**4.1. EGF**

vascular endothelial cells. It belongs to the growth factors family, which includes four ho‐ mologues VEGF-A (commonly referred to as VEGF)-B, -C, -D, -E and placental growth fac‐ tor (PIGF). The biological activity of VEGF is mediated by binding to receptors with tyrosine kinase activity VEGFR-1 (also known as fms-like tyrosine kinase 1, ftl-1), VEGFR-2 (also

When VEGF binds to its receptors it causes receptor dimerization, autophosphorylation, and downstream signaling of different pathways, as v-src sarcoma viral oncogene homolog (Src), phosphoinositol (PI)-3 kinase (PI3K) and phospholipase-C γ (PLCγ) which activate

In animal tumor models, VEGF is produced both by tumor cells and also by stromal tissues [4].

VEGF and its receptor are expressed in tumor cells in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) [36,37]. It is involved in tumor growth by neoangio‐ genesis, lymphangiogenesis and lymph nodal dissemination [38]. High levels of VEGF have been correlated with poor prognosis [39]. But there are several questions about the role of VEGF levels and its various isoforms plays as a potential biomarker, which may be useful in the use and selection of therapies against it. VEGF levels are elevated in lung cancer patients when compared to controls [40]. There is also a correlation between VEGF levels and the clinical stage in NSCLC patients [7,10,13,15] and an inverse correlation between the VEGF serum levels and survival [41]. Low levels of VEGF have shown to be correlated with a good response to chemotherapy [12]. Moreover, a study showed that low levels of VEGF were correlated with a good response to anti-EGFR. Furthermore, levels of VEGF in responders were not significantly different from volunteers, but were different from non-responders [42]. However, it remains unclear whether the clinical effects of anti-EGFR in patients with NSCLC are correlated with reductions in the levels of angiogenic growth factors. Further‐ more, it is unclear whether these factors are correlated with response to anti-EGFR treat‐ ment, blocking EGFR autophosphorylation [43] and the subsequent signal transduction pathways implicated in proliferation, metastasis and inhibition of apoptosis, as well as an‐ giogenesis [44,45]. The inhibition of EGFR has been shown to reduce production of angio‐

Antiangiogenic drugs have demonstrated efficacy in the treatment of NSCLC in the last years. The more tested antiangiogenic drug in lung cancer is bevacizumab, a monoclonal an‐ tibody directed against VEGF, which is the first antiangiogenic approved for treatment of metastatic NSCLC in combination with chemotherapy. Two phase III studies have assessed the efficacy of chemotherapy combinations associated with bevacizumab. The AVAiL study [47] analyzed the combination of cisplatin and gemcitabine with or without bevacizumab in first line treatment for NSCLC. The primary endpoint was reached, showing a benefit in progression-free survival in the bevacizumab arm. The second study [48] compared the ad‐ dition of bevacizumab with carboplatin and paclitaxel regimen, aiming differences in over‐

These detailed studies further in subsequent chapters, show that bevacizumab is an effective

known as kinase-insert domain receptor, KDR) and VEGFR-3 (ftl4).

6 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

genic growth factors in various types of cancer cells [45,46].

all survival, progression-free survival and response rate.

and safe drug in the treatment of advanced NSCLC.

proliferation and angiogenesis.

Members of the EGF family of peptide growth factors serve as agonists for ErbB family re‐ ceptors. They include EGF, TGFα, amphiregulin (AR), betacellulin (BTC), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EPR), epigen (EPG), and the neuregulins (NRGs).

EGF is a polypeptide of 53 amino acids (6 Kda) that appears as a product of proteolytic proc‐ essing of a large protein integral membrane (1207aa). This precursor protein is consisting of 8 domains called EGF-like, of which only one is active. The gene corresponding to this growth factor is located on chromosome 4q25 and stimulates epithelial cell proliferation, on‐ cogenesis and is involved in wound healing. Its three-dimensional structure is characterized by the presence of common domain to other family ligands. This protein shows a strong se‐ quential and functional homology with TGFα, which is a competitor for EGF receptor sites.

Collectively, these agonists regulate the activity of the four ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) family receptors, each of which appears to make a unique set of contributions to a complicated signaling network.

