Deregulated signalling networks in human brain tumours

Deregulated signalling networks in human brain tumours

Biochimica et Biophysica Acta 1804 (2010) 476–483 Contents lists available at ScienceDirect Biochimica et Biophysica Acta j o u r n a l h o m e p a ...

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Biochimica et Biophysica Acta 1804 (2010) 476–483

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a p a p

Review

Deregulated signalling networks in human brain tumours Michal Grzmil ⁎, Brian A. Hemmings ⁎ Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland

a r t i c l e

i n f o

Article history: Received 19 August 2009 Received in revised form 13 October 2009 Accepted 21 October 2009 Available online 29 October 2009 Keywords: Glioblastoma Signalling pathways Kinase inhibitor

a b s t r a c t Despite the variety of modern therapies against human brain cancer, in its most aggressive form of glioblastoma multiforme (GBM) it is a still deadly disease with a median survival of approximately 1 year. Over the past 2 decades, molecular profiling of low- and high-grade malignant brain tumours has led to the identification and molecular characterisation of mechanisms leading to brain cancer development, maintenance and progression. Genetic alterations occurring during gliomagenesis lead to uncontrolled tumour growth stimulated by deregulated signal transduction pathways. The characterisation of hyperactivated signalling pathways has identified many potential molecular targets for therapeutic interference in human gliomas. Overexpressed or mutated and constitutively active kinases are attractive targets for low-molecular-weight inhibitors. Although the first attempts with mono-therapy using a single targeted kinase inhibitor were not satisfactory, recent studies based on the simultaneous targeting of several core hyperactivated pathways show great promise for the development of novel therapeutic approaches. This review focuses on genetic alterations leading to the activation of key deregulated pathways in human gliomas. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The incidence of brain cancer tends to be highest in developed, industrialised countries. Primary brain tumours account for less than 2% of all human cancers but are very often associated with high mortality. Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer, with a median survival of approximately 1 year. In the USA alone, 10,000 new cases are diagnosed each year at the rate

Abbreviations: GBM, glioblastoma multiforme; RTK, receptor tyrosine kinase; EGF (R), epidermal growth factor (receptor); PDGF(R), platelet-derived growth factor (receptor); VEGF(R), vascular endothelial growth factor (receptor); ERBB2, v-erb-b2 erythroblastic leukemia viral oncogene homolog 2; MET, met proto-oncogene (hepatocyte growth factor receptor); PI3K, phosphoinositide-3-kinase; AKT, v-akt murine thymoma viral oncogene; TP53, tumour protein p53; RB1, retinoblastoma 1; CDK 4 (6), cyclin-dependent kinase 4 (6); CDKN2A (B) (C), cyclin-dependent kinase inhibitor 2A, (B) (C); CCND2, cyclin D2; MDM2, Mdm2 p53-binding protein homolog (mouse); MAPK, mitogen-activated protein kinase; RAF1, v-raf-1 murine leukemia viral oncogene homolog 1; ERK, mitogen-activated protein kinase 1; MEK, mitogenactivated protein kinase kinase; FOXO1, forkhead box O1; PTEN, phosphatase and tensin homolog; NF1, neurofibromin 1; MGMT, O-6-methylguanine-DNA methyltransferase; mTOR, mechanistic target of rapamycin; NFk-β, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; BAD, BCL2-associated agonist of cell death; BAX, BCL2-associated × protein; BCL2L12, BCL2-like 12 (proline rich); TGF-α, transforming growth factor, alpha; MCL1, myeloid cell leukemia sequence 1; S6K1, 40S ribosomal protein S6 kinase 1; eIF4EBP1, eukaryotic translation initiation factor 4Ebinding protein 1; HIF1, hypoxia-inducible factor 1; IDH1, isocitrate dehydrogenase 1 ⁎ Corresponding authors. Tel.: +41 61 6974872or6974046; fax: +41 61 6973976. E-mail addresses: [email protected] (M. Grzmil), [email protected] (B.A. Hemmings). 1570-9639/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2009.10.018

