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**Chapter 2**

**Gliomas Biology: Angiogenesis and Invasion**

Glial tumors, within neuroepitelial-derived lesions, are the most common intra-assial neo‐ plastic histotypes. Gliomas account for about 45% of all primary central nervous system (CNS) tumors and 77% of all malignant primary CNS tumors. Gliomas can originate from neural stem cells, progenitor cells, or from de-differentiated mature neural cells transformed into cancer stem cells.  Mortality is still close to 100% and the average survival of patients with glioblastoma multiforme (GBM) is less than 1 year when classical treatment is used. Recent progresses in multimodal treatment has led to only a slight increase in average survival up to 15-18 months [1]. The effectiveness of the actual chemotherapeutic approach and multimodal targeted therapies remains modest

Gliomas are divided into different subtypes based on cell line from which they originate. Gliomas include astrocytomas, glioblastomas, oligodendrogliomas, ependymomas and oth‐ er histological subtypes. The most common gliomas are astrocytomas, oligodendrogliomas and ependymomas. Since 1993, the 4-level grading system proposed by the World Health Organization (WHO), was the most widely accepted and widespread. It is based on histo‐ logic features: nuclear atypia, presence of mitoses, endothelial proliferation. Grade I gliomas are benign with a slow proliferation rate and include pylocitic astrocytoma most common in pediatric age. Grade II gliomas are characterized by a high degree of cellular differentiation and grow diffusely into the normal brain parenchyma and are prone to malignant progres‐ sion. They include astrocytoma, oligodendroglioma and oligoastrocytoma. Grade III lesions

> © 2013 Caffo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Maria Caffo, Valeria Barresi, Gerardo Caruso,

Additional information is available at the end of the chapter

Giuseppe La Fata, Maria Angela Pino, Giuseppe Raudino, Concetta Alafaci and

Francesco Tomasello

**1. Introduction**

in gliomas.

http://dx.doi.org/10.5772/53487

## **Gliomas Biology: Angiogenesis and Invasion**

Maria Caffo, Valeria Barresi, Gerardo Caruso, Giuseppe La Fata, Maria Angela Pino, Giuseppe Raudino, Concetta Alafaci and Francesco Tomasello

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53487

#### **1. Introduction**

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Anticancer Res 2000;20:4457– 4462.

36 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

action. Exp Cell Res 2006;312:594–607.

Cell Res 2005; 307:292–304.

Glial tumors, within neuroepitelial-derived lesions, are the most common intra-assial neo‐ plastic histotypes. Gliomas account for about 45% of all primary central nervous system (CNS) tumors and 77% of all malignant primary CNS tumors. Gliomas can originate from neural stem cells, progenitor cells, or from de-differentiated mature neural cells transformed into cancer stem cells.  Mortality is still close to 100% and the average survival of patients with glioblastoma multiforme (GBM) is less than 1 year when classical treatment is used. Recent progresses in multimodal treatment has led to only a slight increase in average survival up to 15-18 months [1]. The effectiveness of the actual chemotherapeutic approach and multimodal targeted therapies remains modest in gliomas.

Gliomas are divided into different subtypes based on cell line from which they originate. Gliomas include astrocytomas, glioblastomas, oligodendrogliomas, ependymomas and oth‐ er histological subtypes. The most common gliomas are astrocytomas, oligodendrogliomas and ependymomas. Since 1993, the 4-level grading system proposed by the World Health Organization (WHO), was the most widely accepted and widespread. It is based on histo‐ logic features: nuclear atypia, presence of mitoses, endothelial proliferation. Grade I gliomas are benign with a slow proliferation rate and include pylocitic astrocytoma most common in pediatric age. Grade II gliomas are characterized by a high degree of cellular differentiation and grow diffusely into the normal brain parenchyma and are prone to malignant progres‐ sion. They include astrocytoma, oligodendroglioma and oligoastrocytoma. Grade III lesions

© 2013 Caffo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

include anaplastic astrocytoma, anaplastic oligoastrocytoma and anaplastic oligodendro‐ glioma. These tumors show a higher cellular density and a notable presence of atypia and mitotic cells. Grade IV tumors are the most malignant and also the most frequent gliomas and include glioblastoma and gliosarcoma. These tumors presented microvascular prolifera‐ tions and pseudopalisading necrosis.

stream signal transduction pathways, thereby inhibiting growth and progression of cancer. Because of their specificity, targeted therapies should theoretically have better efficacy and

Gliomas Biology: Angiogenesis and Invasion http://dx.doi.org/10.5772/53487 39

In this chapter we evaluated the invasion and angiogenesis in the glioma progression. Moreover, new pharmacological agents on the basis of the more recent literature will be

Approximately 50% of primary brain tumors are gliomas, arising from astrocytes, oligo‐ dendrocytes, or their precursors and ependymal cells. Gliomas are classified from I to IV according to the WHO malignancy scale. Gliomas are divided into different subtypes based on cell line from which they originate. The WHO classification of CNS tumors, now in its fourth edition, is the universal standard for classifying and grading brain neo‐ plasms. Analysis of tumour differentiation, cellularity, cytonuclear atypia, mitotic activity, microvascular proliferation, and necrosis further enables grading of the tumour as grade 2 (diffuse infiltrating low-grade gliomas), 3 (anaplastic gliomas), or 4 (glioblastomas) with increasing aggressiveness. Unfortunately, WHO morphological classification is based on subjective criteria, lacks reproducibility, and remains imperfect in its ability to predict in‐ dividual outcomes. The most recent WHO classification introduced a number of signifi‐ cant changes that include both additions and redefinitions or clarifications of existing entities. Three new tumor types were added to the glioma section. The first of these is angiocentric glioma. Angiocentric gliomas are slowly growing solid hemispheric tumors of children and young adults. They are strongly epileptogenic, with 95% of patients pre‐ senting with intractable seizures. These tumors are characterized histologically by elon‐ gated bipolar glial tumor cells and by their striking perivascular growth pattern. Angiocentric gliomas have a low proliferative potential (MIB-1 between 1% - 5%) and have been designated as WHO grade I neoplasms. The second, pilomyxoid astrocytoma (PMA), is now formally considered as a distinct more aggressive variant of PA. PMAs typically occur at an earlier mean age and are associated with more aggressive behavior than Pas (pilocytic astrocytomas). PMAs are composed of bipolar (piloid or hairlike) cells that lie within a distinct myxoid background matrix. PMAs are currently considered WHO grade II neoplasms. Atypical choroid plexus papilloma (CPP) is the third new tu‐ mor. Cellular atypia and increased mitotic activity are reflected in more aggressive bio‐ logic behavior, with earlier metastases and higher recurrence rates than CPP. An aCPP is designated as a WHO grade II neoplasm. The 2007 WHO version recognized, also, papil‐ lary glioneuronal tumor (PGNT), rosette-forming glioneuronal tumor (RGNT) of the fourth ventricle, and the extraventricular form of neurocytoma. PGNT is a rare relatively well-circumscribed clinically indolent tumor that is exclusively found in the cerebral hemispheres. PGNTs are biphasic tumors, with both astrocytic and neuronal elements. The histologic hallmark of PGNT is the presence of hyalinized vascular pseudopapillae. Although PGNT was not formally assigned a WHO grade in the 2007 revision, this tu‐

safety profiles than systemic cytotoxic chemotherapy or radiotherapy.

evaluated.

**2. Gliomas**

Conventional brain tumor treatments include surgery, radiation therapy and chemotherapy. Surgical treatment is invasive but represents the first approach for the vast majority of brain tumors due to difficulties arising in early stage detection. However, gross total removal is not always achievable in relation to the location of the tumor, to preserve vital nervous or vascular structures. Aggressive treatment modalities have extended the median survival from 4 months to 1 year, but the survival is often associated with significant impairment in the quality of life. Radiation therapy and chemotherapy are non-invasive options often used as adjuvant therapy, but may also be effective for curing early-stage tumors. Radiotherapy seems to be related only to a prolonged progression-free survival, with better control of seiz‐ ures, but with no substantial differences in overall survival. Besides, patients treated with radiotherapy, have high risk to develop some complications such as post radiation leukoen‐ cephalopathy, that is characterized by dementia, gait disturbance, incontinence, deficit in at‐ tention and executive functions. In patients with recurrent GBM, the 6-months progressionfree survival is only 21% after treatment with temozolomide [2]. The effectiveness of systemic chemotherapy is limited by toxic effects on healthy cells, generally resulting in morbidity or mortality of the patient. Moreover, the presence of the blood-brain barrier (BBB) limits the passage of a wide variety of anticancer agents. The high incidence of recur‐ rence and poor prognosis of malignant gliomas compel the development of more powerful anti-cancer treatments. Action of alkylating agents, one of the most common class of chemo‐ therapeutic drugs employed, is limited by activation of methylguanine-methyltransferase. The compromise of the quality of remaining life as well as the limited success of current treatment options in shrinking tumors, raise increasing concerns about the adverse effects of cancer treatment on brain function.

Glioma cell invasion into normal tissue is thought to be a multi-factorial process, consisting of cell interactions with extracellular matrix (ECM) and with adjacent cells, as well as accom‐ panying biochemical processes supportive of active cell movement. Tumor cell invasion re‐ quires four distinct steps: (1) detachment of invading cells from the primary tumor mass, (2) adhesion to ECM, (3) degradation of ECM, and (4) cell motility and contractility. However, growth of tumors necessitates, also, the recruitment of a new blood supply. Angiogenesis, represent a key event in the progression of malignant gliomas. Tumor angiogenesis involves multiple cellular processes including endothelial cell proliferation, migration, reorganization of ECM and tube formation.

