Tumor cells present high levels of glycolysis as a consequence of increased hypoxic microen‐ vironment, which leads to activation of HIF-1α and consequent upregulation of downstream target genes involved in glycolytic metabolism, such as PDK [67]. This enzyme is responsible for the uncoupling between glycolysis and mitochondrial oxidation of glucose, preventing the entry of pyruvate into the mitochondria with consequent increase in glycolytic rates, which confers resistance to apoptosis [67, 68]. Thus, PDK became an important target for glycolytic tumors (Figure 2). Dichloroacetate (DCA), a chemical PDK inhibitor, has been studied in several *in vitro* and *in vivo* models [68]. DCA promotes dephosphorylation of PDH, leading to entry of pyruvate into the mitochondria, decreasing glycolytic rates and lactate production [68, 70]. This leads to activation of oxidative phosphorylation, depolarization of mitochondria and consequent increase in production of reactive oxygen species (ROS), which promotes apoptosis of cancer cells, decreasing tumor cell proliferation [68, 70]. DCA treatment has demonstrated promising results in some tumors, particularly in non-small cell lung, breast and endometrial cancer, either experimentally using *in vitro* and *in vivo* models, as well as in clinical trials [70-73]. In glioblastomas, inhibition of PDK with DCA was evaluated pre-clinically. In C6 glioma cells, it was observed a decrease in lactate production, increase in ROS production, as well as depolarization of mitochondria, which results in a decrease on cell proliferation and induction of apoptosis [74]. *In vivo* it was verified that DCA decreased not only tumor growth but also the angiogenic capacity of glioma cells [74]. The effect of DCA was also tested in a series of glioblastoma patients with congenital acidosis, with a reduction in lactate levels, decrease in HKII localization in the mitochondria, as well as a decrease in mitochondrial polarization, which rendered tumor cells more sensitive to apoptosis [69]. Despite these encouraging results, no exact conclusions can yet be made regarding the efficacy and toxicity of DCA in glioblastoma patients. Thus, a large and randomized clinical study would be important to define the efficacy and toxicity of DCA. Additionally, whether DCA can sensitize GBM cells to temozolomide and radiotherapy remains undetermined.

#### *2.2.2. Isocitrate Dehydrogenase (IDH)*

IDH is an enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketogluta‐ rate, generating NADH in the mitochondria or NADPH in the cytoplasm [75]. It is composed by 5 genes, being the *IDH1* and *IDH2* the most explored and important in gliomas. IDH1 is presentedinthecytoplasmandperoxisome,whereasIDH2ispresentedinthemitochondria[76].

In 2008, recurrent somatic hotspot mutations of *IDH1* and *IDH2* were found in low grade gliomas and secondary glioblastomas [77, 78]. These mutations cause amino acid single change in one of the two alleles of the gene (arginine 132 for *IDH1* and arginine 172 for *IDH2*), being classified has a dominant mutation [79]. It is described that the arginine mutation occurs in the binding site of the substrate isocitrate [42]. *IDH1* mutations are reported in more than 80% secondary glioblastomas, but only 5% in primary glioblastomas [76, 80, 81]. Additionally, it occurs in 80% of diffuse astrocytomas (WHO grade II). These mutations are more frequent in younger patient secondary glioblastomas, associated with a proneural subtype and also with increased survival [82]. It was reported that the presence of IDH mutations leads to a decrease in α-ketoglutarate that is required for prolyl hydroxylase (PHD) activity that promote degradation of HIF-1α (ref). Thus, downregulation of intracellular α-ketoglutarate contributes to stabilization of HIF-1α, leading to pseudohypoxia [83]. Nevertheless, subsequent studies did not verify an alteration in α-ketoglutarate on mutant IDH1/2, instead, a gain of function activity it was found, which converts α-ketoglutarate to 2-hydroxyglutarate (Figure 2) [84, 85]. The latter has been recognized as an oncometabolite, which inhibits enzymes involved in the α-ketoglutarate pathway. Additionally, it was described that 2-hydroxyglutarate is involved in epigenetic regulation, promoting a hypermethylator phenotype in gliomas [86] and also keep cells in an undifferentiated *status*, or stem cell-like, which can be more permissive to transformation [17]. The presence of pseudohypoxia, due to the constitutive stabilization of HIF-1α, indicates that *IDH1/2* mutations are involved in HIF-1α signaling pathway, which promotes glucose metabolism, angiogenesis and invasion [83]. These results suggest the paramount role of IDH mutations on the metabolic remodeling of glioblastomas, contributing to the "Warburg effect". Therefore, study the involvement of *IDH1/2* mutations in metabolic remodelling and in aerobic glycolysis opens a new window for investigation. Overall, IDH1 is an attractive target for therapy (Figure 2) since they are early events in the progression from low grade to high grade gliomas.

#### **2.3. Glutamine metabolism and lipid synthesis in brain tumors**

Like glucose, glutamine is a source of energy for tumor cells, functioning as a nitrogen donor [87, 88]. Glutamine metabolism has been reported to be upregulated in some tumors, being crucial for the biosynthetic processes, namely synthesis of cholesterol and fatty acids [14, 89, 90]. The shift to glutamine metabolism to produce the precursor acetyl-CoA for lipid biosyn‐ thesis is a mechanism of adaptation to glycolytic metabolism that prevents the entry of pyruvate into mitochondria, due to upregulation of PDK [91]. In fact, it has been observed an increased expression of glutaminase (GLS) enzyme in tumor cells. GLS is located in the mitochondria and catalyzes the conversion of glutamine to glutamate being transcriptionally regulated by the oncogenes *c-Myc* and *NFκB*. [92-94], An increased concentration of glutamine in glioblastomas compared to normal brain tissue was demonstrated by nuclear magnetic resonance (NMR) [95]. Additionally, there is a low expression of glutamine synthase that correlates with a better prognosis in glioblastomas [96].

Beyond the altered glycolytic and glutamine metabolism in tumor cells, the alteration in lipid metabolism is also recognized as a component of the metabolic reprogramming. It has been observed that tumor cells present reactivation of *de novo* fatty acid synthesis, impor‐ tant for the biogenesis of cellular membranes [97, 98]. Glioblastomas contain higher levels of unsaturated fatty acids compared to normal brain, indicating the presence of exacerbat‐

ed lipogenesis, which is regulated by several key genes, such as SREBP-1 and its down‐ stream-targeted genes acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and lowdensity lipoprotein receptor (LDLR), which are upregulated in these tumors [99]. Importantly, the EGFR/PI3K/AkT signaling pathway regulates the metabolic reprogram‐ ming in glioblastomas [100]. Cholesterol is also an important component of cell mem‐ branes and cholesterol esters have been found to be abundantly present in high grade gliomas, but undetectable in normal tissues by NMR techniques [101, 102]. Recently, low density lipoprotein receptor (LDLR) has been described to be upregulated in GBM patients, xenografts and cell lines, and this upregulation was correlated with high levels of cholester‐ ol esters in GBM cells [103]. Interestingly, LDLR is also be upregulated by EGFR/ PI3K/Akt signaling, which was been shown to be mediated by SREBP-1 in GBMs [100]. However, little is known about the altered lipid metabolism in cancer cells, namely glioblastomas, and their role in the tumor context, being possible that lipogenesis in cancer cells could support the cell growth located within nutrient-limited areas, thereby contribu‐ ting to symbiotic relationships within tumors. Once more, lipid metabolism, as well as glutamine metabolism, and their key enzymes are interesting targets in glioblastomas (Figure 2)

## **3. Lactate transport and pH regulation in brain tumors**

A constitutive increase in the glycolytic phenotype of cancer cells leads to acute and chronic acidification of tumor microenvironment. Important proteins involved in acidification of the extracellular space are monocarboxylate transporters (MCTs) that co-transport H+and lactate, and carbonic anhydrases (CAIXs), which are activated by growth factors, oncogenic transfor‐ mation, hypoxia, and low intracellular pH [21]. As it is known, tumor acidity is associated with cancer cell invasion behavior, i.e. increased migration, invasion and metastasis [104]. Further, tumor acidosis and lactate contributes to several features of tumor progression and malig‐ nancy, like immune escape, angiogenesis, and radioresistance, making lactate a key player in cancer aggressiveness. [105]. Still in line with a potential involvement of lactate in the invasion behavior, it has been shown that lactate up-regulates the expression of transforming growth factor (TGF-β2), which is associated with increased migration in glioblastomas [106].

#### **3.1. Monocarboxylate transporters**

classified has a dominant mutation [79]. It is described that the arginine mutation occurs in the binding site of the substrate isocitrate [42]. *IDH1* mutations are reported in more than 80% secondary glioblastomas, but only 5% in primary glioblastomas [76, 80, 81]. Additionally, it occurs in 80% of diffuse astrocytomas (WHO grade II). These mutations are more frequent in younger patient secondary glioblastomas, associated with a proneural subtype and also with increased survival [82]. It was reported that the presence of IDH mutations leads to a decrease in α-ketoglutarate that is required for prolyl hydroxylase (PHD) activity that promote degradation of HIF-1α (ref). Thus, downregulation of intracellular α-ketoglutarate contributes to stabilization of HIF-1α, leading to pseudohypoxia [83]. Nevertheless, subsequent studies did not verify an alteration in α-ketoglutarate on mutant IDH1/2, instead, a gain of function activity it was found, which converts α-ketoglutarate to 2-hydroxyglutarate (Figure 2) [84, 85]. The latter has been recognized as an oncometabolite, which inhibits enzymes involved in the α-ketoglutarate pathway. Additionally, it was described that 2-hydroxyglutarate is involved in epigenetic regulation, promoting a hypermethylator phenotype in gliomas [86] and also keep cells in an undifferentiated *status*, or stem cell-like, which can be more permissive to transformation [17]. The presence of pseudohypoxia, due to the constitutive stabilization of HIF-1α, indicates that *IDH1/2* mutations are involved in HIF-1α signaling pathway, which promotes glucose metabolism, angiogenesis and invasion [83]. These results suggest the paramount role of IDH mutations on the metabolic remodeling of glioblastomas, contributing to the "Warburg effect". Therefore, study the involvement of *IDH1/2* mutations in metabolic remodelling and in aerobic glycolysis opens a new window for investigation. Overall, IDH1 is an attractive target for therapy (Figure 2) since they are early events in the progression from

90 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

low grade to high grade gliomas.

**2.3. Glutamine metabolism and lipid synthesis in brain tumors**

correlates with a better prognosis in glioblastomas [96].

Like glucose, glutamine is a source of energy for tumor cells, functioning as a nitrogen donor [87, 88]. Glutamine metabolism has been reported to be upregulated in some tumors, being crucial for the biosynthetic processes, namely synthesis of cholesterol and fatty acids [14, 89, 90]. The shift to glutamine metabolism to produce the precursor acetyl-CoA for lipid biosyn‐ thesis is a mechanism of adaptation to glycolytic metabolism that prevents the entry of pyruvate into mitochondria, due to upregulation of PDK [91]. In fact, it has been observed an increased expression of glutaminase (GLS) enzyme in tumor cells. GLS is located in the mitochondria and catalyzes the conversion of glutamine to glutamate being transcriptionally regulated by the oncogenes *c-Myc* and *NFκB*. [92-94], An increased concentration of glutamine in glioblastomas compared to normal brain tissue was demonstrated by nuclear magnetic resonance (NMR) [95]. Additionally, there is a low expression of glutamine synthase that

Beyond the altered glycolytic and glutamine metabolism in tumor cells, the alteration in lipid metabolism is also recognized as a component of the metabolic reprogramming. It has been observed that tumor cells present reactivation of *de novo* fatty acid synthesis, impor‐ tant for the biogenesis of cellular membranes [97, 98]. Glioblastomas contain higher levels of unsaturated fatty acids compared to normal brain, indicating the presence of exacerbat‐ The MCT family comprises 14 members with similar topology; however, only 4 isoforms (MCT1–MCT4) are proton-linked monocarboxylate transporters, performing the transmem‐ brane transport of monocarboxylates, such as lactate, coupled with a proton, in an equimolar manner [107, 108].

In the last years, several studies reported up-regulation of MCTs in different human solid tumors, showing the importance of MCTs in cancer biology [109].  Strong expression of MCT1 in the plasma membrane was found in high grade gliomas compared with low-grade lesions and normal adjacent tissues, which exhibited negative or weak MCT1 staining [110, 111],. A study in neuroblastomas showed, by mRNA quantification, that MCT1 was differently expressed and that its activity was highly associated with MYCN amplification, leading to the hypothesis that expression of MCT1 could be associated with higher malignancy [112]. Further, expression analysis revealed that SLC16A1 transcript, encoding MCT1, was elevated in 90% of the medulloblastomas analyzed [113]. It was also reported that inhibition of MCT activity, particularly MCT1, decreased the glycolytic phenotype (low glucose consumption and lactate production), cell proliferation and invasion, promoting increase in cell death [111, 114, 115]. This elucidates the importance of MCT1 activity in intracellular pH homeostasis and tumor aggressiveness of glioblastomas.

Although MCTs are not the major H+ transporters, the data available in the literature support the hypothesis of a major contribution of MCTs to the hyper-glycolytic and acid-resistant phenotype, as major adaptation to the hypoxic microenvironment [116]. Thus, MCT inhibition may be a useful therapeutic approach in brain tumors (Figure 2). Actually, it was demonstrated that *in vitro* MCT1 inhibition decreases intracellular pH, leads to cell death and, importantly, enhances cancer cell radiosensivity in gliomas [114, 115]. Importantly, promising results using *in vivo* models have also been reported, where treatment with the chemical inhibitor CHC retarded tumor growth, rendered tumor cells sensitive to radiation and decreased invasion [114, 117]. However, CHC is not a specific MCT inhibitor, having also other targets. Recently, novel MCT1 inhibitors have been designed and may constitute an effective strategy to block MCT1 activity in cancer [118]. A orally administered related compound, AZD3965 (AstraZe‐ neca), is currently in Phase I/II clinical trials for advanced solid tumors [119].

#### **3.2. Carbonic anhydrases**

Carbonic anhydrase catalyzes the conversion of extracellular bicarbonate to CO2 and protons (H+ ), thereby contributing to extracellular acidification [120]. This family is composed by 15 isoforms described in mammals, which differ in cellular localization, catalytic activity and susceptibility to different class of inhibitors. Two carbonic anhydrases are overex‐ pressed in many solid tumors, namely CAIX and CAXII, being associated with tumor progression and response to therapy [121]. It is verified that CAIX is mostly negative in normal tissues but increase in the corresponding tumor tissues, whereas CAXII present a diffuse distribution in healthy tissues [122, 123]. Glioblastomas present high levels of intratumoral hypoxia, with consequent HIF-1α activation which contributes to increased expression of glycolysis-related genes [124], including CAIX [125]. CAIX is overexpressed in these tumors with focal plasma membrane expression close to peri-necrotic regions (hypoxic) [126], being negative in normal adjacent tissues, making it a feasible treatment target [127]. Furthermore, it has been described that CAIX is associated to poor overall survival, because it confers resistance to chemotherapy, radiotherapy and anti-angiogenic therapy [128]. Increased expression of CAIX in advanced stages/grades of many tumor types also suggests its association with dedifferentiation [129].