of 3 per 100,000 persons [1,2]. Despite the available multimodality treatments, no cure or effective therapy for malignant gliomas has been developed to date. In a population-based study of glioblastomas in Switzerland, 17.7% of patients survived for 1 year, whereas only 3.3% of patients with newly diagnosed GBM survived for 2 years [3]. Current standard-of-care therapy for newly diagnosed malignant gliomas includes surgical resection, radiotherapy and temozolomide (TMZ), administered both during and after radiotherapy. Complete or nearly total surgical resection together with post-surgical radiation increases survival rate and, more recently, radiotherapy plus concomitant and adjuvant TMZ significantly improved survival of GBM patients without reduction in quality of life [4,5]. Temozolomide is an alkylating agent that crosses the blood–brain barrier; however, its activity can be antagonised by the DNA-repair enzyme O6methylguanine-DNA methyltransferase (MGMT), which removes a methyl group from DNA and thus triggers a mechanism in GBM cells leading to TMZ resistance [6]. Interestingly, hypermethylation of the MGMT promoter occurs in multiple gliomas and, more important, its transcriptional silencing has been associated with significantly longer survival in cases of GBM and lower-grade gliomas treated by irradiation and alkylating agents, including TMZ [7,8]. Nevertheless, the survival rate for GBM remains very low and most patients develop fatal tumour recurrence or progression within 1 year of the treatment. Thus, there is a clear need for a more efficient treatment that overcomes the resistance of malignant brain tumours to conventional therapies and significantly improves survival rate. The recent identification and characterisation of genetic and molecular mechanisms driving brain tumour development and progression has allowed

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the introduction of novel molecularly targeted therapies that represent promising avenues for therapeutic interference in human brain tumours. 2. Genetic alterations during glioma development and progression To understand the mechanisms of gliomagenesis and the resistance to treatment, many studies have focused on gene expression profiling of brain tumours of different grades. According to the WHO scale, human brain tumours are graded as I to IV depending on malignancy as determined from tumour histopathologic features [9]. Grade I are benign tumours that can be cured by surgical resection. Low-grade (II) tumours show diffuse infiltration of surrounding tissue, whereas grade III tumours are characterised by increased proliferation and anaplasia and are more rapidly fatal. Grade IV tumours are the most aggressive and malignant, exhibiting vascular proliferation, necrosis and resistance to radiation and chemotherapy. GBM is grade IV and such tumours arise by at least two different pathways: from a previous lower-grade astrocytoma (secondary GBM) or de novo from precursor cells (primary GBM) [10]. As shown in Fig. 1, these clinical variants seem to have different molecular profiles but are not clearly different in prognosis, with median survival of 12–15 months. Secondary GBMs are quite rare (ca. 10%) and tend to affect younger patients (below 45 years), whereas primary GBMs account for the great majority of GBM cases (90%) in older patients. Brain tumour profiling analysis together with other recent results [11–13] have revealed major molecular alterations during the genesis of human GBM. Deregulated core pathways promoting brain tumour development and progression include growth factor signalling via activation of receptor tyrosine kinases (RTK), phosphatidylinositol-3-OH kinase (PI3K) and AKT-signalling, as well as the inactivation of p53 and retinoblastoma tumour suppressor pathways. 2.1. Growth- and survival-promoting pathways 2.1.1. Receptor tyrosine kinases Many hyperactivated cellular receptors in human gliomas belong to the RTK group. These kinases, together with deregulated non-

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receptor tyrosine kinases, activate several signalling pathways involved in cellular growth and survival as well as angiogenesis and invasion. Epidermal growth factor receptor (EGFR) gene amplification is very common in GBM, occurring in approximately 50% of cases, and many GBM patients also express a constitutively active, truncated isoform of EGFR lacking the extracellular binding domain [14]. Approximately 20–30% of GBM patients express a mutant EGFR lacking exons 2–7 (EGFRvIII), which results in ligand-independent tyrosine kinase activity that stimulates downstream survival and growth pathways [15,16]. Moreover, gliomas may release the EGFR ligands EGF and TGF-alpha, thus supporting EGFR activation and cellular growth in an autocrine manner [17]. High-grade malignant brain tumours show enhanced resistance to death- and apoptosis-stimulating agents. Hyperactivated RTKs and their downstream PI3K/AKT pathway not only stimulate growth but also contribute to an increase in anti-apoptotic features of glioma cells by various mechanisms. The reported correlation between antiapoptotic protein BCL-2 and tumour grade, where it is more abundant in grade III/IV than in grade I/II gliomas, and the association of MCL-1 expression with early tumour recurrence and shorter survival of glioma patients, clearly indicate that cancer cells acquire resistance to apoptosis via overexpression of anti-apoptotic proteins during gliomagenesis [18,19]. Indeed, upregulation of anti-apoptotic protein Bcl-xL was shown to be induced by hyperactivated EGFR pathways in human glioma cells and this upregulation conferred resistance to the chemotherapeutic agent cisplatin [20]. Other relevant induction of RTKs in malignant gliomas includes overexpressed platelet-derived growth factor receptor (PDGFR) and its ligands (PDGFs) in lower-grade astrocytomas. In addition, PDGFs are also highly expressed in high-grade brain tumours as well as in proliferating endothelial cells. Thus the activation of PDGFR in GBM can occur by both autocrine and paracrine mechanisms [21–23]. More recently, The Cancer Genome Atlas (TCGA) pilot project [12] reported major cancer-causing genome alterations by integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 GBMs. The analysed cohort represented predominantly primary glioblastomas, although a small number of progressive secondary GBM were included. In addition to the especially wellstudied EGFR and PDGFR activation, the study reported the genetic