The advent of molecular studies allowed a new evaluation of the biology of gliomas with, a level of precision that promises interesting advances toward the development of specific and effective therapies. The introduction of molecularly targeted agents is one of the most significant advances in cancer therapy in recent years. Targeted therapies block activation of oncogenic pathways, either at the ligand-receptor interaction level or by inhibiting down‐ stream signal transduction pathways, thereby inhibiting growth and progression of cancer. Because of their specificity, targeted therapies should theoretically have better efficacy and safety profiles than systemic cytotoxic chemotherapy or radiotherapy.

In this chapter we evaluated the invasion and angiogenesis in the glioma progression. Moreover, new pharmacological agents on the basis of the more recent literature will be evaluated.

#### **2. Gliomas**

include anaplastic astrocytoma, anaplastic oligoastrocytoma and anaplastic oligodendro‐ glioma. These tumors show a higher cellular density and a notable presence of atypia and mitotic cells. Grade IV tumors are the most malignant and also the most frequent gliomas and include glioblastoma and gliosarcoma. These tumors presented microvascular prolifera‐

Conventional brain tumor treatments include surgery, radiation therapy and chemotherapy. Surgical treatment is invasive but represents the first approach for the vast majority of brain tumors due to difficulties arising in early stage detection. However, gross total removal is not always achievable in relation to the location of the tumor, to preserve vital nervous or vascular structures. Aggressive treatment modalities have extended the median survival from 4 months to 1 year, but the survival is often associated with significant impairment in the quality of life. Radiation therapy and chemotherapy are non-invasive options often used as adjuvant therapy, but may also be effective for curing early-stage tumors. Radiotherapy seems to be related only to a prolonged progression-free survival, with better control of seiz‐ ures, but with no substantial differences in overall survival. Besides, patients treated with radiotherapy, have high risk to develop some complications such as post radiation leukoen‐ cephalopathy, that is characterized by dementia, gait disturbance, incontinence, deficit in at‐ tention and executive functions. In patients with recurrent GBM, the 6-months progressionfree survival is only 21% after treatment with temozolomide [2]. The effectiveness of systemic chemotherapy is limited by toxic effects on healthy cells, generally resulting in morbidity or mortality of the patient. Moreover, the presence of the blood-brain barrier (BBB) limits the passage of a wide variety of anticancer agents. The high incidence of recur‐ rence and poor prognosis of malignant gliomas compel the development of more powerful anti-cancer treatments. Action of alkylating agents, one of the most common class of chemo‐ therapeutic drugs employed, is limited by activation of methylguanine-methyltransferase. The compromise of the quality of remaining life as well as the limited success of current treatment options in shrinking tumors, raise increasing concerns about the adverse effects of

Glioma cell invasion into normal tissue is thought to be a multi-factorial process, consisting of cell interactions with extracellular matrix (ECM) and with adjacent cells, as well as accom‐ panying biochemical processes supportive of active cell movement. Tumor cell invasion re‐ quires four distinct steps: (1) detachment of invading cells from the primary tumor mass, (2) adhesion to ECM, (3) degradation of ECM, and (4) cell motility and contractility. However, growth of tumors necessitates, also, the recruitment of a new blood supply. Angiogenesis, represent a key event in the progression of malignant gliomas. Tumor angiogenesis involves multiple cellular processes including endothelial cell proliferation, migration, reorganization

The advent of molecular studies allowed a new evaluation of the biology of gliomas with, a level of precision that promises interesting advances toward the development of specific and effective therapies. The introduction of molecularly targeted agents is one of the most significant advances in cancer therapy in recent years. Targeted therapies block activation of oncogenic pathways, either at the ligand-receptor interaction level or by inhibiting down‐

tions and pseudopalisading necrosis.

38 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

cancer treatment on brain function.

of ECM and tube formation.

Approximately 50% of primary brain tumors are gliomas, arising from astrocytes, oligo‐ dendrocytes, or their precursors and ependymal cells. Gliomas are classified from I to IV according to the WHO malignancy scale. Gliomas are divided into different subtypes based on cell line from which they originate. The WHO classification of CNS tumors, now in its fourth edition, is the universal standard for classifying and grading brain neo‐ plasms. Analysis of tumour differentiation, cellularity, cytonuclear atypia, mitotic activity, microvascular proliferation, and necrosis further enables grading of the tumour as grade 2 (diffuse infiltrating low-grade gliomas), 3 (anaplastic gliomas), or 4 (glioblastomas) with increasing aggressiveness. Unfortunately, WHO morphological classification is based on subjective criteria, lacks reproducibility, and remains imperfect in its ability to predict in‐ dividual outcomes. The most recent WHO classification introduced a number of signifi‐ cant changes that include both additions and redefinitions or clarifications of existing entities. Three new tumor types were added to the glioma section. The first of these is angiocentric glioma. Angiocentric gliomas are slowly growing solid hemispheric tumors of children and young adults. They are strongly epileptogenic, with 95% of patients pre‐ senting with intractable seizures. These tumors are characterized histologically by elon‐ gated bipolar glial tumor cells and by their striking perivascular growth pattern. Angiocentric gliomas have a low proliferative potential (MIB-1 between 1% - 5%) and have been designated as WHO grade I neoplasms. The second, pilomyxoid astrocytoma (PMA), is now formally considered as a distinct more aggressive variant of PA. PMAs typically occur at an earlier mean age and are associated with more aggressive behavior than Pas (pilocytic astrocytomas). PMAs are composed of bipolar (piloid or hairlike) cells that lie within a distinct myxoid background matrix. PMAs are currently considered WHO grade II neoplasms. Atypical choroid plexus papilloma (CPP) is the third new tu‐ mor. Cellular atypia and increased mitotic activity are reflected in more aggressive bio‐ logic behavior, with earlier metastases and higher recurrence rates than CPP. An aCPP is designated as a WHO grade II neoplasm. The 2007 WHO version recognized, also, papil‐ lary glioneuronal tumor (PGNT), rosette-forming glioneuronal tumor (RGNT) of the fourth ventricle, and the extraventricular form of neurocytoma. PGNT is a rare relatively well-circumscribed clinically indolent tumor that is exclusively found in the cerebral hemispheres. PGNTs are biphasic tumors, with both astrocytic and neuronal elements. The histologic hallmark of PGNT is the presence of hyalinized vascular pseudopapillae. Although PGNT was not formally assigned a WHO grade in the 2007 revision, this tu‐ mor generally behaves in a benign grade I fashion. RGNTs are rare slow-growing tumors of young and middle-aged adults. RGNTs contain both neurocytic and astrocytic ele‐ ments. Their histologic hallmark is the formation of neurocytic perivascular pseudoro‐ settes. RGNTs are designated as WHO grade I neoplasms. The term "central neurocytoma" describes a neuronal tumor with preferential location in the lateral ventri‐ cle body. These tumors comprise fibrillary areas mimicking neuropil plus collections of uniform round cells that have immunohistochemical and ultrastructural evidence of neu‐ ronal differentiation. A low proliferation rate is typical. Similar neoplasms have been re‐ ported outside the ventricular system, and the WHO 2007 designated the term "EVNCT" (extraventricular neurocytoma) for these uncommon tumors. Because the only distin‐ guishing feature is location EVNCTs are included in the same histopathologic code as central neurocytoma. Both central and extraventricular neurocytomas are designated as WHO grade II neoplasms. Two new tumors of the pineal region were codified in the WHO 2007 classification, papillary tumor of the pineal region (PTPR) and pineal paren‐ chymal tumor of intermediate differentiation (PPTID). PTPR is a rare neuroepithelial tu‐ mor that arises from the subcommissural organ and exhibits ependymal differentiation. Macroscopically, PTPRs are indistinguishable from pineocytomas. Microscopically, the tu‐ mors are easily distinguished. PTPRs show papillary architecture with pseudostratified columnar epithelium. Ultrastructural features suggesting ependymal differentiation are present. Immunohistochemistry is positive for cytokeratins. While grading of PTPRs has yet to be defined, most neuropathologists consider these as WHO grade II or III neo‐ plasms. PPTID is a newly recognized tumor intermediate in malignancy between pineo‐ cytoma and pineoblastoma. Mild-to-moderate nuclear atypia and low-to-moderate mitotic activity result in either WHO grade II or III. PPTIDs have a much more aggressive course than pineocytomas and may warrant adjuvant therapy. PPTIDs are more common than previously recognized, accounting for up to 20% of all pineal parenchymal neo‐ plasms. Recent reports emphasize their large size and focal invasion of adjacent struc‐ tures as features that distinguish them from pineocytoma [3].

**2.1. Genetics of malignant gliomas**

and function.

not mutated in GBM [8].