*In vitro* and *in vivo* approaches have demonstrated the potential of CAIX inhibition (Figure 2). Knockdown of CAIX decreased tumor cell ATP levels under hypoxic and glycolytic

conditions [126]. In addition, the susceptibility of U251 glioblastoma cells to chemothera‐ py and radiation treatment was strongly enhanced after CAIX downregulation, which is supported by a recent *in vivo* study [126]. Similarly, CAIX inhibition enhanced the effect of anti-angiogenic therapy with the anti-VEGF antibody bevacizumab [130]. Some inhibitors have been developed to inhibit CA activity, particularly CAIX and CAXII. Acetazolo‐ mide, enhances the apoptotic response of glioma cells to temozolomide [131] and an *in vivo* study using derivatives of acetazolamide showed retardation of mice carcinoma xenograft growth after 1 month of treatment [131]. Other studies have identified coumarins as CA inhibitors, however they were not tested yet in the cancer context [132, 133]. Further‐ more, specific monoclonal antibodies for the mostly expressed isoforms in tumors, namely CAIX, have been developed, *i.e,* the M75 and WX-G250 for colorectal cancer and renal cell carcinoma, respectively [134, 135].

## **4. Future perspectives and conclusions**

Metabolic transformation plays a major role in gliomas development, tumor progression and adaptation to tumor microenvironment. The interplay between tumor angiogenesis, hypoxia, pH regulation and energy metabolism, glycolysis related enzymes and transporters, as well as pH regulator transporters, may provide promising molecular targets for drug development. In addition to glycolysis, glutaminolysis and fatty acid synthesis represent key metabolic events with potentially interesting drug targets. Furthermore, mutations in *IDH1/2*, detected in a genome wide screen on GBMs, point to new specific transforming events in gliomas. These metabolic pathways are tightly linked and also controlled by signaling events often deregu‐ lated in gliomas, underlying the flexibility of glioma cells to develop adaptive mechanisms when exposed to oxygen or nutrient deprivation. This highlights the need of targeting several pathways simultaneously and linking the metabolic targets to the genetic makeup of GBM tumors.

## **Author details**

lesions and normal adjacent tissues, which exhibited negative or weak MCT1 staining [110, 111],. A study in neuroblastomas showed, by mRNA quantification, that MCT1 was differently expressed and that its activity was highly associated with MYCN amplification, leading to the hypothesis that expression of MCT1 could be associated with higher malignancy [112]. Further, expression analysis revealed that SLC16A1 transcript, encoding MCT1, was elevated in 90% of the medulloblastomas analyzed [113]. It was also reported that inhibition of MCT activity, particularly MCT1, decreased the glycolytic phenotype (low glucose consumption and lactate production), cell proliferation and invasion, promoting increase in cell death [111, 114, 115]. This elucidates the importance of MCT1 activity in intracellular pH homeostasis and tumor

92 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

Although MCTs are not the major H+ transporters, the data available in the literature support the hypothesis of a major contribution of MCTs to the hyper-glycolytic and acid-resistant phenotype, as major adaptation to the hypoxic microenvironment [116]. Thus, MCT inhibition may be a useful therapeutic approach in brain tumors (Figure 2). Actually, it was demonstrated that *in vitro* MCT1 inhibition decreases intracellular pH, leads to cell death and, importantly, enhances cancer cell radiosensivity in gliomas [114, 115]. Importantly, promising results using *in vivo* models have also been reported, where treatment with the chemical inhibitor CHC retarded tumor growth, rendered tumor cells sensitive to radiation and decreased invasion [114, 117]. However, CHC is not a specific MCT inhibitor, having also other targets. Recently, novel MCT1 inhibitors have been designed and may constitute an effective strategy to block MCT1 activity in cancer [118]. A orally administered related compound, AZD3965 (AstraZe‐

Carbonic anhydrase catalyzes the conversion of extracellular bicarbonate to CO2 and protons

*In vitro* and *in vivo* approaches have demonstrated the potential of CAIX inhibition (Figure 2). Knockdown of CAIX decreased tumor cell ATP levels under hypoxic and glycolytic

), thereby contributing to extracellular acidification [120]. This family is composed by 15 isoforms described in mammals, which differ in cellular localization, catalytic activity and susceptibility to different class of inhibitors. Two carbonic anhydrases are overex‐ pressed in many solid tumors, namely CAIX and CAXII, being associated with tumor progression and response to therapy [121]. It is verified that CAIX is mostly negative in normal tissues but increase in the corresponding tumor tissues, whereas CAXII present a diffuse distribution in healthy tissues [122, 123]. Glioblastomas present high levels of intratumoral hypoxia, with consequent HIF-1α activation which contributes to increased expression of glycolysis-related genes [124], including CAIX [125]. CAIX is overexpressed in these tumors with focal plasma membrane expression close to peri-necrotic regions (hypoxic) [126], being negative in normal adjacent tissues, making it a feasible treatment target [127]. Furthermore, it has been described that CAIX is associated to poor overall survival, because it confers resistance to chemotherapy, radiotherapy and anti-angiogenic therapy [128]. Increased expression of CAIX in advanced stages/grades of many tumor types

neca), is currently in Phase I/II clinical trials for advanced solid tumors [119].

also suggests its association with dedifferentiation [129].

aggressiveness of glioblastomas.

**3.2. Carbonic anhydrases**

(H+

Vera Miranda Gonçalves1,2, Fátima Baltazar1,2 and Rui Manuel Reis1,2,3\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal

2 ICVS/3B's-PT Government Associate Laboratory, Braga/Guimarães, Portugal

3 Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil

#### **References**

[16] Vander Heiden, M.G., L.C. Cantley, and C.B. Thompson, *Understanding the Warburg effect: the metabolic requirements of cell proliferation.* Science, 2009. 324(5930): p. 1029-33.

**References**

2005. 109(1): p. 93-108.

Acta Neuropathol, 2007. 114(2): p. 97-109.

*ma.* N Engl J Med, 2005. 352(10): p. 987-96.

*blastoma.* N Engl J Med, 2014. 370(8): p. 709-22.

Mol Sci, 2009. 10(1): p. 184-212.

29(2): p. 142-8.

2014. 6: p. 46.

2010. 12(9): p. 985-90.

2014. 9(6): p. e99198.

2010. 70(22): p. 9243-52.

col Rep, 2010. 23(6): p. 1655-62.

*and proliferation.* Cell Metab, 2008. 7(1): p. 11-20.

*ic outliers teach us?* Cell, 2012. 148(6): p. 1132-44.

233-42.

[1] Ohgaki, H. and P. Kleihues, *Epidemiology and etiology of gliomas.* Acta Neuropathol,

94 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

[2] Huse, J.T. and E.C. Holland, *Targeting brain cancer: advances in the molecular pathology of malignant glioma and medulloblastoma.* Nat Rev Cancer, 2010. 10(5): p. 319-31.

[3] Louis, D.N., et al., *The 2007 WHO classification of tumours of the central nervous system.*

[4] Riemenschneider, M.J. and G. Reifenberger, *Molecular neuropathology of gliomas.* Int J

[5] Stupp, R., et al., *Radiotherapy plus concomitant and adjuvant temozolomide for glioblasto‐*

[6] Lai, A., et al., *Phase II study of bevacizumab plus temozolomide during and after radiation therapy for patients with newly diagnosed glioblastoma multiforme.* J Clin Oncol, 2011.

[7] Chinot, O.L., et al., *Bevacizumab plus radiotherapy-temozolomide for newly diagnosed glio‐*

[8] Weathers, S.P. and M.R. Gilbert, *Advances in treating glioblastoma.* F1000Prime Rep,

[9] de Groot, J.F., et al., *Tumor invasion after treatment of glioblastoma with bevacizumab: ra‐ diographic and pathologic correlation in humans and mice.* Neuro Oncol, 2010. 12(3): p.

[10] Narayana, A., et al., *Bevacizumab in recurrent high-grade pediatric gliomas.* Neuro Oncol,

[11] Mesti, T., et al., *Metabolic impact of anti-angiogenic agents on U87 glioma cells.* PLoS One,

[12] Gaspar, N., et al., *MGMT-independent temozolomide resistance in pediatric glioblastoma cells associated with a PI3-kinase-mediated HOX/stem cell gene signature.* Cancer Res,

[13] Costa, B.M., et al., *Prognostic value of MGMT promoter methylation in glioblastoma pa‐ tients treated with temozolomide-based chemoradiation: a Portuguese multicentre study.* On‐

[14] DeBerardinis, R.J., et al., *The biology of cancer: metabolic reprogramming fuels cell growth*

[15] DeBerardinis, R.J. and C.B. Thompson, *Cellular metabolism and disease: what do metabol‐*

[48] Khalid, M.H., et al., *Inhibition of tumor growth and prolonged survival of rats with intra‐ cranial gliomas following administration of clotrimazole.* J Neurosurg, 2005. 103(1): p. 79-86.

 Br J Biomed

[35] Wilson, J.E., *Isozymes of mammalian hexokinase: structure, subcellular localization and met‐*

[36] Pedersen, P.L., et al., *Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention.* Biochim Bi‐

[37] Wolf, A., et al., *Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme.* J Exp Med, 2011. 208(2): p. 313-26.

[38] Agnihotri, S., et al., *A GATA4-regulated tumor suppressor network represses formation of*

[39] Mathupala, S.P., Y.H. Ko, and P.L. Pedersen, *Hexokinase-2 bound to mitochondria: can‐ cer's stygian link to the "Warburg Effect" and a pivotal target for effective therapy.* Semin

[40] Mellinghoff, I.K., et al., *Molecular determinants of the response of glioblastomas to EGFR*

[41] Pastorino, J.G., N. Shulga, and J.B. Hoek, *Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis.* J Biol Chem, 2002. 277(9): p. 7610-8.

[42] Wolf, A., S. Agnihotri, and A. Guha, *Targeting metabolic remodeling in glioblastoma mul‐*

[43] Wolf, A., et al., *Developmental profile and regulation of the glycolytic enzyme hexokinase 2 in normal brain and glioblastoma multiforme.* Neurobiol Dis, 2011. 44(1): p. 84-91.

[44] Ko, Y.H., et al., *Advanced cancers: eradication in all cases using 3-bromopyruvate therapy*

[45] Oudard, S., et al., *Phase II study of lonidamine and diazepam in the treatment of recurrent*

[46] Carapella, C.M., et al., *The potential role of lonidamine (LND) in the treatment of malig‐*

[47] Liu, H., Y. Li, and K.P. Raisch, *Clotrimazole induces a late G1 cell cycle arrest and sensitiz‐ es glioblastoma cells to radiation in vitro.* Anticancer Drugs, 2010. 21(9): p. 841-9.

*to deplete ATP.* Biochem Biophys Res Commun, 2004. 324(1): p. 269-75.

*glioblastoma multiforme.* J Neurooncol, 2003. 63(1): p. 81-6.

*nant glioma. Phase II study.* J Neurooncol, 1989. 7(1): p. 103-8.

*malignant human astrocytomas.* J Exp Med, 2011. 208(4): p. 689-702.

*kinase inhibitors.* N Engl J Med, 2005. 353(19): p. 2012-24.

*sion, and as a therapeutic target.* J Neurooncol, 2009. 92(3): p. 317-35.

96 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

*tabolism.* Expert Opin Ther Targets, 2011. 15(10): p. 1227-39.

*abolic function.* J Exp Biol, 2003. 206(Pt 12): p. 2049-57.

Sci, 2000. 57(2): p. 170-8.

ophys Acta, 2002. 1555(1-3): p. 14-20.

Cancer Biol, 2009. 19(1): p. 17-24.

*tiforme.* Oncotarget, 2010. 1(7): p. 552-62.

[82] Verhaak, R.G., et al., *Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.* Cancer Cell, 2010. 17(1): p. 98-110.

[65] Seyfried, T.N. and P. Mukherjee, *Targeting energy metabolism in brain cancer: review and*

98 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

[66] Seyfried, T.N., et al., *Metabolic management of brain cancer.* Biochim Biophys Acta,

[67] Kim, J.W., et al., *HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia.* Cell Metab, 2006. 3(3): p. 177-85.

[68] Michelakis, E.D., L. Webster, and J.R. Mackey, *Dichloroacetate (DCA) as a potential met‐*

[69] Michelakis, E.D., et al., *Metabolic modulation of glioblastoma with dichloroacetate.* Sci

[70] Bonnet, S., et al., *A mitochondria-K+channel axis is suppressed in cancer and its normaliza‐ tion promotes apoptosis and inhibits cancer growth.* Cancer Cell, 2007. 11(1): p. 37-51.

[71] Cairns, R.A., et al., *Metabolic targeting of hypoxia and HIF1 in solid tumors can enhance cytotoxic chemotherapy.* Proc Natl Acad Sci U S A, 2007. 104(22): p. 9445-50.

[72] Cao, W., et al., *Dichloroacetate (DCA) sensitizes both wild-type and over expressing Bcl-2*

[73] Wong, J.Y., et al., *Dichloroacetate induces apoptosis in endometrial cancer cells.* Gynecol

[74] Duan, Y., et al., *Antitumor activity of dichloroacetate on C6 glioma cell: in vitro and in vivo*

[75] Reitman, Z.J. and H. Yan, *Isocitrate dehydrogenase 1 and 2 mutations in cancer: alterations at a crossroads of cellular metabolism.* J Natl Cancer Inst, 2010. 102(13): p. 932-41.

[76] Kim, W. and L.M. Liau, *IDH mutations in human glioma.* Neurosurg Clin N Am, 2012.

[77] Parsons, D.W., et al., *An integrated genomic analysis of human glioblastoma multiforme.*

[78] Yan, H., et al., *IDH1 and IDH2 mutations in gliomas.* N Engl J Med, 2009. 360(8): p.

[79] Fu, Y., et al., *Glioma-derived mutations in IDH: from mechanism to potential therapy.* Bio‐

[80] DeAngelis, L.M. and I.K. Mellinghoff, *Virchow 2011 or how to ID(H) human glioblasto‐*

[81] Watanabe, T., et al., *IDH1 mutations are early events in the development of astrocytomas*

*prostate cancer cells in vitro to radiation.* Prostate, 2008. 68(11): p. 1223-31.

*abolic-targeting therapy for cancer.* Br J Cancer, 2008. 99(7): p. 989-94.

*hypothesis.* Nutr Metab (Lond), 2005. 2: p. 30.

2011. 1807(6): p. 577-94.

Transl Med, 2010. 2(31): p. 31ra34.

Oncol, 2008. 109(3): p. 394-402.

Science, 2008. 321(5897): p. 1807-12.

*ma.* J Clin Oncol, 2011. 29(34): p. 4473-4.

23(3): p. 471-80.

765-73.

*evaluation.* Onco Targets Ther, 2013. 6: p. 189-98.

chem Biophys Res Commun, 2010. 397(2): p. 127-30.

*and oligodendrogliomas.* Am J Pathol, 2009. 174(4): p. 1149-53.

[115] Colen, C.B., et al., *Metabolic remodeling of malignant gliomas for enhanced sensitization during radiotherapy: an in vitro study.* Neurosurgery, 2006. 59(6): p. 1313-23; discussion 1323-4.

[98] Abramson, H.N., *The lipogenesis pathway as a cancer target.* J Med Chem, 2011. 54(16):

100 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

[99] Gopal, K., et al., *Lipid Composition of Human Intracranial Tumors: A Biochemical Study.*

[100] Guo, D., et al., *EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy.*  J Lipid Res, 1979. 20(4): p.