Fig. 1. Major genetic alterations leading to glioblastoma development. Primary and secondary glioblastoma multiforme (GBM) develop from precursor cells, including astrocytes or glial precursors, as a result of de novo pathway or progressive pathway from low-grade astrocytomas (diffuse WHO grade II and anaplastic with grade III), respectively. Although primary and secondary GBM are classified as grade IV and have similar patient prognoses, many profiling analyses have revealed genetic and molecular differences between these two types of tumours. Genes that are inactivated via mutation or deletion are depicted in blue, and genes that are induced and hyperactivated during gliomagenesis by various mechanisms, including gene amplification, overexpression or mutation, in red. ⁎Less frequent inactivation in secondary as compared to primary GBM.

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alteration of other RTKs, including ERBB2/HER2 mutations and MET amplifications in 8% and 4% of analysed GBMs, respectively. Many studies have identified activated ERBB2 via mutations, amplifications or overexpression in different epithelial tumours, particularly in breast cancers, and hyperactivated HER2 is now considered to be a major characteristic of invasive breast cancer and a target for therapy. As key players in cancer development and progression, induced ERBB2/HER2 can interact and activate numerous signal transducers, leading to activation of growth and survival pathways [24,25]. Similarly, MET receptor tyrosine kinase is also mutated or overexpressed in many epithelial cancers, including lung cancer or mesothelioma, and its targeting has been extensively investigated with promising results [26,27]. During gliomagenesis an increase in the size of brain tumour leads to hypoxia and further progression then depends on neovascularisation. A key response to lack of oxygen in the tumour is the stabilization of hypoxia-inducible factor 1 (HIF1), which increases transcription of vascular endothelial growth factor (VEGF) and promotes growth [28]. In high-grade brain tumours, VEGFs are very often overexpressed and activation of their receptors VEGFR1/2 seems to play a critical role in blood vessel formation and tumour oxygenation [29]. Induction of VEGFR signalling is regulated by a variety of intracellular pathways, including PTEN/PI3/AKT, MAPK/ ERK and nitric oxide, and further RTKs ligands such as EGF, PDGF-BB and bFGF increase expression and secretion of VEGFs, supporting angiogenesis and growth in human glioma cells [30–33]. Interestingly, the most recent studies reported mutations in the active site of isocitrate dehydrogenase 1 (IDH1) which occurred in over 70% of cases in the grade II and III gliomas and these mutations were associated with an increase in overall survival. Many of the brain tumours that lacked IDH1 mutations were found to have IDH2 mutations [13,34]. Furthermore, forced expression of mutant IDH1 in cells cultured in vitro reduced its enzymatic activity and increased level of HIF-1α suggesting that IDH1 acts as a novel tumour suppressor that negatively regulates HIF-1 and consequently VEGFactivated pathways [35]. 2.1.2. RAS activation Activation of receptor tyrosine kinases leads to dimerisation and autophosphorylation of RTKs and to phosphorylation of the signalling molecules on specific tyrosine residues [36]. As a result of these phosphorylation events, a number of intracellular signal transduction cascades are initiated, including RAS/MAPK, PI3K/AKT and the protein kinase C (PKC) pathway supporting glioma cell growth, survival and invasion. Perhaps the most important consequence of RTK activation is an increase in active RAS-GTP. This activates a number of downstream molecules that can modulate the activity of various transcription factors and other cellular proteins, including RAF and the MAP kinase (MAPK) pathway [37]. Constitutively activated, mutated forms of RAS are found in the majority of human cancers. Although only a few RAS mutations have been found in human gliomas [12], high levels of active RAS have been reported in highgrade astrocytomas [38]. This indicates that hyperactivation of RAS is rather a consequence of elevated RTK signalling that occurs very often in human gliomas. In addition, inactivation of neurofibromin 1 (NF1) gene via mutations and homozygous deletions has been demonstrated in 18% of GBM patients [12]. NF1 protein negatively regulates RAS signalling by stimulating GTP hydrolysis on RAS proteins. Thus NF1 inactivation increases the active level of GTP-bound RAS in human gliomas [39,40]. Furthermore, a recent study described a non-genetic mechanism of NF1 loss in which its proteasomal degradation was triggered by hyperactivation of PKC in sporadic gliomagenesis [41]. 2.1.3. PI3K/AKT pathways The activation of RTKs in GBM leads to activation of class IA phosphatidylinositol 3-kinases (PI3Ks) and the recruitment and