Cancers originate as the result of hereditary or somatic alterations in genes that control criti‐ cal biological processes. The accumulation of such genetic damage over time permits the survival and progressive transformation of abnormal cell populations that eventually lead to the formation of a tumor. Consistent with their high malignant potential, gliomas exhibit a vast array of well-documented genetic changes that likely contribute to their phenotype. Gliomagenesis is characterized by several biological events, such as activated growth factor receptor signaling pathways, downregulation of many apoptotic mechanisms and unbal‐ ance among proangiogenic and antiangiogenic factors. Several growth factor receptors, such epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDRGF), C-Kit, vascular endhotelial growth factor receptor (VEGFR) and others growth factors recep‐ tors, are overexpressed, amplified and/or mutated in gliomas. Features of the glioma cells are the loss of tumor suppressor genes, which are critical for the cell growth, differentiation

Gliomas Biology: Angiogenesis and Invasion http://dx.doi.org/10.5772/53487 41

The TP53 tumor suppressor gene was identified as the main driver of chromosome 17 altera‐ tions in glioblastoma, and further studies indicated that p53 plays a critical role in monitor‐ ing the genome for DNA damage and can control cell cycle arrest to permit DNA repair or can trigger apoptosis to eliminate the damaged cell [7]. It acts as a transcriptional activator and it repress the transcription of other genes, such as IL-6, c-fos, c-jun and other oncogenes. The levels of p53 increase in G1 and S phases of the cell cycle. Cells transfected with the nor‐ mal gene p53 block their growth in G1, suppressing the passage S-phase. The expression of the p53 gene cause apoptosis (programmed cell death). The effects of its mutation are the loss of cell cycle regulation, through the inactivation of p16ink-4a, the super expression of cy‐ cline dependent kinase 4 Cdk4 and Cdk6 and of ubiquitin ligase Mdm2 and Mdm4. A simi‐ lar mechanism is created after mutation of retinoblastoma (Rb), a tumor suppressor gene. Probably, TP53 is a gene responsible for the progression from low-grade to high-grade glio‐ mas, but its mutation is an early event. Many glioma cell lines expressing p53 mutations. The mutated form of p53 has a crucial role in the regulation of neovascularization. It induces increased levels of VEGF and FGFβ through an activation of the transcription of the corre‐ sponding genes. The deletion of NFKBIA (encoding nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor-α), an inhibitor of the EGFR-signaling pathway, promotes tumorigenesis in glioblastomas that do not have alterations of EGFR. Bredel et al. analyzed 790 human GBMs for deletions, mutations, or expression of NFKBIA and EGFR. they stud‐ ied the tumor-suppressor activity of NFKBIA in tumor-cell culture and compared the molec‐ ular results with the outcome of GBM in 570 affected persons. NFKBIA is often deleted but

Epidermal growth factor receptor mutations include amplifications, point mutations and de‐ letions, with the most common alteration being the variant III deletion of the extracellular domain (EGFR-vIII mutant) [9]. Platelet-derived growth factor receptor and its ligands PDGF-A and PDGF-B are also commonly over-expressed in some glioma cells, raising the possibility of autocrine or paracrine activation [10]. Other RTK genes, such as ERBB2, anoth‐ er member of the EGF receptor family, and MET, which encodes the hepatocyte growth fac‐

According to WHO low grade gliomas include diffuse astrocytomas (fibrillar, gemistio‐ cytic, protoplasmatic, mixed), grade II oligodendrogliomas and oligoastrocytomas. Diag‐ nosis of anaplastic glioma is based on the presence of mitotic activity, while vascular proliferation and necrosis are typical features of GBM. The definition of low grade glio‐ ma does not refer perhaps to I grade astrocytomas, that differ from other gliomas in rela‐ tion to their biological behaviour, prognosis and genetic profile [4]. Low grade gliomas, tend perhaps to dedifferentiate: a global therapeutic approach, related to gross total (> 90%) surgical removal, radiotherapy e chemiotherapy with temozolamide, leads only to a slight prolongation of overall survival, calculated from 3 to 7 years [5]. This variability depends from various factors: age at diagnosis, tumor volume, tumor crossing the mid‐ line, neurological deficits before treatment and tumor's histology [6]. Anyway, mortality is near to 100% and only 35% of patients with glioblastoma multiforme have a median survival of one year or more.

#### **2.1. Genetics of malignant gliomas**

mor generally behaves in a benign grade I fashion. RGNTs are rare slow-growing tumors of young and middle-aged adults. RGNTs contain both neurocytic and astrocytic ele‐ ments. Their histologic hallmark is the formation of neurocytic perivascular pseudoro‐ settes. RGNTs are designated as WHO grade I neoplasms. The term "central neurocytoma" describes a neuronal tumor with preferential location in the lateral ventri‐ cle body. These tumors comprise fibrillary areas mimicking neuropil plus collections of uniform round cells that have immunohistochemical and ultrastructural evidence of neu‐ ronal differentiation. A low proliferation rate is typical. Similar neoplasms have been re‐ ported outside the ventricular system, and the WHO 2007 designated the term "EVNCT" (extraventricular neurocytoma) for these uncommon tumors. Because the only distin‐ guishing feature is location EVNCTs are included in the same histopathologic code as central neurocytoma. Both central and extraventricular neurocytomas are designated as WHO grade II neoplasms. Two new tumors of the pineal region were codified in the WHO 2007 classification, papillary tumor of the pineal region (PTPR) and pineal paren‐ chymal tumor of intermediate differentiation (PPTID). PTPR is a rare neuroepithelial tu‐ mor that arises from the subcommissural organ and exhibits ependymal differentiation. Macroscopically, PTPRs are indistinguishable from pineocytomas. Microscopically, the tu‐ mors are easily distinguished. PTPRs show papillary architecture with pseudostratified columnar epithelium. Ultrastructural features suggesting ependymal differentiation are present. Immunohistochemistry is positive for cytokeratins. While grading of PTPRs has yet to be defined, most neuropathologists consider these as WHO grade II or III neo‐ plasms. PPTID is a newly recognized tumor intermediate in malignancy between pineo‐ cytoma and pineoblastoma. Mild-to-moderate nuclear atypia and low-to-moderate mitotic activity result in either WHO grade II or III. PPTIDs have a much more aggressive course than pineocytomas and may warrant adjuvant therapy. PPTIDs are more common than previously recognized, accounting for up to 20% of all pineal parenchymal neo‐ plasms. Recent reports emphasize their large size and focal invasion of adjacent struc‐

40 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

tures as features that distinguish them from pineocytoma [3].

survival of one year or more.

According to WHO low grade gliomas include diffuse astrocytomas (fibrillar, gemistio‐ cytic, protoplasmatic, mixed), grade II oligodendrogliomas and oligoastrocytomas. Diag‐ nosis of anaplastic glioma is based on the presence of mitotic activity, while vascular proliferation and necrosis are typical features of GBM. The definition of low grade glio‐ ma does not refer perhaps to I grade astrocytomas, that differ from other gliomas in rela‐ tion to their biological behaviour, prognosis and genetic profile [4]. Low grade gliomas, tend perhaps to dedifferentiate: a global therapeutic approach, related to gross total (> 90%) surgical removal, radiotherapy e chemiotherapy with temozolamide, leads only to a slight prolongation of overall survival, calculated from 3 to 7 years [5]. This variability depends from various factors: age at diagnosis, tumor volume, tumor crossing the mid‐ line, neurological deficits before treatment and tumor's histology [6]. Anyway, mortality is near to 100% and only 35% of patients with glioblastoma multiforme have a median Cancers originate as the result of hereditary or somatic alterations in genes that control criti‐ cal biological processes. The accumulation of such genetic damage over time permits the survival and progressive transformation of abnormal cell populations that eventually lead to the formation of a tumor. Consistent with their high malignant potential, gliomas exhibit a vast array of well-documented genetic changes that likely contribute to their phenotype. Gliomagenesis is characterized by several biological events, such as activated growth factor receptor signaling pathways, downregulation of many apoptotic mechanisms and unbal‐ ance among proangiogenic and antiangiogenic factors. Several growth factor receptors, such epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDRGF), C-Kit, vascular endhotelial growth factor receptor (VEGFR) and others growth factors recep‐ tors, are overexpressed, amplified and/or mutated in gliomas. Features of the glioma cells are the loss of tumor suppressor genes, which are critical for the cell growth, differentiation and function.

The TP53 tumor suppressor gene was identified as the main driver of chromosome 17 altera‐ tions in glioblastoma, and further studies indicated that p53 plays a critical role in monitor‐ ing the genome for DNA damage and can control cell cycle arrest to permit DNA repair or can trigger apoptosis to eliminate the damaged cell [7]. It acts as a transcriptional activator and it repress the transcription of other genes, such as IL-6, c-fos, c-jun and other oncogenes. The levels of p53 increase in G1 and S phases of the cell cycle. Cells transfected with the nor‐ mal gene p53 block their growth in G1, suppressing the passage S-phase. The expression of the p53 gene cause apoptosis (programmed cell death). The effects of its mutation are the loss of cell cycle regulation, through the inactivation of p16ink-4a, the super expression of cy‐ cline dependent kinase 4 Cdk4 and Cdk6 and of ubiquitin ligase Mdm2 and Mdm4. A simi‐ lar mechanism is created after mutation of retinoblastoma (Rb), a tumor suppressor gene. Probably, TP53 is a gene responsible for the progression from low-grade to high-grade glio‐ mas, but its mutation is an early event. Many glioma cell lines expressing p53 mutations. The mutated form of p53 has a crucial role in the regulation of neovascularization. It induces increased levels of VEGF and FGFβ through an activation of the transcription of the corre‐ sponding genes. The deletion of NFKBIA (encoding nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor-α), an inhibitor of the EGFR-signaling pathway, promotes tumorigenesis in glioblastomas that do not have alterations of EGFR. Bredel et al. analyzed 790 human GBMs for deletions, mutations, or expression of NFKBIA and EGFR. they stud‐ ied the tumor-suppressor activity of NFKBIA in tumor-cell culture and compared the molec‐ ular results with the outcome of GBM in 570 affected persons. NFKBIA is often deleted but not mutated in GBM [8].

