**Role of the Centrosomal MARK4 Protein in Gliomagenesis**

Ivana Magnani, Chiara Novielli and Lidia Larizza *Università degli Studi di Milano Italy* 

#### **1. Introduction**

130 Glioma – Exploring Its Biology and Practical Relevance

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CD133: valuable stem cell-specific markers for determining clinical outcome of glioma patients. *J Exp Clin Cancer Res.,* Vol.27, (December 2008), pp. 85, ISSN 0392Human gliomas are the most frequent tumours of the central nervous system (Kleihues & Cavenee, 2000). They are of neuroectodermal origin and present as different histological types and malignancy grades (Louis et al., 2007).

According to the WHO (world health organization) system, astrocytoma, oligodendroglioma and mixed oligoastrocytoma are classified as differentiated gliomas, while anaplastic glioma and glioblastoma show increasing grades of malignancy (Box 1).

Box 1.

Gliomas are composed of different cell types displaying, even within low-grade tumours, a wide spectrum of heterogeneity regarding morphology, genotype, invasive potentiality, and treatment sensitivity (Noble & Dietrich, 2004). The development and progression of glioma malignancies is driven by accumulation of genomic alterations, including both mutations and chromosomal instability (CIN).

#### **2. Chromosomal instability (CIN) in glioma**

CIN refers to the rate of lost or gained chromosomes and/or structural chromosome anomalies and ploidy changes during cell divisions (Geigl et al., 2008; Lengauer et al., 1998). Structural chromosome anomalies (translocations, deletions, insertions, inversion and additions) may be balanced or unbalanced and involve one or more chromosomes (Bayani et al., 2007). Chromosomal instability in glioma is mainly characterized by aneuploidy (Bigner et al., 1988; Hecht et al., 1995; Jenkins et al., 1989; Lindstrom et al., 1991; Magnani et al., 1994; Park et al., 1995; Thiel et al., 1992) affecting in particular glioblastoma, the most

Role of the Centrosomal MARK4 Protein in Gliomagenesis 133

increased in the latter and multiple chromosomal rearrangements are also present. The finding of common abnormalities associated to both low- and high-grade glioma has suggested a progressive chromosomal evolution during tumour growth (Bigner et al., 1988; Jenkins et al., 1989; Magnani et al., 1994; Thiel et al., 1992) even though it has been demonstrated that a subset of glioblastomas arises clonally *de novo*, further emphasizing the genetic heterogeneity of glioma (Kleihues & Ohgaki, 2000; von Deimling et al., 1993). Given that numerical CIN features many cancer cells, it has been hypothesized that it may have a primary role in tumorigenesis (Duesberg et al., 2006; Weaver et al., 2007). Recently it has been shown that the main pathway to aneuploidy in cancer cells is triggered by extra centrosomes that, increasing improper merotelic attachments of kinetochores to spindle microtubules, cause chromosome mis-segregation (Meraldi et al., 2002) (Figure 3) (see Box 3

Fig. 2. (a) Chromosome 1 rearrangements of both p and q arms observed in different glioma cell lines by G-banding. (b) Rearrangements of chromosome 9p, sharing the loss of p21

At early mitosis, the merotelic orientation escapes the spindle mitotic checkpoint thus representing the major mechanism of chromosome mis-segregation in non-cancer cells. Usually these errors are corrected before cells enter anaphase, to preserve genome stability

band, observed in different glioblastoma cell lines by G-banding.

(Cimini et al., 2004).

for centrosomes and Box 4 for mitotic spindle).

malignant glioma. Gliomas frequently display near-diploid (2n+/-) and/or near-tritetraploid (3n+/-)/(4n+/-) karyotypes, implicating aberrant mitotic divisions, in addition to chromosomal rearrangements. Highly polyploid subpopulations and the presence of apoptotic nuclei are also reported (Figures 1a-d).

Fig. 1. (a) The G-banded, near diploid karyotype of MI-4 GBM (glioblastoma multiforme) cell line (Magnani et al., 1994), showing trisomy of chromosome 7, monosomy of chromosome 10 and a complex rearrangement involving chromosomes 1, 9 and 19. (b) The G-banded, near tetraploid karyotype of MI-4 cell line, displaying several chromosome losses and structural rearrangements including marker chromosomes. (c) Representative polyploid metaphase from MI-60 GBM cell line, characterized by a high frequency of hyperdiploid cells. (d) Apoptotic and large nuclei of MI-60 cell line.

Low-grade astrocytomas and oligodendrogliomas (WHO grades I-II) show a number of chromosome aberrations quite low. When present, they involve the gain of chromosome 7, the loss of chromosomes 10, 22 and one sex chromosome (see Figures 1a, b), while structural changes affect in particular 1p (Figure 2a) and 9p (Figure 2b) chromosome arms.

These chromosome abnormalities are qualitatively similar to those found in anaplastic astrocytoma (WHO grade III) and glioblastoma (WHO grade IV), but their frequency is

malignant glioma. Gliomas frequently display near-diploid (2n+/-) and/or near-tritetraploid (3n+/-)/(4n+/-) karyotypes, implicating aberrant mitotic divisions, in addition to chromosomal rearrangements. Highly polyploid subpopulations and the presence of

Fig. 1. (a) The G-banded, near diploid karyotype of MI-4 GBM (glioblastoma multiforme)

chromosome 10 and a complex rearrangement involving chromosomes 1, 9 and 19. (b) The G-banded, near tetraploid karyotype of MI-4 cell line, displaying several chromosome losses and structural rearrangements including marker chromosomes. (c) Representative polyploid metaphase from MI-60 GBM cell line, characterized by a high frequency of hyperdiploid

Low-grade astrocytomas and oligodendrogliomas (WHO grades I-II) show a number of chromosome aberrations quite low. When present, they involve the gain of chromosome 7, the loss of chromosomes 10, 22 and one sex chromosome (see Figures 1a, b), while structural

These chromosome abnormalities are qualitatively similar to those found in anaplastic astrocytoma (WHO grade III) and glioblastoma (WHO grade IV), but their frequency is

cell line (Magnani et al., 1994), showing trisomy of chromosome 7, monosomy of

changes affect in particular 1p (Figure 2a) and 9p (Figure 2b) chromosome arms.

cells. (d) Apoptotic and large nuclei of MI-60 cell line.

apoptotic nuclei are also reported (Figures 1a-d).

increased in the latter and multiple chromosomal rearrangements are also present. The finding of common abnormalities associated to both low- and high-grade glioma has suggested a progressive chromosomal evolution during tumour growth (Bigner et al., 1988; Jenkins et al., 1989; Magnani et al., 1994; Thiel et al., 1992) even though it has been demonstrated that a subset of glioblastomas arises clonally *de novo*, further emphasizing the genetic heterogeneity of glioma (Kleihues & Ohgaki, 2000; von Deimling et al., 1993). Given that numerical CIN features many cancer cells, it has been hypothesized that it may have a primary role in tumorigenesis (Duesberg et al., 2006; Weaver et al., 2007). Recently it has been shown that the main pathway to aneuploidy in cancer cells is triggered by extra centrosomes that, increasing improper merotelic attachments of kinetochores to spindle microtubules, cause chromosome mis-segregation (Meraldi et al., 2002) (Figure 3) (see Box 3 for centrosomes and Box 4 for mitotic spindle).

Fig. 2. (a) Chromosome 1 rearrangements of both p and q arms observed in different glioma cell lines by G-banding. (b) Rearrangements of chromosome 9p, sharing the loss of p21 band, observed in different glioblastoma cell lines by G-banding.

At early mitosis, the merotelic orientation escapes the spindle mitotic checkpoint thus representing the major mechanism of chromosome mis-segregation in non-cancer cells. Usually these errors are corrected before cells enter anaphase, to preserve genome stability (Cimini et al., 2004).

Role of the Centrosomal MARK4 Protein in Gliomagenesis 135

Fig. 4. Immunofluorescence with anti-tubulin antibody (red) of representative glioblastoma cell lines, showing (a) multiple centrosomes; (b) multipolar spindles; (c) a mitotic bipolar spindle in which centrosomes are larger than the normal one (likely extra centrosomes clustered into two spindle poles), a condition that favours mitotic stability and neoplastic growth; (d) normal centrosomes and a mitotic bipolar spindle configuration. The nuclei are

Fig. 5. Regression analysis between aneuploidies and centrosome aberrations in glioma cell lines, showing a statistically significant positive correlation. OA: oligoastrocytomas; A:

astrocytomas; GBM: glioblastoma multiforme; GC-GBM: giant cell-GBM.

counterstained with DAPI (4',6-di amidino-2-phenyl indole) (blue).

Fig. 3. Proposed events of lagging chromosomes in cancer cells with extra centrosomes through merotelic kinetochore orientation. (top) In the presence of extra centrosomes (three instead of two, as example), merotelic kinetochore orientation may occur: one kinetochore is bound by spindle microtubules from two centrosomes (right) instead of just one (left). (bottom) As cells move to mitosis and cluster extra centrosomes in a bipolar spindle, many attachment errors persist into anaphase, leading to lagging chromosomes.

#### **3. Tetraploidy, centrosome amplification and spontaneous chromosomal instability in glioma**

A relationship between extra centrosomes and the formation of multipolar spindles in cancer cells has been proposed by different authors (Basto et al., 2008; Cimini et al., 2004; Saunders, 2005; Sluder & Nordberg, 2004). Multipolarity in cancer cells is considered an essential transient stage prior to clustering extra centrosomes in a bipolar fashion (Brinkley, 2001). Multiple centrosomes have been detected in many types of cancer cells including glioma (Figure 4) and strongly linked to aneuploidy in a variety of studies (D'Assoro et al., 2002; Ganem et al., 2009; Ghadimi et al., 2000; Katsetos et al., 2006; Lingle et al., 2002; Magnani et al., 2009; Pihan et al., 1998).

A positive linear correlation between the percentage of cells with supernumerary centrosomes and the extent of aneuploidy within a panel of glioblastoma cell lines is shown in Figure 5.

In tumour development, aneuploidy is frequently preceded by tetraploidy, often with prolonged tetraploid precancerous status, a feature that makes it of central importance to cancer research (Margolis et al., 2003). It has been proposed that failure of cytokinesis is a key step in the formation of tetraploid karyotypes and in tumour initiation (Fujiwara et al., 2005). A tetraploid cell inherits twice the normal complement of centrosomes, a condition assessed to generate chromosomes mis-segregation in subsequent cell divisions (Ganem et al., 2007). However, tetraploid cells are observed in some normal tissues including liver and heart, indicating that cytokinesis is physiologically regulated. The possible fate of a tetraploid progeny is shown in Figure 6.

Fig. 3. Proposed events of lagging chromosomes in cancer cells with extra centrosomes through merotelic kinetochore orientation. (top) In the presence of extra centrosomes (three instead of two, as example), merotelic kinetochore orientation may occur: one kinetochore is bound by spindle microtubules from two centrosomes (right) instead of just one (left). (bottom) As cells move to mitosis and cluster extra centrosomes in a bipolar spindle, many

**3. Tetraploidy, centrosome amplification and spontaneous chromosomal** 

A relationship between extra centrosomes and the formation of multipolar spindles in cancer cells has been proposed by different authors (Basto et al., 2008; Cimini et al., 2004; Saunders, 2005; Sluder & Nordberg, 2004). Multipolarity in cancer cells is considered an essential transient stage prior to clustering extra centrosomes in a bipolar fashion (Brinkley, 2001). Multiple centrosomes have been detected in many types of cancer cells including glioma (Figure 4) and strongly linked to aneuploidy in a variety of studies (D'Assoro et al., 2002; Ganem et al., 2009; Ghadimi et al., 2000; Katsetos et al., 2006; Lingle et al., 2002;

A positive linear correlation between the percentage of cells with supernumerary centrosomes and the extent of aneuploidy within a panel of glioblastoma cell lines is shown

In tumour development, aneuploidy is frequently preceded by tetraploidy, often with prolonged tetraploid precancerous status, a feature that makes it of central importance to cancer research (Margolis et al., 2003). It has been proposed that failure of cytokinesis is a key step in the formation of tetraploid karyotypes and in tumour initiation (Fujiwara et al., 2005). A tetraploid cell inherits twice the normal complement of centrosomes, a condition assessed to generate chromosomes mis-segregation in subsequent cell divisions (Ganem et al., 2007). However, tetraploid cells are observed in some normal tissues including liver and heart, indicating that cytokinesis is physiologically regulated. The possible fate of a

attachment errors persist into anaphase, leading to lagging chromosomes.

**instability in glioma** 

in Figure 5.

Magnani et al., 2009; Pihan et al., 1998).

tetraploid progeny is shown in Figure 6.

Fig. 4. Immunofluorescence with anti-tubulin antibody (red) of representative glioblastoma cell lines, showing (a) multiple centrosomes; (b) multipolar spindles; (c) a mitotic bipolar spindle in which centrosomes are larger than the normal one (likely extra centrosomes clustered into two spindle poles), a condition that favours mitotic stability and neoplastic growth; (d) normal centrosomes and a mitotic bipolar spindle configuration. The nuclei are counterstained with DAPI (4',6-di amidino-2-phenyl indole) (blue).

Fig. 5. Regression analysis between aneuploidies and centrosome aberrations in glioma cell lines, showing a statistically significant positive correlation. OA: oligoastrocytomas; A: astrocytomas; GBM: glioblastoma multiforme; GC-GBM: giant cell-GBM.

Role of the Centrosomal MARK4 Protein in Gliomagenesis 137

such as nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere endfusions determining the furrow regression, and nuclear buds (NBUDSs), a biomarker of

Fig. 8. Photomicrographs of glioma cell lines showing (a) typical binucleated cells with nucleoplasmic bridges and (b) binucleated cells with micronuclei and nuclear buds.

subsequent rounds of aberrant mitosis.

**4. Cytogenomics of gliomas** 

Figure 2).

10).

shown in Figure 9.

Thus, binucleated tetraploid cells may be transmitted to the progeny and enhance

Chromosomal instability can be detected by different techniques, including conventional karyotyping, fluorescence *in situ* hybridization (FISH), spectral karyotyping (SKY) and

The classic assay to monitor and quantify chromosome aberrations is karyotyping (see

The *in situ* hybridization technique with fluorescently labelled probes targeting specific chromosomes is commonly applied on fixed glioma cells, allowing the analysis of chromosomes of interest cell by cell. Examples of FISH analysis in glioma cell lines are

Aneuploidies are rapidly detectable by interphase FISH as well as by quantification of micronuclei formed by chromosomes that lagged behind during a previous mitosis (Figure

The technique of array-CGH is considered the most powerful tool for identifying copy number changes of genetic material, since it combines high resolution and large scale genomic analysis, characteristics that are not combined by conventional approaches. Since it allows a quantification of amplifications and deletions, pointing through human genome databases directly to the affected genes, aCGH technology is more and more used in the

study of tumours for the identification of potentially causative cancer genes.

array-based comparative genomic hybridization (aCGH) analyses.

elimination of amplified DNA and/or DNA repair complexes (Figure 8a, b).

Fig. 6. Fate of a tetraploid cell: if extra centrosomes coalesce, a bipolar spindle assures the progeny maintains a tetraploid set, while lack of this *escamotage* gives rise to aneuploid progeny through a multipolar mitosis.

Binucleated tetraploid cells with multiple centrosomes are frequently observed in glioma cell lines, as illustrated by a representative image in Figure 7.

Fig. 7. Immunofluorescence with anti-tubulin antibody (red) of a binucleated, tetraploidderived glioblastoma cell line, showing coalesced centrosomes in one (left) of the two nuclei. Nuclei are counterstained with DAPI (blue).

To measure the occurrence of DNA damage in once-divided binucleated (BN) cells, the cytokinesis-block micronucleus cytome (CBMN Cyt) assay, an established biomarker to detect spontaneous genomic instability (Fenech, 2007), can be used. Application of CBMN Cyt to a series of glioma cell lines evidenced a high rate of micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, and chromosome aberrations

Fig. 6. Fate of a tetraploid cell: if extra centrosomes coalesce, a bipolar spindle assures the progeny maintains a tetraploid set, while lack of this *escamotage* gives rise to aneuploid

Binucleated tetraploid cells with multiple centrosomes are frequently observed in glioma

Fig. 7. Immunofluorescence with anti-tubulin antibody (red) of a binucleated, tetraploidderived glioblastoma cell line, showing coalesced centrosomes in one (left) of the two nuclei.

To measure the occurrence of DNA damage in once-divided binucleated (BN) cells, the cytokinesis-block micronucleus cytome (CBMN Cyt) assay, an established biomarker to detect spontaneous genomic instability (Fenech, 2007), can be used. Application of CBMN Cyt to a series of glioma cell lines evidenced a high rate of micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, and chromosome aberrations

progeny through a multipolar mitosis.

Nuclei are counterstained with DAPI (blue).

cell lines, as illustrated by a representative image in Figure 7.

such as nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere endfusions determining the furrow regression, and nuclear buds (NBUDSs), a biomarker of elimination of amplified DNA and/or DNA repair complexes (Figure 8a, b).

Fig. 8. Photomicrographs of glioma cell lines showing (a) typical binucleated cells with nucleoplasmic bridges and (b) binucleated cells with micronuclei and nuclear buds.

Thus, binucleated tetraploid cells may be transmitted to the progeny and enhance subsequent rounds of aberrant mitosis.

### **4. Cytogenomics of gliomas**

Chromosomal instability can be detected by different techniques, including conventional karyotyping, fluorescence *in situ* hybridization (FISH), spectral karyotyping (SKY) and array-based comparative genomic hybridization (aCGH) analyses.

The classic assay to monitor and quantify chromosome aberrations is karyotyping (see Figure 2).

The *in situ* hybridization technique with fluorescently labelled probes targeting specific chromosomes is commonly applied on fixed glioma cells, allowing the analysis of chromosomes of interest cell by cell. Examples of FISH analysis in glioma cell lines are shown in Figure 9.

Aneuploidies are rapidly detectable by interphase FISH as well as by quantification of micronuclei formed by chromosomes that lagged behind during a previous mitosis (Figure 10).

The technique of array-CGH is considered the most powerful tool for identifying copy number changes of genetic material, since it combines high resolution and large scale genomic analysis, characteristics that are not combined by conventional approaches. Since it allows a quantification of amplifications and deletions, pointing through human genome databases directly to the affected genes, aCGH technology is more and more used in the study of tumours for the identification of potentially causative cancer genes.

Role of the Centrosomal MARK4 Protein in Gliomagenesis 139

aCGH studies have been applied to gliomas and have successfully complemented previously published metaphase-CGH, SKY and LOH (loss of heterozigosity) analyses (Bredel et al, 2005; Cowell et al., 2004a, 2004b; Kitange et al., 2005; Nigro et al., 2005). Integration of the results has demonstrated an excellent correlation between the findings obtained through this genomic approach and those obtained by alternative techniques, stressing the usefulness and overall accuracy of aCGH as compared to classic previously widely employed analyses (Cowell et al., 2004a, 2004b). Comparative analysis of elaborated aCGH data led to identify copy number changes shared by various glioma grades as well as aberrations apparently related to progression to glioblastoma (GBM) (Roversi et al., 2006).

**5. Non-random chromosomal aberrations in gliomas: The 19q13 abnormalities**  Over the last decade, molecular approaches including mutation screening, LOH and aCGH analyses have led to identify the most frequently recurring genomic imbalances associated with each WHO glioma subtype (Kitange et al., 2005; Koschny et al., 2002; Shapiro, 2002) and hence the driver genes acting in pathways involved in glioma development, either in the initiation stages (Tp53 and Ras by PDGF-NF1) or in malignant progression (Rb-CDKN2- CDK4) (Collins, 2004; Zhu & Parada, 2002). Comprehensive genomic characterization by integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in >200 glioblastomas has then refined the definition of human glioblastoma genes and core pathways (The Cancer Genome Atlas [TGCA] Research Network, 2008). Deletion of chromosome 19q is nevertheless of particular interest, as it is shared by all three glioma subtypes, occurring in approximately 75% of oligodendrogliomas, 45% of mixed oligoastrocytomas and 40% of astrocytomas (von Deimling et al., 1992, 1994), where it is associated with the transition from low-grade to anaplastic tumours (Ohgaki et al., 1995;

At the cytogenetic level, chromosome 19q abnormalities are more frequently detectable in GBM than in low grade glioma, with 19q13 as the most affected region, as shown in Table1.

Ritland et al., 1995; Smith et al., 1999) (Box 2).

Box 2. TSGs: tumour suppressor genes.

Fig. 9. Partial karyotype of MI-4 GBM cell line displaying chromosome 1 alterations by (a) whole chromosome 1 painting probe and (b) dual colour FISH of YACs 745h6, spanning the 1p36.3 breakpoint (green), and 957f12, mapping to 1p36.1 (red), showing a transposition of 1p36.1 material to der (1)(p22). DNA is counterstained with DAPI (blue). Interphase dual colour FISH of RP11-111p21, mapping to 3p21 control clone (red), and RP11-172g5, mapping to 3q26.3 (green), (c) in a normal diploid cell and (d) in MI-60 GBM cell line showing amplification of the region targeted by RP22-172g5 (green).