[103] Rudling, M.J., et al., *Low density lipoprotein receptor activity in human intracranial tumors and its relation to the cholesterol requirement.* Cancer Res, 1990. 50(3): p. 483-7.

[104] Dhup, S., et al., *Multiple biological activities of lactic acid in cancer: influences on tumor growth, angiogenesis and metastasis.* Curr Pharm Des, 2012. 18(10): p. 1319-30.

[105] Hirschhaeuser, F., U.G. Sattler, and W. Mueller-Klieser, *Lactate: a metabolic key player*

[106] Baumann, F., et al., *Lactate promotes glioma migration by TGF-beta2-dependent regulation*

[107] Halestrap, A.P. and N.T. Price, *The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation.* Biochem J, 1999. 343 Pt 2: p. 281-99.

[108] Enerson, B.E. and L.R. Drewes, *Molecular features, regulation, and function of monocar‐ boxylate transporters: implications for drug delivery.* J Pharm Sci, 2003. 92(8): p. 1531-44.

[109] Pinheiro, C., et al., *Role of monocarboxylate transporters in human cancers: state of the art.*

[110] Froberg, M.K., et al., *Expression of monocarboxylate transporter MCT1 in normal and neo‐*

[111] Miranda-Goncalves, V., et al., *Monocarboxylate transporters (MCTs) in gliomas: expres‐ sion and exploitation as therapeutic targets.* Neuro Oncol, 2013. 15(2): p. 172-88.

[112] Fang, J., et al., *The H+-linked monocarboxylate transporter (MCT1/SLC16A1): a potential therapeutic target for high-risk neuroblastoma.* Mol Pharmacol, 2006. 70(6): p. 2108-15.

[113] Li, K.K., et al., *miR-124 is frequently down-regulated in medulloblastoma and is a negative*

[114] Colen, C.B., et al., *Metabolic targeting of lactate efflux by malignant glioma inhibits inva‐ siveness and induces necrosis: an in vivo study.* Neoplasia, 2011. 13(7): p. 620-32.

*of matrix metalloproteinase-2.* Neuro Oncol, 2009. 11(4): p. 368-80.

*plastic human CNS tissues.* Neuroreport, 2001. 12(4): p. 761-5.

*regulator of SLC16A1.* Hum Pathol, 2009. 40(9): p. 1234-43.

*magnetic resonance spectroscopy.* Biopolymers, 2001. 62(6): p. 297-306.

p. 5615-38.

428-36.

Acta Neurochir (Wien), 1963. 11: p. 333-47.

*in cancer.* Cancer Res, 2011. 71(22): p. 6921-5.

J Bioenerg Biomembr, 2012. 44(1): p. 127-39.

**Chapter 5**

**BMI Transcription Factor as a Novel Target for the Treatment of Brain Tumors**

Mohammad M. Hossain, Bárbara Meléndez, Juan A. Rey, Javier S. Castresana and Mehdi H. Shahi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59194

## **1. Introduction**

[130] McIntyre, A., et al., *Carbonic anhydrase IX promotes tumor growth and necrosis in vivo and inhibition enhances anti-VEGF therapy.* Clin Cancer Res, 2012. 18(11): p. 3100-11.

102 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

[131] De Simone, G., et al., *Carbonic anhydrase inhibitors: Hypoxia-activatable sulfonamides in‐ corporating disulfide bonds that target the tumor-associated isoform IX.* J Med Chem, 2006.

[132] Maresca, A., et al., *Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a*

[133] Maresca, A., et al., *Deciphering the mechanism of carbonic anhydrase inhibition with cou‐*

[134] Chrastina, A., S. Pastorekova, and J. Pastorek, *Immunotargeting of human cervical carci‐ noma xenograft expressing CA IX tumor-associated antigen by 125I-labeled M75 monoclonal*

[135] Siebels, M., et al., A clinical phase I/II trial with the monoclonal antibody cG250 (RENCAREX(R)) and interferon-alpha-2a in metastatic renal cell carcinoma patients.

*new class of suicide inhibitors.* J Am Chem Soc, 2009. 131(8): p. 3057-62.

*marins and thiocoumarins.* J Med Chem, 2010. 53(1): p. 335-44.

*antibody.* Neoplasma, 2003. 50(1): p. 13-21.

World J Urol, 2011. 29(1): p. 121-6.

49(18): p. 5544-51.

Recent studies have hypothesized that brain tumor stem cells (BTSCs) are responsible for the poor survival outcome of brain tumor patients. Sonic hedgehog (Shh) is one of the crucial signaling pathways to regulate stem cell self-renewable capacity. Disruption of this pathway activate Gli transcription factors, which further activate other downstream target genes including BMI1 to promote brain tumor development (medulloblastoma and glioblastoma among other tumors) (Leung *et al.,* 2004; Bruggeman *et al.,* 2007; Godlewski *et al.,* 2008). BMI1 is a polycomb complex protein also known as polycomb group RING finger protein 4 (PCGF4) or RING finger protein 51 (RNF51). BMI1 gene (B cell-specific Moloney Murin leukemia virus integration site 1) is located on human chromosome 10 (10p13). Interestingly, BMI1 showed high expression in medulloblastoma and glioblastoma (Leung *et al.,* 2004; Natsume *et al.,* 2011). Shh treatment induced both BMI1 and Gli1 expression. Gli1 overexpression also promoted high expression of BMI1. High expression of BMI1 in tumor cells indicates high capacity of self-renewing characteristics (Hemmati *et al.,* 2003). Most of the BTSCs showed high expression of BMI1. Inhibition of Gli with specific inhibitor GANT61 and Gli siRNAs mediated knock‐ down inhibited brain tumor cell proliferation and also decreased the expression of BMI1 in medulloblastoma and glioblastoma (Shahi *et al.,* unpublished). Therefore, all these studies suggest that BMI1 would be a novel therapeutic transcription factor to target BTSCs and enhance the survival of brain tumor patients.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. BMI1: An oncogene and stem cell marker**

BMI1 is one of the crucial genes for the development of various tissues including the nervous system. This gene is located at chromosome 10p13 in humans. BMI1 was initially identified as a murin leukemia viral oncogene [1]. BMI1 was the first gene reported to belong to the Polycomb-group of genes [2]. These genes express the proteins which form a large multimeric structure to silence other genes via modification in chromatin organization [3]. Polycomb repressive complexes are divided into two groups.

Polycomb repressive complex 1 (PRC1)

Polycomb repressive complex 2 (PRC2)

PRC1 includes BMI1, and PRC2 includes enhancer of zeste homolog 2 (EZHZ) to facilitate stable silencing of gene expression [4]

## **3. Role of BMI1 in normal development**

BMI1 polycomb protein is essential for the self-renewal of stem cells of different tissues of the body besides the central nervous system (CNS) [5]. One study showed that Nestin-BMI1-GFP transgenic mice cells increased the development of neural stem cell colonies and the selfrenewal capability of fetal and adult CNS cells *(He et al., 2009).* Ink4a and Arf genes encode tumor suppressor proteins p16Ink4a and p19Arf respectively, which are involved in the inhibition of cell cycle progression [6]. Interestingly, BMI1 promotes the maintenance of CNS stem cells from the embryonic stage to adulthood by suppressing Ink4a and Arf genes [5]. BMI1 is also involved in Shh signaling mediated postnatal cerebellar neurogenesis by binding to the promoter of p21waf1/cip1 [7]. Moreover, BMI1 is a crucial gene for the development of embryonic and adult stem cells and brain tumors. Interestingly, selective conditional knockout of BMI1 based on Cre/LoXP in transgenic mouse showed that BMI1 has potential to induce neural stem cells proliferation and self-renewal both *in vitro* and *in vivo* [8]. In this process BMI1 down-regulates both Ink4a/ARF and p21/FoxG1. Moreover, increased ectopic expression of BMI1 in progenitors committed to a neuronal lineage during embryonic cortical develop‐ ment, triggered apoptosis via a survivin-mediated mechanism, and caused a reduction in brain size [8]. However, the self-renewable capability of adult neural stem progenitor cells is independent of FoxG1, while apoptosis resistance of neural progenitor cells depends on high expression of BMI1 [8].

## **4. Role of BMI1 in glioblastoma development**

Gliomas are the most common brain tumors of the central nervous system comprising astrocytic gliomas, oligodendrogliomas, or a mixture of both. The most malignant glioma is glioblastoma multiforme (GBM) (WHO grade IV) comprising about 50% of glioma. Post-

therapy survival rate of GBM patients is 24% for 1 year and 12% for 2 years only [9]. There are ample amount of evidence suggesting that gliomas have stem-like cells called glioma initiating cells (GICs). These cells have self-renewal capability and cause gliomagenesis. The GICs are under the regulation of several signaling pathways including Shh, Notch, Wnt and BMI1. Effective targeting of GICs could become a novel strategy to target glioma [10]. The 10p13 region is highly significant in brain tumorigenesis especially glioblastoma. BMI1 is part of the polycomb repressive complex 1, which epigenetically regulates gene expression by acetyla‐ tion, methylation, and mono-ubiquitination of histones [11]. These modifications cause transcriptional repression of differentiation and pluripotency of embryonic stem cells [12]. BMI1 is essential for the proliferation and transformation of primary glial cells. BMI1 deficient glial cells have less proliferating and tumorigenic capacities. BMI1 is also involved in pathways including proliferation, adhesion, and differentiation. All these pathways play a significant contribution to stem cell renewal and glioblastoma development [13]. One report demonstrates a high copy number of BMI1 in glioma samples [11]. Even, a murine tumor model showed a role for BMI1 in the genesis of glioma and high fold expression of BMI1 in high-grade gliomas [13]. BMI1 shows functional diversity in different cell types including embryonic stem cells and mature neurons [12]. Moreover, BMI1 sometimes behaves as a tumor suppressor gene or oncogene in different tumors. According to one study most of the glioma samples showed BMI1 gene allelic imbalance. BMI1 negatively regulates p16 in astrocytoma. However, they also suggest that BMI1 is not very significant for prognosis of astrocytic tumors [2]. Interest‐ ingly, BMI1 is considered as a transgene, which helps MYC to promote hematopoietic malignancies [14-16]. High expression of BMI1 has been noted in brain tumors irrespective of tumor grades [5]. BMI1 has a role in glioma and glioma stem cell growth which is both dependent and independent of InK4a-Arf [13]. GBM showed an association of BMI1 overex‐ pression and enrichment in CD133+ cells [Xia *et al.,* 2012]. Stable knockdown of BMI1 in GBM reveals that BMI1 prevented the clonogenic nature of GBM cells and also these cells were not able to develop brain tumors *in vivo.* Accordingly, BMI1 is a potent inhibitor of apoptosis in CD133+ cells and of differentiation into neurons and astrocytes [17]. BMI1 is also capable to inhibit alternate tumor suppressor pathways that attempt to compensate for INK4A-ARF/p53 deletion and hyperactivity of the PI3K/AKT pathway [17]. Interestingly, one study suggests that BMI1 causes apoptotic resistance to glioma cells through the activation of IKK-Nuclear factor-kB pathway and could therefore be a good prognostic factor for glioma [20]. IKK-Nuclear factor-kB pathway is very active in high grade GBM and glioma cell lines [18, 19]. High expression of BMI1 protects brain tumor cells from cytotoxic reagents-induced apoptosis, while attenuated BMI1 expression promotes apoptosis inducer factors. BMI1 also controls apoptosis in glioma cell lines by activation of the IKK-NF-kB pathway and promoting antiapoptotic genes [20]. Co-expression of BMI1 and p65 protein, a subunit of NF-kB in glioma is in favour of BMI1 and NF-kB pathways involvement in glioma chemoresistance. GBM is resistant to all types of therapies. It has been postulated that most CD133+ cells are resistant to gamma radiation via activation of DNA double-strand break (DSB) response mechanism, including the participation of the ataxia-telangiectasia-mutated kinase gene (ATM). After purification of BMI1 with DNA DSB responsive factors and nonhomologous end joining (NHEJ) repair proteins in GBM cells [21], a BMI1 enrichment was observed after irradiation,

**2. BMI1: An oncogene and stem cell marker**

repressive complexes are divided into two groups.

**3. Role of BMI1 in normal development**

**4. Role of BMI1 in glioblastoma development**

Polycomb repressive complex 1 (PRC1) Polycomb repressive complex 2 (PRC2)

stable silencing of gene expression [4]

expression of BMI1 [8].

BMI1 is one of the crucial genes for the development of various tissues including the nervous system. This gene is located at chromosome 10p13 in humans. BMI1 was initially identified as a murin leukemia viral oncogene [1]. BMI1 was the first gene reported to belong to the Polycomb-group of genes [2]. These genes express the proteins which form a large multimeric structure to silence other genes via modification in chromatin organization [3]. Polycomb

104 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

PRC1 includes BMI1, and PRC2 includes enhancer of zeste homolog 2 (EZHZ) to facilitate

BMI1 polycomb protein is essential for the self-renewal of stem cells of different tissues of the body besides the central nervous system (CNS) [5]. One study showed that Nestin-BMI1-GFP transgenic mice cells increased the development of neural stem cell colonies and the selfrenewal capability of fetal and adult CNS cells *(He et al., 2009).* Ink4a and Arf genes encode tumor suppressor proteins p16Ink4a and p19Arf respectively, which are involved in the inhibition of cell cycle progression [6]. Interestingly, BMI1 promotes the maintenance of CNS stem cells from the embryonic stage to adulthood by suppressing Ink4a and Arf genes [5]. BMI1 is also involved in Shh signaling mediated postnatal cerebellar neurogenesis by binding to the promoter of p21waf1/cip1 [7]. Moreover, BMI1 is a crucial gene for the development of embryonic and adult stem cells and brain tumors. Interestingly, selective conditional knockout of BMI1 based on Cre/LoXP in transgenic mouse showed that BMI1 has potential to induce neural stem cells proliferation and self-renewal both *in vitro* and *in vivo* [8]. In this process BMI1 down-regulates both Ink4a/ARF and p21/FoxG1. Moreover, increased ectopic expression of BMI1 in progenitors committed to a neuronal lineage during embryonic cortical develop‐ ment, triggered apoptosis via a survivin-mediated mechanism, and caused a reduction in brain size [8]. However, the self-renewable capability of adult neural stem progenitor cells is independent of FoxG1, while apoptosis resistance of neural progenitor cells depends on high

Gliomas are the most common brain tumors of the central nervous system comprising astrocytic gliomas, oligodendrogliomas, or a mixture of both. The most malignant glioma is glioblastoma multiforme (GBM) (WHO grade IV) comprising about 50% of glioma. Postbeing colocalized and co-purified with ATM and histone gammaH2AX. Deficient BMI1 glioma cells showed inactive DNA DSB response, which promoted sensitivity of radiation in glioma cells. Overexpressed BMI1 modulated the neural stem cells radiation resistance by enhancing the ATM activity. Therefore, a combined effect of BMI1 inhibition together with radiation therapy might efficiently target GBM stem cells [21]. Interestingly, a recent report suggested that expression of BMI1 and c-Myc correlated in glioma. This finding further reveals that c-Myc, either directly or indirectly, activates several epigenetic modulators including acetylase GCN5 and polycomb-group (PcG) gene BMI1 [22, 23]. It is interesting to know that BMI1 promoter contains functional E-box and c-Myc binding regions [24, 25]. Moreover, c-Myc was able to activate BMI1; and BMI1 further facilitated the oncogenic expression of c-Myc in glioblastoma development [17]. It has been speculated that c-Myc and BMI1 might be good biomarkers of glioblastoma due to their high expression and involvement in gliomagenesis. Inhibition of both genes could give glioblastoma a greater sensitivity to combined anticancer therapy. A recent study revealed that Tamoxifen (TAM) has the ability to reduce the expression of neural stem cell markers, like Nestin, Bmi1 and Vimentin, in glioma cell lines. Moreover, the action of TAM in glioma cells apotosis is assisted by prostate apoptosis response-4(par-4) [26]. It has been published that AR-A 014418 inhibits GSK3 beta kinase activation which further regulates the cancer escape pathway via downregulation of anti-apoptotic gene BMI1 [27].