Epidermal growth factor receptor mutations include amplifications, point mutations and de‐ letions, with the most common alteration being the variant III deletion of the extracellular domain (EGFR-vIII mutant) [9]. Platelet-derived growth factor receptor and its ligands PDGF-A and PDGF-B are also commonly over-expressed in some glioma cells, raising the possibility of autocrine or paracrine activation [10]. Other RTK genes, such as ERBB2, anoth‐ er member of the EGF receptor family, and MET, which encodes the hepatocyte growth fac‐ tor receptor, have also been found to be mutated in GBMs [9]. RTKs mediate cell growth and proliferation via downstream effectors such as Ras and phosphatidylinositide-3-kinase (PI3K). Activity of these proteins is tightly regulated, especially by the tumor suppressors Nf1 and Pten. The PI3K pathway is another essential survival pathway for a variety of can‐ cer cells. The tumor suppressor Pten (phosphatase and tensin homologue on chromosome 10) negatively regulates the PI3K pathway by dephosphorylating phosphatidylinositol-3,4,5 triphosphate (PIP3) back to phosphatidylinositol-4,5-biphosphate (PIP2) [11]. Mutations in PTEN frequently involve the phosphatase domain, and mutations in PI3K typically involve the catalytic (p110α) and regulatory (p85α) domains.

Studies suggest that mutant IDH1 loses its normal enzymatic activity in tumors while gain‐ ing a new pro-oncogenic activity, leading to the production of an onco-metabolite [18-20]. Other studies characterizing the genomic make-up of human glioblastoma have provided further insight into the genetic changes, core pathways and molecular subtypes underlying this disease [20-22]. Based on their gene expression profiles, The Cancer Genome Atlas Net‐ work (TCGA) further classified GBMs into four subtypes termed: proneural, neural, classi‐ cal, and mesenchymal [9]. While the status of gene expression and mutation in EGFR, NF1, PDGFA, and IDH1 were defining components of these subtypes, it was also found that re‐ sponse to therapies was different for each subtype, suggesting that personalized treatment based on genomic alterations could lead to a more favorable outcome for this disease [23].

Gliomas Biology: Angiogenesis and Invasion http://dx.doi.org/10.5772/53487 43

The cellular and molecular events that initiate and promote malignant glioma development are not completely understood. Vasculogenesis and angiogenesis potentially play distinct roles in the etiology of primary and recurrent malignant gliomas, suggesting that patient therapy should perhaps be tailored specifically against the predominant vasculature path‐ way at a given specific stage of gliomagenesis [24]. Cerebral gliomas show a unique pattern of invasion and, with rare exceptions, do not metastasize outside of the brain. Invading glio‐ ma cells normally migrate to distinct anatomical structures. These structures include the basement membrane (BM) of blood vessels, the subependymal space, the glial limitans ex‐ terna, and parallel and intersecting nerve fiber tracts in the white matter. Invasion of tumor cells into normal tissue is thought to be a multifactorial process, consisting of cell interac‐ tions with ECM and with adjacent cells, as well as accompanying biochemical processes supportive of active cell movement. Several cell types aim active movement during various stages of embryonal development, during wound healing, and in the course of immune re‐ sponses. This innate activity is regulated in a very rigid manner, suggesting that the reap‐ pearance of a motile phenotype in cancer cells results from the loss or cessation of normal inhibitory controls [25]. Critical factors in tumor cell invasion, include the detachment of in‐ vading cells from the primary tumor mass, the synthesis and deposition of ECM compo‐ nents by tumor cells and mesenchymal cells, the release of ECM-degrading activities for remodeling interstitial space, and the expression of adhesion molecules on glioma cell surfa‐

The detachment of invading glioma cells from the primary tumor mass involves several events, including, destabilization and disorganization of the cadherin-mediated junctions that hold the primary mass together, loss of expression of neural cell adhesion molecule, which promotes adhesion to the primary tumor mass through homophilic binding, cleavage of CD44, which anchors the primary mass to the ECM by the metalloproteinase ADAM.

Cadherin superfamily are important adhesion molecules associated with glioma invasion. Cadherins (E-, P-, and N-cadherin) form adherent junctions and may function as suppres‐ sors of tumor growth and invasion. Desmosomal cadherins interact heterotypically and pro‐

**3. Molecular biology of glioma invasion**

ces that specifically recognize and adhere to ECM components.

Classically, GBM has been described as result endpoint from two main gliomagenesis path‐ ways [12-13]. Primary GBM shows amplification of the EGFR, deletion or mutation of homo‐ zygous cyclin-dependent kinase (CDK) inhibitor p16INK4A/(CDKN2A), alterations in tumor-suppressor PTEN on chromosome 10, and deletion in the INK4a gene with loss of p14 and p16 [14-15]. Progression from low-grade to high-grade astrocytomas involves inac‐ tivating mutations of tumor-suppressor gene TP53 and elevated expression of PDGF ligands and receptors, accumulation of genetic alteration of retinoblastoma-associated cell cycle reg‐ ulatory pathways, including deletion or mutations of cyclin-dependent kinase inhibitor p16INK4A/(CDKN2A) or the retinoblastoma susceptibility locus 1 (pRB1), as well as ampli‐ fication or overexpression of cyclin-dependent kinase 4 (CDK4) and human double minute 2 (HDM2). Evolution to secondary GBM is associated with deletion of chromosome 10, which includes tumor-suppressor phosphatase and tensin homologue (PTEN). PTEN phosphatase were identified as the tumor suppressors lost on chromosomes 19 and 10, respectively. p16 can slow down cell cycle progression, whereas PTEN is a negative regulator of the phos‐ phoinositide 3-kinase (PI3K) pathway, [16] a major signaling pathway that stimulates cellu‐ lar proliferation in response to growth factor stimulation. Interestingly, only 1 copy of the gene is mutated in the tumors, suggesting that the mutations do not result in a simple loss of function. The mutation is very specific and leads to a single amino acid change (arginine 132 usually becomes histidine) in the IDH1 active site, whereby the enzyme loses its ability to catalyze conversion of isocitrate to α-ketoglutarate. It was proposed that this might have an indirect oncogenic effect through the activation of the hypoxia-inducible factor pathway, [17] a critical step in the metabolic adaptation of tumors to anaerobic growth and for the for‐ mation of new blood vessels through the angiogenic process.

Recently, attention has been focused on the role of an enzyme, isocitrate dehydrogenase 1 (IDH1), present in cytoplasm and peroxisomes, catalyzing the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, reducing NADP+ to NADPH. Studies performed with seri‐ ated biopsies from gliomas, revealed that mutation of IDH1 is always found before TP53 mutation or, in case of oligodendrogliomas, before their typical deletions of chromosomes 1p and 19q. Moreover, mutation of IDH1 is quite specific, since it is extremely rare in I grade gliomas, non glioma brain tumors and in other cancers. Yan et al. studied genetic alterations of both IDH1 and of its related gene, IDH2. Genomic analysis, showed mutation of amino acid 132 of IDH1 in over 70% of patients with both low or high grade gliomas, and many of the cases negative for IDH1 mutations, had mutation involving amino acid 172 of IDH2 [18]. Studies suggest that mutant IDH1 loses its normal enzymatic activity in tumors while gain‐ ing a new pro-oncogenic activity, leading to the production of an onco-metabolite [18-20]. Other studies characterizing the genomic make-up of human glioblastoma have provided further insight into the genetic changes, core pathways and molecular subtypes underlying this disease [20-22]. Based on their gene expression profiles, The Cancer Genome Atlas Net‐ work (TCGA) further classified GBMs into four subtypes termed: proneural, neural, classi‐ cal, and mesenchymal [9]. While the status of gene expression and mutation in EGFR, NF1, PDGFA, and IDH1 were defining components of these subtypes, it was also found that re‐ sponse to therapies was different for each subtype, suggesting that personalized treatment based on genomic alterations could lead to a more favorable outcome for this disease [23].