Fig. 10. (a) Interphase FISH with centromeric probes of chromosomes 7 and 10 showing trisomy of chromosome 7 and monosomy of chromosome 10 in MI-4 GBM cell line. (b) Interphase FISH with whole chromosome 19 painting probe showing a micronucleus labelled by chromosome 19 material. DNA is counterstained with DAPI (blue).

Fig. 9. Partial karyotype of MI-4 GBM cell line displaying chromosome 1 alterations by (a) whole chromosome 1 painting probe and (b) dual colour FISH of YACs 745h6, spanning the 1p36.3 breakpoint (green), and 957f12, mapping to 1p36.1 (red), showing a transposition of 1p36.1 material to der (1)(p22). DNA is counterstained with DAPI (blue). Interphase dual colour FISH of RP11-111p21, mapping to 3p21 control clone (red), and RP11-172g5, mapping

to 3q26.3 (green), (c) in a normal diploid cell and (d) in MI-60 GBM cell line showing

Fig. 10. (a) Interphase FISH with centromeric probes of chromosomes 7 and 10 showing trisomy of chromosome 7 and monosomy of chromosome 10 in MI-4 GBM cell line. (b) Interphase FISH with whole chromosome 19 painting probe showing a micronucleus labelled by chromosome 19 material. DNA is counterstained with DAPI (blue).

amplification of the region targeted by RP22-172g5 (green).

aCGH studies have been applied to gliomas and have successfully complemented previously published metaphase-CGH, SKY and LOH (loss of heterozigosity) analyses (Bredel et al, 2005; Cowell et al., 2004a, 2004b; Kitange et al., 2005; Nigro et al., 2005). Integration of the results has demonstrated an excellent correlation between the findings obtained through this genomic approach and those obtained by alternative techniques, stressing the usefulness and overall accuracy of aCGH as compared to classic previously widely employed analyses (Cowell et al., 2004a, 2004b). Comparative analysis of elaborated aCGH data led to identify copy number changes shared by various glioma grades as well as aberrations apparently related to progression to glioblastoma (GBM) (Roversi et al., 2006).

#### **5. Non-random chromosomal aberrations in gliomas: The 19q13 abnormalities**

Over the last decade, molecular approaches including mutation screening, LOH and aCGH analyses have led to identify the most frequently recurring genomic imbalances associated with each WHO glioma subtype (Kitange et al., 2005; Koschny et al., 2002; Shapiro, 2002) and hence the driver genes acting in pathways involved in glioma development, either in the initiation stages (Tp53 and Ras by PDGF-NF1) or in malignant progression (Rb-CDKN2- CDK4) (Collins, 2004; Zhu & Parada, 2002). Comprehensive genomic characterization by integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in >200 glioblastomas has then refined the definition of human glioblastoma genes and core pathways (The Cancer Genome Atlas [TGCA] Research Network, 2008). Deletion of chromosome 19q is nevertheless of particular interest, as it is shared by all three glioma subtypes, occurring in approximately 75% of oligodendrogliomas, 45% of mixed oligoastrocytomas and 40% of astrocytomas (von Deimling et al., 1992, 1994), where it is associated with the transition from low-grade to anaplastic tumours (Ohgaki et al., 1995; Ritland et al., 1995; Smith et al., 1999) (Box 2).


Box 2. TSGs: tumour suppressor genes.

At the cytogenetic level, chromosome 19q abnormalities are more frequently detectable in GBM than in low grade glioma, with 19q13 as the most affected region, as shown in Table1.

Role of the Centrosomal MARK4 Protein in Gliomagenesis 141

Furthermore, similarly to oligodendroglioma, combined LOH of 1p and 19q was found to define a small subset of GBM patients with a significantly better survival, even if their tumours were not morphologically distinguishable from the bulk of GBMs (Schmidt et al., 2002). This finding has been translated into significant advance in the prognosis and treatment of oligodendrogliomas (van den Bent, 2004). A candidate tumour suppressor region has been assigned by LOH to 19q13.3 (Hartmann et al., 2002), but no positional or

Only recently an integrated analysis of human glioblastoma multiforme with the application of next generation sequencing technology disclosed a new marker associated with an increase in overall survival, represented by recurrent mutations in the active site of isocitrate dehydrogenase 1 (*IDH1*) in a large fraction of young patients with secondary GBM (Parsons

**6. Identification of** *MARK4* **gene through refined FISH mapping of 19q13** 

FISH studies of structural 19q chromosomal rearrangements in glioma (Magnani et al., 1999) and a detailed analysis of the breakpoints underlying the 19q13 alterations in the MI-4 glioblastoma cell line, led to identify a 19q13.2 intrachromosomal duplication of the MAP/microtubule affinity-regulating kinase *4 (MARK4)* gene (Beghini et al., 2003) (Figure 11). Genomic profiling by means of array-CGH interrogation of 25 primary glioma cell lines including the MI-4 GBM cell line (Roversi et al., 2006) revealed that the BAC clone encompassing *MARK4* at 19q13.2 (Figure 12) is included in a "gain" region in a few of the tested cell lines and confirmed *MARK4* duplication in the MI-4 glioblastoma cell line

Fig. 11. 19q13.2 intrachromosomal duplication of *MARK4* in the MI-4 GBM cell line detected by G-banding and FISH analysis using a whole chromosome painting 19 probe and a

functional candidate gene in this band has yet been appointed.

et al., 2008).

**breakpoints** 

(Figure 13).

*MARK4*-specific cosmid clone.


Table 1. Cytogenetic alterations of chromosome 19 in gliomas; 19q13 alterations are marked by red stars. GBM: glioblastoma multiforme; AMG: anaplastic mixed glioma; PA: pilocytic astrocytoma; O: oligodendroglioma; AA: anaplastic astrocytoma; AO: anaplastic oligodendroglioma; PXA: pleomorphic xanthoastrocytoma; nr: not reported.

Table 1. Cytogenetic alterations of chromosome 19 in gliomas; 19q13 alterations are marked by red stars. GBM: glioblastoma multiforme; AMG: anaplastic mixed glioma; PA: pilocytic

astrocytoma; O: oligodendroglioma; AA: anaplastic astrocytoma; AO: anaplastic oligodendroglioma; PXA: pleomorphic xanthoastrocytoma; nr: not reported.

Furthermore, similarly to oligodendroglioma, combined LOH of 1p and 19q was found to define a small subset of GBM patients with a significantly better survival, even if their tumours were not morphologically distinguishable from the bulk of GBMs (Schmidt et al., 2002). This finding has been translated into significant advance in the prognosis and treatment of oligodendrogliomas (van den Bent, 2004). A candidate tumour suppressor region has been assigned by LOH to 19q13.3 (Hartmann et al., 2002), but no positional or functional candidate gene in this band has yet been appointed.

Only recently an integrated analysis of human glioblastoma multiforme with the application of next generation sequencing technology disclosed a new marker associated with an increase in overall survival, represented by recurrent mutations in the active site of isocitrate dehydrogenase 1 (*IDH1*) in a large fraction of young patients with secondary GBM (Parsons et al., 2008).

#### **6. Identification of** *MARK4* **gene through refined FISH mapping of 19q13 breakpoints**

FISH studies of structural 19q chromosomal rearrangements in glioma (Magnani et al., 1999) and a detailed analysis of the breakpoints underlying the 19q13 alterations in the MI-4 glioblastoma cell line, led to identify a 19q13.2 intrachromosomal duplication of the MAP/microtubule affinity-regulating kinase *4 (MARK4)* gene (Beghini et al., 2003) (Figure 11). Genomic profiling by means of array-CGH interrogation of 25 primary glioma cell lines including the MI-4 GBM cell line (Roversi et al., 2006) revealed that the BAC clone encompassing *MARK4* at 19q13.2 (Figure 12) is included in a "gain" region in a few of the tested cell lines and confirmed *MARK4* duplication in the MI-4 glioblastoma cell line (Figure 13).

Fig. 11. 19q13.2 intrachromosomal duplication of *MARK4* in the MI-4 GBM cell line detected by G-banding and FISH analysis using a whole chromosome painting 19 probe and a *MARK4*-specific cosmid clone.

Role of the Centrosomal MARK4 Protein in Gliomagenesis 143

The combined FISH and array-CGH results provided the rationale for investigating a possible role of the serine-threonine kinase MARK4 in glioma. It's worth of note that this gene, belonging to the so called "kinome", maps at the centromeric boundary of the 19q13.3

MARK4 (MAP/microtubule affinity-regulating kinase 4) is a member of the MARKs family, constituted in mammals by four serine-threonine kinases (MARK1-4) which are able to phosphorylate the microtubule-associated proteins (MAPs, including Tau, MAP2, MAP4 and doublecortin) (Drewes et al., 1997). Microtubules (MTs) are cytoskeleton cylindrical structures formed by α and β tubulin dimers; dimers can quickly assemble or disassemble, causing the microtubules to grow or shorten and making them very dynamic. MAPs association stabilizes the MTs; when MARK kinases link a phosphate group to MAPs (phosphorylation), MAPs cannot associate to MTs any longer, thus microtubules become

Fig. 14. Schematic representation of microtubules. Assembled α and β tubulin dimers form the microtubules, stabilized by MAP association. When MAPs are phosphorylated, they are

All MARK proteins have a very conserved structure, consisting of six sequence segments

 the catalytic or kinase domain, containing both the activation/inactivation loop (MARK kinases are in turn activated/inactivated by phosphorylation/dephosphorylation) and the catalytic loop, by which MARKs transfer a phosphate group to substrate proteins; a linker, that is a highly and negatively charged motif resembling the common docking

 the UBA domain, a small globular domain with sequence homology to ubiquitinassociated proteins; it may exert an autoregulatory function through interaction with

a spacer, the most variable region among MARK members; it is probably important for

 the C-terminal tail, consisting of the kinase-associated (KA1) domain, whose function is still uncertain. It is characterized by a hydrophobic portion surrounded by positively

LOH region in glioma.

**7. The family of MARK kinases** 

more instable and disassemble (Figure 14).

no more able to bind microtubules, which disassemble.

the N-terminal header, whose role is unknown;

(CD) site in MAP kinases; it may bind interactors;

regulating MARK activity since it holds phosphorylation sites;

**7.1 MARKs protein structure** 

(Marx et al., 2010) (Figure 15):

the catalytic domain;

Fig. 12. *MARK4* genomic region (http://genome.ucsc.edu/). Clones of chromosome 19q full coverage (blue) overlapping *MARK4* gene are circled in red: the gene is entirely encompassed by BAC clones RP11-746H08, RP11-568L16, RP11-202G02, RP11-752G09 RP11- 584B04, RP13-647G21 and partially encompassed by RP11-577I16.

Fig. 13. (left) Chromosome 19q array-CGH of MI-4 GBM cell line, showing duplicated *MARK4* gene (red star) and the common LOH region in glioma. (right) Schematic representation of *MARK4* position on chromosome 19, at the boundary of 19q13.3 LOH region.

The combined FISH and array-CGH results provided the rationale for investigating a possible role of the serine-threonine kinase MARK4 in glioma. It's worth of note that this gene, belonging to the so called "kinome", maps at the centromeric boundary of the 19q13.3 LOH region in glioma.

### **7. The family of MARK kinases**

142 Glioma – Exploring Its Biology and Practical Relevance

Fig. 12. *MARK4* genomic region (http://genome.ucsc.edu/). Clones of chromosome 19q full

encompassed by BAC clones RP11-746H08, RP11-568L16, RP11-202G02, RP11-752G09 RP11-

Fig. 13. (left) Chromosome 19q array-CGH of MI-4 GBM cell line, showing duplicated *MARK4* gene (red star) and the common LOH region in glioma. (right) Schematic representation of *MARK4* position on chromosome 19, at the boundary of 19q13.3 LOH

region.

coverage (blue) overlapping *MARK4* gene are circled in red: the gene is entirely

584B04, RP13-647G21 and partially encompassed by RP11-577I16.

MARK4 (MAP/microtubule affinity-regulating kinase 4) is a member of the MARKs family, constituted in mammals by four serine-threonine kinases (MARK1-4) which are able to phosphorylate the microtubule-associated proteins (MAPs, including Tau, MAP2, MAP4 and doublecortin) (Drewes et al., 1997). Microtubules (MTs) are cytoskeleton cylindrical structures formed by α and β tubulin dimers; dimers can quickly assemble or disassemble, causing the microtubules to grow or shorten and making them very dynamic. MAPs association stabilizes the MTs; when MARK kinases link a phosphate group to MAPs (phosphorylation), MAPs cannot associate to MTs any longer, thus microtubules become more instable and disassemble (Figure 14).

Fig. 14. Schematic representation of microtubules. Assembled α and β tubulin dimers form the microtubules, stabilized by MAP association. When MAPs are phosphorylated, they are no more able to bind microtubules, which disassemble.

#### **7.1 MARKs protein structure**

All MARK proteins have a very conserved structure, consisting of six sequence segments (Marx et al., 2010) (Figure 15):


Role of the Centrosomal MARK4 Protein in Gliomagenesis 145

MARK4 is the less characterized member among MARK proteins. It has been discovered by Kato and colleagues in 2001 among a few genes whose expression resulted significantly increased in hepatocarcinoma cells with elevated β-catenin levels in their nucleus (Kato et

*MARK4* gene is located on chromosome 19q13.2, consists of 18 exons and encodes at least two isoforms, namely MARK4S and MARK4L, originated by alternative splicing (Kato et al., 2001) (Figure 16). mRNA splicing is a complex process consisting in the removal of introns, which are non-coding sequences, and in the joining of exons, the coding sequences, to

 MARK4S ("short") protein is the native isoform, consisting of all the 18 exons, and is 688 aminoacid-long with predicted molecular weight of 75.3 kilo Daltons (kDa); MARK4L protein derives from skipping of exon 16, which causes a shift of the reading frame1 with a downstream stop codon, originating a longer protein (752 aminoacids;

Fig. 16. Alternative splicing of exon 16 gives origin to MARK4 isoforms. When exon 16 is included in the mRNA, the stop codon is inside exon 18 and the encoded protein, MARK4S, lacks the KA1 domain at the C-terminal tail (left); when exon 16 is skipped, a shift of the reading frame occurs, changing the stop codon and generating a longer MARK4L protein,

Both MARK4L and S share the same protein structure of MARKs, with 90% sequence homology in the kinase domain. The two isoforms differ in the C-terminal tail, since MARK4L includes the kinase-associated 1 domain as the other MARK proteins, whereas MARK4S contains a domain with no homology to any known structure (Kato et al., 2001; Moroni et al., 2006) (Figure 16). Actually, MARK4 has less sequence homology in the Cterminus compared to the other MARKs; nevertheless MARK4L tail seems to fold in a similar shape, suggesting that the role of the C-terminal region may apply also to MARK4L

<sup>1</sup> The mRNA sequence is "read" by an enzyme which matches a determinate "codon", made by three nucleotides, with its respective aminoacid. There are two particular codons, namely the start and the

generate the "edited" mRNA ready to be translated into a protein.

predicted molecular weight: 82.5 kDa).

which has the classical KA1 domain (right).

stop codon, which mark the beginning and the end of the protein.

(Marx et al., 2010).

**8. MARK4** 

al., 2001).

charged residues, which may interact with negatively charged regions of cytoskeletal proteins, MARK catalytic domain or MARK CD domain (Tochio et al., 2006) with an inhibitory effect. It has been proposed it could be involved in protein localization to the membrane, being identified as a domain that binds membrane anionic phospholipids, in particular phosphatidylserine (Moravcevic et al., 2010).

Fig. 15. Schematic representation of MARK protein structure. Boxes are not drawn to scale.

#### **7.2 MARKs regulation**

Being composed of several domains, MARK proteins are regulated by multiple mechanisms. All MARKs are activated by liver kinase B (LKB1) and MARK kinase (MARKK) by phosphorylation on the threonine residue in the activation loop (Timm et al., 2008); in addition, phosphorylation by CaMKI (calcium/calmodulin-dependent protein kinase I) activates MARK2 (Matenia & Mandelkow, 2009). On the contrary, phosphorylation by the glycogen synthase kinase 3β (GSK3β) on the serine residue in the activation loop, by aPKC (atypical protein kinase C) in the spacer region or by Pim1 kinase, down-regulates MARK activity (Matenia & Mandelkow, 2009; Timm et al., 2008). Finally, interaction between MARK catalytic domain and other proteins/MARK domains (such as 14-3-3 proteins, PAK5, MARK UBA and KA1 domains) inhibits MARK activity (Marx et al., 2010).

#### **7.3 MARKs functions**

Since MARK kinases regulate the affinity between MAPs and MTs, they are implicated in several cellular processes involving the microtubules, such as cytoskeleton dynamics, neuron motility (Schaar et al., 2004), and microtubule-dependent transport of proteins, vesicles and organelles (Mandelkow et al., 2004). Microtubules also play an important role in centrosome formation (Box 3) and in the correct distribution of the chromosomes in the two daughter cells during cell division (mitosis and cytokinesis; Box 4).

*Tau* is a microtubule-associated protein particularly expressed in the central nervous system. The aggregation of hyperphosphorylated *Tau* has been demonstrated to form insoluble neurofibrillary tangles (Chin et al., 2000; Gamblin et al., 2003) which are characteristic of Alzheimer's disease. MARKs role in this pathology has been evaluated in many studies, demonstrating, as an example, MARK co-localization with neurofibrillary tangles (Chin et al., 2000).

MARK2 is involved in establishing cell polarity, cooperating in the organization of the epithelial structure of liver, kidney and stomach (Cohen et al., 2004; Matenia & Mandelkow, 2009), and regulating axon formation in neuronal cells (Chen et al., 2006). Experiments in mice demonstrated that MARK2 is also implicated in many physiological functions, such as fertility, homeostasis of the immune system, memory, growth and metabolism (Bessone et al., 1999; Hurov et al., 2001; Hurov & Piwnica-Worms, 2007; Segu et al., 2008). MARK3 plays an important role in cell signaling and cell cycle control: phosphorylation of some proteins by MARK3 induces their binding to 14-3-3 proteins thus regulating many cellular pathways (Bachmann et al., 2004; Müller et al., 2001).

### **8. MARK4**

144 Glioma – Exploring Its Biology and Practical Relevance

Fig. 15. Schematic representation of MARK protein structure. Boxes are not drawn to scale.

Being composed of several domains, MARK proteins are regulated by multiple mechanisms. All MARKs are activated by liver kinase B (LKB1) and MARK kinase (MARKK) by phosphorylation on the threonine residue in the activation loop (Timm et al., 2008); in addition, phosphorylation by CaMKI (calcium/calmodulin-dependent protein kinase I) activates MARK2 (Matenia & Mandelkow, 2009). On the contrary, phosphorylation by the glycogen synthase kinase 3β (GSK3β) on the serine residue in the activation loop, by aPKC (atypical protein kinase C) in the spacer region or by Pim1 kinase, down-regulates MARK activity (Matenia & Mandelkow, 2009; Timm et al., 2008). Finally, interaction between MARK catalytic domain and other proteins/MARK domains (such as 14-3-3 proteins, PAK5,

Since MARK kinases regulate the affinity between MAPs and MTs, they are implicated in several cellular processes involving the microtubules, such as cytoskeleton dynamics, neuron motility (Schaar et al., 2004), and microtubule-dependent transport of proteins, vesicles and organelles (Mandelkow et al., 2004). Microtubules also play an important role in centrosome formation (Box 3) and in the correct distribution of the chromosomes in the

*Tau* is a microtubule-associated protein particularly expressed in the central nervous system. The aggregation of hyperphosphorylated *Tau* has been demonstrated to form insoluble neurofibrillary tangles (Chin et al., 2000; Gamblin et al., 2003) which are characteristic of Alzheimer's disease. MARKs role in this pathology has been evaluated in many studies, demonstrating, as an example, MARK co-localization with neurofibrillary tangles (Chin et

MARK2 is involved in establishing cell polarity, cooperating in the organization of the epithelial structure of liver, kidney and stomach (Cohen et al., 2004; Matenia & Mandelkow, 2009), and regulating axon formation in neuronal cells (Chen et al., 2006). Experiments in mice demonstrated that MARK2 is also implicated in many physiological functions, such as fertility, homeostasis of the immune system, memory, growth and metabolism (Bessone et al., 1999; Hurov et al., 2001; Hurov & Piwnica-Worms, 2007; Segu et al., 2008). MARK3 plays an important role in cell signaling and cell cycle control: phosphorylation of some proteins by MARK3 induces their binding to 14-3-3 proteins thus regulating many cellular pathways

MARK UBA and KA1 domains) inhibits MARK activity (Marx et al., 2010).

two daughter cells during cell division (mitosis and cytokinesis; Box 4).

in particular phosphatidylserine (Moravcevic et al., 2010).

**7.2 MARKs regulation** 

**7.3 MARKs functions** 

al., 2000).

