Antibodies neutralizing cytokine levels
Cytokine function in an inflammatory disease state can be successfully targeted with antibodies that will neutralize cytokines or their receptors. For example, anti-TNF antibodies infliximab or adalimumab are approved for the treatment of rheumatoid arthritis and investigated for an array of other inflammatory disorders, including cancers (Larkin et al., 2010; Diaz et al., 2011). Similarly, monoclonal antibody against IL-6, siltuximab, has been trialled for ovarian, prostate and renal cell cancer (Rossi et al., 2010; Coward et al., 2011; Fizazi et al., 2012). In GBM, IL-6R antibody tocilizumab has only been tested in vitro and exerted anti-proliferative effects in U87MG cells (Kudo et al., 2009). The only antibody against a protein in the tumour microenvironment investigated in GBM clinical trials is bevacizumab (Avastin). Avastin targets VEGF and is the only molecularly targeted drug that is FDA-approved for use in recurrent GBM. Unfortunately, although Avastin has potent anti-angiogenic efficacy in GBM patients, it strongly promotes tumour infiltration (Bai et al., 2011), which does not support its use in GBM therapy.
Large molecules like antibodies have limited brain penetration due to their limited ability to cross the blood–brain barrier (BBB). The anti-angiogenic efficacy of bevacizumab suggests that systematically administered antibodies may be useful as anti-glioblastoma therapies. However, bevacizumab targets VEGF, which has crossed from the brain into the vasculature, and the primary target for signal transduction inhibition appear to be endothelial cells lining the capillary walls and not the tumours cells themselves (Patel et al., 2012). Hence, one must be careful when interpreting the therapeutic potential of monoclonal antibodies, and promising results seen with IL-6 antibody tested against GBM in the absence of brain-specific delivery restrictions (Wang et al., 2009) are difficult to translate into the clinic. Along these lines, limited penetration of the EGFR inhibitor gefitinib into the CNS has been documented in GBM patients (Lassman et al., 2005). Hence, although BBB might be disrupted and leaky at the site of surgical resection and radiotherapy, the delivery of drugs to GBM tumours appears to be facing the challenge of all CNS-targeted drugs. Therefore, clinical development of antibodies focuses on novel drug delivery techniques to bypass the BBB by using viral vectors, liposomal carriers, nanoparticles or regional drug delivery to the brain (Allhenn et al., 2012). Alternatively, small molecules with BBB permeability might offer a more promising way to control inflammation in GBM. These agents could inhibit the interconnected circuits of inflammatory signal transduction pathways such as JAK–STAT, p38 MAPK, JNK and/or NF-κB. In this context, one of the most investigated pathways linking cancer with inflammation is the NF-κB pathway, which has been reviewed in great detail elsewhere (Grivennikov and Karin, 2010; Nogueira et al., 2011). In the following, we will summarize the potential of JAK–STAT3, p38 MAPK and JNK inhibitors for GBM therapy.
The thorough investigation of IL-6 induced JAK–STAT signalling (Figure 1) in GBM pathophysiology and the availability of efficacious JAK–STAT inhibitors resulted in considerable pharmacological investigation for GBM therapy. JAK inhibitor JSI-124 (cucurbitacin I, Table 2) potently inhibits growth of U251 and A172 GBM cells where decreased levels of activated STAT3 led to down-regulation of cyclin B1 and cdc2 expression and induced apoptosis and cell cycle arrest (Su et al., 2008). The same inhibitor proved efficacious in inhibiting STAT3 activation in GBM stem cells and inhibited GSCs proliferation and survival (Wang et al., 2009). In vivo, inhibition of JAK–STAT3 activity by curcumin (administered as curcumin-fortified diet) reduced the proliferation of tumour cells as well as growth and midline crossing of intracranially implanted tumours (Weissenberger et al., 2010). The inhibitor AZD1480 (Table 2) also effectively blocked JAK1, JAK2 and STAT3 phosphorylation in GBM cells, leading to a decrease in cell proliferation (Ioannidis et al., 2011). In vivo, AZD1480 inhibits the growth of subcutaneous tumours and increases survival of mice bearing human intracranial GBM tumours (McFarland et al., 2011). WP1066 (Table 2) inhibits the STAT3 pathway by targeting JAK2 (Verstovsek et al., 2008). In vitro, this compound induced apoptosis and potently inhibited viability of U87MG and U373MG cell lines. Systematic i.p. administration of WP1066 in mice attenuated the growth of s.c. GBM xenografts. Immunohistochemical analysis of excised tumours confirmed that the anti-growth efficacy resulted from inhibiting the STAT3 activity as levels of activated STAT3 (p-STAT3) in the treatment group remained inhibited 3 weeks after initial WP1066 injection, whereas tumours from the control group continuously expressed high levels of p-STAT3 (Iwamaru et al., 2007).