#### **3. Molecular biology of glioma invasion**

tor receptor, have also been found to be mutated in GBMs [9]. RTKs mediate cell growth and proliferation via downstream effectors such as Ras and phosphatidylinositide-3-kinase (PI3K). Activity of these proteins is tightly regulated, especially by the tumor suppressors Nf1 and Pten. The PI3K pathway is another essential survival pathway for a variety of can‐ cer cells. The tumor suppressor Pten (phosphatase and tensin homologue on chromosome 10) negatively regulates the PI3K pathway by dephosphorylating phosphatidylinositol-3,4,5 triphosphate (PIP3) back to phosphatidylinositol-4,5-biphosphate (PIP2) [11]. Mutations in PTEN frequently involve the phosphatase domain, and mutations in PI3K typically involve

Classically, GBM has been described as result endpoint from two main gliomagenesis path‐ ways [12-13]. Primary GBM shows amplification of the EGFR, deletion or mutation of homo‐ zygous cyclin-dependent kinase (CDK) inhibitor p16INK4A/(CDKN2A), alterations in tumor-suppressor PTEN on chromosome 10, and deletion in the INK4a gene with loss of p14 and p16 [14-15]. Progression from low-grade to high-grade astrocytomas involves inac‐ tivating mutations of tumor-suppressor gene TP53 and elevated expression of PDGF ligands and receptors, accumulation of genetic alteration of retinoblastoma-associated cell cycle reg‐ ulatory pathways, including deletion or mutations of cyclin-dependent kinase inhibitor p16INK4A/(CDKN2A) or the retinoblastoma susceptibility locus 1 (pRB1), as well as ampli‐ fication or overexpression of cyclin-dependent kinase 4 (CDK4) and human double minute 2 (HDM2). Evolution to secondary GBM is associated with deletion of chromosome 10, which includes tumor-suppressor phosphatase and tensin homologue (PTEN). PTEN phosphatase were identified as the tumor suppressors lost on chromosomes 19 and 10, respectively. p16 can slow down cell cycle progression, whereas PTEN is a negative regulator of the phos‐ phoinositide 3-kinase (PI3K) pathway, [16] a major signaling pathway that stimulates cellu‐ lar proliferation in response to growth factor stimulation. Interestingly, only 1 copy of the gene is mutated in the tumors, suggesting that the mutations do not result in a simple loss of function. The mutation is very specific and leads to a single amino acid change (arginine 132 usually becomes histidine) in the IDH1 active site, whereby the enzyme loses its ability to catalyze conversion of isocitrate to α-ketoglutarate. It was proposed that this might have an indirect oncogenic effect through the activation of the hypoxia-inducible factor pathway, [17] a critical step in the metabolic adaptation of tumors to anaerobic growth and for the for‐

Recently, attention has been focused on the role of an enzyme, isocitrate dehydrogenase 1 (IDH1), present in cytoplasm and peroxisomes, catalyzing the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, reducing NADP+ to NADPH. Studies performed with seri‐ ated biopsies from gliomas, revealed that mutation of IDH1 is always found before TP53 mutation or, in case of oligodendrogliomas, before their typical deletions of chromosomes 1p and 19q. Moreover, mutation of IDH1 is quite specific, since it is extremely rare in I grade gliomas, non glioma brain tumors and in other cancers. Yan et al. studied genetic alterations of both IDH1 and of its related gene, IDH2. Genomic analysis, showed mutation of amino acid 132 of IDH1 in over 70% of patients with both low or high grade gliomas, and many of the cases negative for IDH1 mutations, had mutation involving amino acid 172 of IDH2 [18].

the catalytic (p110α) and regulatory (p85α) domains.

42 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

mation of new blood vessels through the angiogenic process.

The cellular and molecular events that initiate and promote malignant glioma development are not completely understood. Vasculogenesis and angiogenesis potentially play distinct roles in the etiology of primary and recurrent malignant gliomas, suggesting that patient therapy should perhaps be tailored specifically against the predominant vasculature path‐ way at a given specific stage of gliomagenesis [24]. Cerebral gliomas show a unique pattern of invasion and, with rare exceptions, do not metastasize outside of the brain. Invading glio‐ ma cells normally migrate to distinct anatomical structures. These structures include the basement membrane (BM) of blood vessels, the subependymal space, the glial limitans ex‐ terna, and parallel and intersecting nerve fiber tracts in the white matter. Invasion of tumor cells into normal tissue is thought to be a multifactorial process, consisting of cell interac‐ tions with ECM and with adjacent cells, as well as accompanying biochemical processes supportive of active cell movement. Several cell types aim active movement during various stages of embryonal development, during wound healing, and in the course of immune re‐ sponses. This innate activity is regulated in a very rigid manner, suggesting that the reap‐ pearance of a motile phenotype in cancer cells results from the loss or cessation of normal inhibitory controls [25]. Critical factors in tumor cell invasion, include the detachment of in‐ vading cells from the primary tumor mass, the synthesis and deposition of ECM compo‐ nents by tumor cells and mesenchymal cells, the release of ECM-degrading activities for remodeling interstitial space, and the expression of adhesion molecules on glioma cell surfa‐ ces that specifically recognize and adhere to ECM components.

The detachment of invading glioma cells from the primary tumor mass involves several events, including, destabilization and disorganization of the cadherin-mediated junctions that hold the primary mass together, loss of expression of neural cell adhesion molecule, which promotes adhesion to the primary tumor mass through homophilic binding, cleavage of CD44, which anchors the primary mass to the ECM by the metalloproteinase ADAM.

Cadherin superfamily are important adhesion molecules associated with glioma invasion. Cadherins (E-, P-, and N-cadherin) form adherent junctions and may function as suppres‐ sors of tumor growth and invasion. Desmosomal cadherins interact heterotypically and pro‐

vide a linkage to the intermediate filament network through association with cytosolic proteins (desmoplakin, plakoglobin). Cadherins are linked to the actin-cytoskeleton via cate‐ nins (α-, β-catenin, plakoglobin), thereby establishing molecular lines of communication to other cell-cell junctions and to cell-substratum junctions [26]. During glioma progression de‐ creased cadherin function is correlated with de-differentiation, metastasis and poor progno‐ sis [27]. Due to its participation in processes such as morphological differentiation and contact inhibition of growth and motility, cadherins may function as suppressors of tumor growth and invasion [25]. Perego and co-worker, showed that only glioma cells with imma‐ ture adherens junctions are capable of migrating and invading through poly-1-lysine coated filters. In the process of maturation of adhesions, the Lin-7 protein forms a complex with cadherin and β-catenin [28].

kD. The β subunits have 4 cysteine-rich repeat sequences. Both the α subunit β both bind many divalent cations. There are many ways to classify the integrins. For example, a sub‐ class of the chains α has a structural domain in more inserted at the network termination NH2, the so-called alpha-domain A. The integrins that possess this characteristic usually form collagen (α1β1 integrins, and α2β1), or act as adhesion molecules between cell and cell (β2 integrin family). This domain α-1 is the active site for links to various integrins. Those who do not carry this domain, however, have a domain A in their active site, but this is in the β chain. In both cases, the domains A are connected to three active sites consisting of divalent cations. One of these is permanently occupied by physiological concentrations of divalent cations, and binds to a calcium ion or magnesium. The other two sites are occupied by cations when establishing ties - at least for the bonds involving an amino acid, aspartic acid, typically - in their interaction sites. An amino acid characterized in the active site of the integrin many proteins ECM, as part of the amino acid sequence Arginine-Glycine-Aspartic acid. The two main functions of integrins are cell adhesion to ECM and signal transduction from the ECM to the cell. They interact with two large groups of ligands: a variety of ECM proteins, such as fibronectin, vitronectin, fibrinogen, and cell surface molecules, that are members of the immunoglobulin supergene family, such as intracellular adhesion molecules (ICAM-1, ICAM-2) and vascular cell adhesion molecule (VCAM-1). In particular, the integ‐ rin ανβ3, which binds to fibronectin, vitronectin, and tenascin-C in ECM, is thought to play a central role in glioma invasion [36]. Increased expression of integrin ανβ3 leads to in‐ creased motility in human glioma cells with a concomitant decrease in apoptosis sensitivity.

Gliomas Biology: Angiogenesis and Invasion http://dx.doi.org/10.5772/53487 45

Integrins combine the ECM out of the cell to the cytoskeleton (in particular microfilaments) within the cell. What site can bind the ECM is usually decided by the integrin α and β subu‐ nit which is composed of the same integrin. Bonds between the integrin are fibronectin, vi‐ tronectin, collagen, and laminin. The connection between the cell and the ECM makes the cell able to withstand the forces tie rods without being thrown out of the ECM. The connec‐ tions between the integrin and the links in the ECM and microfilaments inside the cell are indirect: they are connected by proteins "armor" as talin, paxillin, and alpha-actinin. These act by regulating the kinase such as FAK (focal adhesion kinase - focal adhesion kinase) and the family of Src kinases to phosphorylated substrates such as the p130CAS or recruiting signal adapters such as Crk. The connection of the cell to the extracellular matrix is a basic requirement to form a multicellular organism. Integrins link the cytoskeleton to the extracel‐ lular matrix, and are recognized to be key regulators of tissue structure. They provide adhe‐ sive, migratory, and survival cues to tumor cells and to cells of the tumor microenvironment, including angiogenic endothelial cells. The integrins ανβ3 and ανβ5, among others, are highly expressed not only on the tumor vasculature and angiogenic endo‐ thelial cells, but also on tumor cells, including gliomas. Consequently, integrins have been considered as a promising therapeutic target in cancer. Monoclonal antibodies and peptidebased integrin inhibitors are being investigated for their potential therapeutic activity in var‐ ious tumor types. This strategy is in advanced stage clinical development in glioblastoma, a

highly vascular primary brain tumor [37].