(Bachmann et al., 2004; Müller et al., 2001).

charged residues, which may interact with negatively charged regions of cytoskeletal proteins, MARK catalytic domain or MARK CD domain (Tochio et al., 2006) with an inhibitory effect. It has been proposed it could be involved in protein localization to the membrane, being identified as a domain that binds membrane anionic phospholipids,

MARK4 is the less characterized member among MARK proteins. It has been discovered by Kato and colleagues in 2001 among a few genes whose expression resulted significantly increased in hepatocarcinoma cells with elevated β-catenin levels in their nucleus (Kato et al., 2001).

*MARK4* gene is located on chromosome 19q13.2, consists of 18 exons and encodes at least two isoforms, namely MARK4S and MARK4L, originated by alternative splicing (Kato et al., 2001) (Figure 16). mRNA splicing is a complex process consisting in the removal of introns, which are non-coding sequences, and in the joining of exons, the coding sequences, to generate the "edited" mRNA ready to be translated into a protein.


Fig. 16. Alternative splicing of exon 16 gives origin to MARK4 isoforms. When exon 16 is included in the mRNA, the stop codon is inside exon 18 and the encoded protein, MARK4S, lacks the KA1 domain at the C-terminal tail (left); when exon 16 is skipped, a shift of the reading frame occurs, changing the stop codon and generating a longer MARK4L protein, which has the classical KA1 domain (right).

Both MARK4L and S share the same protein structure of MARKs, with 90% sequence homology in the kinase domain. The two isoforms differ in the C-terminal tail, since MARK4L includes the kinase-associated 1 domain as the other MARK proteins, whereas MARK4S contains a domain with no homology to any known structure (Kato et al., 2001; Moroni et al., 2006) (Figure 16). Actually, MARK4 has less sequence homology in the Cterminus compared to the other MARKs; nevertheless MARK4L tail seems to fold in a similar shape, suggesting that the role of the C-terminal region may apply also to MARK4L (Marx et al., 2010).

<sup>1</sup> The mRNA sequence is "read" by an enzyme which matches a determinate "codon", made by three nucleotides, with its respective aminoacid. There are two particular codons, namely the start and the stop codon, which mark the beginning and the end of the protein.

Role of the Centrosomal MARK4 Protein in Gliomagenesis 147

lines, it did not evidence *MARK4* copy number variations, except for the MI-4 GBM cell line (Roversi et al., 2006). Only a few *MARK4* alterations are reported in the literature, namely two missense mutations (aminoacidic substitution) in exon 12 (R377Q and R418C in the spacer region), two silent mutations (no aminoacidic substitution) in exons 5 (Y137Y) and 9 (I286I) (kinase domain), while one intronic mutation (exon 8 +5 C>T; kinase domain) has been found in a few tumour samples (Greenman et al., 2007). In addition, only a splice-site mutation (exon 13 +1 G>A; spacer region) has been identified in one among 91 glioblastoma samples (TGCA Research Network, 2008). However, CpG methylation and/or promoter amplification have not yet been investigated. Based on this evidence, neither amplification nor mutations of *MARK4* gene seem to be the cause of its reported sustained expression in

Fig. 17. (a) Semi-quantitative Reverse Transcription-PCR of MARK4S and MARK4L

**10. MARK4 sub-cellular localization in glioma cell lines** 

isoforms in whole normal brain (WNB) and in 32 glioma cell lines, subdivided according to WHO grade (A: astrocytoma; AA: anaplastic astrocytoma; OA: oligoastroctytoma; AOA: anaplastic oligoastrocytoma; O: oligodendroglioma; GBM: glioblastoma multiforme). (b) Downregulation of MARK4L expression during glial differentiation of human neural progenitors: semi-quantitative RT-PCR (top) and graph representation (bottom) of MARK4L expression in neural progenitors at times 0, 10 and 28 days of induced differentiation.

Recently, immunofluorescence analyses with a specific anti-MARK4L antibody highlighted multiple sub-cellular localizations for the endogenous MARK4L protein in glioma cell lines

glioma samples.

(Magnani et al., 2009).

#### **8.1 MARK4 regulation**

Phosphorylation by LKB1, in the activation loop, activates MARK4, while polyubiquitination of MARK4 inhibits the kinase activation (Al-Hakim et al., 2008). Furthermore, as MARK4 interacts with aPKC (Brajenovic et al., 2008), it could be phosphorylated and inactivated by this kinase as reported for MARK2 and MARK3.

#### **8.2 MARK4 interactors and hypothetical functions**

By tandem affinity purification and immunoprecipitation experiments, near twenty proteins have been identified as putative MARK4 interactors (Brajenovic et al., 2008). Among them, PKCλ and Cdc42 are implicated in cell polarity control and TGFβIAF (transforming growth factor β-inducing anti-apoptotic factor) is thought to be a hortologue of Miranda, a protein involved in the asymmetric division of neuroblasts in *Drosophila*. MARK4 interacts with the 14-3-3η isoform (Angrand et al., 2006; Brajenovic et al., 2008) of 14-3-3 proteins, which control multiple cellular processes by binding phosphorylated proteins and could directly regulate MARK4 or act as bridges among different pathways. Other MARK4 interactors are ARHGEF2, a cytoskeleton binding protein, and Phosphatase 2A, which is associated to microtubules and regulates *Tau* (Brajenovic et al., 2008). MARK4 protein has been also found to co-localize and co-precipitate in complex with α, β, and γ tubulin, myosin and actin (Brajenovic et al., 2008; Trinczek et al., 2004).

As the other MARK members, MARK4 phosphorylates MAPs, increasing microtubule dynamics; therefore, as also suggested by the interactions above reported, MARK4 may be involved in many processes involving microtubules, such as cytoskeleton dynamics.

### **9. Up-regulation of MARK4L in glioma**

*MARK4* gene is expressed ubiquitously in human tissues, with particularly elevated levels in brain and testis (Kato et al., 2001).

Few *MARK4* expression studies are reported in literature; they were performed with nonquantitative methods, such as northern blot (Kato et al., 2001; Schneider et al., 2004; Trinczek et al., 2004) and semi-quantitative competitive PCR (polymerase chain reaction) (Moroni et al., 2006), on different organisms (human, rat and mouse tissues) not always allowing to discern between the two MARK4 isoforms. MARK4 transcriptional variants are differentially regulated in human tissues, especially in the central nervous system: MARK4S is the predominant isoform in mouse and human brain, while MARK4L has been found highly expressed in neural progenitors and in gliomas (Beghini et al., 2003; Moroni et al., 2006).

By a semi-quantitative approach MARK4L has been found up-regulated in glioma tissue samples (fragments of glial tumours excised from patients) and glioma cell lines, of different malignancy grades, including the MI-4 GBM cell line carrying the *MARK4* duplicated gene as detected by FISH and aCGH analysis. MARK4L has been also found highly expressed in neural progenitors and down-regulated during their glial differentiation into astrocytes, suggesting that it might be necessary for proliferation, being thus highly enriched in proliferating or undifferentiated cells (Beghini et al., 2003) (Figure 17).

Protein kinase activation, often caused by gene amplification and/or mutation, is frequently associated to cancer initiation and progression, as most kinases are involved in cell proliferation. Although array-CGH analyses on glioma cell lines showed that the BAC clone encompassing *MARK4* at 19q13.2 is included in a "gain" region in a few of the tested cell

Phosphorylation by LKB1, in the activation loop, activates MARK4, while polyubiquitination of MARK4 inhibits the kinase activation (Al-Hakim et al., 2008). Furthermore, as MARK4 interacts with aPKC (Brajenovic et al., 2008), it could be

By tandem affinity purification and immunoprecipitation experiments, near twenty proteins have been identified as putative MARK4 interactors (Brajenovic et al., 2008). Among them, PKCλ and Cdc42 are implicated in cell polarity control and TGFβIAF (transforming growth factor β-inducing anti-apoptotic factor) is thought to be a hortologue of Miranda, a protein involved in the asymmetric division of neuroblasts in *Drosophila*. MARK4 interacts with the 14-3-3η isoform (Angrand et al., 2006; Brajenovic et al., 2008) of 14-3-3 proteins, which control multiple cellular processes by binding phosphorylated proteins and could directly regulate MARK4 or act as bridges among different pathways. Other MARK4 interactors are ARHGEF2, a cytoskeleton binding protein, and Phosphatase 2A, which is associated to microtubules and regulates *Tau* (Brajenovic et al., 2008). MARK4 protein has been also found to co-localize and co-precipitate in complex with α, β, and γ tubulin, myosin and actin

As the other MARK members, MARK4 phosphorylates MAPs, increasing microtubule dynamics; therefore, as also suggested by the interactions above reported, MARK4 may be

*MARK4* gene is expressed ubiquitously in human tissues, with particularly elevated levels

Few *MARK4* expression studies are reported in literature; they were performed with nonquantitative methods, such as northern blot (Kato et al., 2001; Schneider et al., 2004; Trinczek et al., 2004) and semi-quantitative competitive PCR (polymerase chain reaction) (Moroni et al., 2006), on different organisms (human, rat and mouse tissues) not always allowing to discern between the two MARK4 isoforms. MARK4 transcriptional variants are differentially regulated in human tissues, especially in the central nervous system: MARK4S is the predominant isoform in mouse and human brain, while MARK4L has been found highly expressed in neural progenitors and in gliomas (Beghini et al., 2003; Moroni et al.,

By a semi-quantitative approach MARK4L has been found up-regulated in glioma tissue samples (fragments of glial tumours excised from patients) and glioma cell lines, of different malignancy grades, including the MI-4 GBM cell line carrying the *MARK4* duplicated gene as detected by FISH and aCGH analysis. MARK4L has been also found highly expressed in neural progenitors and down-regulated during their glial differentiation into astrocytes, suggesting that it might be necessary for proliferation, being thus highly enriched in

Protein kinase activation, often caused by gene amplification and/or mutation, is frequently associated to cancer initiation and progression, as most kinases are involved in cell proliferation. Although array-CGH analyses on glioma cell lines showed that the BAC clone encompassing *MARK4* at 19q13.2 is included in a "gain" region in a few of the tested cell

proliferating or undifferentiated cells (Beghini et al., 2003) (Figure 17).

involved in many processes involving microtubules, such as cytoskeleton dynamics.

phosphorylated and inactivated by this kinase as reported for MARK2 and MARK3.

**8.2 MARK4 interactors and hypothetical functions** 

(Brajenovic et al., 2008; Trinczek et al., 2004).

**9. Up-regulation of MARK4L in glioma** 

in brain and testis (Kato et al., 2001).

2006).

**8.1 MARK4 regulation** 

lines, it did not evidence *MARK4* copy number variations, except for the MI-4 GBM cell line (Roversi et al., 2006). Only a few *MARK4* alterations are reported in the literature, namely two missense mutations (aminoacidic substitution) in exon 12 (R377Q and R418C in the spacer region), two silent mutations (no aminoacidic substitution) in exons 5 (Y137Y) and 9 (I286I) (kinase domain), while one intronic mutation (exon 8 +5 C>T; kinase domain) has been found in a few tumour samples (Greenman et al., 2007). In addition, only a splice-site mutation (exon 13 +1 G>A; spacer region) has been identified in one among 91 glioblastoma samples (TGCA Research Network, 2008). However, CpG methylation and/or promoter amplification have not yet been investigated. Based on this evidence, neither amplification nor mutations of *MARK4* gene seem to be the cause of its reported sustained expression in glioma samples.

Fig. 17. (a) Semi-quantitative Reverse Transcription-PCR of MARK4S and MARK4L isoforms in whole normal brain (WNB) and in 32 glioma cell lines, subdivided according to WHO grade (A: astrocytoma; AA: anaplastic astrocytoma; OA: oligoastroctytoma; AOA: anaplastic oligoastrocytoma; O: oligodendroglioma; GBM: glioblastoma multiforme). (b) Downregulation of MARK4L expression during glial differentiation of human neural progenitors: semi-quantitative RT-PCR (top) and graph representation (bottom) of MARK4L expression in neural progenitors at times 0, 10 and 28 days of induced differentiation.

#### **10. MARK4 sub-cellular localization in glioma cell lines**

Recently, immunofluorescence analyses with a specific anti-MARK4L antibody highlighted multiple sub-cellular localizations for the endogenous MARK4L protein in glioma cell lines (Magnani et al., 2009).

Role of the Centrosomal MARK4 Protein in Gliomagenesis 149

The endogenous MARK4L localizes both at normal interphase centrosomes (Figure 18) as well as at the aberrant centrosomes frequently observed in glioma cell lines (see Figure 4), suggesting a possible link between the alternatively spliced kinase and the mitotic instability frequently observed in human glioma. Two abnormal centrosome configurations are reported: a random one (multiple centrosomes randomly distributed) and a clustered one (multiple centrosomes collected in a single large aggregate) (Magnani et al., 2009), as

Fig. 19. Anti-MARK4L (green; left) and anti-γtubulin (red; middle) antibodies showing colocalization signals in abnormal centrosomes of glioma cell lines. Both the abnormal centrosome configurations are reported: the random one (top) and the clustered one

The centrosome association is maintained during the entire course of mitosis, as MARK4L co-localizes with γtubulin in all the cell cycle phases. The anti-MARK4L antibody is also detected in the midbody, a microtubule structure forming at the contact point between the two daughter cells at the end of the cell division. These data demonstrate that the kinase is endogenously associated with the centrosomes during the whole cell cycle and concentrates thereafter into the midbody during cytokinesis (Magnani et al., 2009) (Figure 20) (Box 4).

Fig. 20. Co-localization of MARK4L (green) and γtubulin (red) proteins at the midbody (arrow) during the cytokinesis of a glioma cell. The nuclei are counterstained with DAPI

(bottom). The nuclei are counterstained with DAPI (blue, right).

depicted in Figure 19.

**10.2 Midbody localization** 

(blue).

#### **10.1 Centrosome localization**

It has been assessed that, under microtubule-stabilizing conditions, MARK4L localizes in the perinuclear region of glioma cell lines. By co-localization experiments with both anti-MARK4L and anti-γtubulin (the main centrosomal protein) antibodies, this perinuclear localization has been demonstrated to correspond to the centrosome (Magnani et al., 2009), as shown in Figure 18 (Box 3). This result confirms previous data referring to exogenous MARK4 protein conjugated to GFP (green fluorescent protein), which has been shown to co-localize with microtubules and centrosomes of CHO (Chinese hamster ovary) and neuroblastoma cell lines (Trinczek et al., 2004), in contrast to MARK1, MARK2 and MARK3 that exhibit uniform cytoplasmic localization. Furthermore, it has been demonstrated that the association with the centrosome is independent from microtubules, since it is not abolished when microtubules are depolimerized by nocodazole treatment (Magnani et al., 2009).

Fig. 18. Anti-MARK4L (green; left) and anti-γtubulin (red; right) antibodies showing colocalization signals in interphase (top) and mitotic (bottom) centrosomes of glioma cell lines.


It has been assessed that, under microtubule-stabilizing conditions, MARK4L localizes in the perinuclear region of glioma cell lines. By co-localization experiments with both anti-MARK4L and anti-γtubulin (the main centrosomal protein) antibodies, this perinuclear localization has been demonstrated to correspond to the centrosome (Magnani et al., 2009), as shown in Figure 18 (Box 3). This result confirms previous data referring to exogenous MARK4 protein conjugated to GFP (green fluorescent protein), which has been shown to co-localize with microtubules and centrosomes of CHO (Chinese hamster ovary) and neuroblastoma cell lines (Trinczek et al., 2004), in contrast to MARK1, MARK2 and MARK3 that exhibit uniform cytoplasmic localization. Furthermore, it has been demonstrated that the association with the centrosome is independent from microtubules, since it is not abolished when microtubules are

Fig. 18. Anti-MARK4L (green; left) and anti-γtubulin (red; right) antibodies showing colocalization signals in interphase (top) and mitotic (bottom) centrosomes of glioma cell lines.

**10.1 Centrosome localization** 

Box 3.

depolimerized by nocodazole treatment (Magnani et al., 2009).

The endogenous MARK4L localizes both at normal interphase centrosomes (Figure 18) as well as at the aberrant centrosomes frequently observed in glioma cell lines (see Figure 4), suggesting a possible link between the alternatively spliced kinase and the mitotic instability frequently observed in human glioma. Two abnormal centrosome configurations are reported: a random one (multiple centrosomes randomly distributed) and a clustered one (multiple centrosomes collected in a single large aggregate) (Magnani et al., 2009), as depicted in Figure 19.

Fig. 19. Anti-MARK4L (green; left) and anti-γtubulin (red; middle) antibodies showing colocalization signals in abnormal centrosomes of glioma cell lines. Both the abnormal centrosome configurations are reported: the random one (top) and the clustered one (bottom). The nuclei are counterstained with DAPI (blue, right).

#### **10.2 Midbody localization**

The centrosome association is maintained during the entire course of mitosis, as MARK4L co-localizes with γtubulin in all the cell cycle phases. The anti-MARK4L antibody is also detected in the midbody, a microtubule structure forming at the contact point between the two daughter cells at the end of the cell division. These data demonstrate that the kinase is endogenously associated with the centrosomes during the whole cell cycle and concentrates thereafter into the midbody during cytokinesis (Magnani et al., 2009) (Figure 20) (Box 4).

Fig. 20. Co-localization of MARK4L (green) and γtubulin (red) proteins at the midbody (arrow) during the cytokinesis of a glioma cell. The nuclei are counterstained with DAPI (blue).

Role of the Centrosomal MARK4 Protein in Gliomagenesis 151

The overall immunofluorescence data on endogenous MARK4L protein confirm the previous evidence on its centrosome association and highlight two novel localization sites of

Immunoblotting with anti-MARK4L antibody on centrosomes, midbody and nucleoli isolated by biochemical fractionation from glioblastoma cell lines confirmed the presence of MARK4L protein in each fraction, validated by antibodies specific for each cell structure: anti-γtubulin antibody for centrosomes, anti-βtubulin for the midbody (βtubulin, together with αtubulin, accounts for 30% of midbody proteins) and anti-nucleolin for the nucleolus

The localization pattern of MARK4L delineated by the above studies suggests that the kinase may take part in cell cycle progression and influence the microtubules, particularly

MARK4L association with the nucleolus in glial tumours is very interesting, since MARK4L could have a functional impact on this organelle, being requested for its building and maintenance like other protein kinases, as well as it could be spatially regulated by alternate translocation in and out the nucleolus. Many proteins are indeed sequestered in the nucleolus and then released according to a temporally regulated activity, since they must exert their function in certain phases of the cell cycle (Visintin & Amon, 2000). Last, the nucleolar localization of a protein may also influence its stability, protecting the protein from proteasomal degradation, since proteasomes are present in the nucleoplasm but not in

MARK4L: the nucleolus and the midbody (Magnani et al., 2009).

Box 5.

(Magnani et al., 2009).

those affecting the centrosome and midbody.

nucleoli (Wojcik & DeMartino, 2003).

Box 4.

#### **10.3 Nucleolar localization**

Under standard immunofluorescence conditions, anti-MARK4L antibody is also detected in the nucleoli (Box 5).

Silver-colloid method, which allows visualizing the nucleolar organizing regions (NORs), and co-localization experiments with anti-nucleolin (a nucleolar protein) antibody allowed to assess that the nuclear structures bound by MARK4L antibody are indeed the nucleoli (Magnani et al., 2009) (Figure 21) (Box 5).

Fig. 21. Co-localization of MARK4L (green) and nucleolin (red) proteins in the nucleoli of glioma cells. The nuclei are counterstained with DAPI (blue).


Box 5.

150 Glioma – Exploring Its Biology and Practical Relevance

Under standard immunofluorescence conditions, anti-MARK4L antibody is also detected in

Silver-colloid method, which allows visualizing the nucleolar organizing regions (NORs), and co-localization experiments with anti-nucleolin (a nucleolar protein) antibody allowed to assess that the nuclear structures bound by MARK4L antibody are indeed the nucleoli

Fig. 21. Co-localization of MARK4L (green) and nucleolin (red) proteins in the nucleoli of

glioma cells. The nuclei are counterstained with DAPI (blue).

Box 4.

**10.3 Nucleolar localization** 

(Magnani et al., 2009) (Figure 21) (Box 5).

the nucleoli (Box 5).

The overall immunofluorescence data on endogenous MARK4L protein confirm the previous evidence on its centrosome association and highlight two novel localization sites of MARK4L: the nucleolus and the midbody (Magnani et al., 2009).

Immunoblotting with anti-MARK4L antibody on centrosomes, midbody and nucleoli isolated by biochemical fractionation from glioblastoma cell lines confirmed the presence of MARK4L protein in each fraction, validated by antibodies specific for each cell structure: anti-γtubulin antibody for centrosomes, anti-βtubulin for the midbody (βtubulin, together with αtubulin, accounts for 30% of midbody proteins) and anti-nucleolin for the nucleolus (Magnani et al., 2009).

The localization pattern of MARK4L delineated by the above studies suggests that the kinase may take part in cell cycle progression and influence the microtubules, particularly those affecting the centrosome and midbody.