assembly of receptor–PI3K complexes that localise at the membrane. There the catalytic subunit p110 of PI3K activates the conversion of phospholipid PtdIns(4,5)P2 (PIP2) to PtdIns(3,4,5)P3 (PIP3), which serves as a second messenger involved in the activation of AKT kinase [42]. Amplification and elevated expression of PIK3C2B, the overexpression of PIK3CD genes encoding PI3K catalytic subunits, as well as the amplification of AKT3 have also been reported in human GBM [43,12]. Furthermore, newly identified mutations in PI(3)K regulatory subunit PIK3R1 in 18% of human GBMs appear to lead to constitutive PI3K activation by relieving the inhibitory effect of regulatory subunit p85α on catalytic subunit p110α [12]. Activation of PI3K/AKT signalling is also enhanced by loss of the tumour suppressor PTEN gene, located at 10q23.3 that encodes PIP3 3-phosphatase. PTEN directly antagonises the activity of PI3K [44] and it is inactivated in approximately 50% of high-grade brain tumours by mutations, deletions or epigenetic mechanisms leading to promoter methylation and gene silencing [3,43,45-47]. Phosphorylated AKT triggers downstream pathways that regulate and support cellular growth, survival and proliferation by various mechanisms, including the phosphorylation and activation of mTOR kinase, transcription factor NFk-β, and MDM2 E3 ubiquitin ligase, as well as the inactivation of pro-apoptotic protein BAD and FOXO1 transcription factor [48]. Perhaps one of the most extensively investigated AKT downstream pathways in human cancers, including GBM, is activation of mTORC1, which induces phosphorylation of 40S ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (eIF4EBP1). This supports protein synthesis, cell proliferation and survival [49]. Interestingly, active mTORC1 can negatively regulate AKT via phosphorylation and the inhibition of insulin receptor substrate 1 (IRS1), which activates PI3K. Therefore, mono-therapy using an mTORC1-specific inhibitor disrupts such a feedback regulation and leads to an increase in PI3Kmediated AKT activation and subsequent failure of the treatment [50]. During gliomagenesis, AKT-signalling pathways also regulate transcription factors. Activation of NFk-β leads to cytokine secretion, increased resistance to apoptosis, and tumour cell invasion. Moreover, a significant correlation has been demonstrated between the constitutive activation of AKT and NFk-β, which contributes to the progression of diffuse gliomas [51,52]. On the other hand, AKTmediated phosphorylation can inactivate the FOXO1 transcription factor, reducing its nuclear abundance and resulting in decreased expression of the CDK inhibitors p21WAF1/CIP1 and p27KIP1. AKT can also directly phosphorylate and inactivate these CDK inhibitors, which contributes to cell cycle progression of GBM cells [48,53,54]. In addition, a recent study [12] has identified mutations in FOXO1 gene in 1% of human GBMs that contribute to its inactivation during gliomagenesis. Overexpression of anti-apoptotic proteins in GBMs [55] supports cancer cell survival. Similarly phosphorylated AKT plays an important role in apoptosis inhibition. It can directly activate MDM2, leading to the degradation of pro-apoptotic protein p53. Furthermore, AKTsignalling inactivates several other pro-apoptotic proteins, including BAD, BAX or caspase-3 [48].