#### **5. BMI1 and miRNAs**

Low expression of miRNA-128 was reported in GBM samples. High expression of miRNA-128 in GBM down-regulates ARP5 (ANGPTL 6), BMI1 and E2F-3a, factors which are key regulator for brain cell proliferation [28]. miRNA-128 also targets BMI1 and down-regulates its expres‐ sion. Most of the glioma samples show less expression of miRNA-128. Overexpression of miRNA-128 shows inhibition of GICs proliferation and self-renewable capacity [10]. Normal brain has abundant expression of miRNA-124, however, miRNA-124 expression is diminished during the development of glioma. Interestingly, overexpression of miRNA-124 reduced the formation of neurospheres, the CD133+ cell subpopulation, and the expression of stem cells markers like BMI1, Nanog and Nestin [29]. Smoothened inhibitor NPV-LDE-225 (Erismode‐ gib) inhibits BMI1 in GICs via upregulation of miRNA-128, miRNA-21 and miRNA-200 [30]. Interestingly, NPV-LDE-225 was used in topical cream for basal cell carcinoma treatment and it inhibited the Shh pathway [31]. A recent study suggested that NPV-LDE-225 mediated inhibition of Shh signaling downregulates Bmi1 via upregulation of miRNA-128 [30]. Another study also suggested that polycomb repressor complex BMI1 is targeted by miRNA-128 in glioma stem cells [32]. Another miRNA-218 also inhibited the expression of BMI1 which further retarded glioma invasion, migration, and glioma stem cell renewal capability. Most gliomas showed less expression of miRNA-218 and overexpression of miRNA-128, which further inhibited glioma tumor characteristics. It was assumed that BMI1 is the downstream target gene of miRNA-218. miRNA-218 regulates many genes which are involved in glioma tumorigenesis [33]. Interestingly, we illustrate a model diagram for the role of BMI1 in gliomagenesis (Figure 1).

**Figure 1.** BMI1 networking in gliomagenesis

being colocalized and co-purified with ATM and histone gammaH2AX. Deficient BMI1 glioma cells showed inactive DNA DSB response, which promoted sensitivity of radiation in glioma cells. Overexpressed BMI1 modulated the neural stem cells radiation resistance by enhancing the ATM activity. Therefore, a combined effect of BMI1 inhibition together with radiation therapy might efficiently target GBM stem cells [21]. Interestingly, a recent report suggested that expression of BMI1 and c-Myc correlated in glioma. This finding further reveals that c-Myc, either directly or indirectly, activates several epigenetic modulators including acetylase GCN5 and polycomb-group (PcG) gene BMI1 [22, 23]. It is interesting to know that BMI1 promoter contains functional E-box and c-Myc binding regions [24, 25]. Moreover, c-Myc was able to activate BMI1; and BMI1 further facilitated the oncogenic expression of c-Myc in glioblastoma development [17]. It has been speculated that c-Myc and BMI1 might be good biomarkers of glioblastoma due to their high expression and involvement in gliomagenesis. Inhibition of both genes could give glioblastoma a greater sensitivity to combined anticancer therapy. A recent study revealed that Tamoxifen (TAM) has the ability to reduce the expression of neural stem cell markers, like Nestin, Bmi1 and Vimentin, in glioma cell lines. Moreover, the action of TAM in glioma cells apotosis is assisted by prostate apoptosis response-4(par-4) [26]. It has been published that AR-A 014418 inhibits GSK3 beta kinase activation which further regulates the cancer escape pathway via downregulation of anti-apoptotic gene BMI1 [27].

106 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

Low expression of miRNA-128 was reported in GBM samples. High expression of miRNA-128 in GBM down-regulates ARP5 (ANGPTL 6), BMI1 and E2F-3a, factors which are key regulator for brain cell proliferation [28]. miRNA-128 also targets BMI1 and down-regulates its expres‐ sion. Most of the glioma samples show less expression of miRNA-128. Overexpression of miRNA-128 shows inhibition of GICs proliferation and self-renewable capacity [10]. Normal brain has abundant expression of miRNA-124, however, miRNA-124 expression is diminished during the development of glioma. Interestingly, overexpression of miRNA-124 reduced the formation of neurospheres, the CD133+ cell subpopulation, and the expression of stem cells markers like BMI1, Nanog and Nestin [29]. Smoothened inhibitor NPV-LDE-225 (Erismode‐ gib) inhibits BMI1 in GICs via upregulation of miRNA-128, miRNA-21 and miRNA-200 [30]. Interestingly, NPV-LDE-225 was used in topical cream for basal cell carcinoma treatment and it inhibited the Shh pathway [31]. A recent study suggested that NPV-LDE-225 mediated inhibition of Shh signaling downregulates Bmi1 via upregulation of miRNA-128 [30]. Another study also suggested that polycomb repressor complex BMI1 is targeted by miRNA-128 in glioma stem cells [32]. Another miRNA-218 also inhibited the expression of BMI1 which further retarded glioma invasion, migration, and glioma stem cell renewal capability. Most gliomas showed less expression of miRNA-218 and overexpression of miRNA-128, which further inhibited glioma tumor characteristics. It was assumed that BMI1 is the downstream target gene of miRNA-218. miRNA-218 regulates many genes which are involved in glioma tumorigenesis [33]. Interestingly, we illustrate a model diagram for the role of BMI1 in

**5. BMI1 and miRNAs**

gliomagenesis (Figure 1).

## **6. Role of BMI1 in medulloblastoma**

Medulloblastoma is a primitive neuroectodermal tumor of the cerebellum. Medulloblastoma is the most common pediatric malignant brain tumor, representing 20% of newly identified CNS tumors in children [34]. Medulloblastoma is driven by several signaling pathways, among which, Shh plays a critical role for the majority of these tumors [35, 36]. Mutations of the Shh pathway regulators are present in about 20-25% of medulloblastomas [37]. Shh-driven medulloblastoma showed high expression of BMI1. A recent study reveals the role of BMI1 in Shh-driven medulloblastoma. Transgenic mice showed the expression of Shh signaling activator SmoA1 with the help of glial fibrillary acidic protein (GFAP) promoter. They found that SmoA1/BMI1+/+; SmoA1/BMI1+/-postnatal mice (p=26) between days P14 to P26 showed prominent potential to develop medulloblastoma compared to SmoA1/BMI1-/-post natal mice (n=6) [38]. Interestingly, cells with BMI1 deficiency BMI1-/- even in the presence of SmoA1 were non-proliferative compared to BMI1+/+ cells [38]. Two down-stream genes which are inversely regulated by BMI1 in Shh-driven medulloblastoma development were reported. Cyclin D1 expression was downregulated and cyclin-dependent kinase inhibitor p19Arf was upregulated. Moreover, it was concluded that BMI1 is crucial for the Shh-driven medullo‐ blastoma development and that BMI1 facilitates medulloblastoma development de novo [38]. During embryonic development Shh signaling pathway regulates proliferation of granular neuron precursors (GNPs). GNPs are progenitors for medulloblastoma development, com‐ prising the transient external granular layer of cerebellum [39]. BMI1 seems to promote the expression of downstream target genes during cerebellum development [40]. BMI1-null mice developed reduced cerebellum and impaired production of granular neurons. Altered expression of BMI1 in medulloblastoma as well as correlation of BMI1 expression and Shh activation was reported in medulloblastoma [38, 41]. It was suggested that polycomb gene expression could be used as a predictor of poor clinical outcome in medulloblastoma [42]. BMI1 overexpression caused cell proliferation and assisted Shh signaling driven tumorigenesis [43]. BMI1 expression and Shh ligand concentration were positively correlated during develop‐ ment. Chromatin immunoprecipitation experiments revealed that Shh signaling pathway main transcriptional activator Gli1 preferentially binds to the promoter regions of BMI1. Moreover, overexpression and downregulation of Gli1 controls high and low expression of BMI1 respectively. Interestingly, BMI1 is not only a Shh-Gli1 downstream target gene but also promotes a feedback mechanism which further activates Shh-Gli1 signaling. This finding suggested that both BMI1 and Shh signaling pathways are mutually indispensable pathways in brain tumor initiating cells (BTICs) of medulloblastoma [43]. One study reported that overexpressed BMI1 was unable to induce tumors in mice from granule cell progenitors (GCPs). Therefore, it was concluded that overexpression of BMI1 in GCP-derived human medulloblastoma, could promote later stages of tumorigenesis and further sustain tumor cell survival [44]. Apart from cell proliferation other characteristic of tumors include anti-apoptotic nature and sustaining of high metabolic rate, both supported by high BMI1 and low TP53 levels of expression which are characteristic of group 4 human medulloblastoma [44]. BMI1 overex‐ pression alone was not sufficient to induce medulloblastoma; however BMI1 overexpression and loss of p53 induced medulloblastoma in mice, producing similar tumors to group 4 human medulloblastomas [44]. Recently, medulloblastomas have been categorized in 4 subgroups on the basis of prognosis and predicted therapeutics (Kool et al., 2012, Ramaswamy et al., 2013 and Gottardo et al., 2014). Group 1 and group 2 are under the good clinical outcome and regulated by Shh and Wnt signaling, respectively [45]. Groups 3 and 4 medulloblastoma are not Shh/Wnt signaling mediated tumors, have metastatic potencial, and poor patient outcome and lack of known molecular pathways. Current gene expression analysis is unable to detect self-renewal gene and brain tumor-initiating cells (BTIC) in group 3 and group 4 medullo‐ blastoma. BTICs constitute a minority of the tumor mass, and their detection can be difficult in medulloblastoma. High BTIC promoted tumors to increase tumor aggressiveness and poor patient outcome. They investigated the potential stem cells candidate genes among the different subgroups of 251 human medulloblastoma samples from the 4 overlapping MB transcriptional data bases (Amsterdam, Memphis, Toronto and Boston) and 74 nano-string sub-grouped medulloblastoma (Vancouver) [45]. This analysis showed two crucial genes BMI1 and FoxG1, which presented abundant expression in non-Shh/wnt medulloblastoma groups. These genes are responsible to promote MB stem cells and tumor initiation in mice [45]. We also depicted a model for the different group of medulloblastoma development on the basis of cell signaling pathways and gene expression profile (Figure 3). This study also identified a reciprocal promoter in CD15+ medulloblastoma stem cells. The finding could be used as a novel target therapy against BTIC self-renewal. They also found BMI1 is a downstream target of FoxG1 and further promotes tumorigenicity. BMI1 also exerts feedback to FoxG1 expression and facilitates *in vivo* tumor malignancy and enhances in vitro stem cell self-renewal capability. Moreover, high expression of BMI1 can be considered as a strong molecular prognostic marker in pediatric brain tumors. We attempted to show a model for the role of BMI1 in medullo‐ blastoma development (Figure 2).

BMI Transcription Factor as a Novel Target for the Treatment of Brain Tumors http://dx.doi.org/10.5772/59194 109

**Figure 2.** BMI1 networking in medulloblastoma development

expression of BMI1 in medulloblastoma as well as correlation of BMI1 expression and Shh activation was reported in medulloblastoma [38, 41]. It was suggested that polycomb gene expression could be used as a predictor of poor clinical outcome in medulloblastoma [42]. BMI1 overexpression caused cell proliferation and assisted Shh signaling driven tumorigenesis [43]. BMI1 expression and Shh ligand concentration were positively correlated during develop‐ ment. Chromatin immunoprecipitation experiments revealed that Shh signaling pathway main transcriptional activator Gli1 preferentially binds to the promoter regions of BMI1. Moreover, overexpression and downregulation of Gli1 controls high and low expression of BMI1 respectively. Interestingly, BMI1 is not only a Shh-Gli1 downstream target gene but also promotes a feedback mechanism which further activates Shh-Gli1 signaling. This finding suggested that both BMI1 and Shh signaling pathways are mutually indispensable pathways in brain tumor initiating cells (BTICs) of medulloblastoma [43]. One study reported that overexpressed BMI1 was unable to induce tumors in mice from granule cell progenitors (GCPs). Therefore, it was concluded that overexpression of BMI1 in GCP-derived human medulloblastoma, could promote later stages of tumorigenesis and further sustain tumor cell survival [44]. Apart from cell proliferation other characteristic of tumors include anti-apoptotic nature and sustaining of high metabolic rate, both supported by high BMI1 and low TP53 levels of expression which are characteristic of group 4 human medulloblastoma [44]. BMI1 overex‐ pression alone was not sufficient to induce medulloblastoma; however BMI1 overexpression and loss of p53 induced medulloblastoma in mice, producing similar tumors to group 4 human medulloblastomas [44]. Recently, medulloblastomas have been categorized in 4 subgroups on the basis of prognosis and predicted therapeutics (Kool et al., 2012, Ramaswamy et al., 2013 and Gottardo et al., 2014). Group 1 and group 2 are under the good clinical outcome and regulated by Shh and Wnt signaling, respectively [45]. Groups 3 and 4 medulloblastoma are not Shh/Wnt signaling mediated tumors, have metastatic potencial, and poor patient outcome and lack of known molecular pathways. Current gene expression analysis is unable to detect self-renewal gene and brain tumor-initiating cells (BTIC) in group 3 and group 4 medullo‐ blastoma. BTICs constitute a minority of the tumor mass, and their detection can be difficult in medulloblastoma. High BTIC promoted tumors to increase tumor aggressiveness and poor patient outcome. They investigated the potential stem cells candidate genes among the different subgroups of 251 human medulloblastoma samples from the 4 overlapping MB transcriptional data bases (Amsterdam, Memphis, Toronto and Boston) and 74 nano-string sub-grouped medulloblastoma (Vancouver) [45]. This analysis showed two crucial genes BMI1 and FoxG1, which presented abundant expression in non-Shh/wnt medulloblastoma groups. These genes are responsible to promote MB stem cells and tumor initiation in mice [45]. We also depicted a model for the different group of medulloblastoma development on the basis of cell signaling pathways and gene expression profile (Figure 3). This study also identified a reciprocal promoter in CD15+ medulloblastoma stem cells. The finding could be used as a novel target therapy against BTIC self-renewal. They also found BMI1 is a downstream target of FoxG1 and further promotes tumorigenicity. BMI1 also exerts feedback to FoxG1 expression and facilitates *in vivo* tumor malignancy and enhances in vitro stem cell self-renewal capability. Moreover, high expression of BMI1 can be considered as a strong molecular prognostic marker in pediatric brain tumors. We attempted to show a model for the role of BMI1 in medullo‐

108 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

blastoma development (Figure 2).