MARK4L association with the nucleolus in glial tumours is very interesting, since MARK4L could have a functional impact on this organelle, being requested for its building and maintenance like other protein kinases, as well as it could be spatially regulated by alternate translocation in and out the nucleolus. Many proteins are indeed sequestered in the nucleolus and then released according to a temporally regulated activity, since they must exert their function in certain phases of the cell cycle (Visintin & Amon, 2000). Last, the nucleolar localization of a protein may also influence its stability, protecting the protein from proteasomal degradation, since proteasomes are present in the nucleoplasm but not in nucleoli (Wojcik & DeMartino, 2003).

Role of the Centrosomal MARK4 Protein in Gliomagenesis 153

Glioma cell lines were derived from primary tumour post-surgery specimens and subsequently maintained by serial passages in RPMI 1640 medium containing 5% Fetal Calf Serum at 37°C in a 5% CO2 atmosphere. Most of the cell lines were used within the first 30

Metaphase spreads were obtained on both fresh tumours and cultured cell lines, harvested when "peak" mitotic activity was observed; usually, a 16-hour treatment with Colcemid at a

Fluorescence hybridization with genomic DNA has proven to be a powerful tool for identification of chromosome rearrangements in cancer cells. Potential applications include detection of chromosome-specific aneuploidy in metaphase and interphase cells, quantification of the frequency of chromosome translocations and/or aneuploidy as a measure of genetic damage, and detection of diagnostically and prognostically relevant chromosomal lesions. Detection of translocations between human metaphase chromosomes is possible by using cocktails of chromosome-specific sequences that hybridize more or less uniformly along the chromosome. Depending on the aberration, its detection may be by visual fluorescence microscopy (see Figures 9, 10). In brief: slides carrying interphase or metaphase spreads are washed in 2x SSC (1 x SSC is 0.15 M NaCl/0.015 M sodium citrate), dehydrated in an ethanol series and denatured [70% (vol/vol) formamide/2x SSC (final concentration), pH 7, at 70°C for 2 min]. The hybridization mix consists of (final concentrations) 50% formamide, 2x SSC, 20% dextran sulfate, carrier DNA (sonicated herring sperm DNA), and biotin-labeled human genomic DNA. The mixture is applied to the slides under a glass coverslip. After overnight incubation at 37°C, the slides are washed at 45°C (50% formamide/2x SSC, pH 7), and immersed in BN buffer (0.1 M sodium bicarbonate, 0.05% Nonidet P-40, pH 8). The slides are never allowed to dry after this step. The coverslips are then removed and fluorescein-avidin DCS is applied. The coverslips are put back in their original places and the slides incubated 20 min at 37°C. They are then washed in BN buffer at 45°C. The intensity of biotin fluorescence is amplified by adding a layer of biotinylated goat anti-avidin antibody followed, after washing as above, by another layer of fluorescein-avidin DCS. After washing in BN buffer a fluorescence anti-fade solution is added. The DNA counterstain [4,6-diamidino-2-phenylindole (DAPI) or propidium iodide] is included in the anti-fade solution (Magnani et al., 1999; Pinkel et al.,

Immunofluorescence analyses enable to visualize, by fluorescence microscopy, the subcellular localization of a specific protein in cultured cells. The target protein is recognized by an antibody, which in turn is conjugated to a fluorochrome emitting fluorescent light. Briefly, cells are grown on glass chamber slides, then permeabilized (with solvents that extract lipids from the membranes allowing antibodies to reach a sub-cellular structure) and fixed (in order to protect the cell structure from eventual damages and to "freeze" cells in their current state). Afterwards, cells are incubated with bovine serum albumin (BSA) to block non-specific binding of antibodies. Glass slides are then incubated with a primary

**13.1 Cell cultures and preparation of human metaphase chromosomes** 

final concentration of 0.01-0.02 mg/ml is employed (Magnani et al., 1994).

**13.2 Fluorescence** *in situ* **hybridization (FISH) analysis** 

**13. Methods** 

passages.

1986).

**13.3 Immunofluorescence** 

#### **11. Conclusion**

A few remarks can be drawn from the above synthesis on cytogenomics of human gliomas and the *MARK4* cell cycle gene as a likely "player" in gliomagenesis.

Gliomas are one of the most intractable tumours due to their "complex identity": as it has been beautifully underlined, the generation - since the earliest glioma stages - of multiple cell populations with different genotypic and phenotypic features makes unlikely to succeed therapeutic strategies targeting only clones with "dominant" or "average" characteristics of the cell population (Noble & Dietrich, 2004). The intrinsic genomic heterogeneity of human glioma has first been disclosed cytogenetically, as documented by a huge number of studies which across two decades have used the cytogenetic tools suitable to monitor the intratumour cell heterogeneity and to discern "recurrent" and potentially causative chromosomal rearrangements. A few of these rearrangements entered the diagnostic and prognostic flow chart of gliomas, others allowed to identify crucial genes which mutations or imbalance are the signature of a specific glioma type or glioma malignancy stage. In line with a research pathway that has been reiterated for several genes of relevance in cancer, focus on *MARK4* has been pinpointed by cytogenetics and deepened by multiple tools ranging from gene-targeted molecular to genomic and cytogenomic analyses. Despite its nature of serine-threonine kinase gene, *MARK4* has not be found mutated or affected by copy number alterations in glioma, while its encoded proteins represented by two different isoforms, MARK4S and MARK4L, could be featured as a potential target of dysregulation in tumours due to its dual nature. The latter isoform, produced by alternative splicing, has been found up-regulated in glioma and shown to display sub-cellular localizations, namely the centrosome, the midbody and the nucleolus, which strictly associate it with the process of cell division. Interestingly, alternative mRNA splicing has been considered a mechanism not only increasing proteomic complexity but also involved in cancer, through mechanisms of oncogenes/tumour suppressors activation/inactivation or through the generation of CIN (López-Saavedra & Herrera, 2010). CIN is a general property of aneuploid cancer cells and is generated by defects in different processes, among which the regulation of the number of centrosomes, the dynamics of microtubules attachment to the kinetochores and the overall control of cell cycle. Defects in centrosomal number and structure have been well documented in gliomas (D'Assoro et al., 2002; Katsetos et al., 2006; Magnani et al., 2009) raising the issue whether the increased MARK4L isoform, a gene involved in microtubule dynamics, may concur to errors in chromosomal segregation driving gliomagenesis.

Recent application of multidimensional technological approaches has comprehensively highlighted the scenario of glioma genes and core pathways. However, despite the impressive advances, the links between genes alteration and cellular behavior are yet hampered by the multiplicity of the genetic lesions and the interconnections among the different affected pathways. Hopefully ongoing and next years research will compose the puzzle promising to translate into the clinical set the unraveled glioma pathomechanisms.

#### **12. Acknowledgement**

We thank the *Associazione italiana per la ricerca sul cancro* (AIRC) for supporting this work (grant n 4217 to LL for 2008).

#### **13. Methods**

152 Glioma – Exploring Its Biology and Practical Relevance

A few remarks can be drawn from the above synthesis on cytogenomics of human gliomas

Gliomas are one of the most intractable tumours due to their "complex identity": as it has been beautifully underlined, the generation - since the earliest glioma stages - of multiple cell populations with different genotypic and phenotypic features makes unlikely to succeed therapeutic strategies targeting only clones with "dominant" or "average" characteristics of the cell population (Noble & Dietrich, 2004). The intrinsic genomic heterogeneity of human glioma has first been disclosed cytogenetically, as documented by a huge number of studies which across two decades have used the cytogenetic tools suitable to monitor the intratumour cell heterogeneity and to discern "recurrent" and potentially causative chromosomal rearrangements. A few of these rearrangements entered the diagnostic and prognostic flow chart of gliomas, others allowed to identify crucial genes which mutations or imbalance are the signature of a specific glioma type or glioma malignancy stage. In line with a research pathway that has been reiterated for several genes of relevance in cancer, focus on *MARK4* has been pinpointed by cytogenetics and deepened by multiple tools ranging from gene-targeted molecular to genomic and cytogenomic analyses. Despite its nature of serine-threonine kinase gene, *MARK4* has not be found mutated or affected by copy number alterations in glioma, while its encoded proteins represented by two different isoforms, MARK4S and MARK4L, could be featured as a potential target of dysregulation in tumours due to its dual nature. The latter isoform, produced by alternative splicing, has been found up-regulated in glioma and shown to display sub-cellular localizations, namely the centrosome, the midbody and the nucleolus, which strictly associate it with the process of cell division. Interestingly, alternative mRNA splicing has been considered a mechanism not only increasing proteomic complexity but also involved in cancer, through mechanisms of oncogenes/tumour suppressors activation/inactivation or through the generation of CIN (López-Saavedra & Herrera, 2010). CIN is a general property of aneuploid cancer cells and is generated by defects in different processes, among which the regulation of the number of centrosomes, the dynamics of microtubules attachment to the kinetochores and the overall control of cell cycle. Defects in centrosomal number and structure have been well documented in gliomas (D'Assoro et al., 2002; Katsetos et al., 2006; Magnani et al., 2009) raising the issue whether the increased MARK4L isoform, a gene involved in microtubule

dynamics, may concur to errors in chromosomal segregation driving gliomagenesis.

Recent application of multidimensional technological approaches has comprehensively highlighted the scenario of glioma genes and core pathways. However, despite the impressive advances, the links between genes alteration and cellular behavior are yet hampered by the multiplicity of the genetic lesions and the interconnections among the different affected pathways. Hopefully ongoing and next years research will compose the puzzle promising to translate into the clinical set the unraveled glioma

We thank the *Associazione italiana per la ricerca sul cancro* (AIRC) for supporting this work

and the *MARK4* cell cycle gene as a likely "player" in gliomagenesis.

**11. Conclusion** 

pathomechanisms.

**12. Acknowledgement** 

(grant n 4217 to LL for 2008).

#### **13.1 Cell cultures and preparation of human metaphase chromosomes**

Glioma cell lines were derived from primary tumour post-surgery specimens and subsequently maintained by serial passages in RPMI 1640 medium containing 5% Fetal Calf Serum at 37°C in a 5% CO2 atmosphere. Most of the cell lines were used within the first 30 passages.

Metaphase spreads were obtained on both fresh tumours and cultured cell lines, harvested when "peak" mitotic activity was observed; usually, a 16-hour treatment with Colcemid at a final concentration of 0.01-0.02 mg/ml is employed (Magnani et al., 1994).

#### **13.2 Fluorescence** *in situ* **hybridization (FISH) analysis**

Fluorescence hybridization with genomic DNA has proven to be a powerful tool for identification of chromosome rearrangements in cancer cells. Potential applications include detection of chromosome-specific aneuploidy in metaphase and interphase cells, quantification of the frequency of chromosome translocations and/or aneuploidy as a measure of genetic damage, and detection of diagnostically and prognostically relevant chromosomal lesions. Detection of translocations between human metaphase chromosomes is possible by using cocktails of chromosome-specific sequences that hybridize more or less uniformly along the chromosome. Depending on the aberration, its detection may be by visual fluorescence microscopy (see Figures 9, 10). In brief: slides carrying interphase or metaphase spreads are washed in 2x SSC (1 x SSC is 0.15 M NaCl/0.015 M sodium citrate), dehydrated in an ethanol series and denatured [70% (vol/vol) formamide/2x SSC (final concentration), pH 7, at 70°C for 2 min]. The hybridization mix consists of (final concentrations) 50% formamide, 2x SSC, 20% dextran sulfate, carrier DNA (sonicated herring sperm DNA), and biotin-labeled human genomic DNA. The mixture is applied to the slides under a glass coverslip. After overnight incubation at 37°C, the slides are washed at 45°C (50% formamide/2x SSC, pH 7), and immersed in BN buffer (0.1 M sodium bicarbonate, 0.05% Nonidet P-40, pH 8). The slides are never allowed to dry after this step. The coverslips are then removed and fluorescein-avidin DCS is applied. The coverslips are put back in their original places and the slides incubated 20 min at 37°C. They are then washed in BN buffer at 45°C. The intensity of biotin fluorescence is amplified by adding a layer of biotinylated goat anti-avidin antibody followed, after washing as above, by another layer of fluorescein-avidin DCS. After washing in BN buffer a fluorescence anti-fade solution is added. The DNA counterstain [4,6-diamidino-2-phenylindole (DAPI) or propidium iodide] is included in the anti-fade solution (Magnani et al., 1999; Pinkel et al., 1986).

#### **13.3 Immunofluorescence**

Immunofluorescence analyses enable to visualize, by fluorescence microscopy, the subcellular localization of a specific protein in cultured cells. The target protein is recognized by an antibody, which in turn is conjugated to a fluorochrome emitting fluorescent light. Briefly, cells are grown on glass chamber slides, then permeabilized (with solvents that extract lipids from the membranes allowing antibodies to reach a sub-cellular structure) and fixed (in order to protect the cell structure from eventual damages and to "freeze" cells in their current state). Afterwards, cells are incubated with bovine serum albumin (BSA) to block non-specific binding of antibodies. Glass slides are then incubated with a primary

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antibody specific to the target protein, then with a secondary antibody conjugated to the fluorochrome and finally observed under the microscope (Magnani et al., 2009).

#### **13.4 Biochemical fractionation and immunoblotting**

By biochemical fractionation we mean the whole techniques that allow to separate and isolate intact cellular components. It usually consists in carefully breaking the cell membrane with homogenizers and isotonic/hypotonic solutions, so that intact organelles can come out, and in separating cellular components by centrifugation, on the basis of differences in their mass and specific weight. Centrosome, midbody and nucleoli isolation protocols are described in Magnani et al., 2009 and based on methods respectively by Moudjou & Bornens, 1994; Chu & Sisken, 1977; Muramatsu et al., 1963. In particular, for midbody isolation cells are synchronized in mitosis by nocodazole treatment and then released from mitotic arrest in nocodazole-free medium, so that after 30 minutes near 90% of cells had formed the midbody.

After membrane breaking, all the passages are done at 4°C and with protease inhibitors, in order to prevent protein degradation, possibly exerted by released proteases. Proteins extracted from centrosome, midbody and nucleolus fractions are then analyzed by immunoblotting. Proteins are first separated, according to their molecular weight, by SDS-PAGE (Sodium Dodecyl Sulphate – PolyAcrilamide Gel Electrophoresis): this technique allows proteins to migrate, driven by electric current, in a porous gel, with speed depending exclusively on their size. Afterwards, separated proteins are transferred onto a membrane, incubated with a blocking solution (BSA or milk) to prevent non-specific binding of antibodies and then incubated with appropriate antibodies (immunoblotting). The primary antibody is specific to the target protein and is recognized by the secondary antibody conjugated to HRP (horse radish peroxidase). Antibodies are detected by covering the membrane with a peroxide/enhancer solution, which is oxidized by HRP and emits light signals.

#### **14. References**


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After membrane breaking, all the passages are done at 4°C and with protease inhibitors, in order to prevent protein degradation, possibly exerted by released proteases. Proteins extracted from centrosome, midbody and nucleolus fractions are then analyzed by immunoblotting. Proteins are first separated, according to their molecular weight, by SDS-PAGE (Sodium Dodecyl Sulphate – PolyAcrilamide Gel Electrophoresis): this technique allows proteins to migrate, driven by electric current, in a porous gel, with speed depending exclusively on their size. Afterwards, separated proteins are transferred onto a membrane, incubated with a blocking solution (BSA or milk) to prevent non-specific binding of antibodies and then incubated with appropriate antibodies (immunoblotting). The primary antibody is specific to the target protein and is recognized by the secondary antibody conjugated to HRP (horse radish peroxidase). Antibodies are detected by covering the membrane with a peroxide/enhancer solution, which is oxidized by HRP and emits light

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**8** 

*Italy* 

**New Insight on the Role of Transient** 

Giorgio Santoni1, Maria Beatrice Morelli1,2, Consuelo Amantini1,

*1School of Pharmacy, Section of Experimental Medicine, University of Camerino* 

Gliomas are primary brain tumours believed to arise from glial cells or their progenitors. They account for 78% of malignant brain tumours (Shwartzbaum et al., 2006). The vast majority of gliomas is high-grade glioblastoma multiforme (GBM)*,* and is characterized by almost unrestrained growth. Consequently, the median survival of patients with GBM was approximately 12 months (Huncharek & Muscat, 1998). While research has generated abundant information regarding the growth characteristics of these cancers, clinical care remains palliative and the prognosis dismal (Butowski et al*.,* 2006). Gliomagenesis and progression are complex processes only partly understood. At molecular level, tumor progression and the associated heterogeneity is likely the result of multiple mutations in certain key signaling proteins (Furnari et al., 2007). Among these proteins, the Transient Receptor Potential (TRP) channel family has been identified to profoundly affect a variety of physiological and pathological processes (Kiselyov et al., 2007; Nilius et al., 2007). Members of TRP channels control cellular homeostasis by regulating calcium flux, cell proliferation, differentiation and apoptosis; moreover, in the last years an additional role for TRP ion channel family in malignant cancer growth and progression has been recognized (Xu et al., 2001; Wisnoskey et al., 2003; Xin et al., 2005; Bidaux et al., 2007; Prevarskaya et al., 2007; Gkika & Prevarskaya, 2009). Approximately thirty TRPs have been identified to date, and are classified in seven different families: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane

The expression levels and activity of members of the TRPC, TRPM, and TRPV families have been correlated with malignant growth and progression (Duncan et al., 1998; Tsavaler et al., 2001; Wissenbach et al., 2001; Thebault et al., 2006; Amantini et al., 2007; Caprodossi et al., 2008; Nabissi et al., 2010). TRP channels may regulate glioma growth and progression at different levels by controlling cell proliferation, inhibiting apoptosis, stimulating angiogenesis and triggering the migration and the invasion during tumor

**1. Role of TRP channels in glioma growth and progression** 

protein) and TRPN (NomPC-like) (Montell, 2003) (Fig.1).

progression (Table 1).

**Receptor Potential (TRP) Channels in Driven Gliomagenesis Pathways** 

*2Department of Molecular Medicine, Sapienza University, Rome* 

Matteo Santoni1 and Massimo Nabissi1


## **New Insight on the Role of Transient Receptor Potential (TRP) Channels in Driven Gliomagenesis Pathways**

Giorgio Santoni1, Maria Beatrice Morelli1,2, Consuelo Amantini1, Matteo Santoni1 and Massimo Nabissi1 *1School of Pharmacy, Section of Experimental Medicine, University of Camerino 2Department of Molecular Medicine, Sapienza University, Rome Italy* 

#### **1. Role of TRP channels in glioma growth and progression**

162 Glioma – Exploring Its Biology and Practical Relevance

Zhang, H.; Ma, X.; Shi, T.; Song, Q.; Zhao, H. & Ma, D. (2010). NSA2, a novel nucleolus

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*Nature Reviews Cancer* , Vol. 2, pp. 616-626 , ISSN 1474-175X

protein regulates cell proliferation and cell cycle. *Biochemical and Biophysical Research* 

Gliomas are primary brain tumours believed to arise from glial cells or their progenitors. They account for 78% of malignant brain tumours (Shwartzbaum et al., 2006). The vast majority of gliomas is high-grade glioblastoma multiforme (GBM)*,* and is characterized by almost unrestrained growth. Consequently, the median survival of patients with GBM was approximately 12 months (Huncharek & Muscat, 1998). While research has generated abundant information regarding the growth characteristics of these cancers, clinical care remains palliative and the prognosis dismal (Butowski et al*.,* 2006). Gliomagenesis and progression are complex processes only partly understood. At molecular level, tumor progression and the associated heterogeneity is likely the result of multiple mutations in certain key signaling proteins (Furnari et al., 2007). Among these proteins, the Transient Receptor Potential (TRP) channel family has been identified to profoundly affect a variety of physiological and pathological processes (Kiselyov et al., 2007; Nilius et al., 2007). Members of TRP channels control cellular homeostasis by regulating calcium flux, cell proliferation, differentiation and apoptosis; moreover, in the last years an additional role for TRP ion channel family in malignant cancer growth and progression has been recognized (Xu et al., 2001; Wisnoskey et al., 2003; Xin et al., 2005; Bidaux et al., 2007; Prevarskaya et al., 2007; Gkika & Prevarskaya, 2009). Approximately thirty TRPs have been identified to date, and are classified in seven different families: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like) (Montell, 2003) (Fig.1).

The expression levels and activity of members of the TRPC, TRPM, and TRPV families have been correlated with malignant growth and progression (Duncan et al., 1998; Tsavaler et al., 2001; Wissenbach et al., 2001; Thebault et al., 2006; Amantini et al., 2007; Caprodossi et al., 2008; Nabissi et al., 2010). TRP channels may regulate glioma growth and progression at different levels by controlling cell proliferation, inhibiting apoptosis, stimulating angiogenesis and triggering the migration and the invasion during tumor progression (Table 1).