2.1.4. PKC signalling Activated RTK-pathways (such as EGFR or PDGFR) in human gliomas also act via protein kinase C (PKC). Overexpression or hyperactivity of PKC are among the most distinguishing characteristics of malignant CNS tumours. The PKC family is comprised of 14 serine-threonine kinases involved in a wide range of signal transduction pathways supporting cell growth, survival and invasion. PKCsignalling stimulates the RAF/MEK/ERK pathway supporting growth and regulates anti-apoptotic protein BCL2 [56]. Furthermore, there is a link between PKC and PI3-K/AKT-signalling. The recent study demonstrates that PI3-kinase inhibitor LY294002 blocks PKC-

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mediated activation of AKT but not mTOR, suggesting that PKC can independently activate AKT and mTOR in GBM cells [57]. Moreover, active PKC is involved in signal transduction from the cytoplasm to the nucleus that results in expression of genes supporting degradation of extracellular matrix and invasiveness of glioma cells including membrane-type matrix metalloproteinase 1 (MT1-MMP) and MMP2 [56]. 2.2. Activation of cell cycle progression TP53 is a well-studied tumour suppressor gene involved in human cancers that encodes transcription factor p53. This protein regulates cell cycle progression and apoptosis in response to a wide variety of stress signals, including DNA damage. In response to various genotoxic agents, p53 is stabilized and binds to the promoters of effector genes. This leads to the expression of cell cycle checkpoint proteins, including p21WAF1/CIP1 [58]. Inactivation of TP53 is common in human gliomas. Mutations of the tumour suppressor gene TP53 are most frequent in diffuse astrocytomas and anaplastic astrocytoma GBM and are already present in approximately 60% of precursor lowgrade astrocytomas. They are thought to be hallmarks of secondary GBM [59]. Inactivation of TP53 in primary GBM can occur via amplification or overexpression of the p53 negative regulators MDM2 and MDM4. MDM2 is a p53 ubiquitin ligase targeting p53 for proteasome degradation, whereas MDM4 inhibits p53 transcription and enhances the activity of MDM2 [60–63]. Further common genetic alterations in primary GBM are mutations or deletions in the tumour suppressor gene CDKN2A that drives expression of two alternatively spliced transcripts for p14ARF and p16INK4A proteins [12,64,65]. Under conditions of oncogenic stress, p14ARF stabilizes p53 activity by binding and neutralizing MDM2. p16INK4A, a further protein encoded by CDKN2A, inhibits the cyclin D-CDK4/CDK6 complexes that phosphorylate pRb, inducing release of the E2F transcription factor that activates genes involved in G1/S transition [66]. RB activity is also frequently lost via mutations, as found in approximately 30% of highgrade astrocytomas. RB promoter methylation and gene silencing is more frequently found in secondary GBMs (43%) than primary glioblastoma (14%) [67,68]. Inactivation of pRB via phosphorylation is also supported by amplification and overexpression of CDK4/6 and Cyclin D1/3 in secondary and primary GBMs, respectively [69–71]. A recent Cancer Genome Atlas project [12] identified homozygous deletions of CDKN2A, CDKN2B and CDKN2C in 52%, 47% and 2% of GBMs, respectively, and amplification of CDK4, CCND2 encoding Cyclin D2 and CDK6 in 18%, 2% and 1% of glioblastoma patients. Thus, the identified genetic alterations again support cell cycle progression via RB inactivation. 2.3. Key deregulated signalling pathways The frequent inactivation of the p53 and pRB pathways found in human gliomas occurs directly by mutations, deletions or promoter methylations at the TP53 and RB loci, as well as indirectly by inactivation of positive regulators (p14ARF or p16INK4A). Furthermore, during brain cancer development, the amplification or overexpression of genes involved in p53 degradation (MDM2/4) or RB inactivation via phosphorylation (CDK2/6 and cyclin D) further decreases the activity of these tumour suppressive pathways. The activation of receptor tyrosine kinase receptors during gliomagenesis (mostly EGFR and PDGFR but also ERBB2/HER2, MET and VEGFR) happens by various mechanisms, including gene amplification, overexpression or the gain of activating mutations or deletions, as well as by overexpression of their ligands. Hyperactivated RTKs transduce growth and survival signals via phosphorylation events and the activation of downstream pathways, including RAS/MAPK, RAS/PI3K, PI3K/AKT and PKC signalling (Fig. 2). Moreover, in human gliomas, activation of the many components from these pathways is further increased by

