high degree of resistance to multiple established therapeutic modalities. The resistance of GBM cells to undergo death processes has, in part, been associated with mutations of specific oncogenes and altered expression of other signaling molecules that lead to reduced capacities to activate multiple apoptosis pathways as well as altered rates of DNA repair and autophagy in response to cytotoxic drugs and cellular stresses. This review will examine how gene therapeutic approaches have been used in the past and are continuing to be used alongside cell signaling modulators and DNA damaging agents as clinical tools to treat GBM. The concerted use of established and novel signal transduction modulatory agents on GBM survival may have potential to lower the apoptotic threshold and facilitate killing in this lethal malignancy.     

免疫治疗联合放化疗治疗胶质母细胞瘤的研究进展

目前胶质母细胞瘤（glioblastoma,GBM）的标准治疗方法仍然是在最大安全程度的手术切除基础上辅以放化疗,但其5年生存率仍＜10%。免疫治疗如树突状细胞(dendritic cell,DC)疫苗、表皮生长因子受体突变体(EGFRvIII)疫苗、热休克蛋白（heat shock proteins,HSPs）疫苗等在临床试验中已经取得了巨大成就,临床III期试验也证明了免疫治疗与放化疗有协同作用。细胞毒性电离辐射是一种引起促炎信号级联免疫活化辅助细胞死亡的治疗方法,借此可以利用免疫治疗抗肿瘤。肿瘤免疫治疗的发展,使得免疫治疗可能成为继手术、放化疗后GBM治疗的另一个有效方法。寻找新的免疫治疗方法是未来GBM治疗的主要研究方向。本文就目前GBM的治疗策略及困境和放化疗联合免疫检查点抑制剂、DC疫苗以及EGFRvIII疫苗等免疫治疗研究进展作一综述。     

ATP binding cassette (ABC) transporters: expression and clinical value in glioblastoma

ATP-binding cassette transporters (ABC transporters) regulate traffic of multiple compounds, including chemotherapeutic agents, through biological membranes. They are expressed by multiple cell types and have been implicated in the drug resistance of some cancer cells. Despite significant research in ABC transporters in the context of many diseases, little is known about their expression and clinical value in glioblastoma (GBM). We analyzed expression of 49 ABC transporters in both commercial and patient-derived GBM cell lines as well as from 51 human GBM tumor biopsies. Using The Cancer Genome Atlas (TCGA) cohort as a training dataset and our cohort as a validation dataset, we also investigated the prognostic value of these ABC transporters in newly diagnosed GBM patients, treated with the standard of care. In contrast to commercial GBM cell lines, GBM-patient derived cell lines (PDCL), grown as neurospheres in a serum-free medium, express ABC transporters similarly to parental tumors. Serum appeared to slightly increase resistance to temozolomide correlating with a tendency for an increased expression of ABCB1. Some differences were observed mainly due to expression of ABC transporters by microenvironmental cells. Together, our data suggest that the efficacy of chemotherapeutic agents may be misestimated in vitro if they are the targets of efflux pumps whose expression can be modulated by serum. Interestingly, several ABC transporters have prognostic value in the TCGA dataset. In our cohort of 51 GBM patients treated with radiation therapy with concurrent and adjuvant temozolomide, ABCA13 overexpression is associated with a decreased progression free survival in univariate (p?<?0.01) and multivariate analyses including MGMT promoter methylation (p?=?0.05) suggesting reduced sensitivity to temozolomide in ABCA13 overexpressing GBM. Expression of ABC transporters is: (i) detected in GBM and microenvironmental cells and (ii) better reproduced in GBM-PDCL. ABCA13 expression is an independent prognostic factor in newly diagnosed GBM patients. Further prospective studies are warranted to investigate whether ABCA13 expression can be used to further personalize treatments for GBM.     

Abrogation of radioresistance in glioblastoma stem‐like cells by inhibition of ATM kinase

Resistance to radiotherapy in glioblastoma (GBM) is an important clinical problem and several authors have attributed this to a subpopulation of GBM cancer stem cells (CSCs) which may be responsible for tumour recurrence following treatment. It is hypothesised that GBM CSCs exhibit upregulated DNA damage responses and are resistant to radiation but the current literature is conflicting. We investigated radioresistance of primary GBM cells grown in stem cell conditions (CSC) compared to paired differentiated tumour cell populations and explored the radiosensitising effects of the ATM inhibitor KU‐55933.We report that GBM CSCs are radioresistant compared to paired differentiated tumour cells as measured by clonogenic assay. GBM CSC''s display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and repair DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation.Inhibition of ATM kinase by KU‐55933 produced potent radiosensitisation of GBM CSCs (sensitiser enhancement ratios 2.6–3.5) and effectively abrogated the enhanced DSB repair proficiency observed in GBM CSCs at 24 h post irradiation. G2/M checkpoint activation was reduced but not abolished by KU‐55933 in GBM CSCs.ATM kinase inhibition overcomes radioresistance of GBM CSCs and, in combination with conventional therapy, has potential to improve outcomes for patients with GBM.     

Therapeutic advances for glioblastoma multiforme: Current status and future prospects

Glioblastoma multiforme (GBM) is the most common central nervous system malignancy. It is rapidly progressive with rare opportunity for cure. After three decades of laboratory and clinical research, a newly evolved chemoradiotherapy approach using the alkylating agent temozolomide during and after radiotherapy has resulted in the first significant impact on this disease. Here we discuss the basis for this positive interaction as well as potential mechanisms of resistance to it. Additionally, in the context of current and planned research, we explore approaches to take advantage of this combination and the use of targeted therapies to exploit cell signaling alterations found in GBM. We anticipate that a multimodality approach directed at tumor-specific biology will result in more meaningful advancements in the treatment of this fatal disease.