New Insight on the Role of Transient

stimulation

proliferation

expression

activation

[Ca(2+)]i signaling

intracranial model

and angiogenesis

apoptosis

manner

TRPV1 Ca(2+) influx, p38MAPK-dependent

Inhibition of cell survival and

TRPM8 Increases intracellular Ca(2+), BK channel activity, cell migration

Table 1. Expression and function of TRP channels in human gliomas

TRPC1

TRPC3

TRPC6

TRPV2

Receptor Potential (TRP) Channels in Driven Gliomagenesis Pathways 165

Sontheimer, 2010

Wang et al., 2009

Barajas et al., 2008

Nakao et al., 2008

Ding et al., 2010

Grimaldi et al., 2003

Chigurupati et al., 2010

Amantini et al., 2007

Nabissi et al., 2010

Wondergem et al., 2008 Wondergem & Bartley, 2009

Bomben & Sontheimer, 2018 Bomben & Sontheimer, 2010

TRP channel Function/s References

Calcium signaling during cytokinesis (Multinucleated-giant cells), stimulates

Up-regulates hypoxia-induced VEGF

Ca(2+) influx, PAR-1-mediated astrocytic

Increase intracellular Ca(2+) induced by PDGF, stimulates G2/M phase transition and clonogenic ability; increases tumor volume in a subcutaneous mouse model of xenografted human tumors and decreases mean survival in mice in an

Increases [Ca(2+)]i elevation coupled to NFAT activation; stimulates hypoxiainduced Notch1-driven growth, invasion

proliferation, increase sensitivity to Fasinduced apoptosis in an ERK-dependent

TRPM2 ROS-induced cell death Ishii et al., 2007

TRPC activation has been shown downstream of the epidermal growth factor receptor (EGFR) (Odell et al*.*,2005) that is the major growth factor receptor activated in malignant gliomas. Indeed, mutated or amplified EGFR is often observed in malignant gliomas and has been associated with the increased cell proliferation seen in them (Bryant et al*.,* 2004). In Cos-7 cells, EGFR activation causes phosphorylation of TRPC4 and results in channel insertion into the plasma membrane (Odell et al*.,* 2005). Additionally, knockdown of TRPC4 in human corneal epithelial cells suppresses epidermal growth factor (EGF)-induced cell proliferation, again linking proliferation to TRPC channels (Yang et al*.,* 2005). Among TRPC channels, TRPC6 and TRPC1 seem to play a major role in the control of cell cycle and glioma

TRPC4 Histamine-induced Ca(2+) entry Barajas et al., 2008

Histamine-induced Ca(2+) entry

Chemotaxis in response to EGF

Fig. 1. TRP superfamily. TRP subgroups are represented in square, the members are indicated for each subfamily.

#### **2. Role of TRPC and TRPV channels in cell cycle arrest and cytokinesis in malignant glioma**

Growth control of cancer cell populations has been studied extensively over the past decades and research has identified a multitude of transmembrane TRP channels involved in this process (Schönherr, 2005; Santoni et al., 2011) (Fig.2). While our understanding of their exact role in the physiology of cell proliferation remains tentative, many TRP channel agonists or antagonists also stimulate or retard cell population growth, which support the notion that TRP channels are intrinsic component of the cell cycle. In particular, calcium Ca(2+) signaling plays an important role in normal and aberrant cell proliferation, and some members of the Ca(2+)-permeable TRPC family have demonstrated a role in the proliferation of many types of cancer cells (Malarkey et al., 2008). Using a combination of molecular, biochemical and biophysical approaches, it was demonstrated the expression of five TRPC channel proteins (TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6) in patient biopsies and cell lines derived from glioma patients (Tables 1). Activation of TRPC channels typically occurs through the triggering of phospholipase C and this signaling cascade is the target of a number of G-protein-coupled receptors and receptor tyrosine kinases. An important form of

Fig. 1. TRP superfamily. TRP subgroups are represented in square, the members are

**2. Role of TRPC and TRPV channels in cell cycle arrest and cytokinesis in** 

Growth control of cancer cell populations has been studied extensively over the past decades and research has identified a multitude of transmembrane TRP channels involved in this process (Schönherr, 2005; Santoni et al., 2011) (Fig.2). While our understanding of their exact role in the physiology of cell proliferation remains tentative, many TRP channel agonists or antagonists also stimulate or retard cell population growth, which support the notion that TRP channels are intrinsic component of the cell cycle. In particular, calcium Ca(2+) signaling plays an important role in normal and aberrant cell proliferation, and some members of the Ca(2+)-permeable TRPC family have demonstrated a role in the proliferation of many types of cancer cells (Malarkey et al., 2008). Using a combination of molecular, biochemical and biophysical approaches, it was demonstrated the expression of five TRPC channel proteins (TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6) in patient biopsies and cell lines derived from glioma patients (Tables 1). Activation of TRPC channels typically occurs through the triggering of phospholipase C and this signaling cascade is the target of a number of G-protein-coupled receptors and receptor tyrosine kinases. An important form of

indicated for each subfamily.

**malignant glioma** 


Table 1. Expression and function of TRP channels in human gliomas

TRPC activation has been shown downstream of the epidermal growth factor receptor (EGFR) (Odell et al*.*,2005) that is the major growth factor receptor activated in malignant gliomas. Indeed, mutated or amplified EGFR is often observed in malignant gliomas and has been associated with the increased cell proliferation seen in them (Bryant et al*.,* 2004). In Cos-7 cells, EGFR activation causes phosphorylation of TRPC4 and results in channel insertion into the plasma membrane (Odell et al*.,* 2005). Additionally, knockdown of TRPC4 in human corneal epithelial cells suppresses epidermal growth factor (EGF)-induced cell proliferation, again linking proliferation to TRPC channels (Yang et al*.,* 2005). Among TRPC channels, TRPC6 and TRPC1 seem to play a major role in the control of cell cycle and glioma

New Insight on the Role of Transient

viability and proliferation (Nabissi et al., 2010).

Receptor Potential (TRP) Channels in Driven Gliomagenesis Pathways 167

have indicated an association between TRPC1 and RhoA (Mehta et al*.,* 2003) and independently of TRPC6 and RhoA in certain cell types (Singh et al*.,* 2007). Finally, receptors belonging to the TRPV channel family have been found to inhibit *in vitro* glioma cell proliferation. In this regard, we have recently reported that TRPV2 mRNA was expressed in benign astrocyte tissues, and its expression progressively declined in high-grade glioma tissues as histological grade increased. TRPV2 negatively controls glioblastoma survival and proliferation. In U87 glioma cells, silencing of TRPV2 by RNA interference (siRNA) affects several genes controlling cell cycle and proliferation (Nabissi et al., 2010). Downregulation of CD95/Fas and parallel up-regulation of CCNE1, CDK2, E2F1, Raf-1 gene expression was observed in siTRPV2-U87 glioma cells as respect to controls. Moreover, TRPV2 knock-out increased glioblastoma proliferation and survival in an ERK-dependent manner. Inhibition of ERK activation by treatment of siRNA-TRPV2 U87 glioma cells with the specific MEK-1 inhibitor PD98059, promoted Fas expression and restored Akt/PKB pathway activation leading to reduced cell survival and proliferation (Nabissi et al., 2010). Conversely, TRPV2 transfection of primary MZC glioblastoma cells also reduced glioma

Fig. 2. TRP and glioma progression. In each square are represented the members of the TRP

Tumor microvessels are highly tortuous with sluggish flow and diminished gradient for oxygen delivery and increased susceptibility to thrombosis and microhemorragies. The

**3. Role of TRPC and TRPV channels in hypoxia-induced angiogenesis of** 

family, that are involved in the main processes driving glioma progression.

**human gliomas: Role for VEGF and angiopoietin-1** 

cell proliferation. Functional TRPC6 channels were overexpressed in human U251, U87, and T98G glioma cell lines. Moreover, increased TRPC6 expression was found in GBM biopsies compared with normal brain tissue, suggesting a role for TRPC6 in malignant growth of gliomas *in vitro* and *in vivo* (Ding et al., 2010). TRPC6 channels have been implicated in cell proliferation and hypertrophic gene expression through the activation of the calcineurinnuclear factor of activated T-cell (NFAT) pathway in normal (K. Kuwahara et al., 2006; Onohara et al., 2006) and malignant cells (Bomben & Sontheimer, 2008). Because glioma cells lack the expression of voltage-gated calcium channels (Kunzelmann, 2005) and Ca(2+) signaling promotes G1/S phase transition and cell cycle progression in a variety of cell types (Lipskaia & Lopré, 2004; M. Kuwahara et al., 2006), the TRPC6-mediated sustained elevation of [Ca(2+)]i and calcineurin-NFAT pathway activation is vital for the proliferation and malignant growth of gliomas under hypoxia. Consistently, inhibition of hypoxia-induced TRPC6 expression causes a dramatic decrease in NFAT activation (Bucholz & Ellenrieder, 2007). In glioma cells, inhibition of TRPC6 activity or expression by using a dominantnegative mutant TRPC6 (DNC6) or RNA interference, respectively, attenuated the increase in intracellular Ca(2+) induced by platelet-derived growth factor (PDGF), suppressed cell growth and clonogenic ability, induced cell cycle arrest at the G2/M phase, and enhanced the antiproliferative effect of ionizing radiation. Cyclin-dependent kinase 1 (CdK1) activation and cell division cycle 25 homolog C (Cdc25) expression regulated the DNC6 induced cell cycle arrest. Inhibition of TRPC6 activity also significantly reduced tumor volume in a subcutaneous mouse model of xenografted human tumors and increased mean survival in mice in an intracranial model (Ding et al., 2010). In addition to TRPC6 a role for TRPC3 in glioma cell proliferation has been suggested. The TRPC3 channel has been found to cause intracytoplasmic calcium oscillations in rat glial cells (Grimaldi et al., 2003). In rat cortical astrocytes, thrombin via Ca(2+) signal, induces TRPC3 upregulation and enhanced proliferation, and these effects were inhibited by TRPC3 blockers and siTRPC3 RNA (Shirakawa et al., 2010). Ca(2+) mobilization mediated by TRPC3 is associated with thrombin-induced morphological changes in human astrocytoma cells (Nakao et al., 2008). Glioblastoma multiforme proliferates extensively and cells often undergo incomplete cell divisions, resulting in multinucleated cells. Cytokinesis, which begins at the onset of anaphase, is the division of remaining cytoplasmic substances in the cell, aside from the nuclear events of mitosis (Glotzer, 2005; Eggert et al*.,* 2006). Recent evidence (Bomben & Sontheimer, 2010) indicated that the functional loss of TRPC1 channels involved in agonistinduced calcium entry and reloading of intracellular Ca(2+) stores disrupts glioma cytokinesis leading to bizarre and greatly enlarged multinucleated glioma cells (GMGCs) showing slow growth (Palma et al., 1989). Pharmacological inhibition of TRPC1 expression using the continuously administration for up to 4 days of the chronic inhibitor of TRPC channels, SKF96365, or TRPC1 suppression using a doxycycline inducible shRNA knockdown approach, causes loss of functional channels and store-operated calcium entry in glioma cells, and a significant decrease of tumor size, respectively. This effect is associated with reduced cell proliferation and, frequently, with incomplete cell division due to arrest at the G2/M phase of the cell cycle (Stark & Taylor, 2006). Cytokinesis is typically described with two key components being the central spindle and the contractile ring. RhoA guanosine triphosphatase GTPase is one key player in contractile ring formation, which is important for actin nucleation and myosin activation (Bement et al*.,* 2006). Recently reports

cell proliferation. Functional TRPC6 channels were overexpressed in human U251, U87, and T98G glioma cell lines. Moreover, increased TRPC6 expression was found in GBM biopsies compared with normal brain tissue, suggesting a role for TRPC6 in malignant growth of gliomas *in vitro* and *in vivo* (Ding et al., 2010). TRPC6 channels have been implicated in cell proliferation and hypertrophic gene expression through the activation of the calcineurinnuclear factor of activated T-cell (NFAT) pathway in normal (K. Kuwahara et al., 2006; Onohara et al., 2006) and malignant cells (Bomben & Sontheimer, 2008). Because glioma cells lack the expression of voltage-gated calcium channels (Kunzelmann, 2005) and Ca(2+) signaling promotes G1/S phase transition and cell cycle progression in a variety of cell types (Lipskaia & Lopré, 2004; M. Kuwahara et al., 2006), the TRPC6-mediated sustained elevation of [Ca(2+)]i and calcineurin-NFAT pathway activation is vital for the proliferation and malignant growth of gliomas under hypoxia. Consistently, inhibition of hypoxia-induced TRPC6 expression causes a dramatic decrease in NFAT activation (Bucholz & Ellenrieder, 2007). In glioma cells, inhibition of TRPC6 activity or expression by using a dominantnegative mutant TRPC6 (DNC6) or RNA interference, respectively, attenuated the increase in intracellular Ca(2+) induced by platelet-derived growth factor (PDGF), suppressed cell growth and clonogenic ability, induced cell cycle arrest at the G2/M phase, and enhanced the antiproliferative effect of ionizing radiation. Cyclin-dependent kinase 1 (CdK1) activation and cell division cycle 25 homolog C (Cdc25) expression regulated the DNC6 induced cell cycle arrest. Inhibition of TRPC6 activity also significantly reduced tumor volume in a subcutaneous mouse model of xenografted human tumors and increased mean survival in mice in an intracranial model (Ding et al., 2010). In addition to TRPC6 a role for TRPC3 in glioma cell proliferation has been suggested. The TRPC3 channel has been found to cause intracytoplasmic calcium oscillations in rat glial cells (Grimaldi et al., 2003). In rat cortical astrocytes, thrombin via Ca(2+) signal, induces TRPC3 upregulation and enhanced proliferation, and these effects were inhibited by TRPC3 blockers and siTRPC3 RNA (Shirakawa et al., 2010). Ca(2+) mobilization mediated by TRPC3 is associated with thrombin-induced morphological changes in human astrocytoma cells (Nakao et al., 2008). Glioblastoma multiforme proliferates extensively and cells often undergo incomplete cell divisions, resulting in multinucleated cells. Cytokinesis, which begins at the onset of anaphase, is the division of remaining cytoplasmic substances in the cell, aside from the nuclear events of mitosis (Glotzer, 2005; Eggert et al*.,* 2006). Recent evidence (Bomben & Sontheimer, 2010) indicated that the functional loss of TRPC1 channels involved in agonistinduced calcium entry and reloading of intracellular Ca(2+) stores disrupts glioma cytokinesis leading to bizarre and greatly enlarged multinucleated glioma cells (GMGCs) showing slow growth (Palma et al., 1989). Pharmacological inhibition of TRPC1 expression using the continuously administration for up to 4 days of the chronic inhibitor of TRPC channels, SKF96365, or TRPC1 suppression using a doxycycline inducible shRNA knockdown approach, causes loss of functional channels and store-operated calcium entry in glioma cells, and a significant decrease of tumor size, respectively. This effect is associated with reduced cell proliferation and, frequently, with incomplete cell division due to arrest at the G2/M phase of the cell cycle (Stark & Taylor, 2006). Cytokinesis is typically described with two key components being the central spindle and the contractile ring. RhoA guanosine triphosphatase GTPase is one key player in contractile ring formation, which is important for actin nucleation and myosin activation (Bement et al*.,* 2006). Recently reports have indicated an association between TRPC1 and RhoA (Mehta et al*.,* 2003) and independently of TRPC6 and RhoA in certain cell types (Singh et al*.,* 2007). Finally, receptors belonging to the TRPV channel family have been found to inhibit *in vitro* glioma cell proliferation. In this regard, we have recently reported that TRPV2 mRNA was expressed in benign astrocyte tissues, and its expression progressively declined in high-grade glioma tissues as histological grade increased. TRPV2 negatively controls glioblastoma survival and proliferation. In U87 glioma cells, silencing of TRPV2 by RNA interference (siRNA) affects several genes controlling cell cycle and proliferation (Nabissi et al., 2010). Downregulation of CD95/Fas and parallel up-regulation of CCNE1, CDK2, E2F1, Raf-1 gene expression was observed in siTRPV2-U87 glioma cells as respect to controls. Moreover, TRPV2 knock-out increased glioblastoma proliferation and survival in an ERK-dependent manner. Inhibition of ERK activation by treatment of siRNA-TRPV2 U87 glioma cells with the specific MEK-1 inhibitor PD98059, promoted Fas expression and restored Akt/PKB pathway activation leading to reduced cell survival and proliferation (Nabissi et al., 2010). Conversely, TRPV2 transfection of primary MZC glioblastoma cells also reduced glioma viability and proliferation (Nabissi et al., 2010).

Fig. 2. TRP and glioma progression. In each square are represented the members of the TRP family, that are involved in the main processes driving glioma progression.

#### **3. Role of TRPC and TRPV channels in hypoxia-induced angiogenesis of human gliomas: Role for VEGF and angiopoietin-1**

Tumor microvessels are highly tortuous with sluggish flow and diminished gradient for oxygen delivery and increased susceptibility to thrombosis and microhemorragies. The

New Insight on the Role of Transient

Receptor Potential (TRP) Channels in Driven Gliomagenesis Pathways 169

Notch signaling is critical for TRPC6 upregulation, it remains to be determined whether the Notch pathway directly or indirectly, through cross-talk with other transcription factors (Gustafsson et al., 2005; Song et al., 2008), regulates TRPC6 transcription. TRPC6 activity is increased with EGFR activation (Odell et al., 2005), suggesting a link between growth factor response to tumor growth, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces hypoxia-induced glioma progression (Mosieniak et al., 1998; Chigurupati et al., 2010). The activation of TRPC6 by Galphaq induces RhoA activation and increased [Ca(2+)]i that stimulate thrombin-induced increase of actinomyosin-mediated endothelial cell contraction, cell shape change and consequently increased endothelial permeability. Inhibitor of Galphaq or phospholipase C and the Ca(2+) chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. By contrast, activation of TRPC6 by oleoyl-2 acetyl-sn-glycerol (OAG), the membrane permeable analogue of the Galphaq-phospholipase C product, diacylglycerol, induced RhoA activity. Receptor-operated Ca(2+) activation was mediated by TRPC6. Thus, TRPC6 knockdown significantly reduced Ca(2+) entry and prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either OAG or thrombin (Singh et al., 2007). Lysophosphatidylcholine (lysoPC) has been also found to induce a rapid translocation of TRPC6 in endothelial cells, that triggeres calcium influx resulting in externalization of TRPC5. Activation of this novel TRPC6-TRPC5 channel cascade by lysoPC, inhibits endothelial cell migration. TRPC5 siRNA down-regulates the lysoPC-induced rise in [Ca(2+)]i and reverts the inhibition of EC migration (Chaudhuri et al., 2008), suggesting a negative role played by this channel in the regulation of EC migration. Finally, the phosphatase and tensin homologue (PTEN), has been found to serves as a scaffold for TRPC6 channel by enabling cell surface expression of the channel. Ca(2+) entry through TRPC6 induces an increase in endothelial permeability and directly promotes angiogenesis (Kini et al., 2010) (Fig 3). PTEN is a dual lipid-protein phosphatase that catalyzes the conversion of phosphoinositol 3,4,5-triphosphate to phosphoinositol 4,5 bisphosphate and thereby inhibits PI3K-Akt-dependent cell proliferation, migration, and tumor vascularization. Recently, a PTEN phosphatase-independent mechanism in regulating Ca(2+) entry through TRPC6 has been reported. PTEN tail-domain residues 394- 403 permit PTEN to associate with TRPC6, and thrombin promotes this association. Deletion of PTEN residues 394-403 prevents TRPC6 cell surface expression and Ca(2+) entry (Kini et al., 2010). Other TRPC channels have been found to be involved in glioma angiogenesis. Studies in zebrafish, have demonstrated that the involvement of TRPCs channels in angiogenesis represents a reminiscent of the role of TRPC channels in axon guidance (Yu et al., 2010). Activation of TRPC1 seems to be essential for the angiogenesis *in vivo.* Knockdown of TRPC1 by antisense oligonucleotides severely disrupted angiogenic sprouting of intersegmental vessels (ISVs). *In vivo* time-lapse imaging revealed that the angiogenic defect was attributable to impairment of filopodia extension, migration, and proliferation of ISV tip cells. TRPC1 acts synergistically with VEGFA in controlling ISV growth, and appeared to be downstream to VEGFA in controlling angiogenesis (Yu et al., 2010). Recently a role for TRPC1 in hypoxia-induced VEGF expression in U87 glioma cells has been reported. TRPC1 siRNA markedly inhibits hypoxia-induced up-regulation of