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mutation (PI3K, RAS) or gene amplification and overexpression (PKC, AKT). In contrast, signal transduction is also enhanced by the acquisition of inactivating mutations or deletions in genes that antagonise phosphorylation events (PTEN phosphates) or negatively regulate the stability of the active transducers (NF1). During transition from G1 to S phase in human gliomas, lack of cell cycle guardians leads to improper cell division and the accumulation of mutations that, together with hyperactivated mitogenic pathways, promote glioma cell proliferation and survival and lead to brain tumour growth and progression. 3. Kinase inhibitors in the development of therapeutic approaches Recent gene expression-focused analysis has not only improved our understanding of brain tumour formation, its maintenance and progression but has also led to the development of novel therapeutic interventions for human glioblastoma. Over the past decades, glioblastoma profiling has allowed the development of many targeted therapies based on the inactivation of overexpressed or hyperactivated kinases involved in tumour growth and survival [72–74]. The human kinome includes more than 500 protein and lipid kinases and it has been estimated that up to 30% of all human proteins may be modified by kinase activity, making them key regulators of the signal transduction essential to normal development and cell homeostasis [75]. The main goals in the design of drugs that efficiently target kinase activity in the deregulated signalling pathways of cancer cells has been to maximize specificity and minimize toxicity. The enzymatic activity of a kinase involves transferring a phosphate group from ATP or GTP and covalently attaching it to substrates, where it alters their biological activity. Low-molecular-weight inhibitors very often mimic ATP and bind to the ATP-binding pocket in the catalytic domain, which is highly conserved between various groups of kinases [76]. Therefore, many tested inhibitors express off-target effects on kinases other than those predicted. Interestingly, less selective inhibitors have been extensively investigated in recent clinical trails, where their activity against several hyperactivated pathways shows significant promise for the treatment of human gliomas (Table 1). Most induced receptors in human gliomas exert tyrosine activity (e.g. EGFR, PDGF or VEGF) and, therefore, they share features common to downstream signalling. Monoclonal antibodies (MAb) and lowmolecular-weight compounds have been widely used for kinase inhibition in cancer therapies. The small size of low-molecular-weight inhibitors confers an advantage for delivery in clinical trails for brain tumours. However, inhibitors that exceed 400 Da or are lipid insoluble require an alternative strategy to improve delivery [77,78]. Monoclonal antibodies have a high selectivity and affinity for targeted epitopes but their administration may be hindered by the blood–brain barrier, clearly a major obstacle in drug delivery to the brain. Most therapeutic antibodies are delivered locally to a brain tumour or to a resection cavity and novel technologies have been developed recently for overcoming the blood–brain barrier [79]. The potential of endogenous transporters localised within the brain capillary endothelium has been explored for delivery of peptidomimetic MAb to brain tumours. Modified MAbs can also be used as molecular “Trojan horses” to ferry large molecules into the brain, including recombinant proteins, antibodies or RNAi-based drugs [80]. In addition, MAb against overexpressed and circulating ligands that activate receptor tyrosine kinases (e.g. bevacizumab against VEGFs) may be used in the treatment of human gliomas without the need to cross the blood– brain barrier. Recent experimental data suggest that effective therapy of malignant glioma will require combined regimens targeting multiple cellular pathways [81,82]. As seen previously, single-agent targeted therapies have not produced high response rates or durable responses in patients

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Fig. 2. Key deregulated pathways in human gliomas. Although human GBMs are highly heterogeneous, the many different genetic and molecular alterations lead to modifications of the same major signalling pathways that result in brain tumour growth and progression. Direct or indirect depletion of p53 and RB tumour-suppressor pathways (via alterations in CDK4/6, Cyclin D and CDKN2A/p14/p16, CDKN2B/C) promotes G1/S transition during the cell cycle and inhibits apoptosis. Activation of RTK signalling pathways (EGFR, PDGFR, ERRB2, MET and VEGFR) together with hyperactivated downstream pathways, including RAS/MAPK, RAS/PI3K, PI3K/AKT, AKT/mTOR and PKC signalling, contribute significantly to malignancy by promoting glioma growth, proliferation and survival as well as vascularisation and invasion. Activation of RTKs is also supported by overexpression of RTK ligands (EGF, TNF-α, PDGF, and VEGF) in both glioma and associated endothelial cells (ES). In addition, RTK-downstream signalling is increased significantly by inactivation of the negative regulators PTEN and NF1. Genes that are inactivated or hyperactivated by different mechanisms in human GBM are shown in blue and red, respectively.