EGF binds to a specific receptor on the surface of responsive cells known as EGFR (Epider‐ mal growth factor receptor). EGFR is a member of the ErbB family receptors, a subfamily of four closely related to tyrosine kinase receptors: EGFR (ErbB1), Her2/c-neu (ErbB2), Her3 (ErbB3) and Her4 (ErbB4) (Fig.1). The EGF family ligands exhibits a complex pattern of in‐ teractions with the four ErbB family receptors; for example, EGFR can bind eight different EGF family members and Neuregulin 2beta (NRG2β) binds EGFR, ErbB3 and ErbB4. Given that ErbB2 lacks an EGF family ligand, ErbB3 lacks kinase activity, and the four ErbB recep‐ tor display distinct coupling patterns to different signaling effectors in the affinity of a given EGF family member as a key determinant of specificity for the ligand [49].

In response to toxic environmental stimuli, such as ultraviolet irradiation, or to receptor occupation by EGF, the EGFR forms Homo- or Heterodimers with other family mem‐ bers. Binding of EGF to the extracellular domain of EGFR leads to receptor dimerization, activation of the intrinsic PTK (Protein Tyrosine Kinase), tyrosine autophosphorylation, and recruitment of various signaling proteins to these autophosphorylation sites located primarily in the C-terminal tail of the receptor. Tyrosine phosphorylation of the EGFR leads to the recruitment of diverse signaling proteins, including the Adaptor proteins GRB2 (Growth Factor Receptor-Bound Protein-2) and Nck (Nck Adaptor Protein), PLC- &γ; (Phospholipase-C-γ), SHC (Src Homology-2 Domain Containing Transforming Pro‐ tein), STATs (Signal Transducer and Activator of Transcription), and several other proteins and molecules (Fig 2).

Although EGFR plays an important role in maintaining normal cell function, deregula‐ tion of EGFR pathway contributes to the development of malignancy progression, inhibi‐ tion of apoptosis, induction of angiogenesis, promotion of tumor-cell motility and metastasis. Aberrant regulation of the activity or action of EGFR and other members of the RTK family have been involved in multiple cancers, including of brain, lung, breast and ovary. Furthermore, in many tumors EGF-related growth factors are produced either by the tumor cells themselves or are available from surrounding stromal cells, leading to constitutive EGFR activation. In gliomas, EGFR amplification is often accompanied by structural rearrangements that cause in-frame deletions in the extracellular domain of the receptor, the most frequent is the EGFRvIII variant. Somatic mutations in the tyrosine-

Angiogenesis and Lung Cancer http://dx.doi.org/10.5772/54309 9

When mutated, EGFR tyrosine kinase is constitutively activated, resulting in uncontrolled proliferation, invasion and metastasis. Expression of EGFR and their ligands, especially TGFα, by lung cancer cells, indicates the presence of an autocrine (self-stimulatory) growth factor loop. Activating EGFR mutations are observed in approximately 10% of North Ameri‐ can and European populations and 30% to 50% of Asian populations [50] and are signifi‐ cantly more common in never-smokers (100 or less cigarettes per lifetime) or light former smokers (quit 1 year or more ago and less than ten-pack per year smoking history). The leu‐ cine to arginine substitution at position 858 (L858R) in exon 21 and short in-frame deletions in exon 19 are the most common mutations seen in adenocarcinomas of the lung. These mu‐ tations result in prolonged activation of the receptor and downstream signaling through phosphorylated Akt, in the absence of ligand stimulation of the extracellular domain. EGFR mutations are both prognostic for response rate to chemotherapy and survival irrespective of therapy and are predictive of response to specific inhibitors of the EGFR tyrosine kinase.

Platelet-derived growth factor (PDGF) is a major mitogen for fibroblasts, smooth muscle cells (SMCs), and glia cells. Originally, was identified as a constituent of whole blood serum that was absent in cell-free plasma-derived serum, and was subsequently purified from hu‐ man platelets [51]. Although the α-granules of platelets are a major storage site for PDGF, can be synthesized by a number of different cell types including fibroblasts, muscle, bone /

The synthesis is often increased in response to external stimuli, such as exposure to low oxy‐

PDGF is a family of cationic homo- and heterodimers of disulphide-bonded polypeptide chains. In mammals, a total of four different genes encode four PDGF chains (PDGF-A, PDGF-B, PDGF-C, and PDGF-D), which are assembled in five different isoforms known as: AA, AB, BB, CC and DD [53]. All members carry a growth factor core domain containing a conserved set of cysteine residues. The core domain is necessary and sufficient for receptor binding and activation. Classification into PDGFs is based on receptor binding. It has been

gen tension, thrombin, or stimulation with various growth factors and cytokines [52].

generally assumed that PDGF is selective for their owns receptors.

kinase domain of EGFR were also identified in NSCLC.

**4.2. PDGF**

cartilage, and connective tissue cells.