GBM microvasculature provides little support in oxygen/nutrient delivery, paradoxically contributing to exacerbate a metabolic mismatch between supply and demand leading to progressive hypoxia and eventually necrosis. In addition with the poor vascular architecture, endothelial cells associated with tumor vasculature fail to form tight junctions and have few associated pericytes or astrocytic foot processes leaving the integrity of the brain blood barrier compromised. This process requires that endothelial cells respond to a variety of extracellular signals that activate receptors responsible for growth and differentiation. VEGF (Vascular Endothelial Growth Factor), and Angiopoietin are key molecules in the promotion of angiogenesis via activation of the VEGFR (VEGF Receptor), and Tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) expressed on vascular endothelial cells (Lutsenko et al., 2003). The Ca(2+) is another important second messenger and its entry through plasma membrane affects the angiogenesis. VEGF causes an increase in intracellular Ca(2+) concentration in cultured endothelial cells (Criscuolo et al., 1989) through both intracellular Ca(2+) release and extracellular Ca(2+) entry (Brock et al., 1991; Faehling et al., 2001; Wu et al., 1999; Cheng et al., 2006) and up-regulates vascular permeability (Criscuolo et al., 1988). Many of its physiological functions are dependent on Ca(2+) influx (Kawasaki et al., 2000; Faehling et al., 2002) through a store-independent mechanism (Pocock et al., 2000). Vascular permeability has been shown to be dependent on calcium influx, possibly through a TRPC-mediated channels. In particular, recent data indicate that TRPC6 represent an obligatory component of cation channels required for the VEGF-mediated increase in cytosolic calcium and subsequent downstream signaling that leads to processes associated with angiogenesis. The TRPC6 channel can be activated by VEGF. Overexpression of a dominant negative TRPC6 construct in human microvascular endothelial cells (HMVECs) inhibited the VEGF-mediated increase in cytosolic calcium, migration, sprouting, and proliferation. In contrast, overexpression of a wild-type TRPC6 construct increased the proliferation and migration of HMVECs (Hamdollah Zadeh et al., 2008). Inhibition of TRPC6 in HUVECs by pharmacological or genetic approaches arrested HUVECs at G2/M phase and suppressed VEGF-induced HUVEC proliferation and tube formation. Furthermore, inhibition of TRPC6 abolished VEGF-, but not FGF-induced angiogenesis in the chick embryo chorioallantoic membrane (Ge et al., 2009). Reduced oxygen availability (hypoxia) in the surrounding brain tissue is a major driving force behind GBM angiogenesis, and the low oxygen environment in the brain is positively related to GBM aggressiveness and poor prognosis (Hockel & Vaupel, 2001). The role of Hif-1α in tumor growth and invasion is well established (Semenza, 2003). Hif-1α protein was undetectable or low in glioma cells under normoxic conditions but increased markedly under hypoxia. Similarly, Notch1 activity was low in glioma cells but was elevated after the hypoxic switch. In addition to Notch1, other components of the Notch pathway were increased in glioma cells after the hypoxic switch. Specifically, the levels of Jagged-1 protein were increased under hypoxia. The molecular signals that link tissue hypoxia, Hif-1α activation to tumor angiogenesis are poorly understood. In glioma cells, the expression of TRPC6 is low or undetectable. Hypoxia by inducing Notch1 activation, increases TRPC6 expression in primary GBM and cell lines derived from GBM. Knockdown of TRPC6 expression inhibits glioma angiogenesis. Moreover, pharmacologic inhibition of Notch blocked the hypoxiainduced upregulation of TRPC6. The induction of TRPC6 expression in gliomas was TRPC subtype specific because other members of TRPC subfamily were unaffected. Although

GBM microvasculature provides little support in oxygen/nutrient delivery, paradoxically contributing to exacerbate a metabolic mismatch between supply and demand leading to progressive hypoxia and eventually necrosis. In addition with the poor vascular architecture, endothelial cells associated with tumor vasculature fail to form tight junctions and have few associated pericytes or astrocytic foot processes leaving the integrity of the brain blood barrier compromised. This process requires that endothelial cells respond to a variety of extracellular signals that activate receptors responsible for growth and differentiation. VEGF (Vascular Endothelial Growth Factor), and Angiopoietin are key molecules in the promotion of angiogenesis via activation of the VEGFR (VEGF Receptor), and Tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) expressed on vascular endothelial cells (Lutsenko et al., 2003). The Ca(2+) is another important second messenger and its entry through plasma membrane affects the angiogenesis. VEGF causes an increase in intracellular Ca(2+) concentration in cultured endothelial cells (Criscuolo et al., 1989) through both intracellular Ca(2+) release and extracellular Ca(2+) entry (Brock et al., 1991; Faehling et al., 2001; Wu et al., 1999; Cheng et al., 2006) and up-regulates vascular permeability (Criscuolo et al., 1988). Many of its physiological functions are dependent on Ca(2+) influx (Kawasaki et al., 2000; Faehling et al., 2002) through a store-independent mechanism (Pocock et al., 2000). Vascular permeability has been shown to be dependent on calcium influx, possibly through a TRPC-mediated channels. In particular, recent data indicate that TRPC6 represent an obligatory component of cation channels required for the VEGF-mediated increase in cytosolic calcium and subsequent downstream signaling that leads to processes associated with angiogenesis. The TRPC6 channel can be activated by VEGF. Overexpression of a dominant negative TRPC6 construct in human microvascular endothelial cells (HMVECs) inhibited the VEGF-mediated increase in cytosolic calcium, migration, sprouting, and proliferation. In contrast, overexpression of a wild-type TRPC6 construct increased the proliferation and migration of HMVECs (Hamdollah Zadeh et al., 2008). Inhibition of TRPC6 in HUVECs by pharmacological or genetic approaches arrested HUVECs at G2/M phase and suppressed VEGF-induced HUVEC proliferation and tube formation. Furthermore, inhibition of TRPC6 abolished VEGF-, but not FGF-induced angiogenesis in the chick embryo chorioallantoic membrane (Ge et al., 2009). Reduced oxygen availability (hypoxia) in the surrounding brain tissue is a major driving force behind GBM angiogenesis, and the low oxygen environment in the brain is positively related to GBM aggressiveness and poor prognosis (Hockel & Vaupel, 2001). The role of Hif-1α in tumor growth and invasion is well established (Semenza, 2003). Hif-1α protein was undetectable or low in glioma cells under normoxic conditions but increased markedly under hypoxia. Similarly, Notch1 activity was low in glioma cells but was elevated after the hypoxic switch. In addition to Notch1, other components of the Notch pathway were increased in glioma cells after the hypoxic switch. Specifically, the levels of Jagged-1 protein were increased under hypoxia. The molecular signals that link tissue hypoxia, Hif-1α activation to tumor angiogenesis are poorly understood. In glioma cells, the expression of TRPC6 is low or undetectable. Hypoxia by inducing Notch1 activation, increases TRPC6 expression in primary GBM and cell lines derived from GBM. Knockdown of TRPC6 expression inhibits glioma angiogenesis. Moreover, pharmacologic inhibition of Notch blocked the hypoxiainduced upregulation of TRPC6. The induction of TRPC6 expression in gliomas was TRPC subtype specific because other members of TRPC subfamily were unaffected. Although

Notch signaling is critical for TRPC6 upregulation, it remains to be determined whether the Notch pathway directly or indirectly, through cross-talk with other transcription factors (Gustafsson et al., 2005; Song et al., 2008), regulates TRPC6 transcription. TRPC6 activity is increased with EGFR activation (Odell et al., 2005), suggesting a link between growth factor response to tumor growth, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces hypoxia-induced glioma progression (Mosieniak et al., 1998; Chigurupati et al., 2010). The activation of TRPC6 by Galphaq induces RhoA activation and increased [Ca(2+)]i that stimulate thrombin-induced increase of actinomyosin-mediated endothelial cell contraction, cell shape change and consequently increased endothelial permeability. Inhibitor of Galphaq or phospholipase C and the Ca(2+) chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. By contrast, activation of TRPC6 by oleoyl-2 acetyl-sn-glycerol (OAG), the membrane permeable analogue of the Galphaq-phospholipase C product, diacylglycerol, induced RhoA activity. Receptor-operated Ca(2+) activation was mediated by TRPC6. Thus, TRPC6 knockdown significantly reduced Ca(2+) entry and prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either OAG or thrombin (Singh et al., 2007). Lysophosphatidylcholine (lysoPC) has been also found to induce a rapid translocation of TRPC6 in endothelial cells, that triggeres calcium influx resulting in externalization of TRPC5. Activation of this novel TRPC6-TRPC5 channel cascade by lysoPC, inhibits endothelial cell migration. TRPC5 siRNA down-regulates the lysoPC-induced rise in [Ca(2+)]i and reverts the inhibition of EC migration (Chaudhuri et al., 2008), suggesting a negative role played by this channel in the regulation of EC migration. Finally, the phosphatase and tensin homologue (PTEN), has been found to serves as a scaffold for TRPC6 channel by enabling cell surface expression of the channel. Ca(2+) entry through TRPC6 induces an increase in endothelial permeability and directly promotes angiogenesis (Kini et al., 2010) (Fig 3). PTEN is a dual lipid-protein phosphatase that catalyzes the conversion of phosphoinositol 3,4,5-triphosphate to phosphoinositol 4,5 bisphosphate and thereby inhibits PI3K-Akt-dependent cell proliferation, migration, and tumor vascularization. Recently, a PTEN phosphatase-independent mechanism in regulating Ca(2+) entry through TRPC6 has been reported. PTEN tail-domain residues 394- 403 permit PTEN to associate with TRPC6, and thrombin promotes this association. Deletion of PTEN residues 394-403 prevents TRPC6 cell surface expression and Ca(2+) entry (Kini et al., 2010). Other TRPC channels have been found to be involved in glioma angiogenesis. Studies in zebrafish, have demonstrated that the involvement of TRPCs channels in angiogenesis represents a reminiscent of the role of TRPC channels in axon guidance (Yu et al., 2010). Activation of TRPC1 seems to be essential for the angiogenesis *in vivo.* Knockdown of TRPC1 by antisense oligonucleotides severely disrupted angiogenic sprouting of intersegmental vessels (ISVs). *In vivo* time-lapse imaging revealed that the angiogenic defect was attributable to impairment of filopodia extension, migration, and proliferation of ISV tip cells. TRPC1 acts synergistically with VEGFA in controlling ISV growth, and appeared to be downstream to VEGFA in controlling angiogenesis (Yu et al., 2010). Recently a role for TRPC1 in hypoxia-induced VEGF expression in U87 glioma cells has been reported. TRPC1 siRNA markedly inhibits hypoxia-induced up-regulation of

New Insight on the Role of Transient

Receptor Potential (TRP) Channels in Driven Gliomagenesis Pathways 171

Spike in [Ca2+]i entry Prolonged [Ca2+]i entry

Fig. 3. Different modes of TRPC6 activation and cellular response, in glioma cells A) Spike in [Ca2+]i entry induces endothelial cell (EC) contraction, cell shape and permeability; B) while

prolonged [Ca2+]i entry by LysoPC-induced TRPC6 activation inhibits EC migration.

**4. TRPC and TRPM channels stimulate glioma cell migration and invasion** 

Glioblastoma multiforme is extremely invasive and consequently the clinical prognosis for patients is dismal. Invasion by glioma cells into regions of normal brain is driven by a multifactorial process involving cell interactions with ECM and with adjacent cells, as well as accompanying biochemical processes supportive of proteolytic degradation of ECM, and active cell movements (Bomben et al., 2010). These processes bear a striking resemblance to the robust inherent migration potential of glial cells during embryogenesis. Invasion and migration of glial tumors differ from other tumors where local spread is very limited and dissemination occurs hematogenously or via the lymphatic system. As they spread and form metastasis, glioma cells migrate through the narrow extracellular brain spaces often following the path of nerve fiber or blood vessels. Invading glioma cells commonly assume an elongated spindle-shaped morphology, suggesting that the cells have shrunk to fit into the narrow space into the brain (Sontheimer, 2008). Several studies have focused on the understanding of different molecular mechanisms expressed by invading tumor cells. Gliomas utilize a number of proteins and pathways to infiltrate the brain parenchyma including ion channels and calcium signaling pathways. Ion channels have recently involved in glioma invasion as a means to control cell volume or regulating Ca(2+) signaling pathways in invasive cells. Calcium signaling has been shown to play important roles in glioma cell invasion (Komuro & Kumada, 2005). Cell shrinkage by adaptation of cell size

VEGF mRNA and protein levels (Wang et al., 2009). TRPC1-dependent Ca(2+) influx induced by VEGF also increases endothelial permeability. Angiopoietin-1 (Ang1) that exerts a vascular endothelial barrier protective effect by blocking the action of permeabilityincreasing mediators such as VEGF, inhibited the VEGF-induced Ca(2+) influx and increased the endothelial permeability in a concentration-dependent manner. Ang1 interfered with downstream IP3-dependent plasmalemmal Ca(2+) entry. Anti-TRPC1 antibody (Ab) inhibited the VEGF-induced Ca(2+) entry and the increased endothelial permeability. TRPC1 overexpression in endothelial cells augmented the VEGF-induced Ca(2+) entry, and application of Ang1 opposed this effect. Consistent with the coupling hypothesis of Ca(2+) entry, Ang1 by inhibiting the association of IP3 receptor (IP3R) and TRPC1, abrogates the increase in endothelial permeability (Jho et al., 2005). Although the previously reported study has been focused on Ang1 regulation of TRPC1 activation, we cannot rule out the involvement of other relevant TRPC channels. TRPC4 acts as a functional homologue in mouse endothelia to TRPC1 in humans (Nilius et al., 2003; Tiruppathi et al., 2002). For agonist-induced Ca(2+) entry in mouse aortic endothelial cells, TRPC4 was essential as either a channel-forming subunit or a constituent required for channel activation (Freichel et al., 2001). Because TRPC1 and TRPC4 can oligomerize (Hofmann et al., 2002), it is possible that both may be needed for the VEGF-induced Ca(2+) entry. The importance of TRPC4 in regulation of endothelial permeability in mice has been reinforced by the observations that the effects of Ang1 on VEGF-induced Ca(2+) entry and permeability were mimicked by deletion of the TRPC4 gene in mice (Tiruppathi et al., 2002). Finally, VEGF-induced activation of Ca(2+) entry can also occur via TRPC6 which is activated by PLC-generated DAG (Pocock et al., 2001, 2004). TRPC4 has been also found to control thrombospondin-1 (TSP-1) secretion and angiogenesis in renal cell carcinoma (RCC) (Veliceasa et al., 2007). TRPC4 loss has been lead to impaired Ca(2+) intake, misfolding, retrograde transport and diminished secretion of antiangiogenic TSP-1, thus enabling angiogenic switch during RCC progression. TRPC4 has been recently reported to be expressed in glioma cells (Wang et al., 2009), however at present no data on the role of this channel in the inhibition of glioma angiogenesis has been provided so far. Membrane-stretch activated TRPV calcium channels have been known to mediate the orientation of endothelial cells lining blood vessels thus influencing the angiogenesis. So, TRPV4 channels expressed in the plasma membrane of capillary endothelial cells is required for mechanical-induced changes in focal adhesion assembly, cell orientation and directional migration. Recent reports indicate that activation of the mechanosensitive TRPV4 in capillary endothelial cells, stimulates phosphatidylinositol 3-kinase-dependent activation and binding of additional 1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking Abs and knock-down of TRPV4 using siRNA, suppress capillary cell reorientation. Activation of TRPV4 channels by force transfer from integrins and CD98 may enable compartmentalization of calcium signaling within focal adhesions. This early-immediate calcium signaling response required the distal region of the 1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and application of external force to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response (Matthews et al., 2010). Thus, mechanical forces that physically deform extracellular matrix (ECM) guide capillary cell reorientation through an "integrin-to-integrin" signaling mechanism mediated by activation of mechanically gated TRPV4 channels on the cell surface (Thodeti et al., 2009). We have recently reported the expression of TRPV4 channels in glioma cell lines (Santoni et al., 2011), however the potential role of TRPV4 in the migration of endothelial cells during glioma angionenesis is at present unknown.

VEGF mRNA and protein levels (Wang et al., 2009). TRPC1-dependent Ca(2+) influx induced by VEGF also increases endothelial permeability. Angiopoietin-1 (Ang1) that exerts a vascular endothelial barrier protective effect by blocking the action of permeabilityincreasing mediators such as VEGF, inhibited the VEGF-induced Ca(2+) influx and increased the endothelial permeability in a concentration-dependent manner. Ang1 interfered with downstream IP3-dependent plasmalemmal Ca(2+) entry. Anti-TRPC1 antibody (Ab) inhibited the VEGF-induced Ca(2+) entry and the increased endothelial permeability. TRPC1 overexpression in endothelial cells augmented the VEGF-induced Ca(2+) entry, and application of Ang1 opposed this effect. Consistent with the coupling hypothesis of Ca(2+) entry, Ang1 by inhibiting the association of IP3 receptor (IP3R) and TRPC1, abrogates the increase in endothelial permeability (Jho et al., 2005). Although the previously reported study has been focused on Ang1 regulation of TRPC1 activation, we cannot rule out the involvement of other relevant TRPC channels. TRPC4 acts as a functional homologue in mouse endothelia to TRPC1 in humans (Nilius et al., 2003; Tiruppathi et al., 2002). For agonist-induced Ca(2+) entry in mouse aortic endothelial cells, TRPC4 was essential as either a channel-forming subunit or a constituent required for channel activation (Freichel et al., 2001). Because TRPC1 and TRPC4 can oligomerize (Hofmann et al., 2002), it is possible that both may be needed for the VEGF-induced Ca(2+) entry. The importance of TRPC4 in regulation of endothelial permeability in mice has been reinforced by the observations that the effects of Ang1 on VEGF-induced Ca(2+) entry and permeability were mimicked by deletion of the TRPC4 gene in mice (Tiruppathi et al., 2002). Finally, VEGF-induced activation of Ca(2+) entry can also occur via TRPC6 which is activated by PLC-generated DAG (Pocock et al., 2001, 2004). TRPC4 has been also found to control thrombospondin-1 (TSP-1) secretion and angiogenesis in renal cell carcinoma (RCC) (Veliceasa et al., 2007). TRPC4 loss has been lead to impaired Ca(2+) intake, misfolding, retrograde transport and diminished secretion of antiangiogenic TSP-1, thus enabling angiogenic switch during RCC progression. TRPC4 has been recently reported to be expressed in glioma cells (Wang et al., 2009), however at present no data on the role of this channel in the inhibition of glioma angiogenesis has been provided so far. Membrane-stretch activated TRPV calcium channels have been known to mediate the orientation of endothelial cells lining blood vessels thus influencing the angiogenesis. So, TRPV4 channels expressed in the plasma membrane of capillary endothelial cells is required for mechanical-induced changes in focal adhesion assembly, cell orientation and directional migration. Recent reports indicate that activation of the mechanosensitive TRPV4 in capillary endothelial cells, stimulates phosphatidylinositol 3-kinase-dependent activation and binding of additional 1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking Abs and knock-down of TRPV4 using siRNA, suppress capillary cell reorientation. Activation of TRPV4 channels by force transfer from integrins and CD98 may enable compartmentalization of calcium signaling within focal adhesions. This early-immediate calcium signaling response required the distal region of the 1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and application of external force to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response (Matthews et al., 2010). Thus, mechanical forces that physically deform extracellular matrix (ECM) guide capillary cell reorientation through an "integrin-to-integrin" signaling mechanism mediated by activation of mechanically gated TRPV4 channels on the cell surface (Thodeti et al., 2009). We have recently reported the expression of TRPV4 channels in glioma cell lines (Santoni et al., 2011), however the potential role of TRPV4 in the migration of endothelial cells during glioma angionenesis is

at present unknown.

Fig. 3. Different modes of TRPC6 activation and cellular response, in glioma cells A) Spike in [Ca2+]i entry induces endothelial cell (EC) contraction, cell shape and permeability; B) while prolonged [Ca2+]i entry by LysoPC-induced TRPC6 activation inhibits EC migration.

#### **4. TRPC and TRPM channels stimulate glioma cell migration and invasion**

Glioblastoma multiforme is extremely invasive and consequently the clinical prognosis for patients is dismal. Invasion by glioma cells into regions of normal brain is driven by a multifactorial process involving cell interactions with ECM and with adjacent cells, as well as accompanying biochemical processes supportive of proteolytic degradation of ECM, and active cell movements (Bomben et al., 2010). These processes bear a striking resemblance to the robust inherent migration potential of glial cells during embryogenesis. Invasion and migration of glial tumors differ from other tumors where local spread is very limited and dissemination occurs hematogenously or via the lymphatic system. As they spread and form metastasis, glioma cells migrate through the narrow extracellular brain spaces often following the path of nerve fiber or blood vessels. Invading glioma cells commonly assume an elongated spindle-shaped morphology, suggesting that the cells have shrunk to fit into the narrow space into the brain (Sontheimer, 2008). Several studies have focused on the understanding of different molecular mechanisms expressed by invading tumor cells. Gliomas utilize a number of proteins and pathways to infiltrate the brain parenchyma including ion channels and calcium signaling pathways. Ion channels have recently involved in glioma invasion as a means to control cell volume or regulating Ca(2+) signaling pathways in invasive cells. Calcium signaling has been shown to play important roles in glioma cell invasion (Komuro & Kumada, 2005). Cell shrinkage by adaptation of cell size

New Insight on the Role of Transient

chemotherapeutic strategy in GBM patients.