The second step is a decline in the expression of connexin 43, which leads to a reduction in gap junction formation. Connexin 43 is the most abundant gap junction protein in CNS and is expressed primarily in astrocytes [29]. Cell-cell communication is important in growth control and differentiation, and it is partly achieved using gap junctions and via second messengers [30]. Decreased gap junction formation may result in fewer inhibitory signals, facilitating uncontrolled cell division and de-differentiation [31]. Has been demonstrated that reduced gap junction formation is correlated with increased motility of glioma cells in vitro [32]. Increased malignancy of glioma specimens correlates with reduced in situ gap junction formation as well as reduced connexin 43 expression [33]. The third event is cleav‐ age of CD44, which anchors the primary mass to ECM, by the metalloproteinase ADAM. CD44 is a transmembrane glycoprotein belonging to the immunoglobulin receptor super‐ family, which interacts with hyaluronic acid as its ligand. Monoclonal antibodies directed against CD44 decrease intracerebral invasion of glioma cells in vivo and through matrigel matrices in vitro [34]. CD44 can be cleaved by ADAM 10 and 17, and both the extracellular and intracellular cleaved components of CD44 promote cell migration [35].

#### **3.1. Integrins**

Integrins are heterodimeric transmembrane cell surface receptors that play a key role in the crosstalk between the cell and its surrounding stroma. Integrins regulate cell adhesion, mi‐ gration, differentiation, proliferation, and survival during physiological and pathological conditions, including inflammation and cancer. Upon ligation to extracellular ligands (ma‐ trix proteins such as collagens, laminins, vitronectins and fibronectins), integrins activate downstream signaling pathways in concert with growth factor receptors, including PDGFR, EGFR and VEGFR. Preclinical data indicate that integrins play a key role in cancer initiation and progression.

Integrins contain two distinct chains, calls subunit α (alpha) and β (beta). Have been identi‐ fied about 18 α and 8 β subunits. The integrin subunits penetrate the plasma membrane and typically have very short cytoplasmic domains of about 40-70 amino acids. Outside the plas‐ ma membrane, α and β chains protrude to a length of about 23 nm, the last 5 of which thanks to the termination of each chain formed dall'NH2 - a region used to form bonds to the ECM. The molecular mass of the subunits of the integrin may range from 90 kD to 160 kD. The β subunits have 4 cysteine-rich repeat sequences. Both the α subunit β both bind many divalent cations. There are many ways to classify the integrins. For example, a sub‐ class of the chains α has a structural domain in more inserted at the network termination NH2, the so-called alpha-domain A. The integrins that possess this characteristic usually form collagen (α1β1 integrins, and α2β1), or act as adhesion molecules between cell and cell (β2 integrin family). This domain α-1 is the active site for links to various integrins. Those who do not carry this domain, however, have a domain A in their active site, but this is in the β chain. In both cases, the domains A are connected to three active sites consisting of divalent cations. One of these is permanently occupied by physiological concentrations of divalent cations, and binds to a calcium ion or magnesium. The other two sites are occupied by cations when establishing ties - at least for the bonds involving an amino acid, aspartic acid, typically - in their interaction sites. An amino acid characterized in the active site of the integrin many proteins ECM, as part of the amino acid sequence Arginine-Glycine-Aspartic acid. The two main functions of integrins are cell adhesion to ECM and signal transduction from the ECM to the cell. They interact with two large groups of ligands: a variety of ECM proteins, such as fibronectin, vitronectin, fibrinogen, and cell surface molecules, that are members of the immunoglobulin supergene family, such as intracellular adhesion molecules (ICAM-1, ICAM-2) and vascular cell adhesion molecule (VCAM-1). In particular, the integ‐ rin ανβ3, which binds to fibronectin, vitronectin, and tenascin-C in ECM, is thought to play a central role in glioma invasion [36]. Increased expression of integrin ανβ3 leads to in‐ creased motility in human glioma cells with a concomitant decrease in apoptosis sensitivity.

vide a linkage to the intermediate filament network through association with cytosolic proteins (desmoplakin, plakoglobin). Cadherins are linked to the actin-cytoskeleton via cate‐ nins (α-, β-catenin, plakoglobin), thereby establishing molecular lines of communication to other cell-cell junctions and to cell-substratum junctions [26]. During glioma progression de‐ creased cadherin function is correlated with de-differentiation, metastasis and poor progno‐ sis [27]. Due to its participation in processes such as morphological differentiation and contact inhibition of growth and motility, cadherins may function as suppressors of tumor growth and invasion [25]. Perego and co-worker, showed that only glioma cells with imma‐ ture adherens junctions are capable of migrating and invading through poly-1-lysine coated filters. In the process of maturation of adhesions, the Lin-7 protein forms a complex with

44 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

The second step is a decline in the expression of connexin 43, which leads to a reduction in gap junction formation. Connexin 43 is the most abundant gap junction protein in CNS and is expressed primarily in astrocytes [29]. Cell-cell communication is important in growth control and differentiation, and it is partly achieved using gap junctions and via second messengers [30]. Decreased gap junction formation may result in fewer inhibitory signals, facilitating uncontrolled cell division and de-differentiation [31]. Has been demonstrated that reduced gap junction formation is correlated with increased motility of glioma cells in vitro [32]. Increased malignancy of glioma specimens correlates with reduced in situ gap junction formation as well as reduced connexin 43 expression [33]. The third event is cleav‐ age of CD44, which anchors the primary mass to ECM, by the metalloproteinase ADAM. CD44 is a transmembrane glycoprotein belonging to the immunoglobulin receptor super‐ family, which interacts with hyaluronic acid as its ligand. Monoclonal antibodies directed against CD44 decrease intracerebral invasion of glioma cells in vivo and through matrigel matrices in vitro [34]. CD44 can be cleaved by ADAM 10 and 17, and both the extracellular

Integrins are heterodimeric transmembrane cell surface receptors that play a key role in the crosstalk between the cell and its surrounding stroma. Integrins regulate cell adhesion, mi‐ gration, differentiation, proliferation, and survival during physiological and pathological conditions, including inflammation and cancer. Upon ligation to extracellular ligands (ma‐ trix proteins such as collagens, laminins, vitronectins and fibronectins), integrins activate downstream signaling pathways in concert with growth factor receptors, including PDGFR, EGFR and VEGFR. Preclinical data indicate that integrins play a key role in cancer initiation

Integrins contain two distinct chains, calls subunit α (alpha) and β (beta). Have been identi‐ fied about 18 α and 8 β subunits. The integrin subunits penetrate the plasma membrane and typically have very short cytoplasmic domains of about 40-70 amino acids. Outside the plas‐ ma membrane, α and β chains protrude to a length of about 23 nm, the last 5 of which thanks to the termination of each chain formed dall'NH2 - a region used to form bonds to the ECM. The molecular mass of the subunits of the integrin may range from 90 kD to 160

and intracellular cleaved components of CD44 promote cell migration [35].

cadherin and β-catenin [28].

**3.1. Integrins**

and progression.

Integrins combine the ECM out of the cell to the cytoskeleton (in particular microfilaments) within the cell. What site can bind the ECM is usually decided by the integrin α and β subu‐ nit which is composed of the same integrin. Bonds between the integrin are fibronectin, vi‐ tronectin, collagen, and laminin. The connection between the cell and the ECM makes the cell able to withstand the forces tie rods without being thrown out of the ECM. The connec‐ tions between the integrin and the links in the ECM and microfilaments inside the cell are indirect: they are connected by proteins "armor" as talin, paxillin, and alpha-actinin. These act by regulating the kinase such as FAK (focal adhesion kinase - focal adhesion kinase) and the family of Src kinases to phosphorylated substrates such as the p130CAS or recruiting signal adapters such as Crk. The connection of the cell to the extracellular matrix is a basic requirement to form a multicellular organism. Integrins link the cytoskeleton to the extracel‐ lular matrix, and are recognized to be key regulators of tissue structure. They provide adhe‐ sive, migratory, and survival cues to tumor cells and to cells of the tumor microenvironment, including angiogenic endothelial cells. The integrins ανβ3 and ανβ5, among others, are highly expressed not only on the tumor vasculature and angiogenic endo‐ thelial cells, but also on tumor cells, including gliomas. Consequently, integrins have been considered as a promising therapeutic target in cancer. Monoclonal antibodies and peptidebased integrin inhibitors are being investigated for their potential therapeutic activity in var‐ ious tumor types. This strategy is in advanced stage clinical development in glioblastoma, a highly vascular primary brain tumor [37].