**Figure 1.** The binding of specific ligands to the receptor activates EGFR and generates a signal transduction cascade through its 2-way main PI3K/Akt and Ras / Raf / MAPK eventually stimulate proliferation, cell cycle progression, repair, angiogenesis and invasion.

**Figure 2.** Binding specificities of EGF-related peptide growth factors

Although EGFR plays an important role in maintaining normal cell function, deregula‐ tion of EGFR pathway contributes to the development of malignancy progression, inhibi‐ tion of apoptosis, induction of angiogenesis, promotion of tumor-cell motility and metastasis. Aberrant regulation of the activity or action of EGFR and other members of the RTK family have been involved in multiple cancers, including of brain, lung, breast and ovary. Furthermore, in many tumors EGF-related growth factors are produced either by the tumor cells themselves or are available from surrounding stromal cells, leading to constitutive EGFR activation. In gliomas, EGFR amplification is often accompanied by structural rearrangements that cause in-frame deletions in the extracellular domain of the receptor, the most frequent is the EGFRvIII variant. Somatic mutations in the tyrosinekinase domain of EGFR were also identified in NSCLC.

When mutated, EGFR tyrosine kinase is constitutively activated, resulting in uncontrolled proliferation, invasion and metastasis. Expression of EGFR and their ligands, especially TGFα, by lung cancer cells, indicates the presence of an autocrine (self-stimulatory) growth factor loop. Activating EGFR mutations are observed in approximately 10% of North Ameri‐ can and European populations and 30% to 50% of Asian populations [50] and are signifi‐ cantly more common in never-smokers (100 or less cigarettes per lifetime) or light former smokers (quit 1 year or more ago and less than ten-pack per year smoking history). The leu‐ cine to arginine substitution at position 858 (L858R) in exon 21 and short in-frame deletions in exon 19 are the most common mutations seen in adenocarcinomas of the lung. These mu‐ tations result in prolonged activation of the receptor and downstream signaling through phosphorylated Akt, in the absence of ligand stimulation of the extracellular domain. EGFR mutations are both prognostic for response rate to chemotherapy and survival irrespective of therapy and are predictive of response to specific inhibitors of the EGFR tyrosine kinase.

## **4.2. PDGF**

**Figure 1.** The binding of specific ligands to the receptor activates EGFR and generates a signal transduction cascade through its 2-way main PI3K/Akt and Ras / Raf / MAPK eventually stimulate proliferation, cell cycle progression, repair,

angiogenesis and invasion.

**Figure 2.** Binding specificities of EGF-related peptide growth factors

8 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

Platelet-derived growth factor (PDGF) is a major mitogen for fibroblasts, smooth muscle cells (SMCs), and glia cells. Originally, was identified as a constituent of whole blood serum that was absent in cell-free plasma-derived serum, and was subsequently purified from hu‐ man platelets [51]. Although the α-granules of platelets are a major storage site for PDGF, can be synthesized by a number of different cell types including fibroblasts, muscle, bone / cartilage, and connective tissue cells.

The synthesis is often increased in response to external stimuli, such as exposure to low oxy‐ gen tension, thrombin, or stimulation with various growth factors and cytokines [52].

PDGF is a family of cationic homo- and heterodimers of disulphide-bonded polypeptide chains. In mammals, a total of four different genes encode four PDGF chains (PDGF-A, PDGF-B, PDGF-C, and PDGF-D), which are assembled in five different isoforms known as: AA, AB, BB, CC and DD [53]. All members carry a growth factor core domain containing a conserved set of cysteine residues. The core domain is necessary and sufficient for receptor binding and activation. Classification into PDGFs is based on receptor binding. It has been generally assumed that PDGF is selective for their owns receptors.

PDGF isoforms exert their effects on target cells by activating two structurally related pro‐ tein tyrosine kinase receptors. The α and β receptors have molecular sizes of 170 and 180 kda, respectively, after maturation of their carbohydrates. Extracellularly, each receptor con‐ tains five immunoglobulin-like domains, and intracellularly there is a tyrosine kinase do‐ main that contains a characteristic inserted sequence without homology to kinases.