**Figure 3.** Division of medulloblastoma on the basis of gene and cell signaling expression

## **7. Conclusion**

BMI1 is a very significant stem cell marker gene which contributes to the development of glioblastoma and medulloblastoma. Therefore, the treatment of glioblastoma and medullo‐ blastoma would improve with the addition of BMI1 inhibitors. Moreover, high expression of BMI1 could be used as one of the earliest markers to diagnose brain tumors.

## **Acknowledgements**

Authors are thankful to all members of Interdisciplinary Brain Research Centre, Aligarh Muslim University, Aligarh, India for their constant supports.

## **Author details**

Mohammad M. Hossain1 , Bárbara Meléndez2 , Juan A. Rey3 , Javier S. Castresana4 and Mehdi H. Shahi1\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Interdisciplinary Brain Research Centre, Faculty of Medicine, Aligarh Muslim University,Aligarh, , India

2 Molecular Pathology Research Unit, Virgen de la Salud Hospital, Toledo, Spain

3 IdiPaz Research Unit, La Paz University Hospital, Madrid, Spain

4 Department of Biochemistry and Genetics, University of Navarra School of Sciences, Pamplona, Spain

## **References**

[4] Dellino, G.I., et al., *Polycomb silencing blocks transcription initiation*. Mol Cell, 2004. 13(6): p. 887-93.

**7. Conclusion**

**Acknowledgements**

**Author details**

Mehdi H. Shahi1\*

Pamplona, Spain

**References**

Mohammad M. Hossain1

University,Aligarh, , India

204(9): p. 625-31.

BMI1 is a very significant stem cell marker gene which contributes to the development of glioblastoma and medulloblastoma. Therefore, the treatment of glioblastoma and medullo‐ blastoma would improve with the addition of BMI1 inhibitors. Moreover, high expression of

110 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

Authors are thankful to all members of Interdisciplinary Brain Research Centre, Aligarh

, Juan A. Rey3

, Javier S. Castresana4

and

BMI1 could be used as one of the earliest markers to diagnose brain tumors.

Muslim University, Aligarh, India for their constant supports.

\*Address all correspondence to<EMAIL_ADDRESS>
, Bárbara Meléndez2

3 IdiPaz Research Unit, La Paz University Hospital, Madrid, Spain

*cancer.* Nat Rev Cancer, 2006. 6(11): p. 846-56.

1 Interdisciplinary Brain Research Centre, Faculty of Medicine, Aligarh Muslim

2 Molecular Pathology Research Unit, Virgen de la Salud Hospital, Toledo, Spain

4 Department of Biochemistry and Genetics, University of Navarra School of Sciences,

*and senescence through the ink4a locus*. Nature, 1999. 397(6715): p. 164-8.

[1] Jacobs, J.J., et al., *The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation*

[2] Tirabosco, R., et al*., Expression of the Polycomb-Group protein BMI1 and correlation with p16 in astrocytomas an immunohistochemical study on 80 cases*. Pathol Res Pract, 2008.

[3] Sparmann, A. and M. van Lohuizen, *Polycomb silencers control cell fate, development and*

[34] Packer, R.J., et al., *Medulloblastoma: clinical and biologic aspects*. Neuro Oncol, 1999. 1(3): p. 232-50.

[19] Nagai, S., et al., *Aberrant nuclear factor-kappaB activity and its participation in the growth*

[20] Li, J., et al., *Oncoprotein Bmi-1 renders apoptotic resistance to glioma cells through activa‐ tion of the IKK-nuclear factor-kappaB Pathway*. Am J Pathol, 2010. 176(2): p. 699-709.

[21] Facchino, S., et al., *BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery*. J Neurosci, 2010. 30(30): p.

[22] Cole, M.D. and V.H. Cowling, *Transcription-independent functions of MYC: regulation of translation and DNA replication*. Nat Rev Mol Cell Biol, 2008. 9(10): p. 810-5.

[23] Martinato, F., et al., *Analysis of Myc-induced histone modifications on target chromatin*.

[24] Guney, I., S. Wu, and J.M. Sedivy, *Reduced c-Myc signaling triggers telomere-independ‐ ent senescence by regulating Bmi-1 and p16(INK4a)*. Proc Natl Acad Sci U S A, 2006.

[25] Guo, W.J., et al., *Mel-18, a polycomb group protein, regulates cell proliferation and senes‐ cence via transcriptional repression of Bmi-1 and c-Myc oncoproteins*. Mol Biol Cell, 2007.

[26] Jagtap, J.C., et al., *Expression and regulation of prostate apoptosis response-4 (Par-4) in hu‐ man glioma stem cells in drug-induced apoptosis*. PLoS One, 2014. 9(2): p. e88505.

[27] Yadav, A.K., et al., *AR-A 014418 Used against GSK3beta Downregulates Expression of*

[28] Cui, J.G., et al., *Micro-RNA-128 (miRNA-128) down-regulation in glioblastoma targets ARP5 (ANGPTL6), Bmi-1 and E2F-3a, key regulators of brain cell proliferation*. J Neuroon‐

[29] Xia, H., et al., *Loss of brain-enriched miR-124 microRNA enhances stem-like traits and in‐*

[30] Fu, J. and W. Hsu, *Epidermal Wnt controls hair follicle induction by orchestrating dynamic signaling crosstalk between the epidermis and dermis*. J Invest Dermatol, 2013. 133(4): p.

[31] Skvara, H., et al., *Topical treatment of Basal cell carcinomas in nevoid Basal cell carcinoma syndrome with a smoothened inhibitor*. J Invest Dermatol, 2011. 131(8): p. 1735-44.

[32] Peruzzi, P., et al., *MicroRNA-128 coordinately targets Polycomb Repressor Complexes in*

[33] Tu, Y., et al., *MicroRNA-218 inhibits glioma invasion, migration, proliferation, and cancer stem-like cell self-renewal by targeting the polycomb group gene Bmi1*. Cancer Res, 2013.

*hnRNPA1 and SF2/ASF Splicing Factors.* J Oncol, 2014. 2014: p. 695325.

*vasiveness of glioma cells.* J Biol Chem, 2012. 287(13): p. 9962-71.

*glioma stem cells*. Neuro Oncol, 2013. 15(9): p. 1212-24.

*of human malignant astrocytoma*. J Neurosurg, 2002. 96(5): p. 909-17.

112 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

10096-111.

PLoS One, 2008. 3(11): p. e3650.

103(10): p. 3645-50.

18(2): p. 536-46.

890-8.

73(19): p. 6046-55.

col, 2010. 98(3): p. 297-304.

## **Metalloproteinases in Brain Tumors**

Krzysztof Siemianowicz, Wirginia Likus and Jarosław Markowski

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58964

## **1. Introduction**

Metalloproteinases (MMPs) were first described by Gross more than fifty years ago. They are a family of zinc-dependent endopeptidases. They comprise a group of 25 enzymes. MMPs were first described as proteases degrading extracellular matrix (ECM) proteins such as collagens, elastin, proteoglycans and laminins, hence they were named matrix metalloprotei‐ nases. MMPs were divided according to their substrate specificity into collagenases, gelati‐ nases, stromolysins and matrilysins. This classification was later replaced by numbering the enzymes according to the chronology of their identification.

Four metalloproteinases (MMP-14, MMP-15, MMP-16 and MMP-24) have a transmembrane and cytosolic domains. They constitute a subgroup of membrane-type metalloproteinases (MT-MMPs). Recently an intracellular, nuclear localization and functions of metalloprotei‐ nases have been discovered [1-4].

## **2. Physiological role of metalloproteinases**

MMP-1 (collagenase 1) hydrolyzes collagen types I, II, III, VII, VIII, X and XI, as well as gelatin, fibronectin, vitronectin, laminin, tenascin, aggrecan, links protein, myelin basic protein and versican. MMP-2 (gellatinase) degrades collagen types I, II, III, IV, V, VII, X and XI, gelatin, elastin, fibronectin, vitronectin, laminin, entactin, tenascin, SPARC and aggrecan, links protein, galectin-3, versican, decanin and myelin basic protein. One of the most important differences between these two metalloproteinases is the possibility of the hydrolysis of elastin and collagen type IV by MMP-2, but not by MMP-1. Researchers have also focused their interest

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

on MMP-9 which can degrade collagen types IV, V, VII, X and XIV, fibronectin, laminin, nidogen, proteoglycan link protein and versican.

For a long time metalloproteinases have been viewed solely as enzymes of matrix proteins breakdown. Results of researches performed in recent years indicate that there is a group of non-matrix proteins which can be substrates for various MMPs. Metalloproteinases are involved in the activation of latent forms of effective proteins. For example, MMP-2, MMP-3 and MMP-9 can activate interleukin 1β (IL-1β). They can also act on active cytokines, IL-1β undergoes subsequent degradation catalyzed by MMP-3. Metalloproteinases can alter cell surface proteins such as receptors and act on microbial peptides.

Metalloproteinases are not indiscriminately released by cells. They are secreted to or anchored to cell membrane. MT-MMPs have a specific transmembrane domain placing them in a certain position. Other metalloproteinases can be bound by specific cell-MMP interactions. This phenomenon allows an exact localization of their proteolytic activity [1-3].

## **3. Activation of metalloproteinases**

Metalloproteinases are encoded as inactive proenzymes, zymogens. They undergo proteolytic activation. This process can take place either intracellulary or extracellulary. One third of MMPs are activated by intracellular serine protease, furin. This process takes place in trans-Golgi network. A number of MMPs has a cleavage site for other metalloproteinases. MMP-3 activates proMMP-1 and pro-MMP-7. Some metalloproteinases have been described to be activated by kallikrein or plasmin.

*In vivo* studies indicate that reactive oxygen species (ROS) generated by neutrophils can both activate and subsequently inactivate MMPs. Hypochlorus acid (HClO) generated by neutro‐ phil myeloperoxidase and hydroxyl radicals can activate proMMP-1, proMMP-7 and proMMP-9, whereas peroxynitrate can activate proenzymes of MMP-1, MMP-2 and MMP-9. This process enables a control of burst of proteolytic activity within an inflammatory setting.

Like some other proteases, activity of MMPs is controlled also by two other mechanisms, regulation of gene expression and specific inhibitors. MMP-2 is constitutively expressed and regulation of its activity occurs by either activation or inhibition. Expression of a number of metalloproteinases is up-regulated during various pathological conditions. Among them inflammation is the most studied setting. MMPs are inhibited by α-2 macroglobulin and tissue inhibitors of metalloproteinases (TIMPs). There are four TIMPs. Their secretion is also regulated and represents another point in a network of control of the activity of metallopro‐ teinases. TIMP-3 is primarily bond to ECM and allows a regulation of MMPs' activity in the very site of their action. The network of the control of the activity of metalloproteinases is complex and very precise. Sometimes TIMP interacts with proMMP and inactivate other MMP, e.g. a complex of TIMP-1 and proMMP-9 inactivates MMP-3.

Protection from MMP degradation represents the next step in this sophisticated network of diverse interactions. Neutrophil gelatinase-associated lipocalin (NGAL) bounds to MMP-9 protecting this metalloproteinase from its degradation [1-3].

## **4. Metalloproteinases in central nervous system**

on MMP-9 which can degrade collagen types IV, V, VII, X and XIV, fibronectin, laminin,

116 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

For a long time metalloproteinases have been viewed solely as enzymes of matrix proteins breakdown. Results of researches performed in recent years indicate that there is a group of non-matrix proteins which can be substrates for various MMPs. Metalloproteinases are involved in the activation of latent forms of effective proteins. For example, MMP-2, MMP-3 and MMP-9 can activate interleukin 1β (IL-1β). They can also act on active cytokines, IL-1β undergoes subsequent degradation catalyzed by MMP-3. Metalloproteinases can alter cell

Metalloproteinases are not indiscriminately released by cells. They are secreted to or anchored to cell membrane. MT-MMPs have a specific transmembrane domain placing them in a certain position. Other metalloproteinases can be bound by specific cell-MMP interactions. This

Metalloproteinases are encoded as inactive proenzymes, zymogens. They undergo proteolytic activation. This process can take place either intracellulary or extracellulary. One third of MMPs are activated by intracellular serine protease, furin. This process takes place in trans-Golgi network. A number of MMPs has a cleavage site for other metalloproteinases. MMP-3 activates proMMP-1 and pro-MMP-7. Some metalloproteinases have been described to be

*In vivo* studies indicate that reactive oxygen species (ROS) generated by neutrophils can both activate and subsequently inactivate MMPs. Hypochlorus acid (HClO) generated by neutro‐ phil myeloperoxidase and hydroxyl radicals can activate proMMP-1, proMMP-7 and proMMP-9, whereas peroxynitrate can activate proenzymes of MMP-1, MMP-2 and MMP-9. This process enables a control of burst of proteolytic activity within an inflammatory setting. Like some other proteases, activity of MMPs is controlled also by two other mechanisms, regulation of gene expression and specific inhibitors. MMP-2 is constitutively expressed and regulation of its activity occurs by either activation or inhibition. Expression of a number of metalloproteinases is up-regulated during various pathological conditions. Among them inflammation is the most studied setting. MMPs are inhibited by α-2 macroglobulin and tissue inhibitors of metalloproteinases (TIMPs). There are four TIMPs. Their secretion is also regulated and represents another point in a network of control of the activity of metallopro‐ teinases. TIMP-3 is primarily bond to ECM and allows a regulation of MMPs' activity in the very site of their action. The network of the control of the activity of metalloproteinases is complex and very precise. Sometimes TIMP interacts with proMMP and inactivate other MMP,

Protection from MMP degradation represents the next step in this sophisticated network of diverse interactions. Neutrophil gelatinase-associated lipocalin (NGAL) bounds to MMP-9

nidogen, proteoglycan link protein and versican.

**3. Activation of metalloproteinases**

activated by kallikrein or plasmin.

surface proteins such as receptors and act on microbial peptides.

e.g. a complex of TIMP-1 and proMMP-9 inactivates MMP-3.

protecting this metalloproteinase from its degradation [1-3].

phenomenon allows an exact localization of their proteolytic activity [1-3].

Metalloproteinases in central nervous system can be produced by cells constituting it, by cells of blood vessels' wall or by blood cells. The production of MMPs in central nervous system under normal conditions is low, however it can be augmented in several neoplastic and nonneoplastic conditions. The expression of MMP-14 (MT1-MMP) in microglia is very low under physiological conditions. It can be increased in neurodegenerative and neuroinflammatory pathologies, e.g. Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS) or even in a stroke. Astrocytes were reported to secrete MMP-2 and MMP-9 [5,6].

For a long time MMPs were thought to be enzymes acting exclusively in extracellular compart‐ ment. Studies carried in last few years have revealed nontraditional roles for MMPs in extracellular space as well as inthe cytosol andnucleus.MMP-2 andMMP-9 which were largely studied in central nervous system have been shown to present an increased activity in cortex neuronal nuclei after focal cerebral ischemia. These two MMPs, MMP-2 and MMP-9, are also termed gelatinase A and gelatinase B. The increased gelanolytic activity in nucleus occurs to be linked with MMP-dependent cell death triggering neuroinflammatory reactions. MMP-13, named also collagenase-3, was found to be activated mostly in neurons and oligodendro‐ cytes. Its function in cell nucleus may be linked to the apoptosis cascade following ischemic stimulus. MMPs localized in cell nucleus can have a different set of target proteins than MMPs acting in extracellular space. Poly-ADP-ribose polymerase-1 (PARP-1) and X-ray crosscomplementary factor-1 (XRCC-1) can be the substrates for MMP-dependent cleavage [4].