The malignancy of glioma is dependent on diffusive properties of tumor cells that may in‐ vade widely as single cells anywhere in the brain. Integrins are the family of adhesive recep‐ tors that promote invasiveness of glioma. These heterodimeric transcellular membrane receptors are composed of two subunits, α and β, and are the major receptors for the ECM proteins. Immunohistopathological studies revealed an upregulation of α2β1, α3β1, α5β1, α6β1, and αvβ3 integrins on GBM when compared with normal brain [38].

an inhibition of glioma associated angiogenesis at the invasive edge of the tumors [44]. Down-regulated β1 integrin protein levels in vivo probably affect interactions of glioma cells with ECM components, leading to reduced migration along vascular basement mem‐ branes. The integrin α6β1 plays an important role for the regulation of glioma-initiating cells in the perivascular niche. This integrin mediates the interaction of glioma-initiating cells to laminin, an extracellular matrix protein expressed in basement membranes, including those supporting endothelial cells. This interaction provides an anchorage for glioma-initiating

Gliomas Biology: Angiogenesis and Invasion http://dx.doi.org/10.5772/53487 47

Brown et al. evaluate the role of α9β1 integrin interaction with nerve growth factor (NGF) in glioma progression, they selected the two most malignant glioblastoma cell lines: LN229, which expresses α9β1, and LN18,in which does not. Presence of the α9 in‐ tegrin subunit on LN229 and absence on LN18 cell lines were verified using several as‐ says such as monoclonal antibody adhesion microarray, Western blot analysis of cell lysates, flow cytometry, and immunocytostaining [38]. Very high expression of the α9 in‐ tegrin subunit was observed only on the LN229 cell line. Western blot analysis, using polyclonal anti-α9 antibody, showed the absence of this integrin subunit on normal astro‐ cytes. The expression of α9β1 integrin on LN229 and lack of it on LN18 was confirmed by flow cytometry and immunocytostaining analysis. The investigation of cellular re‐ sponses following binding of NGF to α9β1 integrin on LN229 and LN18 cells required evaluation of the level of two other NGF receptors, TrkA and p75NTR. Western blot analy‐ sis of cell lysates and immunocytostaining revealed negligible expression of TrkA on both types of cells, and a stable expression of p75NTR in comparison with PC12 neuronal cells used as a positive control. Interestingly, the level of p75NTR appeared to be lower on LN18 cells, which are α9β1 integrin deficient, than on LN229 cells, which are α9β1 integ‐ rin positive. This correlation was previously observed in the mRNA level of SW480 cells transfected with α9 integrin subunit. Cells transfected with the α9 subunit showed a po‐ tent increase of p75NTR, whereas mock-transfected cells had a significantly lower expres‐ sion of this NGF receptor. The opposite situation occurred for TrkA, suggesting that this specific high affinity NGF receptor has a functional complementary association with α9β1 integrin. Several factors expressed in glioma cells have been found to regulate integrin expression. Glioma expression of focal adhesion kinase, a nonreceptor cytoplasmic tyro‐ sine kinase, has been shown to increase phosphorylation of the enhancer of filamentation 1, which in turn stimulates PDGF-mediated stimulation of glioma integrin adhesion to

Extracellular proteolytic enzymes are critical for the invasive properties of malignant neo‐ plasms [46]. Barrier to invasion do not appear as restrictive within CNS parenchyma and matrix substrates most responsible for impeding tumor migration have not been identified [47]. Nonetheless, there is a strong evidence that the expression of specific extracellular ma‐ trix proteases promotes invasive behavior by gliomas. These include the matrix metallopro‐ teinases (MMPs), the urokinase-dependent plasminogen activating cascade, and cathepsin

cells within the perivascular niche and supports their tumorigenic potential [37].

ECM [45].

**3.2. Proteases**

Glioblastoma commonly displays enhanced expression of several integrins along with their ECM ligands: αvβ3 and αvβ5 (tenascin and vitronectin receptors), α5β1 (fibronectin recep‐ tor), α2β1 (collagens receptor), and α3β1, α6β4, and α6β1 (laminins receptors). Numerous studies have focused on the αv integrin family. The integrins αvβ3 and αvβ5 are markers of glioblastoma malignancy and influence a variety of processes in glioblastoma progression in vivo, including proliferation, apoptosis, and angiogenesis. Furthermore, cilengitide, an αvβ3 and αvβ5 integrins antagonist, extends mouse survival by delaying the tumor growth and is nowadays in clinical trial for recurrent malignant glioma. Two other integrins, α5β1 and α3β1, have been shown to be implicated in glioma cell adhesion and migration in vitro. The α6 integrin subunit associates with β1 or β4 subunits to form functional heterodimers that selectively bind laminins. The α6β4 integrin is essential for the organization and mainte‐ nance of epithelial hemidesmosomes that link the intermediate filaments with the extracellu‐ lar matrix. The major ligand of α6β4 is the laminin-332, while α6β1 is a well-characterized laminin-111 receptor. Overexpression of α6β1 integrin has been associated with the progres‐ sion of many epithelial tumors. Several studies concerning gliomas and the α6β1 ligand laminin-111 have been reported in the literature. Using immunohistochemistry studies, Gingras et al. showed that α6 integrin was strongly expressed in glioblastoma tissue, where‐ as it was weakly expressed in normal brain [39]. Previtali et al. confirmed that the expres‐ sion of α6 was increased in glioblastoma and in other central nervous system tumors, such as meningioma, astrocytoma, and neuroblastoma, when compared with the autologous nor‐ mal tissue counterpart [40]. In glioblastoma biopsies, laminin-111 is highly expressed on tu‐ mor blood vessels, but also within the brain tumor as punctuate deposits and at the tumor invasion front. In vitro, glioma cells can both secrete laminin-111 and induce its expression in normal brain tissue. Moreover, laminin-111 is one of the most permissive substrates for adhesion and migration of glioma cells in vitro. Additionally, over laminin-111, migrating glioma cells are protected from apoptosis [41].

In malignant glioma, and in particular in GBM, overexpression of αvβ3 integrin is well documented. The integrin αvβ3, which binds to fibronectin, vitronectin, and tenascin-C in ECM, is thought to play a central role in glioma invasion [36]. Increased expression of integ‐ rin αvβ3 leads to increased motility in human glioma cells with a concomitant decrease in apoptosis sensitivity. Importantly, αvβ3 integrin is expressed both on angiogenic endothe‐ lial cells and on tumor cells. Conversely, inhibition of integrin αvβ3 decreases glioma cell motility [42]. Several factors expressed in glioma cells have been found to regulate integrin expression. Particularly, uPA secreted by glioma cells has been shown to upregulate integrin αvβ3 expression by autocrine mechanism [43]. Furthermore, decreasing the in vivo expres‐ sion of the β1 subunit by an antisense strategy in the intracranial C6 glioma model leads to an inhibition of glioma associated angiogenesis at the invasive edge of the tumors [44]. Down-regulated β1 integrin protein levels in vivo probably affect interactions of glioma cells with ECM components, leading to reduced migration along vascular basement mem‐ branes. The integrin α6β1 plays an important role for the regulation of glioma-initiating cells in the perivascular niche. This integrin mediates the interaction of glioma-initiating cells to laminin, an extracellular matrix protein expressed in basement membranes, including those supporting endothelial cells. This interaction provides an anchorage for glioma-initiating cells within the perivascular niche and supports their tumorigenic potential [37].

Brown et al. evaluate the role of α9β1 integrin interaction with nerve growth factor (NGF) in glioma progression, they selected the two most malignant glioblastoma cell lines: LN229, which expresses α9β1, and LN18,in which does not. Presence of the α9 in‐ tegrin subunit on LN229 and absence on LN18 cell lines were verified using several as‐ says such as monoclonal antibody adhesion microarray, Western blot analysis of cell lysates, flow cytometry, and immunocytostaining [38]. Very high expression of the α9 in‐ tegrin subunit was observed only on the LN229 cell line. Western blot analysis, using polyclonal anti-α9 antibody, showed the absence of this integrin subunit on normal astro‐ cytes. The expression of α9β1 integrin on LN229 and lack of it on LN18 was confirmed by flow cytometry and immunocytostaining analysis. The investigation of cellular re‐ sponses following binding of NGF to α9β1 integrin on LN229 and LN18 cells required evaluation of the level of two other NGF receptors, TrkA and p75NTR. Western blot analy‐ sis of cell lysates and immunocytostaining revealed negligible expression of TrkA on both types of cells, and a stable expression of p75NTR in comparison with PC12 neuronal cells used as a positive control. Interestingly, the level of p75NTR appeared to be lower on LN18 cells, which are α9β1 integrin deficient, than on LN229 cells, which are α9β1 integ‐ rin positive. This correlation was previously observed in the mRNA level of SW480 cells transfected with α9 integrin subunit. Cells transfected with the α9 subunit showed a po‐ tent increase of p75NTR, whereas mock-transfected cells had a significantly lower expres‐ sion of this NGF receptor. The opposite situation occurred for TrkA, suggesting that this specific high affinity NGF receptor has a functional complementary association with α9β1 integrin. Several factors expressed in glioma cells have been found to regulate integrin expression. Glioma expression of focal adhesion kinase, a nonreceptor cytoplasmic tyro‐ sine kinase, has been shown to increase phosphorylation of the enhancer of filamentation 1, which in turn stimulates PDGF-mediated stimulation of glioma integrin adhesion to ECM [45].