Table 2. Overview of kinase inhibitors profiled in the review
|Inhibitor||Structure||Primary Targets||Activity in GBM-related models|
|JSI-124|| || |
(Blaskovich et al., 2003)
Inhibits growth of U251 and A172 GBM cells (Su et al., 2008)
Inhibits GSCs proliferation and survival (Wang et al., 2009)
Sensitizes GBM cells to gefitinib and temozolomide (Lo et al., 2008)
|AZD1480|| || || |
Inhibits GBM cell proliferation (Ioannidis et al., 2011)
Inhibits growth of subcutaneous GBM tumours
|WP1066|| || |
in vitro IC50 not available
Induces apoptosis, inhibits viability of U87MG and U373MG cells, attenuates the growth of s.c. GBM xenografts (Iwamaru et al., 2007)
Potentiates TMZ efficacy in TMZ-resistant GBM cells (Kohsaka et al., 2012)
|Ruxolitinib|| || |
(Quintás-Cardama et al., 2010)
|FDA approved for treatment of myeloproliferative neoplasms|
|SB202190|| || |
(kinase activity assay) (Gallagher et al., 1997)
Abolishes the GBM-conditioned media-triggered increase in microglial expression of membrane type 1 MMP (MT1-MMP) (Markovic et al., 2009)
Abrogates the GBM conditioned media-induced MT1-MMP activity (Markovic et al., 2009)
|SB203580|| || |
(kinase activity assay) (Gallagher et al., 1997)
Sensitizes mismatch repair-proficient tumour cells to temozolomide (Hirose et al., 2003)
Inhibits synthesis of IL-6, IL-8, VEGF in GBM cells (Yoshino et al., 2006; Yeung et al., 2012)
Inhibits LPA-induced GBM cell migration (Malchinkhuu et al., 2005)
|LY479754|| || || |
Sensitizes non-migrating GBM cells to cytotoxic therapy with temozolomide (Demuth et al., 2007)
Reduces tumour growth in vivo (Campbell et al., 2005)
|069A|| || ||Inhibits inflammatory response, migration and invasiveness of U251 GBM cells (Yeung et al., 2012) |
|BIRB796|| || || |
|SP600125|| || || |
Inhibits LPA-induced migration and invasiveness of GBM cells (Malchinkhuu et al., 2005)
Inhibits adenosine receptor stimulated increase of MMP-9 (Gessi et al., 2010)
Amplifies senescence in TMZ-treated U87MG cells (Ohba et al., 2009)
Inhibits IL-1β-induced IL-6, VEGF and sphingosine kinase 1 upregulation (Yoshino et al., 2006; Paugh et al., 2009; Tanabe et al., 2011)
Blocks IL-8 promoter activity and expression in EGFRvIII-bearing cells (Bonavia et al., 2011)
In the context of GBM development, it is important to note that STAT3 is activated not only by JAKs but also by EGFR and EGFRvIII (Figure 1), which are overexpressed in 40–50% of GBMs (see above). STAT3 activation and de-regulated EGFR correlated in 27.2% of investigated tumours. Thus, targeting JAKs alone might not be sufficient to halt growth of tumours with increased EGFR activity. Sorafenib, an oral inhibitor targeting several receptor tyrosine kinases including EGFR, inhibited proliferation in primary and established GBM