The detailed expression patterns of the individual PDGF ligands and receptors are complex. There are some general patterns, however: PDGF-B is mainly expressed in vascular endothelial cells, megakaryocytes, and neurons. PDGF-A and PDGF-C are ex‐ pressed in epithelial cells, muscle, and neuronal progenitors. PDGF-D expression is less well characterized, but it has been observed in fibroblasts and SMCs at certain lo‐ cations (possibly suggesting autocrine functions via PDGFR-β). PDGFR-α is expressed in mesenchymal cells. Particularly strong expression of PDGFR-α has been noticed in subtypes of mesenchymal progenitors in lung, skin, and intestine and in oligodendro‐ cyte progenitors (OPs). PDGFR-β is expressed in mesenchyme, particularly in vascular

Angiogenesis and Lung Cancer http://dx.doi.org/10.5772/54309 11

PDGF biosynthesis and processing are controlled at multiple levels and differ for the differ‐ ent PDGFs. PDGF-A and PDGF-B become disulphide-linked into dimers already as propep‐ tides. PDGF-C and PDGF-D have been less studied on this regard. PDGF-A and PDGF-B contain N-terminal pro-domains that are removed intracellularly by furin or related propro‐ tein convertases. Likely, PDGF-B also requires N-terminal propeptide removal to become ac‐ tive. In contrast, PDGF-C and PDGF-D are not processed intracellularly but are instead secreted as latent (conditionally inactive) ligands. Activation in the extracellular space re‐

Dimerization is the key event in PDGF receptor activation as it allows for receptor auto‐ phosphorylation on tyrosine residues in the intracellular domain. Autophosphorylation activates the receptor kinase and provides docking sites for downstream signaling mole‐ cules and further signal propagation involves protein–protein interactions through specif‐ ic domains; e.g., Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains recognizing phosphorylated tyrosines, SH3 domains recognizing proline-rich regions, pleckstrin homology (PH) domains recognizing membrane phospholipids, and PDZ do‐ mains recognizing C terminal specific sequences. Most of the PDGFR effectors bind to specific sites on the phosphorylated receptors through their SH2 domains. Both PDGFRα and PDGFR-β engage several well-characterized signaling pathways, e.g. Ras-MAPK, PI3K and PLC-γ, which are known to be involved in multiple cellular and developmen‐

The PDGFR is expressed on capillary endothelial cells and PDGF has been shown to have an angiogenic effect. The effect is, however, weaker than that of fibroblast growth factors or VEGF, and PDGF does not appear to be of importance for the initial formation of blood ves‐ sels. PDGF B-chain produced by capillaries may have an important role to recruit pericytes that is likely to be required to promote the structural integrity of the vessels. PDGF has also

PDGF functions have been implicated in a broad range of diseases. For a few of them, i.e., some cancers, there is a strong evidence for a causative role of PDGF signaling in this hu‐ man disease process. In these cases, genetic aberrations cause uncontrolled PDGF signaling

been implicated in the regulation of the tonus of blood vessels [57].

SMCs (vSMCs) and pericytes.

tal responses [56].

in the tumor cells.

quires dissociation of the growth factor domain.

The human α-receptor gene is localized on chromosome 4q12, close to the genes for the SCF (stem cell factor) receptor and VEGF receptor-2, and the β-receptor gene is on chromosome 5 close to the *CSF*-1 (colony stimulating factor-1) receptor gene [54].

Because PDGF isoforms are dimeric molecules, they bind two receptors simultaneously and dimerize receptors upon binding. The α receptor binds both the A and B chains of PDGF with high affinity, whereas the β receptor binds only the B chain with high affinity. There‐ fore, PDGF-AA induces αα receptor homodimers, PDGF-AB αα receptor homodimers or αβ receptor heterodimers, and PDGF-BB all three dimeric combinations of α and β receptors (Fig 3). General mesenchymal expression of PDGFRs is low in vivo, but increases dramati‐ cally during inflammation and in culture. Several factors induce PDGFR expression, includ‐ ing TGF-β, estrogen (probably linked to hypertrophic smooth muscle responses in the pregnant uterus), interleukin-1α (IL-1α), basic fibroblast growth factor-2 (FGF-2), tumor ne‐ crosis factor-β, and lipopolysaccharide [55].

**Figure 3.** adapted from J Andrae 2008): PDGF–PDGFR interactions. Each chain of the PDGF dimer interacts with one receptor subunit. The active receptor configuration is therefore determined by the ligand dimer configuration. The top panel shows the interactions that have been demonstrated in cell culture. Hatched arrows indicate weak interac‐ tions or conflicting results.