#### **3.2. Proteases**

The malignancy of glioma is dependent on diffusive properties of tumor cells that may in‐ vade widely as single cells anywhere in the brain. Integrins are the family of adhesive recep‐ tors that promote invasiveness of glioma. These heterodimeric transcellular membrane receptors are composed of two subunits, α and β, and are the major receptors for the ECM proteins. Immunohistopathological studies revealed an upregulation of α2β1, α3β1, α5β1,

Glioblastoma commonly displays enhanced expression of several integrins along with their ECM ligands: αvβ3 and αvβ5 (tenascin and vitronectin receptors), α5β1 (fibronectin recep‐ tor), α2β1 (collagens receptor), and α3β1, α6β4, and α6β1 (laminins receptors). Numerous studies have focused on the αv integrin family. The integrins αvβ3 and αvβ5 are markers of glioblastoma malignancy and influence a variety of processes in glioblastoma progression in vivo, including proliferation, apoptosis, and angiogenesis. Furthermore, cilengitide, an αvβ3 and αvβ5 integrins antagonist, extends mouse survival by delaying the tumor growth and is nowadays in clinical trial for recurrent malignant glioma. Two other integrins, α5β1 and α3β1, have been shown to be implicated in glioma cell adhesion and migration in vitro. The α6 integrin subunit associates with β1 or β4 subunits to form functional heterodimers that selectively bind laminins. The α6β4 integrin is essential for the organization and mainte‐ nance of epithelial hemidesmosomes that link the intermediate filaments with the extracellu‐ lar matrix. The major ligand of α6β4 is the laminin-332, while α6β1 is a well-characterized laminin-111 receptor. Overexpression of α6β1 integrin has been associated with the progres‐ sion of many epithelial tumors. Several studies concerning gliomas and the α6β1 ligand laminin-111 have been reported in the literature. Using immunohistochemistry studies, Gingras et al. showed that α6 integrin was strongly expressed in glioblastoma tissue, where‐ as it was weakly expressed in normal brain [39]. Previtali et al. confirmed that the expres‐ sion of α6 was increased in glioblastoma and in other central nervous system tumors, such as meningioma, astrocytoma, and neuroblastoma, when compared with the autologous nor‐ mal tissue counterpart [40]. In glioblastoma biopsies, laminin-111 is highly expressed on tu‐ mor blood vessels, but also within the brain tumor as punctuate deposits and at the tumor invasion front. In vitro, glioma cells can both secrete laminin-111 and induce its expression in normal brain tissue. Moreover, laminin-111 is one of the most permissive substrates for adhesion and migration of glioma cells in vitro. Additionally, over laminin-111, migrating

In malignant glioma, and in particular in GBM, overexpression of αvβ3 integrin is well documented. The integrin αvβ3, which binds to fibronectin, vitronectin, and tenascin-C in ECM, is thought to play a central role in glioma invasion [36]. Increased expression of integ‐ rin αvβ3 leads to increased motility in human glioma cells with a concomitant decrease in apoptosis sensitivity. Importantly, αvβ3 integrin is expressed both on angiogenic endothe‐ lial cells and on tumor cells. Conversely, inhibition of integrin αvβ3 decreases glioma cell motility [42]. Several factors expressed in glioma cells have been found to regulate integrin expression. Particularly, uPA secreted by glioma cells has been shown to upregulate integrin αvβ3 expression by autocrine mechanism [43]. Furthermore, decreasing the in vivo expres‐ sion of the β1 subunit by an antisense strategy in the intracranial C6 glioma model leads to

α6β1, and αvβ3 integrins on GBM when compared with normal brain [38].

46 Evolution of the Molecular Biology of Brain Tumors and the Therapeutic Implications

glioma cells are protected from apoptosis [41].

Extracellular proteolytic enzymes are critical for the invasive properties of malignant neo‐ plasms [46]. Barrier to invasion do not appear as restrictive within CNS parenchyma and matrix substrates most responsible for impeding tumor migration have not been identified [47]. Nonetheless, there is a strong evidence that the expression of specific extracellular ma‐ trix proteases promotes invasive behavior by gliomas. These include the matrix metallopro‐ teinases (MMPs), the urokinase-dependent plasminogen activating cascade, and cathepsin B. Wild-Bode et al. [48] found that MMP-2 and MMP-3 levels and MMP-2/MMP-9 activity correlated with glioma cell migration and invasion. NF-jB, uPA, low-density lipoprotein re‐ ceptor-related protein 1, and insulin-like growth factor binding protein-2 are known to upre‐ gulate MMP expression. Induced expression of tissue inhibitor of metalloproteinases-3 (TIMP-3), a putative inhibitor of MMP activity, has been shown to suppress infiltration and also to induce apoptosis in cancer cell lines. MMPs play an important role in human brain tumor invasion, probably due to an imbalance between the production of MMPs and tissue inhibitor of metalloproteinases-1 (TIMP-1) by the tumor cells. MMP-1 is the crucial enzyme able to initiate breakdown of the interstitial collagen types; in this way, it activates the other MMPs, which allows the glioma cells to infiltrate normal brain tissue.

teinases (TIMPs) and RECK (reversion-inducing cysteine-rich protein with kazal motifs) [49]. Numerous studies have investigated the expression of selected MMPs in human GBM cell lines. As it has already been reported that there are differences in the expression pat‐ terns of MMPs in different cell-lines [57]. A number of different techniques have been used to detect protease expression patterns. This may have led to dissimilar conclusions, due to disparate sensitivities and due to comparing mRNA expression with protein expression or protein activity. Variations in MMP expression may be due to in vitro selection processes or karyotype evolution, where the transcription of either the enzyme and/or its inhibitor may be affected which ultimately leads to an imbalance in the MMP-regulatory network [57]. MMP-9's production is dependent on a regulation by extracellular signal-regulated kinase (ERK), PKCα/NF-κB and jun amino-terminal kinase (JNK) signaling cascades. Glioblastomas are highly hypoxic and hypoxia upregulates MMP-2 mRNA expression in U87, U251, U373 and LN18 glioblastoma cell-lines by activation of the HIF-1α transcription factor, thereby en‐ hancing their invasive potential. Migration and invasion of U87 and T98G GBM cells is also facilitated by NO, which can be found in high concentrations in glioblastoma tissue. NO stimulates MMP-1 expression and activity. EGF raises MMP14 expression in U251 cells, but does not influence MMP-15, -16 or MMP-24. MMP-2 expression and secretion is induced by IL-6 in U87 cells. However, IL-6 action seems to be cell-line specific, since U343 cells were

Gliomas Biology: Angiogenesis and Invasion http://dx.doi.org/10.5772/53487 49

The inflammatory cytokine TNF-α and the immune-suppressive cytokine TGF-β have been implicated in migration and invasion of glioma cells in vitro. In U251 and in U373 cells, TNF-α stimulated the expression of MMP-9 and MMP-19. MMP-1 mRNA expression was significantly increased in U373 cells by TNF-α, whereas its expression in U251 cells re‐ mained unaffected. This may be due to the high basal level of MMP-1 expression displayed by U251 cells, where a further increase is not possible, or else it could also be a cell- line spe‐ cific effect. Such an effect has been observed for MMP-1, -2, -3 and MMP-7 regulation by TNF-α and TGF-β1, which only caused a marked induction of expression in some GBM celllines. TNF-α enhances the invasiveness of T98G cells through an induction of MMP-3, but has no effect on MMP-1, -2 or MMP-9. However, in U251 cells TNF-α inhibits MMP-2 and decreases invasiveness into the extracellular matrix. In A172 cells, TNF-α induces gene ex‐ pression and protein secretion of MMP-9. TGF-β1 alone had no effect on MMP-9 production. However, when it was added together with TNF-α a significant dose-dependent inhibition of MMP-9 secretion was observed. TGF-β1 displayed inconsistent effects on adhesion and invasiveness, depending on the cell-line examined. The invasive potential of U138 cells was markedly reduced, whereas U373 cell invasion remained unchanged. TGF-β1 caused a sig‐ nificant induction of MMP-11 and MMP-24 expression in U373 cells, whereas there was no impact on MMP expression in U251 cells. In U87 and LN229 cells, TGF-β upregulates MMP-2. Thus, the transcriptional modulation of MMP genes in response to TNF-α or TGF-β is not consistent, but extremely cell-line specific [57]. Elevated levels of several MMPs were also found in malignant glioma tissue samples. MMP-1 expression was increased in surgical specimens of GBM compared to low grade astrocytomas and normal brain. This increased expression is probably due to a single nucleotide polymorphism in the MMP-1 promoter at position-1607, creating a functional binding site for members of the ETS family of transcrip‐

not affected [57].

Matrix metalloproteinases comprise a large family of zinc-dependent endoproteinases, col‐ lectively capable of degrading all ECM components. A total of 23 families of MMPs are known and these are numbered in the sequence of their discovery [49]. Originally, MMPs were classified according to their respective substrate specificity, now they are divided into eight structural subgroups, five of which are secreted and three of which are transmem‐ brane MMPs. A signal peptide leads them to the secretory pathway. Then, these enzymes can be secreted from the cell or anchored to the plasma membrane, thereby confining their catalytic activity to the extracellular space or to the cell surface, respectively. Their function is to degrade different protein components of the ECM and of basement membranes, there‐ fore they are essential for the interaction of individual cells with their surrounding and for the development and function of multicellular organisms [50]. Between the various sub‐ strates of MMPs we include collagens, non collagens glicoproteins, proteoglycans and other ECM components like tenascin, fibronectin and laminin, which often show tumor specific expression are also substrates [51]. The proteolytic activities of MMPs influence essential cel‐ lular processes like cell proliferation, migration and adhesion, as well as many fundamental physiological events involving tissue remodeling, such as angiogenesis, bone development, wound healing, and uterine and mammary involution [52]. However, the increasing expres‐ sion of this enzymes is associated with many pathological conditions [53-54] such as rheu‐ matoid arthritis, cardiovascular diseases or cancer progression [55]. MMP expression and activity can be regulated at different levels including gene transcription, proenzyme activa‐ tion and endogenous inhibition, which act in a coordinated manner to confine the diverse MMP proteolytic activities to those conditions and locations where they are necessary. Un‐ fortunately, these restrictive regulatory mechanisms are frequently lost in multiple patho‐ logical conditions [56]. MMPs expression is induced by different factors like EGF, TGF-β, PDGF, and various inflammation's mediated, included TNFα and IL-1β.