cell lines. Effects of sorafenib were associated with inhibiting STAT phosphorylation via inhibiting JAK1 and JAK2 (Yang et al., 2010). Combination of EGFR inhibitor gefitinib (Iressa) and JAK2 inhibitor JSI-124 synergistically suppressed STAT3 activation and potently killed GBM cells that expressed EGFR or EGFRvIII. JSI-124 also sensitized GBM cells to TMZ, an alkylating agent used in GBM therapy (Lo et al., 2008). Kohsaka et al. (2012) observed increased sensitivity towards TMZ and reported significant positive correlation between expression levels of MGMT and p-STAT3 in 44 GBM specimens. In vitro, up-regulation of IL-6, STAT3 and MGMT was accompanied with acquisition of TMZ resistance. Importantly, STAT3 inhibitor WP1066 potentiated TMZ efficacy in TMZ-resistant GBM cell lines by post-transcriptionally suppressing MGMT protein levels. However, the study failed to show in vivo efficacy of a WP1066–TMZ combination, suggesting that fast metabolism of STAT3 inhibitor and short half-life of TMZ hindered in vivo evaluation.
Together, the encouraging results from these studies indicate that pharmacological inhibition of the JAK2-STAT3 pathway could be considered for the treatment of GBM patients. Availability of ruxolitinib (Table 2), safe and efficacious JAK2 inhibitor recently approved by FDA for the treatment of myelofibrosis (Verstovsek et al., 2010; 2012; Mesa et al., 2012) and currently in clinical trials for the treatment of other malignancies (invasive metastatic breast cancer, multiple myeloma), is a promising starting point for anti-inflammatory GBM therapy.
p38 MAPK inhibitors
Limited studies have yet addressed the therapeutic potential of p38 MAPK inhibition in GBM, but rather focussed on improving the understanding of the role of p38 MAPK isoforms (α, β, γ, δ) in various aspects of GBM pathophysiology.
p38 MAPK activity contributes to the highly invasive capacity of GBM cell lines and regulates the expression of MMPs, which are responsible for the proteolytic degradation of extracellular matrix, which is the major obstacle for cell motility. MMP secretion from GBM cells stimulates the migratory response in a p38 MAPK-dependent manner. Accordingly, inhibition of p38 MAPK blocked MMP secretion and invasion of GBM cells (Park et al., 2002; Malchinkhuu et al., 2005). IR-induced EGFR activation, which triggers p38 MAPK activation, along with Akt and PI3K signalling, also increased MMP2 expression and heightened invasiveness of PTEN deficient GBM cells (Park et al., 2006). Furthermore, GBM-released factors manipulated the tumour-associated microglia via toll-like receptor induced p38 MAPK signalling, which up-regulates membrane type 1-MMP to activate pro-MMP2 and thereby support GBM expansion (Markovic et al., 2009).