## **5. Metalloproteinases in brain tumors**

Gliomas are the most common malignant tumors in the brain, and the overall prognosis for patients suffering from this neoplasm is poor. Glioblastoma multiforme (GBM) is the most aggressive type of glioma. Molecular mechanisms of invasiveness of this neoplasm have been most widely studied. Many factors are involved in the migration and invasiveness of GBM. MMPs have gained a large interest of researchers. The role of MMPs is significant in the degradation of ECM, thereby facilitating tumor cell invasion into surrounding stroma. Neoplastic cell invades in three steps. The first one is the attachment of tumor cell to the basement membrane through binding of neoplastic cell receptors to the basement membrane receptors. The next one is the secretion of hydrolytic enzymes, MMPs, which locally degrade ECM by extracellular proteolysis. The third step comprises the movement of the tumor cell to the free space obtained by degradation of ECM. MMP-13 is involved in the initiation of progression of invasion due to its proteolytic activity. Expression of MMP-13 is higher in glioma than in the surrounding normal brain tissue. High expression of this MMP is more often detected in advanced grades of glioma. Some researchers suggest that MMP-13 can be used as a biomarker of GBM progression. A study of Hsieh *et al.* revealed that GBM cells express higher amount of endothelin-1 receptors ETA and ETB. The stimulation of one of the most invasive glioma cell lines *in vivo*, U251, with endothelin resulted in an increased expres‐ sion of MMP-13, MMP-9 and increased cell migration. The addition of MMP-13 and MMP-9 inhibitors attenuated this increased cell migration [7-9].

Other scientists have shown that GBM cells can present an increased expression of other metalloproteinases: MMP-1, MMP-2, MMP-3, MMP-7, MMP-9 and MMP-14. Some researchers tried to reveal the molecular markers of invasiveness in gliomas. In the results of their studies various MMPs can be found. Bakalova *et al.* found that patients in terminal stages of brain tumors had elevated plasma levels of MMP-2 and MMP-9. Mariani *et al.* measured levels of MMP-2 and MMP-9 in cerebrospinal fluid (CSF) of dogs with intracranial tumors. Latent, but not active form of MMP-2 was found in all samples. MMP-9 was found in CSF of a minority of studied animals. Xu *et al.* analyzed brain samples obtained from patients undergoing surgery for GBM and non-malignant condition, epilepsy. Overexpression of both MMP-1 and vascular endothelial growth factor-1 (VEGF-1) was an independent poor prognostic factor in gliomas. Wang and co-workers analyzed frozen glioma samples and found out an increased expression of stromal periostin (POSTN) gene. This protein took part in both cell invasion and migration. In glioma cells POSTN signaling led to increased MMP-9 expression. The expres‐ sion of POSTN correlated with both grade and progression of glioma being a poor prognostic factor [10-13].

MT1-MMP (MMP-14) activates directly proMMP-2 and indirectly MMP-2 and MMP-9. Expression of MMP-14 was shown to correlate with invasiveness of glioma and to increase with glioma grade. MMP-14 expression was also shown to correlate with brain tumor pro‐ gression. This metalloproteinase, MMP-14, has been proposed as a biomarker to determine the type and grade of specific tumor. MMP-14 has a very interesting set of digested proteins. Apart from ECM proteins it can hydrolyze the most potent central nervous myelin inhibitory proteins, including BN-220. MMP-14 can also digest some proteins having adhesion functions. MMP-14 can also be involved in some intracellular processes. It can be trafficked along the tubulin cytoskeleton and be involved in intracellular recycling pathway. MMP-14 expression abnormalities were linked to mitotic spindle aberrations and chromosome instability leading to malignant transformation of neoplastic cells. MMP-14 may also be involved in regulation of VEGF-A expression. VEGF-A induces angiogenesis and inhibits apoptosis. MMP-14 seems to promote malignant glioma transformation, invasion and metastasis through intracellular signaling pathways [14].

### **6. Metalloproteinases and intracellular signaling pathways**

Increased expression of various MMPs observed in brain tumors is a result of multiple intracellular events which may be termed as dysregulated pathways. These intracellular molecular mechanisms leading to increased invasion of neoplasmatic cells have focused scientists' interest. Understanding these complex mechanisms may be a key to design a molecular targeted therapy for patients with brain tumors. Signaling pathways leading to increased expression of MMPs are of special interest.

Tsai *et al.* observed that inhibition of focal adhesion kinase (FAK) phosporylation by osthole reduced MMP-13 expression in human glioma cells. This inhibition led to a reduction of cells migration even in a subgroup of glioma cells selected for high migratory ability. Lee *et al.* observed GMB U251 cell line presented increased FAK activation which led to an augmented expression of MMP-2. This study also revealed that examined GBM cells had an increased Bclw (B-cell lymphoma-w) expression. This protein is a prosurvival member of Bcl-2 family. The augmented expression of this protein is associated with infiltration properties and aggres‐ siveness of various cancers. Bcl-w promotes the mesenchymal traits of glioblastoma cells by inducing vimentin expression of transcription factors, β-catenin, Twist1 and Snail. The increased expression of MMP-2 is accompanied by and results from the FAK activation, i.e. phosphorylation, via the PI3K-p-Akt-p-GSK3β-β-catenin pathway. The role of Bcl-w in promoting invasiveness of GBM by increasing MMP-2 activation was also confirmed in another study of this researchers team which proposed Bcl-w induced activation of β-catenin, also termed specificity protein-1 (Sp1), as a putative marker for aggressiveness of GBM. Nuclear factor of activated T cell (NFAT) family has been identified as a group of regulators of oncogenic transformation in several human malignancies. NFAT1 (NFATc2) is the prevalent family member expressed in peripheral T lymphocytes and many other cells outside the immune system. It is associated with tumor cell survival, apoptosis, migration and invasion. Clustering analysis of microarray data revealed that in glioma cells the expression of invasion related genes, cyclooxygenase-2 (COX-2), MMP-7 and MMP-9, was correlated with the expression of NFAT1. In vitro analysis confirmed the role of NFAT1, as in a specific NFAT1 knock down in U87 glioma cell line led to a marked reduction of COX-2, MMP-7 and MMP-9 expression [7,15-17].

sion of MMP-13, MMP-9 and increased cell migration. The addition of MMP-13 and MMP-9

118 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

Other scientists have shown that GBM cells can present an increased expression of other metalloproteinases: MMP-1, MMP-2, MMP-3, MMP-7, MMP-9 and MMP-14. Some researchers tried to reveal the molecular markers of invasiveness in gliomas. In the results of their studies various MMPs can be found. Bakalova *et al.* found that patients in terminal stages of brain tumors had elevated plasma levels of MMP-2 and MMP-9. Mariani *et al.* measured levels of MMP-2 and MMP-9 in cerebrospinal fluid (CSF) of dogs with intracranial tumors. Latent, but not active form of MMP-2 was found in all samples. MMP-9 was found in CSF of a minority of studied animals. Xu *et al.* analyzed brain samples obtained from patients undergoing surgery for GBM and non-malignant condition, epilepsy. Overexpression of both MMP-1 and vascular endothelial growth factor-1 (VEGF-1) was an independent poor prognostic factor in gliomas. Wang and co-workers analyzed frozen glioma samples and found out an increased expression of stromal periostin (POSTN) gene. This protein took part in both cell invasion and migration. In glioma cells POSTN signaling led to increased MMP-9 expression. The expres‐ sion of POSTN correlated with both grade and progression of glioma being a poor prognostic

MT1-MMP (MMP-14) activates directly proMMP-2 and indirectly MMP-2 and MMP-9. Expression of MMP-14 was shown to correlate with invasiveness of glioma and to increase with glioma grade. MMP-14 expression was also shown to correlate with brain tumor pro‐ gression. This metalloproteinase, MMP-14, has been proposed as a biomarker to determine the type and grade of specific tumor. MMP-14 has a very interesting set of digested proteins. Apart from ECM proteins it can hydrolyze the most potent central nervous myelin inhibitory proteins, including BN-220. MMP-14 can also digest some proteins having adhesion functions. MMP-14 can also be involved in some intracellular processes. It can be trafficked along the tubulin cytoskeleton and be involved in intracellular recycling pathway. MMP-14 expression abnormalities were linked to mitotic spindle aberrations and chromosome instability leading to malignant transformation of neoplastic cells. MMP-14 may also be involved in regulation of VEGF-A expression. VEGF-A induces angiogenesis and inhibits apoptosis. MMP-14 seems to promote malignant glioma transformation, invasion and metastasis through intracellular

**6. Metalloproteinases and intracellular signaling pathways**

increased expression of MMPs are of special interest.

Increased expression of various MMPs observed in brain tumors is a result of multiple intracellular events which may be termed as dysregulated pathways. These intracellular molecular mechanisms leading to increased invasion of neoplasmatic cells have focused scientists' interest. Understanding these complex mechanisms may be a key to design a molecular targeted therapy for patients with brain tumors. Signaling pathways leading to

inhibitors attenuated this increased cell migration [7-9].

factor [10-13].

signaling pathways [14].

MMP-2 has been discovered to posses intracellular activity and play some role in cell nucleus. A study by Kesanakurti *et al.* put new light on a role of MMP-2 in molecular mechanisms engaged in aggressiveness of glioma cells. p21 activated kinase 4 (PAK4) is one of down stream effectors of small GTPases Rac1 and Cdc42 which have diverse cellular functions by regulating cytoskeletal reorganization, cell survival and angiogenesis. Abberant PAK4 expression was found to be associated with enhanced tumor progression in various carcinomas. MMP-2 directly interacts with PAK-4 and augments the activation of αvβ3-mediated phosphoepidermal growth factor receptor (phospho-EGFR) in GBM. MMP-2 is supposed to bind to PAK4 and the complex PAK4/MMP-2 is supposed to regulate integrin mediated pathways in gliomas. Earlier study of Kesanakurti study group revealed that MMP-2 knock down glioma cells entered on apoptosis pathway [18-20].

Understanding the molecular pathways enhancing aggressiveness of glioma cells may lead to introducing a complex therapy focused on several targets which may give a better effectiveness.

## **7. Metalloproteinases in other cells supporting tumor and metastasis development**

In last few years scientists have paid more attention to interactions between glioma cells and microglia as well as on inreactions between metastatic cancer cells and astrocytes. EllertMiklaszewska *et al.* observed that glioma attracted microglia and polarized them into tumorsupporting cells that participated in matrix-remodeling, invasion, angiogenesis and suppression of adaptative immunity. In her experiment rat microglial cultures exposed to glioma conditioned medium polarized into pro-inflammatory or alternatively activated cells. Glioma derived factors increased cell motility, phagocytosis and sustained proliferation. Glioma induced activation of microglia was associated with induction of expression of several genes. One of them was MT1-MMP. Vinnakota *et al.* also observed that microglia promoted glioma through upregulation of MT1-MMP. This conversion of microglia into glioma suppor‐ tive phenotype was dependent on activation of Toll-like receptor 2 (TLR 2) along with TLR 1 and or TLR 6 signaling [21, 22].

Brain metastasis is a defining component of tumor pathophysiology and underlying mecha‐ nisms urgently need deeper elucidation. The relationship between metastatic cells and astrocytes is crucial for tumor cell sustenance in brain. Some researchers postulate that tumor cell metastasis to the brain are influenced by astrocyte secretome and astrocytes play a direct role in tumor metastasis. Wang *et al.* revealed that astrocyte conditioned tumor cells displayed highly invasive and metastatic behavior both *in vitro* and *in vivo* as well. MMP-2 and MMP-9 were two factors in the astrocyte secretome that were responsible for that response. Blocking these MMPs proteins partially prevented the invasion and metastasis of tumor cells both *in vitro* and *in vivo* as well. A very important question arises. What are the mechanisms by which MMPs secreted by astrocytes trigger invasion of tumor cells? MMP-2 and MMP-9 may increase the permeability of blood-brain barrier and allow the transfer of metastatic cells reaching brain via blood stream. The alternative hypothesis is that latent MMPs substrates on tumor cells or cells of tumor microenvironment may be activated upon cleavage by astrocyte secreted MMP-2 and MMP-9 leading to an invasive phenotype. The precise elucidation of these interactions is urgent as astrocytes may be a novel target for therapy aimed at prevention of brain metastases in patients with various cancers [21-23].

### **8. Blocking MMPs expression and/or activity**

Scientists have widely studied the possibilities of attenuating MMPs expression and activity in order to reduce the invasiveness of gliomas. They efforts have combined various directions.

Atorvastatin is a well known statin, an inhibitor of β-hydroxy-β-methylglutaryl-CoA reduc‐ tase. By inhibiting the key enzyme in a mevalonate synthesis pathway atorvastatin has pleiotropic effects. The main mode of action of this drug is the inhibition of *de novo* cholesterol synthesis. However, this drug has some other advantages resulting in its pleiotropic antia‐ therogenic properties due to the inhibition of synthesis of other biologically important mevalonate pathway derivates: dolichol, ubichinon, farnesyl and geranyl residues. This inhibition leads to disturbances in some signaling pathways. The possible anticancer proper‐ ties of statins have been postulated since a long time.

Yongjun and co-workers have observed that atorvastatin reduced pro-tumorigenic effects of microglia on glioma migration and invasion by reducing microglial expression of MT1-MMP (MMP-14). Mohebbi *et al.* observed that atorvastatin 40 mg administered twice daily seven from days before till three weeks after the neurosurgical procedure led to better outcome after the neurosurgical treatment of brain tumors and raised significantly Karnofsky score. The biochemical analysis showed that two weeks after the surgical treatment patients on atorvas‐ tatin therapy had significantly reduced plasma level of MMP-9 compared to patients receiving placebo [24,25].

Locatelli *et al.* evaluated a composition of polymeric nanoparticles containing a composition of two cytotoxic agents, drug alisertib and nanosilver conjugated with chlorotoxin, a peptide binding specifically to MMP-2. Their experimental *in vitro* and *in vivo* studies showed the tumor reduction [26].

Researchers are trying to investigate drugs aimed at inhibiting MT1-MMP (MMP-14).  Two natural isoflavonoid phytoestrogens, genistein and biochain A, were shown to reduce MMP-14 activity in a dose dependent manner in U87MG cell line. The green tea polyphenol, (Q)-epigallocatechin gallate (EGCg), has been found to inhibit MMP-14 mediated cell migration. This compound also disrupted proMMP-2 activation via downregulation of MMP-14 gene expression. Marimastat, an orally administered MMP inhibitor, was tested in two clinical trials in GBM patients after neurosurgery or irradiation. Marimastat alone did not improve survival, but in conjunction with cytotoxic chemotherapy gave promising results [27-30].

The next point of scientists interest are microRNAs (miRNAs). These small, non-coding RNA molecules containing 18-25 nucleotides in length can inhibit gene expression by binding to the 3' untranslated region of their target genes and suppress translation. Several studies have shown that various miRNAs can inhibit expression of MMP-14, MMP-2 and inhibit tumor cell adhesion, migration, invasion and angiogenesis [31-35].