with malignant gliomas. For example, several studies have been conducted to evaluate the effectiveness of EGFR low-molecular-weight inhibitors using gefitinib or erlotinib. However, the response rates were modest and appeared to depend on expression of PI3-kinase antagonist PTEN [83]. The study clearly suggests that combinatory therapy based on inhibition of the PI3K (or its downstream effectors including mTOR kinase) combined with EGFR inhibitors could promote responsiveness in patients with PTEN-deficient tumours. Indeed, preclinical studies of AEE788 (EGFR and VEGFR2 inhibitor) and mTOR inhibitor RAD001 resulted in greater inhibition of tumour growth and survival than monotherapy in mouse bearing established human malignant glioma xenografts expressing a nonfunctional form of the tumour suppressor PTEN [84]. Another potential therapeutic approach was studied using a novel tyrosine kinase inhibitor GW572016 (lapatinib) that shows dual action on EGFR and ERBB2/HER2 family of receptors. Although this agent has been used for treatment of patients with advanced or metastatic breast cancer, the most recent phase I/II clinical trial demonstrated that lapatinib did not show significant activity in GBM patients [85] suggesting that inhibition of different hyperactivated RTK pathways is not sufficient for GBM treatment. Furthermore, preclinical and clinical studies have identified mechanisms of resistance, such as the compensatory activation of other signalling pathways or the independent activation of intracellular mediators [86]. For example, monotherapy using mTOR inhibitors triggers PI3Kmediated AKT activation by disruption of mTOR-dependent feedback regulation and leads to therapy resistance [50]. Therefore, use of dual inhibitor of PI3K and mTOR kinase may circumvent this resistance mechanism. The recent study [87] identified PI-103 compound that

efficiently inhibited both PI3 kinase α and mTOR. Moreover, PI-103 showed significant activity in human glioma xenografts in vivo with no observable toxicity indicating potentially effective strategy for cancer therapy based on concomitant inhibition of PI3K and mTOR pathways in human GBM. In addition, the most recent report showed that PI-103 also enhanced chemotherapy-induced cell death of GBM cells by inhibiting DNA-PK-mediated DNA repair indicating that inhibition of deregulated networks can also significantly sensitize cancer cells to conventional therapies [88].

Table 1 Targeting deregulated signalling pathways in human gliomas: selected kinase inhibitors and their specificities. Targeted kinase

Agent

Ref.

EGFR EGFR, ERBB2/HER2 EGFR, VEGFR2 EGFR, VEGF, RET PDGFR, FLT-3, c-KIT PDGFR, Bcr/Abl, c-KIT PDGFR, VEGFR, RAF, c-KIT, FLT-3 PDGFR, VEGFR, c-KIT PDGF, VEGF, c-KIT, SRC, EPHA2 PI3K PI3K, mTOR PI3K, mTOR, DNA-PK AKT mTOR PKCβ, GSK3β MEK ½

Gefitinib, Erlotinib, Cetuximab Lapatinib AEE788, Zactima Vandetanib Tandutinib Imatinib Sorafenib Pazopanib Dasatinib PX-866 BEZ235 PI-103 Perifosine Sirolimus, temsirolimus, everolimus, Enzastaurin PD 0325901

[83,89] [85] [84,90] [91] [92] [93] [94] [95] [96] [97] [98] [88] [99] [100] [101] [102]