In addition, p38 MAPK may affect GBM malignancy via modulating cytokines and growth factors in the tumour microenvironment. p38 MAPK has a key role in the production of TNF-α, IL-1β and IL-6 from activated microglia and also regulates signalling of these cytokines in GBM cells (Figure 1), further exacerbating local inflammation. These cytokines exert their pro-tumourigenic effects in GBM pathology by increased expression of other inflammatory (e.g. COX-2; Xu and Shu, 2007), invasiveness-promoting (e.g. MMPs; Markovic et al., 2009; Sarkar and Yong, 2009) or angiogenic (e.g. IL-8 and VEGF; Yoshino et al., 2006) mediators. GBM cells could potentially up-regulate these mediators via p38 MAPK-dependent phosphorylation of transcription factors such as NF-κB (Figure 1). Post-transcriptional gene regulation has also been implicated as one mechanism of the tumour response to the inflammatory microenvironment, and in this regard, p38 MAPK-mediated shuttling of the mRNA stabilizing human antigen R (HuR, Figure 1) protein is linked to increased stability of VEGF, TGF-β, IL-6, IL-8 and TNF-α mRNA in GBM cells (Nabors et al., 2003). HuR depletion led to transcript destabilization, reduced protein expression, a significant decrease in tumour volume and increased sensitivity to chemotherapeutic drugs (Filippova et al., 2011). Further supporting p38 MAPK activity in regulating mRNA stability, p38 MAPK-mediated hyperphosphorylation deactivated the RNA destabilizer tristetraprolin (TTP), resulting in stabilization of IL-8 and VEGF mRNA, and promoted GBM cell proliferation and viability (Suswam et al., 2008). Moreover, MAPK-interacting kinase 1 and 2 (Mnk1/2) are translation-controlling serine/threonine kinases that are activated by p38 MAPK (Figure 1) and Mnk1 knockdown in U87MG reduced tumourigenic activity in nude mice (Ueda et al., 2010). Finally, activity of p38 MAPK influences the response of GBM cells to DNA-alkylating agents and pharmacological inhibition of p38 MAPK sensitized GBM cells to TMZ-induced toxicity (Hirose et al., 2003; 2004).
Despite increasing evidence for p38 MAPK as an anti-GBM target, currently in vivo data are limited in order to validate the effectiveness of p38 MAPK inhibition in GBM. Indirectly supporting the use of p38 MAPK inhibitors for GBM therapy comes from research on minocycline, a semi-synthetic broad-spectrum and lipophilic tetracycline antibiotic approved by the FDA that is able to cross the BBB and inhibit microglial activation and inflammation in CNS disease models. One of the main targets of minocycline is p38 MAPK (Nikodemova et al., 2006), and the anti-inflammatory effect of minocycline correlates with inhibition of microglial p38 MAPK phosphorylation and decreased GBM invasiveness and expansion in vitro and in vivo (Liu et al., 2011; Markovic et al., 2011).
Commonly, the prototypical p38 MAPK inhibitors SB203580 (Table 2) and SB202190 (Table 2) have been utilized to examine p38 MAPK in GBM malignancy (Hirose et al., 2003; Malchinkhuu et al., 2005; Markovic et al., 2009; Bonavia et al., 2011). A better clinical candidate, the p38 MAPK inhibitor LY479754 (Table 2), was shown to inhibit GBM invasiveness in vitro. In addition, LY479754 also sensitized arrested, non-migrating cells to cytotoxic therapy with TMZ (Demuth et al., 2007). We recently showed that a BBB-permeable p38 MAPK inhibitor, 069A (Table 2, Munoz et al., 2007), can potentially target both malignant and non-malignant microglial cells in GBM tumours and attenuate the development of an inflammatory microenvironment and GBM invasiveness (Yeung et al., 2012). Possibly in combination with other treatments, these promising results mandate further development and testing of suitable p38 MAPK inhibitors against invasive glioblastoma in vivo. In clinic, however, BBB permeability might be problematic as p38 MAPK inhibitors have been developed for rheumatoid arthritis therapy and were intentionally made highly polar in order to avoid BBB permeability. This restricts the use of current p38 MAPK inhibitors for CNS-related diseases. Certainly, a p38 MAPK inhibitor with satisfying preclinical safety might be able to control the local inflammation at the tumour site, but a new p38 MAPK inhibitor with improved BBB permeability could have far more potential in GBM therapy.