Lei *et al.* proposed a new strategy combining a cytotoxic drug paclitaxel and RNA interference suppressing MMP-2 expression. This conception was aimed at blocking tumor growth and proliferation by Paclitaxel and blocking tumor angiogenesis and invasion by inhibiting MMP-2 expression [36].

## **9. Conclusions**

Miklaszewska *et al.* observed that glioma attracted microglia and polarized them into tumorsupporting cells that participated in matrix-remodeling, invasion, angiogenesis and suppression of adaptative immunity. In her experiment rat microglial cultures exposed to glioma conditioned medium polarized into pro-inflammatory or alternatively activated cells. Glioma derived factors increased cell motility, phagocytosis and sustained proliferation. Glioma induced activation of microglia was associated with induction of expression of several genes. One of them was MT1-MMP. Vinnakota *et al.* also observed that microglia promoted glioma through upregulation of MT1-MMP. This conversion of microglia into glioma suppor‐ tive phenotype was dependent on activation of Toll-like receptor 2 (TLR 2) along with TLR 1

120 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

Brain metastasis is a defining component of tumor pathophysiology and underlying mecha‐ nisms urgently need deeper elucidation. The relationship between metastatic cells and astrocytes is crucial for tumor cell sustenance in brain. Some researchers postulate that tumor cell metastasis to the brain are influenced by astrocyte secretome and astrocytes play a direct role in tumor metastasis. Wang *et al.* revealed that astrocyte conditioned tumor cells displayed highly invasive and metastatic behavior both *in vitro* and *in vivo* as well. MMP-2 and MMP-9 were two factors in the astrocyte secretome that were responsible for that response. Blocking these MMPs proteins partially prevented the invasion and metastasis of tumor cells both *in vitro* and *in vivo* as well. A very important question arises. What are the mechanisms by which MMPs secreted by astrocytes trigger invasion of tumor cells? MMP-2 and MMP-9 may increase the permeability of blood-brain barrier and allow the transfer of metastatic cells reaching brain via blood stream. The alternative hypothesis is that latent MMPs substrates on tumor cells or cells of tumor microenvironment may be activated upon cleavage by astrocyte secreted MMP-2 and MMP-9 leading to an invasive phenotype. The precise elucidation of these interactions is urgent as astrocytes may be a novel target for therapy aimed at prevention of brain metastases

Scientists have widely studied the possibilities of attenuating MMPs expression and activity in order to reduce the invasiveness of gliomas. They efforts have combined various directions. Atorvastatin is a well known statin, an inhibitor of β-hydroxy-β-methylglutaryl-CoA reduc‐ tase. By inhibiting the key enzyme in a mevalonate synthesis pathway atorvastatin has pleiotropic effects. The main mode of action of this drug is the inhibition of *de novo* cholesterol synthesis. However, this drug has some other advantages resulting in its pleiotropic antia‐ therogenic properties due to the inhibition of synthesis of other biologically important mevalonate pathway derivates: dolichol, ubichinon, farnesyl and geranyl residues. This inhibition leads to disturbances in some signaling pathways. The possible anticancer proper‐

Yongjun and co-workers have observed that atorvastatin reduced pro-tumorigenic effects of microglia on glioma migration and invasion by reducing microglial expression of MT1-MMP

and or TLR 6 signaling [21, 22].

in patients with various cancers [21-23].

**8. Blocking MMPs expression and/or activity**

ties of statins have been postulated since a long time.

In last few years MMPs have been shown to exert new biochemical properties. Their extrac‐ ellular mode of action as well as intracellular, intranuclear activities were shown to be involved in invasiveness of brain tumors, especially gliomas. Inhibiting their expression may be a new therapeutical approach. So far some drugs being MMPs inhibitors have some serious adverse effects. Inhibiting MMPs expression and activity seems to be rather a supplement to chemo‐ therapy, radiotherapy or neurosurgical procedures than a new single method of treatment of brain tumors.

## **Author details**

Krzysztof Siemianowicz1\*, Wirginia Likus2 and Jarosław Markowski<sup>3</sup>

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Biochemistry, School of Medicine in Katowice, Medical University of Silesia, Poland

2 Department of Human Anatomy, School of Medicine in Katowice, Medical University of Silesia, Poland

3 Department of Laryngology, School of Medicine in Katowice, Medical University of Silesia, Poland

Sections 1-3 contain text originally published in [3].

## **References**

[8] Wang J, Li Y, Li C, Yu K, Wang Q. Increased expression of metalloproteinase-13 in glioma is associated with poor overall survival of patients. Medical Oncology 2012; 29, 2432-2437.

**Author details**

Poland

Poland

Silesia, Poland

**References**

Krzysztof Siemianowicz1\*, Wirginia Likus2

\*Address all correspondence to<EMAIL_ADDRESS>
Sections 1-3 contain text originally published in [3].

ogy 2007;26: 587-596.

2014;92(3) 275-286.

and Jarosław Markowski<sup>3</sup>

1 Department of Biochemistry, School of Medicine in Katowice, Medical University of Silesia,

122 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

2 Department of Human Anatomy, School of Medicine in Katowice, Medical University of

3 Department of Laryngology, School of Medicine in Katowice, Medical University of Silesia,

[1] Pearce WH, Shively VP. Abdominal aortic aneurysm as a complex multifactorial dis‐

[2] Ra H-J, Parks WC. Control of matrix metalloproteinase catalytic activity. Matrix Biol‐

[3] Siemianowicz K. The role of metalloproteinases in the development of aneurysm. in:

[4] Mannello F, Medda V. Nuclear localization of matrix metalloproteinases. Progress in

[5] Langenfurth A, Rinnenthal JL, Vinnakota K, Prinz V, Carlo AS, Stadelmann C, Siffrin V, Peaschke S, Endres M, Heppner F, Glass R, Wolf SA, Kettenmann H. Membranetype 1 metalloproteinase is upregulated in microglia/brain macrophages in neurode‐ generative and neuroinflammatory diseases. Journal of Neuroscience Research

[6] Wang L, Cossette SM, Rarick KR, Gershan J, Dwinell MB, Harder DR, Ramchandran R. Astrocytes directly influence tumor cell invasion and metastasis *in vivo*. PLoS ONE

[7] Tsai CF, Yeh WL, Chen JH, Lin C, Huang SS, Lu DY. Osthole suppresses the migrato‐ ry ability of human glioblastoma multiforme cells via inhibition of focal adhesion kinase-mediated matrix metalloproteinase-13 expression. International Journal of

ease. Annals of New York Academy of Sciences 2006;1085 117-132.

"Aneurysm" (ed.) Yasuo Murai, Rijeka, InTech, 2012, p65-74.

Histochemistry and Cytochemistry 2012;47 27-58.

8(12): e80933, doi: 10.1371/journal.pone.0080933.

Molecular Sciences 2014;15 3889-3903.

[29] Annabi B, Lachambre MP, Bousquet-Gagnon N, Page M, Gingras D, Beliveau R. Green tea polyphenol (−)-epigallocatechin 3-gallate inhibits MMP-2 secretionand MT1-MMP-driven migration in glioblastoma cells, Biochim. Biophys. Acta 2002;1542 209-220.

[19] Kesanakurti D, Chetty C, Bhoopathi P, Lakka SS, Gorantla B, Tsung AJ, Rao JS. Sup‐ pression of MMP-2 attenuates TNF-alpha induced NF-kappaB activation and leads

124 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

[20] Kesanakurti D, Chetty C, Dinh DH, Gujrati M, Rao JS. Role of MMP-2 in the regula‐ tion of IL-6/Stat3 survival signaling via interaction with a5b1 integrin in glioma. On‐

[21] Ellert-Miklaszewska A, Dabrowski M, Lipko M, Sliwa M, Maleszewska M, Kaminska B, Molecular definition of the pro-tumorigenic phenotype of glioma-activated micro‐

[22] Vinnakota K, Hu F, Ku MC, Georgieva PB, Szulzewsky F, Polhmann A, Waiczies S, Waiczies H, Niedorf T, Lehnardt S, Hanisch S, Synowitz M, Markovic D, Wolf SA, Glass R, Kettenmann H. Toll-like receptor-2 mediates microglia/brain macrophage MT1-MMP expression and glioma expansion. Neuro-Oncology 2013;15(11)

[23] Wang L, Cossette SM, Rarick KR, Gershan J, Dwinell MB, Harder DR, Ramchandran R. Astrocytes directly influence tumor cell invasion and metastasis in vivo. PLos

[24] Yongjun Y, Shuyun H, Lei C, Xiangrong C, Zhilin Y, Yiquan K. Atorvastatin sup‐ presses glioma invasion and migration by reducing microglial MT1-MMP expres‐

[25] Mohebbi N, Khoshnevisan A, Naderi S, Abdollahzade S, Salamzadeh J, Javadi M, Mojtahezadeh M, Gholami K. Effects of atorvastatin on plasma matrix metalloprotei‐ nase-9 concentration after glial tumor resection; a randomized, double blind, placebo controlled trial. Daru. Journal of Pharmaceutical Sciences 2014;7;22(1): 10. doi:

[26] Locatelli E, Naddaka M, Uboldi C, Loudos G, Fragogeorgi E, Molinari V, Pucci A, Tsotakos T, Psimadas D, Ponti J, Franchini MC. Targeted delivery of silver nanoparti‐ cles and alisertib: in vitro and in vivo synergistic effect against glioblastoma. Nano‐

[27] Devy L, Huang L, Naa L, Yanamandra N, Pieters H, Frans N, Chang E, Tao Q, Van‐ hove M, Lejeune A, van Gool R, Sexton DJ, Kuang G, Rank D, Hogan S, Pazmany C, Ma YL, Schoonbroodt S, Nixon AE, Ladner RC, Hoet R, Henderikx P, Tenhoor C, Rabbani SA, Valentino ML, Wood CR, Dransfield DT. Selective inhibition of matrix metalloproteinase-14 blocks tumor growth, invasion, and angiogenesis. Cancer Re‐

[28] Puli S, Lai JC, Bhushan A. Inhibition of matrix degrading enzymes and invasion in human glioblastoma (U87MG) cells by isoflavones. Journal of Neuro-Oncology

ONE 2013;8(12): e80933. doi:10.1371/journal.pone.0080933.

sion. Journal of Neuroimmunology 2013;260(1-2) 1-8.

to JNK mediated cell death in glioma. PLoS One 2011; 6: e19341.

cogene 2013;32(3) 327-40.

10.1186/2008-2231-22-10.

search 2009;69 1517-1526.

2006;79 135-142.

medicine (Lond) 2014;9(6) 839-49.

1457-1468.

glia. Glia 2013;61(7) 1178-1190.

**Clinical Management of Brain Tumor Patients**

## **Chapter 7**

## **Radiation-Induced Glioma**

Ryuya Yamanaka and Azusa Hayano

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59047

## **1. Introduction**

Radiation therapy is widely used for patients with intracranial tumors. However, there are many complications, including cerebral atrophy, calcifying microangiopathy, radiation necrosis, leukoencephalopathy and development of radiation-induced tumors [1-5]. Radia‐ tion-induced central nervous system (CNS) neoplasms are recognized in patients who have had therapeutic radiotherapy to the head or face [6]. Radiation-induced CNS neoplasms are rare, but the cumulative risk of brain tumor after therapeutic cranial irradiation is reported as 0.5-2.7% at 15 years [7]. Among radiation-induced CNS neoplasms, meningiomas are about 70%, gliomas about 20% and sarcomas less than 10% [4,6,8]. In children, high-grade gliomas are the most common radiation-induced tumor [9]. Type of post-radiation gliomas are glioblastoma (GBM) in 75% and anaplastic astrocytoma in 25% [7]. Brada et al. [6] described a relative risk of secondary glioma of 7.92 times higher than that of the normal non-irradiated population, with an average latency period to glioma diagnosis of 7 years, in 334 patients with pituitary lesions, irradiated to a median dose of 45 Gy for the sellar region. Cahan et al. [10] established criteria to diagnose a radiotherapy induced brain tumor. These criteria were modified in 1972 by Schrantz and Araoz [11] as follows:(1) the tumor must appear within the irradiated field; (2) the tumor was not present prior to the radiotherapy; (3) a sufficient latency period must elapse between irradiation and appearance of the tumor (usually>5 years); (4) the radiation induced tumor must be histologically proven and a different histological type from the original neoplasm treated by the radiation therapy.

## **2. Radiation-induced gliomas in the literature**

In a review of the literature, 191 cases of radiation-induced glioma that analyzed in detail were identified in the period 1960-2014 [9,12-120]. The latency period from the irradiation to the

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

onset of the secondary glioma ranged from 6 months to 50 years, with an average of 11.1 years. More than 40 Gy irradiation was delivered in 50% of cases, with an average of 37.2 Gy. As shown in Table 1, 29 grade II, 37 grade III and 97 grade IV gliomas had been reported, and no specific grade had been shown in other 28 cases. Grade II gliomas developed after 10.7 years, and grade IV gliomas developed after 10.6 years from the time of irradiation. The radiation dose of grade II gliomas for primary lesion is 29.7 Gy, grade III 37.4 Gy and grade IV 37.3 Gy, respectively. There was a significant difference of radiation dose between grade II and grade IV glioma (p<0.05). In Table 2 and 3, the relation of primary lesion and radiation dose, latency to radiation induced glioma occurrence, glioma grade are shown. The latency in acute lymphoblastic leukemia (ALL) / acute myeloblastic leukemia (AML), Hodgkin/non-Hodgkin lymphoma and cancer patients is short compared to that of intracranial and scalp lesion. And, the irradiated dose in ALL/AML patients is rather small compared to that of intracranial lesion. Patients with ALL/AML and Hodgkin/non-Hodgkin lymphoma are usually intensively treated with anticancer agents with carcinogenic effects, so the patients may suffer from glioma by the synergistic effects of prophylactic irradiation and chemotherapy.

**Table 1.** Radiation induced glioma

**Table 2.** Radiation induced glioma of intracranial primary lesion

**Table 3.** Radiation induced glioma of extracranial primary lesion

onset of the secondary glioma ranged from 6 months to 50 years, with an average of 11.1 years. More than 40 Gy irradiation was delivered in 50% of cases, with an average of 37.2 Gy. As shown in Table 1, 29 grade II, 37 grade III and 97 grade IV gliomas had been reported, and no specific grade had been shown in other 28 cases. Grade II gliomas developed after 10.7 years, and grade IV gliomas developed after 10.6 years from the time of irradiation. The radiation dose of grade II gliomas for primary lesion is 29.7 Gy, grade III 37.4 Gy and grade IV 37.3 Gy, respectively. There was a significant difference of radiation dose between grade II and grade IV glioma (p<0.05). In Table 2 and 3, the relation of primary lesion and radiation dose, latency to radiation induced glioma occurrence, glioma grade are shown. The latency in acute lymphoblastic leukemia (ALL) / acute myeloblastic leukemia (AML), Hodgkin/non-Hodgkin lymphoma and cancer patients is short compared to that of intracranial and scalp lesion. And, the irradiated dose in ALL/AML patients is rather small compared to that of intracranial lesion. Patients with ALL/AML and Hodgkin/non-Hodgkin lymphoma are usually intensively treated with anticancer agents with carcinogenic effects, so the patients may suffer from glioma

130 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

by the synergistic effects of prophylactic irradiation and chemotherapy.