The detailed expression patterns of the individual PDGF ligands and receptors are complex. There are some general patterns, however: PDGF-B is mainly expressed in vascular endothelial cells, megakaryocytes, and neurons. PDGF-A and PDGF-C are ex‐ pressed in epithelial cells, muscle, and neuronal progenitors. PDGF-D expression is less well characterized, but it has been observed in fibroblasts and SMCs at certain lo‐ cations (possibly suggesting autocrine functions via PDGFR-β). PDGFR-α is expressed in mesenchymal cells. Particularly strong expression of PDGFR-α has been noticed in subtypes of mesenchymal progenitors in lung, skin, and intestine and in oligodendro‐ cyte progenitors (OPs). PDGFR-β is expressed in mesenchyme, particularly in vascular SMCs (vSMCs) and pericytes.

PDGF isoforms exert their effects on target cells by activating two structurally related pro‐ tein tyrosine kinase receptors. The α and β receptors have molecular sizes of 170 and 180 kda, respectively, after maturation of their carbohydrates. Extracellularly, each receptor con‐ tains five immunoglobulin-like domains, and intracellularly there is a tyrosine kinase do‐

The human α-receptor gene is localized on chromosome 4q12, close to the genes for the SCF (stem cell factor) receptor and VEGF receptor-2, and the β-receptor gene is on chromosome 5

Because PDGF isoforms are dimeric molecules, they bind two receptors simultaneously and dimerize receptors upon binding. The α receptor binds both the A and B chains of PDGF with high affinity, whereas the β receptor binds only the B chain with high affinity. There‐ fore, PDGF-AA induces αα receptor homodimers, PDGF-AB αα receptor homodimers or αβ receptor heterodimers, and PDGF-BB all three dimeric combinations of α and β receptors (Fig 3). General mesenchymal expression of PDGFRs is low in vivo, but increases dramati‐ cally during inflammation and in culture. Several factors induce PDGFR expression, includ‐ ing TGF-β, estrogen (probably linked to hypertrophic smooth muscle responses in the pregnant uterus), interleukin-1α (IL-1α), basic fibroblast growth factor-2 (FGF-2), tumor ne‐

**Figure 3.** adapted from J Andrae 2008): PDGF–PDGFR interactions. Each chain of the PDGF dimer interacts with one receptor subunit. The active receptor configuration is therefore determined by the ligand dimer configuration. The top panel shows the interactions that have been demonstrated in cell culture. Hatched arrows indicate weak interac‐

main that contains a characteristic inserted sequence without homology to kinases.

close to the *CSF*-1 (colony stimulating factor-1) receptor gene [54].

10 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

crosis factor-β, and lipopolysaccharide [55].

tions or conflicting results.

PDGF biosynthesis and processing are controlled at multiple levels and differ for the differ‐ ent PDGFs. PDGF-A and PDGF-B become disulphide-linked into dimers already as propep‐ tides. PDGF-C and PDGF-D have been less studied on this regard. PDGF-A and PDGF-B contain N-terminal pro-domains that are removed intracellularly by furin or related propro‐ tein convertases. Likely, PDGF-B also requires N-terminal propeptide removal to become ac‐ tive. In contrast, PDGF-C and PDGF-D are not processed intracellularly but are instead secreted as latent (conditionally inactive) ligands. Activation in the extracellular space re‐ quires dissociation of the growth factor domain.

Dimerization is the key event in PDGF receptor activation as it allows for receptor auto‐ phosphorylation on tyrosine residues in the intracellular domain. Autophosphorylation activates the receptor kinase and provides docking sites for downstream signaling mole‐ cules and further signal propagation involves protein–protein interactions through specif‐ ic domains; e.g., Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains recognizing phosphorylated tyrosines, SH3 domains recognizing proline-rich regions, pleckstrin homology (PH) domains recognizing membrane phospholipids, and PDZ do‐ mains recognizing C terminal specific sequences. Most of the PDGFR effectors bind to specific sites on the phosphorylated receptors through their SH2 domains. Both PDGFRα and PDGFR-β engage several well-characterized signaling pathways, e.g. Ras-MAPK, PI3K and PLC-γ, which are known to be involved in multiple cellular and developmen‐ tal responses [56].

The PDGFR is expressed on capillary endothelial cells and PDGF has been shown to have an angiogenic effect. The effect is, however, weaker than that of fibroblast growth factors or VEGF, and PDGF does not appear to be of importance for the initial formation of blood ves‐ sels. PDGF B-chain produced by capillaries may have an important role to recruit pericytes that is likely to be required to promote the structural integrity of the vessels. PDGF has also been implicated in the regulation of the tonus of blood vessels [57].

PDGF functions have been implicated in a broad range of diseases. For a few of them, i.e., some cancers, there is a strong evidence for a causative role of PDGF signaling in this hu‐ man disease process. In these cases, genetic aberrations cause uncontrolled PDGF signaling in the tumor cells.