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4. Conclusions and perspectives At present, with a median survival of approximately 1 year for patients diagnosed with glioblastoma, there is a clear need to improve current therapies or develop other more efficient therapeutic strategies to overcome resistance mechanisms. The identification of other deregulated pathways and their targeting, in combination with already studied compounds and treatments, is still an attractive therapeutic perspective. Importantly, as malignant brain tumours tend to be highly heterogeneous, the most appropriate and efficient therapy should be selected for each individual patient. Personalised clinical approaches based on the assessment of deregulated signalling networks for each GBM patient before treatment, and the targeting of specific hyperactivated pathways alone or in combination with standard-of-care therapy may significantly improve treatment. The development of such a treatment plan specific to the molecular fingerprint of each patient is a promising avenue that may lead to new and efficient therapies for human brain cancer. Acknowledgements The research was funded by Oncosuisse CCRP grant (KFP OCS01613-12-2004) to B.A. Hemmings. M. Grzmil was supported by Marie Curie Fellowship (FP7-IEF-236745). We thank P. King (FMI) for editing the manuscript. The FMI is part of the Novartis Research Foundation. References [1] CBTRUS, Statistical report: primary brain tumors in the United States, 1998– 2002, Central Brain Tumor Registry of the United States, Hinsdale, IL, USA, 2005. [2] S.A. Grossman, J.F. Batara, Current management of glioblastoma multiforme, Semin. Oncol. 31 (2004) 635–644. [3] H. Ohgaki, P. Dessen, B. Jourde, S. Horstmann, T. Nishikawa, P.L. Di Patre, C. Burkhard, D. Schüler, N.M. Probst-Hensch, P.C. Maiorka, N. Baeza, P. Pisani, Y. Yonekawa, M.G. Yasargil, U.M. Lütolf, P. Kleihues, Genetic pathways to glioblastoma: a population-based study, Cancer Res. 64 (2004) 6892–6899. [4] R. Stupp, W.P. Mason, M.J. van den Bent, M. Weller, B. Fisher, M.J. Taphoorn, K. Belanger, A.A. Brandes, C. Marosi, U. Bogdahn, J. Curschmann, R.C. Janzer, S.K. Ludwin, T. Gorlia, A. Allgeier, D. Lacombe, J.G. Cairncross, E. Eisenhauer, R.O. Mirimanoff, Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma, N. Engl. J. Med. 352 (2005) 987–996. [5] M.J. Taphoorn, R. Stupp, C. Coens, D. Osoba, R. Kortmann, M.J. van den Bent, W. Mason, R.O. Mirimanoff, B.G. Baumert, E. Eisenhauer, P. Forsyth, A. Bottomley, Health-related quality of life in patients with glioblastoma: a randomised controlled trial, Lancet Oncol. 6 (2005) 937–944. [6] S.L. Gerson, Clinical relevance of MGMT in the treatment of cancer, J. Clin. Oncol. 20 (2002) 2388–2399. [7] M.E. Hegi, L. Liu, J.G. Herman, R. Stupp, W. Wick, M. Weller, M.P. Mehta, M.R. Gilbert, Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity, J. Clin. Oncol. 26 (2008) 4189–4199. [8] S. Everhard, G. Kaloshi, E. Crinière, A. Benouaich-Amiel, J. Lejeune, Y. Marie, M. Sanson, M. Kujas, K. Mokhtari, K. Hoang-Xuan, J.Y. Delattre, J. Thillet, MGMT methylation: a marker of response to temozolomide in low-grade gliomas, Ann. Neurol. 60 (2006) 740–743. [9] D.N. Louis, H. Ohgaki, O.D. Wiestler, W.K. Cavenee, P.C. Burger, A. Jouvet, B.W. Scheithauer, P. Kleihues, The 2007 WHO classification of tumours of the central nervous system, IARC Press, Lyon, France, 2007. [10] D. Arjona, J.A. Rey, S.M. Taylor, Early genetic changes involved in low-grade astrocytic tumor development, Curr. Mol. Med. 6 (2006) 645–650. [11] F.B. Furnari, T. Fenton, R.M. Bachoo, A. Mukasa, J.M. Stommel, A. Stegh, W.C. Hahn, K.L. Ligon, D.N. Louis, C. Brennan, L. Chin, R.A. DePinho, W.K. Cavenee, Malignant astrocytic glioma: genetics, biology, and paths to treatment, Genes Dev. 21 (2007) 2683–2710. [12] Cancer Genome Atlas Research Network, Comprehensive genomic characterization defines human glioblastoma genes and core pathways, Nature 455 (2008) 1061–1068. [13] D.W. Parsons, S. Jones, X. Zhang, J.C. Lin, R.J. Leary, P. Angenendt, P. Mankoo, H. Carter, I.M. Siu, G.L. Gallia, A. Olivi, R. McLendon, B.A. Rasheed, S. Keir, T. Nikolskaya, Y. Nikolsky, D.A. Busam, H. Tekleab, L.A. Diaz, J. Hartigan, D.R. Smith, R.L. Strausberg, S.K. Marie, S.M. Shinjo, H. Yan, G.J. Riggins, D.D. Bigner, R. Karchin, N. Papadopoulos, G. Parmigiani, B. Vogelstein, V.E. Velculescu, K.W. Kinzler, An integrated genomic analysis of human glioblastoma multiforme, Science 321 (2008) 1807–1812.

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