**Number of cases**

**Table 2.** Radiation induced glioma of intracranial primary lesion

**Table 1.** Radiation induced glioma

Optic glioma/ Retinoblastoma

**WHO grade Number of cases Irradiated dose (Gy) Latency (years)**

Grade II 29 29.7±18.4 10.7±7.7 Grade III 37 37.4±15.9 12.6±8.6 Grade IV 97 37.3±17.5 10.7±6.6

**Irradiated dose**

Pituitary adenoma 20 52.2±12.7 13.5±7.5 Grade III/IV 16, II 3 Craniopharyngioma 15 63.9±7.0 11.7±7.0 Grade III/IV 13, II 1 Medulloblastoma 16 44.9±9.5 15.8±12.2 Grade III/IV 12, II 4 Germ cell tumor 10 44.9±8.5 15.2±13.4 Grade III/IV 9, II 1

Menigioma/Neurinoma 6 38.4±14.2 10±5.7 Grade III/IV 5, II 1 Low grade glioma 12 47.0±7.8 13.5±10.1 Grade III/IV 11, II 1

**(Gy) Latency (years) Radiation induced glioma**

6 46.5±10.9 7.2±2.4 Grade IV 6

## **3. Genetic characteristics of radiation-induced glioma**

In a patient reported by Gessi [36], the genetic alterations were p53 mutation (C to G transition at codon 176 of Exon 5), loss of heterozygosity (LOH) of 17p and 19q, O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation, and no amplification of epidermal growth factor receptor (EGFR). In Yang's case [115], p53 mutation (deletion at codon 233 of Exon 7 and a C to G transition at codon 278 of Exon 8) and no amplification of EGFR were reported. In Tada's case [104], 3-bp homozygous deletion in exon 7 of the p53 gene was described. In Kon's case [58], although LOH and amplification of EGFR, phosphatase and tensin analog (PTEN) was not observed, LOH of 1p, K-ras, p16, p53 were observed. In Alexous's two cases, LOH of 1p was found in both cases [15]. Nine radiation-induced highgrade gliomas were studied for possible molecular alterations in p53, PTEN, K-ras, EGFR, and p16 by Brat [121]. Exon 8 of p53 gene mutation (G to A substitution in codon 285) is detected in one case, EGFR amplification in 2 cases and p16/ methylthioadenosine phosphorylase (MTAP) gene deletion in 2 cases [121]. However, genetic alterations similar to those described in spontaneous, sporadic primary GBM, except the absence of PTEN mutations in the radia‐ tion-induced group were found. Radiation-induced GBMs have a lower percent of EGFR and p16 alterations than primary GBM [121]. Donson et al. [122] demonstrated by gene expression analysis genetic homogenity relative to de-novo gliomas, suggesting a common precursor cell for radiation-induced gliomas. It is not well known about the molecular alteration of radiationinduced gliomas, due to the limited number of cases and limited genes were analyzed.

## **4. Therapeutic implications**

Radiation-induced glioma is difficult to treat; radiotherapy is not always a therapeutic option because the patient has already been exposed to radiation. However there are several patients

reported who had a sustained remission following chemotherapy alone or radiochemothera‐ py. About the reports of treatment of radiation-induced glioma, a dramatic response and prolonged survival by carmustine, nimustine hydrochloride and temozolomide were reported [52, 58, 71, 75]. These tumors have a poor prognosis due to their intrinsic resistance to treatment and the difficulty using aggressive therapies in previously irradiated patients. However, vigorous chemotherapeutic approaches may yield prolonged disease control in some patients with radiation-induced glioma. The relationship of 1p LOH and chemosensitivity in oligo‐ dendroglial tumors is well known [123]. 1p LOH may account for the marked response to chemotherapy [52,58], although the reason of the chemosensitivity is not discussed in other case. In Fukui's case [32], 40 Gy of local radiotherapy and chemotherapy with nimustine hydrochloride and Interferone-β yielded dramatic response. The patient received 15 Gy of whole brain radiotherapy 7 years prior to the onset of radiation-induced glioma. Although the tolerable radiation dose is not well known after initial radiation therapy, additional radio‐ therapeutic approaches may yield prolonged disease control in some patients with radiationinduced glioma. The marked chemo and radiosensitivity should be further investigated for the development of glioma therapy.

## **5. Conclusion**

In case that intracranial and extracranial lesions are treated by standard fractionated radiation or stereotatic radiosurgery, radiation-induced gliomas should be considered as possible longterm side effect. And the patients should be followed for a long term, even long after the period of risk for relapse of the primary site has passed.

## **Author details**

Ryuya Yamanaka\* and Azusa Hayano

\*Address all correspondence to<EMAIL_ADDRESS>
Graduate School for Health Care Science, Kyoto Prefectural University of Medicine, Kyoto, Japan

### **References**

[3] Dooms GC, Hecht S, Brant-Zawadzki M, Berthiaume Y, Norman D, Newton TH. Brain radiation lesions. MR imaging. Radiology 1986;158(1):149-55.

reported who had a sustained remission following chemotherapy alone or radiochemothera‐ py. About the reports of treatment of radiation-induced glioma, a dramatic response and prolonged survival by carmustine, nimustine hydrochloride and temozolomide were reported [52, 58, 71, 75]. These tumors have a poor prognosis due to their intrinsic resistance to treatment and the difficulty using aggressive therapies in previously irradiated patients. However, vigorous chemotherapeutic approaches may yield prolonged disease control in some patients with radiation-induced glioma. The relationship of 1p LOH and chemosensitivity in oligo‐ dendroglial tumors is well known [123]. 1p LOH may account for the marked response to chemotherapy [52,58], although the reason of the chemosensitivity is not discussed in other case. In Fukui's case [32], 40 Gy of local radiotherapy and chemotherapy with nimustine hydrochloride and Interferone-β yielded dramatic response. The patient received 15 Gy of whole brain radiotherapy 7 years prior to the onset of radiation-induced glioma. Although the tolerable radiation dose is not well known after initial radiation therapy, additional radio‐ therapeutic approaches may yield prolonged disease control in some patients with radiationinduced glioma. The marked chemo and radiosensitivity should be further investigated for

132 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

In case that intracranial and extracranial lesions are treated by standard fractionated radiation or stereotatic radiosurgery, radiation-induced gliomas should be considered as possible longterm side effect. And the patients should be followed for a long term, even long after the period

Graduate School for Health Care Science, Kyoto Prefectural University of Medicine, Kyoto,

[1] Mikhael MA. Radiation necrosis of the brain: correlation between patterns on com‐ puted tomography and dose of radiation. J Comput Assist Tomogr 1979;3(2):241-9.

[2] Jacoby CG, Tewfik HH, Blackwelder JT. Cerebellar atrophy developing after cranial

the development of glioma therapy.

of risk for relapse of the primary site has passed.

and Azusa Hayano

\*Address all correspondence to<EMAIL_ADDRESS>
irradiation. J Comput Assist Tomogr 1982;6(1):159-62.

**5. Conclusion**

**Author details**

Ryuya Yamanaka\*

Japan

**References**

[31] Fontana M, Stanton C, Pompili A, Amadori S, Mandelli F, Meloni G, Riccio A, Rubin‐ stein LJ. Late multifocal gliomas in adolescents previously treated for lymphoblastic leukaemia. Cancer 1987; 60:1510–8.

[17] Bachman DS, Ostrow PT. Fatal long-term sequela following radiation "cure" for

134 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

[18] Balasubramaniam A, Shannon P, Hodaie M, Laperriere N, Michaels H, Guha A. Glio‐ blastoma multiforme after stereotactic radiotherapy for acoustic neuroma: case re‐

[19] Barnes ARE, Liwnicz BH, Schellhas HE, Altshuller G, Aron BS, Lippert WR. Success‐ ful treatment of placental choriocarcinoma metastatic to brain followed by primary

[20] Bazan C, New PZ, Kaghan-Hallet KS. MRI of radiation-induced spinal cord glioma.

[21] Berman EL, Eade TN, Brown D, Weaver M, Glass J, Zorman G, Feigenberg SJ. Radia‐ tion-induced tumor after stereotactic radiosurgery for an arteriovenous malforma‐

[22] Beute BJ, Fobben ES, Hubschmann O, Zablow A, Eanelli T, Solitare G. Cerebellar gliosarcoma: report of a probable radiation-induced neoplasm. AJNR 1991;12:554–6.

[23] Bonilha L, Borges G, Fernandes YB, Ramina R, Carelli EF, Alvarenga M. Pilocytic as‐ trocytoma following radiotherapy for craniopharyngioma: case report. Arq Neuro‐

[24] Chung CK, Stryker JA, Cruse R, Vanucci R, Towfighi J. Glioblastoma multiforme fol‐ lowing prophylactic cranial irradiation and intrathecal methotrexate in a child with

[25] Clifton MD, Amromin GD, Perry MC, Abadir R, Watts C, Levy N. Spinal cord glio‐

[26] Cohen MS, Kushner MJ, Dell S. Frontal lobe astrocytoma following radiotherapy for

[27] Diersse G, Alvarez G, Figols J. Anaplastic astrocytoma associated with previous radi‐

[28] Doskaliyev A, Yamasaki F, Kenjo M, Shrestha P, Saito T, Hanaya R, Sugiyama K, Kurisu K. Secondary anaplastic oligodendroglioma after cranial irradiation: a case re‐

[29] Edwards MK, Terry JG, Montebello JF, Hornback NB, Kuharik MA. Gliomas in chil‐ dren following radiation therapy for lymphoblastic leukemia. Acta Radiol Suppl

[30] Enchev Y, Ferdinandov D, Kounin G, Encheva E, Bussarsky V. Radiation-induced gliomas following radiotherapy for craniopharyngiomas: a case report and review of

ma following irradiation for Hodgkin's disease. Cancer 1980; 45:2051–5.

port and review of the literature. Neuro Oncol 2007;9(4):447-53.

ependymoma. Ann Neurol 1978; 4:319–21.

brain glioblastoma. Gynecol Oncol 1982;13:108–14.

tion: case report. Neurosurgery 2007;61(5):E1099.

acute lymphoblastic leukaemia. Cancer 1980; 47:2563–6.

otherapy: report of three cases. Neurosurgery 1988; 6:1095–7.

the literature. Clin Neurol Neurosurg 2009;111(7):591-6.

medulloblastoma. Neurology 1988; 31:616–9.

port. J Neurooncol 2008;88(3):299-303.

1986;369:651-3.

Neuroradiology 1990;32:331–3.

psiquiatr 2000;58(3A):731-5.

[58] Kon T, Natsumeda M, Taki T, Takahashi H, Fujii Y, Yamanaka R. Radiation-induced glioblastoma following radiotherapy for pituitary adenomas: marked response to chemotherapy. J Neurol Neurophysiol 2013;4:155.

[45] Judge MR, Eden OB, O'Neill P. Cerebral glioma after cranial prophylaxis for acute

[46] Kaido T, Hoshida T, Uranishi R, Akita N, Kotani A, Nishi N, Sakaki T. Radiosurgery-

[47] Kamide T, Nakada M, Hayashi Y, Suzuki T, Hayashi Y, Uchiyama N, Kijima T, Ham‐ ada J. Radiation-induced cerebellar high-grade glioma accompanied by meningioma and cavernoma 29 years after the treatment of medulloblastoma: a case report. J Neu‐

[48] Kaschten B, Flandroy P, Reznik M, Hainaut H, Stevenaert A. Radiation-induced glio‐ sarcoma: case report and review of the literature. J Neurosurg 1995;83:154–62.

[49] Kato N, Kayama T, Sakurada K, Saino M, Kuroki A. Radiation-induced glioblastoma:

[50] Kawaguchi S, Kashiwaba T, Koiwa M, Shimoyama M, Kobayashi N, Fukushi Y, To‐ kuda K. Two autopsied cases of radiation-induced gliosarcoma. No Shinkei Geka

[51] Kawanabe Y, Sawada M, Yukawa H, Ueda S, Sasaki N, Koizumi T, Kihara S, Hoshi‐ maru M. Radiation-induced spinal cord anaplastic astrocytoma subsequent to radio‐

[52] Khoo HM, Kishima H, Kinoshita M, Goto Y, Kagawa N, Hashimoto N, Maruno M, Yoshimine T. Radiation-induced anaplastic ependymoma with a remarkable clinical

[53] Kikkawa Y, Suzuki SO, Nakamizo A, Tsuchimochi R, Murakami N, Yoshitake T, Aishima S, Okubo F, Hata N, Amano T, Yoshimoto K, Mizoguchi M, Iwaki T, Sasaki T. Radiation-induced spinal cord glioblastoma with cerebrospinal fluid dissemina‐ tion subsequent to treatment of lymphoblastic lymphoma. Surg Neurol Int. 2013;4:27.

[54] Kitanaka C, Shitara N, Nakagomi T, Nakamura H, Genka S, Nakagawa K, Akanuma A, Aoyama H, Takakura K. Postradiation astrocytoma: report of two cases. J Neuro‐

[55] Kleriga E, Sher JH, Nallainathan SK, Stein SC, Sacher M. Development of cerebellar malignant astrocytoma at site of medulloblastoma treated 11 years earlier. J Neuro‐

[56] Komaki S, Komaki R, Chol H, Correa-Paz F. Radiation and drug-induced intracranial neoplasm with angiographic demonstration. Neurol Med Chir (Tokio) 1977;17:55–62.

[57] Komatsu F, Kawaguchi H, Tsugu H, Oshiro S, Komatsu M, Fukushima T, Nabeshima K, Inoue T. Radiation-induced astrocytoma with rapid malignant transformation:

case report. Neurol Med Chir (Tokyo) 2011;51(3):243-6.

therapy for testicular seminoma. Neurol Med Chir (Tokyo) 2012;52(9):675-8.

response to temozolomide: a case report. Br J Neurosurg 2013;27(2):259-61.

lymphoblastic leukaemia. Br Med J (Clin Res Ed) 1984; 289:1038–9.

136 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

induced brain tumor: case report. J Neurosurg 2001;95:710–3.

rooncol 2010;100(2):299-303.

1991;19(3):285-90.

surg 1989;70:469–74.

surg 1979;49:445–9.

a case report. No To Shinkei 2000;52:413–8.

[87] Rittinger O, Kranzinger M, Jones R, Jones N. Malignant astrocytoma arising 10 years after combined treatment of craniopharyngioma. J Pediatr Endocrinol Metab 2003; 16: 97–101.

[73] Muzumdar DP, Desai K, Goel A. Glioblastoma multiforme following prophylactic cranial irradiation and intrathecal methotrexate on a child with acute lymphoblastic

138 Molecular Considerations and Evolving Surgical Management Issues in the Treatment of Patients with a Brain Tumor

[74] Ng C, Fairhall J, Rathmalgoda C, Stening W, Smee R. Spinal cord glioblastoma multi‐ forme induced by radiation after treatment for Hodgkin disease. Case report. J Neu‐

[75] Nicolardi L, DeAngelis LM. Response to chemotherapy of a radiation-induced glio‐

[76] Nishio S, Morioka T, Inamura T, Takeshita I, Fukui M, Sasaki M, Nakamura K, Waki‐ saka S. Radiation-induced brain tumors: potential late complications of radiation