JNKs are an evolutionarily conserved sub-group of MAPKs activated by MAPK kinases 4 and 7 (MMK4 and MKK7), which integrate a wide array of stimuli to phosphorylate and stimulate JNKs. The major JNK target is the transcription factor AP-1, which is composed of Fos and c-Jun family members. Thereby, the JNK/c-Jun pathway modulates expression of a plethora of AP-1 target genes that control inflammatory response, cell proliferation, apoptosis as well as invasion. Besides regulating proto-oncogenes Jun, Fos and Myc; JNKs have also been linked to p53-dependent senescence and apoptosis. Overall, pro- and anti-apoptotic effects of JNK signalling on tumour development appear to be determined by stimuli and tissue specificity, signal intensity and crosstalk between JNK isoforms (Wagner and Nebreda, 2009).
In GBM, JNKs and c-Jun phosphorylation correlate with the grade of malignancy and patients' age (Antonyak et al., 2002). JNK2 is the major activated JNK isoform in GBM (Tsuiki et al., 2003). This isoform is unique among all MAPKs as it possesses autophosphorylation activity and constitutive substrate kinase activity in vitro and in vivo (Nitta et al., 2008). JNK2 is also activated by oncogenic EGFRvIII (Figure 1) (Antonyak et al., 1998) and supports tumourigenesis in vivo through increased proliferation and tumour formation (Cui et al., 2006). In addition to its role in apoptosis, JNKs have been linked to glial-derived neurotrophic factor (Lu et al., 2010) and lysophosphatidic acid-induced migration and invasiveness of GBM (Malchinkhuu et al., 2005). SP600125-mediated JNK inhibition antagonized adenosine receptor stimulated increase of MMP-9 levels (Gessi et al., 2010). Furthermore, SP600125 (Table 2) amplified senescence in TMZ-treated U87MG cells, suggesting that SP600125 potentiates TMZ-dependent cell death pathways (Ohba et al., 2009).
Within the tumour microenvironment, JNKs likely potentiate carcinogenesis by promoting local inflammatory responses. For example, IL-1β-induced IL-6 production, which contributes to increased tumour development and invasiveness, is inhibited by SP600125 in an inflammatory model of GBM (Tanabe et al., 2011). Inhibition of JNK signalling also suppressed angiogenesis and invasiveness of GBM cell via inhibiting IL-1β-induced VEGF production and sphingosine kinase 1 upregulation, respectively (Yoshino et al., 2006; Paugh et al., 2009). Together, these studies indicate that therapeutic manipulation of oncogenic IL-1β activities can be managed with JNK inhibitors; however, the JNK isoforms responsible for these effects have yet to be identified. Interference with the JNK pathway also decreased EGFR-dependent tissue factor expression (Rong et al., 2009). TF is the main cellular initiator of coagulation that significantly contributes to two fundamental features of GBM, namely hypoxia and necrosis. EGFR-mediated TF expression depends on AP-1 transcriptional activity and is associated with JNK and JunD activation. These mechanisms are likely to work in vivo as elevated/mutated EGFR highly correlated with TF expression in GBM specimen (Rong et al., 2009). Recently, Bonavia et al. (2011) showed that the pharmacological targeting of the JNK–NF-κB pathway with SP600125 efficiently blocked IL-8 promoter activity and expression in EGFRvIII-bearing cells. However, interpretation of results derived from the pan-JNK inhibitor SP600125 should have taken into consideration the vast number of off-target kinases that this compound inhibits (Bain et al., 2007).
A significant number of potent and selective JNK inhibitors have recently been developed, some with BBB permeability (He et al., 2011; Plantevin Krenitsky et al., 2012). These inhibitors either target peripheral JNK1 in obesity and diabetes, or the JNK3 isoform expressed in CNS to reduce neurodegeneration. Yet JNK2 appears to be the main JNK isoform involved in GBM pathophysiology and JNK2 selective inhibitors are yet to be developed. Interestingly, BIRB796 (Table 2), a potent type II inhibitor of p38 MAPK that also binds with high affinity to JNK2 (Gruenbaum et al., 2009), could provide an opportunity as dual-kinase inhibitor.