Receptor Potential (TRP) Channels in Driven Gliomagenesis Pathways 173

malignant phenotypes in GBM remain poorly characterized. Notch signaling mediates hypoxia-induced tumor migration and invasion under hypoxic environment (Sahlgren et al., 2008). TRPC6 has been found to markedly inhibited glioma cell migration and invasion in response to hypoxia by regulating actin cytoskeleton assembling and disassembling which control cell shape, allowing the cell to move along the surface. The last step of invasion requires cytoskeletal rearrangements and formation of lamillipodia and fillopodia for which the family of Rho GTPases plays an important role. Most Rho proteins, cycle between GTPbound active and GDP-bound inactive state. From the family members, Rho stimulates formation of stress fibres and focal adhesion, Rac is required for the formation of lamellipodia and Cdc42 regulates cell polarity and fillopodia formation (Teodorczyk & Martin-Villalba, 2009). A role for TRPC6 in Rho activation and actin cytoskeleton rearrangements has been suggested (Albert & Large, 2003). The TRPC6-mediated Ca(2+) entry may contribute to invasion by promoting actin-myosin interactions and the formation and disassembly of cell-substratum adhesions that are important for glioma migration (Kim & Saffen, 2005). Moreover, a role for TRPC3 activation has been also proposed. Thus, Ca(2+) entry in type I astrocytes and rat C6 glioma cells induced by OAG was InsP3-independent and inhibited by a TRPC3 antisense (Grimaldi et al., 2003). In addition, TRPC3 is functionally involved in Ca(2+) entry and thrombin stimulated morphological changes (cell rounding) induced by PAR-1 activation in 1321N1 human astrocytoma cells (Nakao et al., 2008). Finally, GBM cells express TRPM8 mRNA and protein, and its involvement in menthol and hepatocyte growth factor/scatter factor (HGF/SF) increase of [Ca(2+)]i and glioma cell migration has been reported (Wondergem et al., 2008). Menthol a TRPM8 agonist, stimulated influx of Ca(2+), membrane current, and migration of human glioblastoma DBTRG cells. The effects on Ca(2+) and migration were enhanced by pretreatment with HGF/SF. The effects on Ca(2+) also were greater in migrating cells compared with non-migrating cells. 2-Aminoethoxydiphenyl borate inhibited all menthol stimulations. In addition, menthol, by increasing [Ca(2+)]i, in human glioblastoma cells, resulted in activation of the large-conductance Ca(2+)-activated K+ membrane ion channels (BK channels). Kinetic analysis showed that menthol increased channel open probability and mean open frequency after 5 min, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca(2+)-free external solution. In addition, inhibition of BK channels by paxillin reverses menthol-stimulated increase of [Ca(2+)]i and cell migration. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or tetraethylammonium ion (Wondergem & Bartley, 2009). Invasion and metastasis are biologic hallmarks of malignant tumour. The invasion of ECM requires active degradation of ECM components. Tumour cells themselves secrete proteolytic enzymes (metalloproteinases, MMPs) or induce host cells to elaborate proteases (Pluda, 1997; Price et al., 1997; Liotta & Kohn, 1997). Glioma cells secrete MMPs to degradate the ECM surrounding invading cells (Levicar et al., 2003). In this regard, cannabidiol (CBD) has been found to impair the migration of U87 glioma cells in a cannabinoid receptorindependent manner (Vaccani et al., 2005), by increasing the tissue inhibitor of MMP1, (TIMP-1) (Ramer et al., 2010) and down-regulating the MMP-2 expression (Blazquez et al., 2008). Since CBD represents a specific ligand for TRPV2 (Qin et al., 2008), and being TRPV2 downregulated in the more invasive malignat gliomas (Nabissi et al., 2010), activation of this channel may represent an important target in anti-invasive

and volume to fit into narrow spaces is a prerequisite for cell movement and migration. Most immature cells that can migrate are well equipped to accumulate and release intracellular ions to shrink. How cell movement and invasion are coupled to the controlled activation of Ca(2+) channels is only partially understood (Mcferrin & Sontheimer, 2006). In glioma cells, invasion appears to involve a coordinated reduction in cell volume, which is mediated by the efflux of Cl− and K+ through ion channels. The Cl− efflux is accompanied by the movement of K+ ions. The principal pathway for K+ efflux from glioma cells appears to be via Ca(2+)-activated bradykinin (BK) channels, which have the unique ability to couple changes in intracellular Ca(2+) to changes in membrane K+ conductance and are expressed highly in glioma cells (Ransom & Sontheimer, 2001). In glioma cells, migration is accompanied by oscillatory changes in intracellular Ca(2+) in response to different stimuli (Grimaldi et al., 2003), which activate BK K+ channels, and the velocity of cell migration of glioma cells correlates with oscillatory changes in intracellular Ca(2+) concentration (Bordey et al., 2000). Among ion channels contributing to Ca(2+) signaling, cytoskeleton changes, movement and migration, the TRPM and TRPC channel families seem to play an important role. Thus, triggering of TRPM8 by the specific agonist, menthol (Wondergem & Bartley, 2009), as TRPC3 and TRPC6 (Kim et al., 2009) increases glioma cell [Ca(2+)]i that in turn activates BK channels. Thus. TRP-mediated activation of Ca(2+) influx appears to be the prerequisite for cell migration and this Ca(2+) signal is instructive with regards to cell volume changes that occur down-stream. Cell shape, adhesion and migration have been regulated by actomyosin contractility. TRPM7-like transcripts current has been identified in rat microglia (Jiang et al., 2003). TRPM7 plays a role in linking receptor-mediated signals to actomyosin remodelling and cell adhesion. Activation of TRPM7 by BK, leads to a Ca(2+) and kinase-dependent interaction with the actomyosin cytoskeleton. Overexpression of TRPM7, by increasing the intracellular Ca(2+) levels resulted in cell spreading, adhesion and formation of focal adhesions (Clark et al., 2006). The effects of TRPM7 on cell morphology is directly dependent on integrin activation or is associated to increase in cytosolic Ca(2+) concentrations that affect the actomyosin cytoskeleton. The integrin activation can lead to the remodeling of the actomyosin cytoskeleton that promotes cell spreading via outside-in signaling pathways. Alternatively, Ca(2+) is an important second messenger in actin remodeling including polymerization, severing of filaments and F-actin–membrane interactions. The TRPC channels play a role in store-operated calcium entry (SOCE), and in particular TRPC1 is involved in SOCE in glioma cells (Bomben & Sontheimer, 2010). TRPC1-dependent migration and chemotaxis have been reported in different cell types such as myoblasts (Louis et al., 2008), renal epithelial (Fabian et al., 2008) and nervous cells (Wang & Poo, 2005) (Fig.2). Recently, (Bomben & Sontheimer, 2010) showed that TRPC1 channel association with lipid rafts is essential for glioma chemotaxis in response to stimuli, such as EGF, but not chemokinesis. EGF stimulation affects both TRPC trafficking (Bezzerides et al., 2004) and activation (Beech, 2005; Liu et al., 2009), and TRPC1 channel localization to the leading edge of migrating glioma cells. TRPC1 channels co-localize with the lipid raft proteins, caveolin-1. Chemotaxis toward EGF was lost when TRPC channels were pharmacologically inhibited or by shRNA knock-down of TRPC1 channels, yet without affecting unstimulated cell motility. Lipid raft integrity was required for gliomas chemotaxis; thus disruption of lipid rafts not only impaired chemotaxis but also impaired TRPC currents and decreased store-operated calcium entry. TRPC6 is markedly upregulated under hypoxia in a manner dependent on Notch activation. The Notch-regulated transcriptional targets that are responsible for the development of the aggressive and

and volume to fit into narrow spaces is a prerequisite for cell movement and migration. Most immature cells that can migrate are well equipped to accumulate and release intracellular ions to shrink. How cell movement and invasion are coupled to the controlled activation of Ca(2+) channels is only partially understood (Mcferrin & Sontheimer, 2006). In glioma cells, invasion appears to involve a coordinated reduction in cell volume, which is mediated by the efflux of Cl− and K+ through ion channels. The Cl− efflux is accompanied by the movement of K+ ions. The principal pathway for K+ efflux from glioma cells appears to be via Ca(2+)-activated bradykinin (BK) channels, which have the unique ability to couple changes in intracellular Ca(2+) to changes in membrane K+ conductance and are expressed highly in glioma cells (Ransom & Sontheimer, 2001). In glioma cells, migration is accompanied by oscillatory changes in intracellular Ca(2+) in response to different stimuli (Grimaldi et al., 2003), which activate BK K+ channels, and the velocity of cell migration of glioma cells correlates with oscillatory changes in intracellular Ca(2+) concentration (Bordey et al., 2000). Among ion channels contributing to Ca(2+) signaling, cytoskeleton changes, movement and migration, the TRPM and TRPC channel families seem to play an important role. Thus, triggering of TRPM8 by the specific agonist, menthol (Wondergem & Bartley, 2009), as TRPC3 and TRPC6 (Kim et al., 2009) increases glioma cell [Ca(2+)]i that in turn activates BK channels. Thus. TRP-mediated activation of Ca(2+) influx appears to be the prerequisite for cell migration and this Ca(2+) signal is instructive with regards to cell volume changes that occur down-stream. Cell shape, adhesion and migration have been regulated by actomyosin contractility. TRPM7-like transcripts current has been identified in rat microglia (Jiang et al., 2003). TRPM7 plays a role in linking receptor-mediated signals to actomyosin remodelling and cell adhesion. Activation of TRPM7 by BK, leads to a Ca(2+) and kinase-dependent interaction with the actomyosin cytoskeleton. Overexpression of TRPM7, by increasing the intracellular Ca(2+) levels resulted in cell spreading, adhesion and formation of focal adhesions (Clark et al., 2006). The effects of TRPM7 on cell morphology is directly dependent on integrin activation or is associated to increase in cytosolic Ca(2+) concentrations that affect the actomyosin cytoskeleton. The integrin activation can lead to the remodeling of the actomyosin cytoskeleton that promotes cell spreading via outside-in signaling pathways. Alternatively, Ca(2+) is an important second messenger in actin remodeling including polymerization, severing of filaments and F-actin–membrane interactions. The TRPC channels play a role in store-operated calcium entry (SOCE), and in particular TRPC1 is involved in SOCE in glioma cells (Bomben & Sontheimer, 2010). TRPC1-dependent migration and chemotaxis have been reported in different cell types such as myoblasts (Louis et al., 2008), renal epithelial (Fabian et al., 2008) and nervous cells (Wang & Poo, 2005) (Fig.2). Recently, (Bomben & Sontheimer, 2010) showed that TRPC1 channel association with lipid rafts is essential for glioma chemotaxis in response to stimuli, such as EGF, but not chemokinesis. EGF stimulation affects both TRPC trafficking (Bezzerides et al., 2004) and activation (Beech, 2005; Liu et al., 2009), and TRPC1 channel localization to the leading edge of migrating glioma cells. TRPC1 channels co-localize with the lipid raft proteins, caveolin-1. Chemotaxis toward EGF was lost when TRPC channels were pharmacologically inhibited or by shRNA knock-down of TRPC1 channels, yet without affecting unstimulated cell motility. Lipid raft integrity was required for gliomas chemotaxis; thus disruption of lipid rafts not only impaired chemotaxis but also impaired TRPC currents and decreased store-operated calcium entry. TRPC6 is markedly upregulated under hypoxia in a manner dependent on Notch activation. The Notch-regulated transcriptional targets that are responsible for the development of the aggressive and malignant phenotypes in GBM remain poorly characterized. Notch signaling mediates hypoxia-induced tumor migration and invasion under hypoxic environment (Sahlgren et al., 2008). TRPC6 has been found to markedly inhibited glioma cell migration and invasion in response to hypoxia by regulating actin cytoskeleton assembling and disassembling which control cell shape, allowing the cell to move along the surface. The last step of invasion requires cytoskeletal rearrangements and formation of lamillipodia and fillopodia for which the family of Rho GTPases plays an important role. Most Rho proteins, cycle between GTPbound active and GDP-bound inactive state. From the family members, Rho stimulates formation of stress fibres and focal adhesion, Rac is required for the formation of lamellipodia and Cdc42 regulates cell polarity and fillopodia formation (Teodorczyk & Martin-Villalba, 2009). A role for TRPC6 in Rho activation and actin cytoskeleton rearrangements has been suggested (Albert & Large, 2003). The TRPC6-mediated Ca(2+) entry may contribute to invasion by promoting actin-myosin interactions and the formation and disassembly of cell-substratum adhesions that are important for glioma migration (Kim & Saffen, 2005). Moreover, a role for TRPC3 activation has been also proposed. Thus, Ca(2+) entry in type I astrocytes and rat C6 glioma cells induced by OAG was InsP3-independent and inhibited by a TRPC3 antisense (Grimaldi et al., 2003). In addition, TRPC3 is functionally involved in Ca(2+) entry and thrombin stimulated morphological changes (cell rounding) induced by PAR-1 activation in 1321N1 human astrocytoma cells (Nakao et al., 2008). Finally, GBM cells express TRPM8 mRNA and protein, and its involvement in menthol and hepatocyte growth factor/scatter factor (HGF/SF) increase of [Ca(2+)]i and glioma cell migration has been reported (Wondergem et al., 2008). Menthol a TRPM8 agonist, stimulated influx of Ca(2+), membrane current, and migration of human glioblastoma DBTRG cells. The effects on Ca(2+) and migration were enhanced by pretreatment with HGF/SF. The effects on Ca(2+) also were greater in migrating cells compared with non-migrating cells. 2-Aminoethoxydiphenyl borate inhibited all menthol stimulations. In addition, menthol, by increasing [Ca(2+)]i, in human glioblastoma cells, resulted in activation of the large-conductance Ca(2+)-activated K+ membrane ion channels (BK channels). Kinetic analysis showed that menthol increased channel open probability and mean open frequency after 5 min, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca(2+)-free external solution. In addition, inhibition of BK channels by paxillin reverses menthol-stimulated increase of [Ca(2+)]i and cell migration. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or tetraethylammonium ion (Wondergem & Bartley, 2009). Invasion and metastasis are biologic hallmarks of malignant tumour. The invasion of ECM requires active degradation of ECM components. Tumour cells themselves secrete proteolytic enzymes (metalloproteinases, MMPs) or induce host cells to elaborate proteases (Pluda, 1997; Price et al., 1997; Liotta & Kohn, 1997). Glioma cells secrete MMPs to degradate the ECM surrounding invading cells (Levicar et al., 2003). In this regard, cannabidiol (CBD) has been found to impair the migration of U87 glioma cells in a cannabinoid receptorindependent manner (Vaccani et al., 2005), by increasing the tissue inhibitor of MMP1, (TIMP-1) (Ramer et al., 2010) and down-regulating the MMP-2 expression (Blazquez et al., 2008). Since CBD represents a specific ligand for TRPV2 (Qin et al., 2008), and being TRPV2 downregulated in the more invasive malignat gliomas (Nabissi et al., 2010), activation of this channel may represent an important target in anti-invasive chemotherapeutic strategy in GBM patients.

New Insight on the Role of Transient

Receptor Potential (TRP) Channels in Driven Gliomagenesis Pathways 175

tumor mass as a therapeutic strategy (Reya et al., 2001). Recent evidences adscript an emergent role of TRP channels in regulating neurogenesis (Tai et al., 2009) as well as neural differentiation (Shin et al., 2010), suggesting that deregulation of specific TRP target genes may be involved in gliomagenesis (Van Meir et al., 2010; Liu et al., 2010). In this regard, the expression of TRPV2 in normal neural stem/progenitor cells (NS/PC) from olfactory bulb and GSC lines derived from GBM patients, and a role of this TRP channel in the regulation of cellular proliferation and differentiation, have been observed (Nabissi et al., personal communication). Stem cells proliferation is maintained by a balance between proliferative and antiproliferative signals and any genetic or biochemical modifications that lead stem cells to become independent of growth signals, could induce an uncontrolled proliferation and possible tumorogenesis (Li & Neaves, 2006). GSCs divide core regulatory pathways with normal neural stem cells (NPSs), sharing developmental programs that lead NSCs to differentiate into astrocytes, oligodendrocytes and neurons (Galli et al., 2004; Singh et al., 2003), but induce in GSCs an aberrant differentiation (Cheng et al., 2010). GSCs are reported to express CD133 and nestin and to differentiate into cells expressing neuronal or glial cell markers upon growth factor depletion (Gunther et al., 2008). In addition to these NSC characteristics, glioma-derived neurospheres or CD133+ cells are tumorigenic and when transplanted into SCID mice formed secondary tumors with phenotypic and cytogenetic similarities to the patient tumor from which they were originally derived (Singh et al., 2003; Lee et al., 2006). Recent findings in GSCs demonstrated that the upregulation of classical pathways associated with neural development, as Notch, WNT, Hedgehog and TGF/BMT pathways (Clark et al., 2007; Silver & Steindler, 2009), induce in GSC-derived GBMs an invasive, angiogenetic, proliferative and chemoresistant phenotype (Sanai et al., 2005). So, modulation of these pathways may represent novel therapeutic approach for GBM. Notch is a family of hetero-dimeric transmembrane receptors composed of an extracellular domain responsible for ligand recognition, a transmembrane domain, and an intracellular domain involved in transcriptional regulation (Stockhausen et al., 2010). Notch proteins (and ligands)contain extracellular EGF-like repeats, which interact with the DSL domain of ligands. Activation of Notch upon ligand binding is accompanied by proteolytic processing that releases an intracellular domain of Notch (NICD) from the membrane tether. The NICD contains the RAM23 domain (RAM), which enhances interaction with the CSL protein, NLS (Nuclear Localization Signals), a CDC10/Ankyrin repeat domain ANK, which mediates interactions with CSL and other proteins, and a PEST domain rich in proline, glutamate, serine and threonine residues (Kopan, 2002). When Notch receptor is triggered by the ligands on the neighboring cells, the intracellular domain of the Notch receptor (NICD) is released from the membrane, after successive proteolytic cleavages by the -secretase complex. NICD then translocates into the nucleus and associates with the transcription factor RBP-J. This complex by secruiting other co-activators, stimulates the expression of downstream genes as Cyclin-D1, EGFR, and MAPK (Mitogen-Activated Protein Kinase) inducing cell proliferation, angiogenesis and chemoresistance, in GSCs (Stockhausen et al., 2010). Regarding the role of Notch signaling in GBM, gene microarray analysis have demonstrated that its expression in brain tumors correlated with good versus poor prognosis (Phillips et al., 2006). Moreover, in GBM tissue samples, high expression of Notch signal has been associated with high nestin levels, suggesting a correlation between GSCs and Notch expression (Purow et al., 2005; Lino et al.,2010; Boulay et al., 2007; Shih & Holland, 2006). Infact, Notch signaling plays a pivotal role in the maintenance of NSCs and leads to GSC-driven brain tumor development ( Lino et al., 2010; Louvi & Artavanis-

#### **5. TRPV and TRPM channels trigger cell death in human glioma cells**

Members of the TRPV and TRPM channels have been found to regulate apoptotic and necrotic cell death processes, respectively, as well as resistance to apoptotic stimuli in glioblastoma cells. In this regard, a role for TRPV1 in the apoptosis of glioma cells has been reported (Amantini et al., 2007). Thus, TRPV1 mRNA and protein expression was evidenced in normal astrocytes and glioma cells and tissues (Contassot et al., 2004; Amantini et al., 2007). TRPV1 expression inversely correlated with glioma grading, with a marked loss of TRPV1 expression in the majority of grade IV glioblastoma tissues. In addition, TRPV1 activation by the synthetic ligand, capsaicin (CPS) induced apoptosis of U373 glioma cells, and involved rise of Ca(2+) influx, p38MAPK activation, mitochondrial permeability transmembrane pore opening and transmembrane potential dissipation and caspase-3 activation (Amantini et al., 2007). Similarly, an other TRPV1 agonist, arachidonylethanolamide (AEA) induces apoptosis of human glioma cells in a TRPV1 dependent-manner (Contassot et al., 2004). Resistance of cancer cells to chemotherapeuticinduced cytotoxicity during tumor progression partially depends by a decrease sensitivity to CD95/Fas-induced apoptosis (Amantini et al., 2009). Induction of cell death by some cytotoxic drugs seems to depend to an intact Fas/FasL system. Tumour progression by exerting selective pressure alters Fas status and subsequently affects the sensitivity of cancer cells to chemotherapy (Sindhwani et al., 2001). Glioblastoma cells are resistant to Fasinduced cell death. We have recently reported that TRPV2 negatively controls glioblastoma survival as well as resistance to Fas/CD95-induced apoptosis in an ERK-dependent manner. Silencing of TRPV2 by RNA interference (siRNA) in U87 glioma cells down-regulated Fas/CD95 and procaspase-8 expression, and up-regulated Bcl-XL mRNA expression. Moreover, TRPV2 siRNA increased glioblastoma survival to Fas/CD95-induced apoptosis in an ERK-dependent manner (Nabissi et al., 2010). Inhibition of ERK activation by treatment of the siRNA-TRPV2 U87 glioma cells with the specific MEK-1 inhibitor PD98059, reduced Bcl-XL protein levels, promoted Fas/CD95 expression and restored Akt/PKB pathway activation leading to reduced cell survival and increased sensitivity to Fas/CD95 induced apoptosis (Nabissi et al., 2010). These events are consistent with previous evidence showing that PI3K pharmacological inhibitors inhibited calcium overload and cell death in TRPV2-transfected mouse cells (Penna et al., 2006). Consistently, TRPV2 transfection of the primary MZC glioblastoma cells also reduced glioma viability and increased spontaneous and Fas/CD95-induced apoptosis, by inducing Fas/CD95 expression (Nabissi et al., 2010). Among TRPM channels, a role for the Ca(2+) permeable TRPM2 channel in glioma cell death has been reported. Thus, insertion of TRPM2 in human A172 glioma cells enhanced cell death induced by H2O2 (Ishi et al., 2007).

#### **6. TRP channels as cross-road of deregulated transcriptional activity in glioma stem like-cells**

Evidence that malignant gliomas may arise from and contain a minority tumour cells with stem cell-like (GSCs) properties has been increased by the demonstration that GSCs maintain the potential for self-renewal and multi-lineage differentiation that recap the phenotype of the original glioma (Galli et al., 2004; Singh et al., 2003; Yuan et al., 2004), Since GSCs has been suggested to play an important role in glioma initiation, growth, and recurrence, it is extremely important to understand the signal pathways that contribute to their formation and maintenance, with the future aims to eliminate GSCs from the bulk

Members of the TRPV and TRPM channels have been found to regulate apoptotic and necrotic cell death processes, respectively, as well as resistance to apoptotic stimuli in glioblastoma cells. In this regard, a role for TRPV1 in the apoptosis of glioma cells has been reported (Amantini et al., 2007). Thus, TRPV1 mRNA and protein expression was evidenced in normal astrocytes and glioma cells and tissues (Contassot et al., 2004; Amantini et al., 2007). TRPV1 expression inversely correlated with glioma grading, with a marked loss of TRPV1 expression in the majority of grade IV glioblastoma tissues. In addition, TRPV1 activation by the synthetic ligand, capsaicin (CPS) induced apoptosis of U373 glioma cells, and involved rise of Ca(2+) influx, p38MAPK activation, mitochondrial permeability transmembrane pore opening and transmembrane potential dissipation and caspase-3 activation (Amantini et al., 2007). Similarly, an other TRPV1 agonist, arachidonylethanolamide (AEA) induces apoptosis of human glioma cells in a TRPV1 dependent-manner (Contassot et al., 2004). Resistance of cancer cells to chemotherapeuticinduced cytotoxicity during tumor progression partially depends by a decrease sensitivity to CD95/Fas-induced apoptosis (Amantini et al., 2009). Induction of cell death by some cytotoxic drugs seems to depend to an intact Fas/FasL system. Tumour progression by exerting selective pressure alters Fas status and subsequently affects the sensitivity of cancer cells to chemotherapy (Sindhwani et al., 2001). Glioblastoma cells are resistant to Fasinduced cell death. We have recently reported that TRPV2 negatively controls glioblastoma survival as well as resistance to Fas/CD95-induced apoptosis in an ERK-dependent manner. Silencing of TRPV2 by RNA interference (siRNA) in U87 glioma cells down-regulated Fas/CD95 and procaspase-8 expression, and up-regulated Bcl-XL mRNA expression. Moreover, TRPV2 siRNA increased glioblastoma survival to Fas/CD95-induced apoptosis in an ERK-dependent manner (Nabissi et al., 2010). Inhibition of ERK activation by treatment of the siRNA-TRPV2 U87 glioma cells with the specific MEK-1 inhibitor PD98059, reduced Bcl-XL protein levels, promoted Fas/CD95 expression and restored Akt/PKB pathway activation leading to reduced cell survival and increased sensitivity to Fas/CD95 induced apoptosis (Nabissi et al., 2010). These events are consistent with previous evidence showing that PI3K pharmacological inhibitors inhibited calcium overload and cell death in TRPV2-transfected mouse cells (Penna et al., 2006). Consistently, TRPV2 transfection of the primary MZC glioblastoma cells also reduced glioma viability and increased spontaneous and Fas/CD95-induced apoptosis, by inducing Fas/CD95 expression (Nabissi et al., 2010). Among TRPM channels, a role for the Ca(2+) permeable TRPM2 channel in glioma cell death has been reported. Thus, insertion of TRPM2 in human A172 glioma cells enhanced

**5. TRPV and TRPM channels trigger cell death in human glioma cells** 

cell death induced by H2O2 (Ishi et al., 2007).

**glioma stem like-cells** 

**6. TRP channels as cross-road of deregulated transcriptional activity in** 

Evidence that malignant gliomas may arise from and contain a minority tumour cells with stem cell-like (GSCs) properties has been increased by the demonstration that GSCs maintain the potential for self-renewal and multi-lineage differentiation that recap the phenotype of the original glioma (Galli et al., 2004; Singh et al., 2003; Yuan et al., 2004), Since GSCs has been suggested to play an important role in glioma initiation, growth, and recurrence, it is extremely important to understand the signal pathways that contribute to their formation and maintenance, with the future aims to eliminate GSCs from the bulk tumor mass as a therapeutic strategy (Reya et al., 2001). Recent evidences adscript an emergent role of TRP channels in regulating neurogenesis (Tai et al., 2009) as well as neural differentiation (Shin et al., 2010), suggesting that deregulation of specific TRP target genes may be involved in gliomagenesis (Van Meir et al., 2010; Liu et al., 2010). In this regard, the expression of TRPV2 in normal neural stem/progenitor cells (NS/PC) from olfactory bulb and GSC lines derived from GBM patients, and a role of this TRP channel in the regulation of cellular proliferation and differentiation, have been observed (Nabissi et al., personal communication). Stem cells proliferation is maintained by a balance between proliferative and antiproliferative signals and any genetic or biochemical modifications that lead stem cells to become independent of growth signals, could induce an uncontrolled proliferation and possible tumorogenesis (Li & Neaves, 2006). GSCs divide core regulatory pathways with normal neural stem cells (NPSs), sharing developmental programs that lead NSCs to differentiate into astrocytes, oligodendrocytes and neurons (Galli et al., 2004; Singh et al., 2003), but induce in GSCs an aberrant differentiation (Cheng et al., 2010). GSCs are reported to express CD133 and nestin and to differentiate into cells expressing neuronal or glial cell markers upon growth factor depletion (Gunther et al., 2008). In addition to these NSC characteristics, glioma-derived neurospheres or CD133+ cells are tumorigenic and when transplanted into SCID mice formed secondary tumors with phenotypic and cytogenetic similarities to the patient tumor from which they were originally derived (Singh et al., 2003; Lee et al., 2006). Recent findings in GSCs demonstrated that the upregulation of classical pathways associated with neural development, as Notch, WNT, Hedgehog and TGF/BMT pathways (Clark et al., 2007; Silver & Steindler, 2009), induce in GSC-derived GBMs an invasive, angiogenetic, proliferative and chemoresistant phenotype (Sanai et al., 2005). So, modulation of these pathways may represent novel therapeutic approach for GBM. Notch is a family of hetero-dimeric transmembrane receptors composed of an extracellular domain responsible for ligand recognition, a transmembrane domain, and an intracellular domain involved in transcriptional regulation (Stockhausen et al., 2010). Notch proteins (and ligands)contain extracellular EGF-like repeats, which interact with the DSL domain of ligands. Activation of Notch upon ligand binding is accompanied by proteolytic processing that releases an intracellular domain of Notch (NICD) from the membrane tether. The NICD contains the RAM23 domain (RAM), which enhances interaction with the CSL protein, NLS (Nuclear Localization Signals), a CDC10/Ankyrin repeat domain ANK, which mediates interactions with CSL and other proteins, and a PEST domain rich in proline, glutamate, serine and threonine residues (Kopan, 2002). When Notch receptor is triggered by the ligands on the neighboring cells, the intracellular domain of the Notch receptor (NICD) is released from the membrane, after successive proteolytic cleavages by the -secretase complex. NICD then translocates into the nucleus and associates with the transcription factor RBP-J. This complex by secruiting other co-activators, stimulates the expression of downstream genes as Cyclin-D1, EGFR, and MAPK (Mitogen-Activated Protein Kinase) inducing cell proliferation, angiogenesis and chemoresistance, in GSCs (Stockhausen et al., 2010). Regarding the role of Notch signaling in GBM, gene microarray analysis have demonstrated that its expression in brain tumors correlated with good versus poor prognosis (Phillips et al., 2006). Moreover, in GBM tissue samples, high expression of Notch signal has been associated with high nestin levels, suggesting a correlation between GSCs and Notch expression (Purow et al., 2005; Lino et al.,2010; Boulay et al., 2007; Shih & Holland, 2006). Infact, Notch signaling plays a pivotal role in the maintenance of NSCs and leads to GSC-driven brain tumor development ( Lino et al., 2010; Louvi & Artavanis-

New Insight on the Role of Transient

promoting cell proliferation.

**7. Conclusions and prospectives** 

Receptor Potential (TRP) Channels in Driven Gliomagenesis Pathways 177

al., 2006b). A series of pathways, including the Sonic hedgehog (Shh) and Notch, have been shown to be implicated in glioma's resistance to alkylating agents and/or the maintenance of brain tumor stem cells (Ulasov et al., 2011; Clement et al., 2007). Moreover, overexpression of *Dkk-1*, a gene encoding for a Wnt antagonist protein, has been shown to sensitize the U87 glioma cells to the cytotoxic effects of bis-chloronitrosourea (BCNU) and cisplatin (Shou et al., 2002). In this regard, an inverse correlation between TRPV2 and SHH and Notch pathways (Phillips et al., 2006; Nabissi et al., 2010) in regulating chemoresistance to the alkylating agent bis-chloronitrosourea (BCNU), can be supposed. TRPV2 expression progressively declined in high-grade glioma tissues as histological grade increased, while Notch and SHH signaling was activated in GBM. Knockdown of TRPV2 gene in gliomas increased the resistance to BCNU cytotoxicity which was associated with Ras/MEK/Erk and Akt overexpression in chemosensitive glioma cells, while TRPV2 overexpression augmented the chemosensitivity of resistant glioma cells to BCNU. In addition, downregulation of TRPV2 reduced Fas expression and Fas-mediated apoptosis (Nabissi et al., 2010). Parallelely, upregulation of Notch 1, increased the resistance of glioma cell to apoptosis (Purow et al., 2005). Finally, forced Notch 1 overexpression in glioma cells increased the proliferation and the formation of nestin-positive, neurosphere-forming stem cells (Zhang et al., 2008). Overall, these data suggest that in gliomas, TRPV2 could be a downstream gene target of Notch signaling rescuing glioma cells to apoptosis and

In this chapter, we have summarized current basic and translational changes and highlight the striking scientific advances regarding the expression and the function of the TRP channel family in glioma growth and progression, that promise to improve the clinical course of this lethal disease. These include a more comprensive view of the interplay between changes in TRP channel expression and functions (e.g., TRPC, TRPM and TRPV family) and alterations in transcriptional and growth factor pathways (e.g., Notch, PTEN, HIF-α, EGFR) driving the uncontrolled cellular proliferation, aberrant angiogenesis, intense migration and invasion, increased resistance to apoptosis. Clearly, the identification of cluster of TRP ion channels altered during glioma progression presents an opportunity for improving the understanding of this cancer. The progress and depth of understanding of the role of ion channels, including the TRP family in glioma, together with truly manipulable experimental models, now offer a real opportunities for the development of effective target therapy (Santoni & Farfariello, 2011). Despite significant gaps in our understanding, a wealth of information now exists about clinical and biological behaviour of these tumours, the genetic pathways involved in gliomagenesis and the nature and the role of their alterations. The challenge is now to integrate all of this knowledge in an interdisciplinary way to full understand this disease and how its heterogenicity contributes to the relatively poor therapeutic responses of GBM patients. In regard to stem cell issue, the fact that the glioma-like stem cells (GSCs) that play an important role in the development and recurrence of malignat glioma, not only express TRP channels, but also show functional alterations in their expression and transcriptional regulation, combined with the evidence that they displayed nearly identical Ca(2+) transients and pharmacological sensitivities to TRP channel antagonists (Nabissi et al., personal communication; Weick et al., 2009), may

Tsakonas, 2006). Recently, has been demonstrated that Notch activation is increased during hypoxia and hypoxia direct GBM to the development of an aggressive phenotype and resistance to radiation and chemotherapy (Flynn et al., 2008). Regarding the relationship between Notch signaling and TRP channels, a direct correlation has been demonstrated in human glioma cell lines where TRPC6 transcripts have been found to be increased under hypoxic condition and the involvement of Notch in hypoxia-induced TRPC6 expression in glioma has been demonstrated. Silencing of Notch1 gene inhibits TRPC6 expression suggesting that Notch1 is required for hypoxia-induced TRPC6 overexpression (Chigurupati et al., 2010). In response to hypoxia, the hypoxia inducing factors (HIF1- and HIF-2) are stabilized and as a consequence VEGF and TGF are upregulated (Birlik et al., 2006). Moreover, hypoxia-induced endothelial cell proliferation is associated with an increase of AP-1 expression, elevated store-operated calcium entry, and enhanced TRPC4 expression (Fantozzi et al., 2003), suggesting that additional TRP channels may regulate angiogenic signals (Fig.4). The interplay between GSCs and the endothelial compartement seems to be critical in gliomagenesis. Thus, GSCs closely interacting with the endothelial cells in vascular niche, promote angiogenesis through VEGF release (Bao et al., 2006a; Folkins et al., 2009). GSCs are reported to express CD133

Fig. 4. The putative role of TRP channels in neural and glioma stem cell-like differentiation and angiogenesis. A schematic representation of different TRP members involved in the regulation of neuro- and glioma-genesis

and nestin (Yuan et al., 2004; Gunther et al., 2008) and have been demonstrated to have multipotent differentiative potential (Galli et al., 2004; Singh et al., 2003). Several authors have hypothesized that CD133+ tumor stem cells are the source of the recurrent tumors after treatment (Chua et al., 2008; Bleau et al., 2009) and the CD133+ cell population was enriched after radiation or chemotherapy and exhibited an increase in DNA repair capacity (Bao et

Tsakonas, 2006). Recently, has been demonstrated that Notch activation is increased during hypoxia and hypoxia direct GBM to the development of an aggressive phenotype and resistance to radiation and chemotherapy (Flynn et al., 2008). Regarding the relationship between Notch signaling and TRP channels, a direct correlation has been demonstrated in human glioma cell lines where TRPC6 transcripts have been found to be increased under hypoxic condition and the involvement of Notch in hypoxia-induced TRPC6 expression in glioma has been demonstrated. Silencing of Notch1 gene inhibits TRPC6 expression suggesting that Notch1 is required for hypoxia-induced TRPC6 overexpression (Chigurupati et al., 2010). In response to hypoxia, the hypoxia inducing factors (HIF1- and HIF-2) are stabilized and as a consequence VEGF and TGF are upregulated (Birlik et al., 2006). Moreover, hypoxia-induced endothelial cell proliferation is associated with an increase of AP-1 expression, elevated store-operated calcium entry, and enhanced TRPC4 expression (Fantozzi et al., 2003), suggesting that additional TRP channels may regulate angiogenic signals (Fig.4). The interplay between GSCs and the endothelial compartement seems to be critical in gliomagenesis. Thus, GSCs closely interacting with the endothelial cells in vascular niche, promote angiogenesis through VEGF release (Bao et al., 2006a; Folkins et al., 2009). GSCs are reported to express CD133

Fig. 4. The putative role of TRP channels in neural and glioma stem cell-like differentiation and angiogenesis. A schematic representation of different TRP members involved in the

and nestin (Yuan et al., 2004; Gunther et al., 2008) and have been demonstrated to have multipotent differentiative potential (Galli et al., 2004; Singh et al., 2003). Several authors have hypothesized that CD133+ tumor stem cells are the source of the recurrent tumors after treatment (Chua et al., 2008; Bleau et al., 2009) and the CD133+ cell population was enriched after radiation or chemotherapy and exhibited an increase in DNA repair capacity (Bao et

regulation of neuro- and glioma-genesis

al., 2006b). A series of pathways, including the Sonic hedgehog (Shh) and Notch, have been shown to be implicated in glioma's resistance to alkylating agents and/or the maintenance of brain tumor stem cells (Ulasov et al., 2011; Clement et al., 2007). Moreover, overexpression of *Dkk-1*, a gene encoding for a Wnt antagonist protein, has been shown to sensitize the U87 glioma cells to the cytotoxic effects of bis-chloronitrosourea (BCNU) and cisplatin (Shou et al., 2002). In this regard, an inverse correlation between TRPV2 and SHH and Notch pathways (Phillips et al., 2006; Nabissi et al., 2010) in regulating chemoresistance to the alkylating agent bis-chloronitrosourea (BCNU), can be supposed. TRPV2 expression progressively declined in high-grade glioma tissues as histological grade increased, while Notch and SHH signaling was activated in GBM. Knockdown of TRPV2 gene in gliomas increased the resistance to BCNU cytotoxicity which was associated with Ras/MEK/Erk and Akt overexpression in chemosensitive glioma cells, while TRPV2 overexpression augmented the chemosensitivity of resistant glioma cells to BCNU. In addition, downregulation of TRPV2 reduced Fas expression and Fas-mediated apoptosis (Nabissi et al., 2010). Parallelely, upregulation of Notch 1, increased the resistance of glioma cell to apoptosis (Purow et al., 2005). Finally, forced Notch 1 overexpression in glioma cells increased the proliferation and the formation of nestin-positive, neurosphere-forming stem cells (Zhang et al., 2008). Overall, these data suggest that in gliomas, TRPV2 could be a downstream gene target of Notch signaling rescuing glioma cells to apoptosis and promoting cell proliferation.

#### **7. Conclusions and prospectives**

In this chapter, we have summarized current basic and translational changes and highlight the striking scientific advances regarding the expression and the function of the TRP channel family in glioma growth and progression, that promise to improve the clinical course of this lethal disease. These include a more comprensive view of the interplay between changes in TRP channel expression and functions (e.g., TRPC, TRPM and TRPV family) and alterations in transcriptional and growth factor pathways (e.g., Notch, PTEN, HIF-α, EGFR) driving the uncontrolled cellular proliferation, aberrant angiogenesis, intense migration and invasion, increased resistance to apoptosis. Clearly, the identification of cluster of TRP ion channels altered during glioma progression presents an opportunity for improving the understanding of this cancer. The progress and depth of understanding of the role of ion channels, including the TRP family in glioma, together with truly manipulable experimental models, now offer a real opportunities for the development of effective target therapy (Santoni & Farfariello, 2011). Despite significant gaps in our understanding, a wealth of information now exists about clinical and biological behaviour of these tumours, the genetic pathways involved in gliomagenesis and the nature and the role of their alterations. The challenge is now to integrate all of this knowledge in an interdisciplinary way to full understand this disease and how its heterogenicity contributes to the relatively poor therapeutic responses of GBM patients. In regard to stem cell issue, the fact that the glioma-like stem cells (GSCs) that play an important role in the development and recurrence of malignat glioma, not only express TRP channels, but also show functional alterations in their expression and transcriptional regulation, combined with the evidence that they displayed nearly identical Ca(2+) transients and pharmacological sensitivities to TRP channel antagonists (Nabissi et al., personal communication; Weick et al., 2009), may

New Insight on the Role of Transient

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**9** 

*Brazil* 

**The Role of Stem Cells in the Glioma Growth** 

Malignant glioma is the most common type of primary brain tumor and represents one of the most lethal cancers. In contrast to the long-standing and well-defined histopathology, the underlying molecular and genetic bases for gliomas are less known. (Collins, 2004; Dai &

As some other human cancers, particularly central nervous tumors are highly heterogeneous. Primarily because of its diffuse nature, relatively little is known about the processes by which they develop (Hulleman & Helin, 2005). Thus, the traditional evolution concept of tumors arising from a single mutated cell has limitations in explaining the

Recent decades have seen only limited progress in treatment trials and basic research on human glioma, the most common central nervous malignancy (Huang et al., 2008). Unfortunately, for such gliomas, tumor recurrence after treatment is the rule due to the infiltrative nature of these tumors and the presence of cellular populations with ability to escape therapies and drive tumor recurrence and progression. At least in some cases, these resistant cells exhibit stem cell properties (Frosina, 2011). For these reasons the comprehension of the current knowledge of cancer stem cells (CSC) in relation to gliomas origin, growth and treatment is crucial. As the stem cells (for glioma, neuronal stem cells) are more susceptible to mutation, they become altered easily for their genetic composition and therefore act as the source of cancer/glioma cells. They are not actually a separate cell type and in most cases they are misinterpreted as cancer stem cells (in brain, they are glioma

For a long time it has been known that there are subpopulations of cells within solid tumors that contain different biological behaviors. Among these subpopulations, accumulating evidence supports the existence of the so-called cancer stem cells (CSCs), because these tumor cells possess stem cell properties, possibly being responsible for the initiation, growth and recurrence of tumors. Apparent similarities with non-transformed stem cells, including high self-renewal capacity and the ability to generate differentiated progeny of several cellular lineages, have led to the proposal that stem cell-like cancer cells may either originate from adult undifferentiated stem and progenitor cells or that these properties are being

**1. Introduction** 

Holland, 2001).

stem cells).

heterogeneity observed in a single tumor nest.

**2. Glioma and the concept of cancer stem cells** 

Sergio Garcia, Vinicius Kannen and Luciano Neder

*Faculty of Medicine of Ribeirao Preto* 

*University of Sao Paulo* 

