Growth Factor Overview

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Growth factors and their receptors are the core components of signal transduction pathways. Growth factors are proteins that bind to receptors on the cell surface and stimulate various cellular functions, including growth and differentiation. Some growth factors stimulate a wide variety of cell types, while others are specific for a given cell type. When these growth-regulating polypeptides bind to their cognate receptors, they induce cell growth or differentiation through receptor stimulation and initiation of the signal transduction cascade or modulation in normal tissues. A number of growth factors have been studied in a great detail, including epidermal growth factor (EGF), platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), transforming growth factors (TGF), fibroblast growth factors (FGF), insulin-like growth factors (IGF), hepatocyte growth factor (HGF), erythropoietin, and nerve growth factor (NGF).

Under certain conditions, growth factors can promote malignancy. In a variety of human cancers, modification of growth factor production, receptor expression, and alterations in the intracellular mitogenic signals play a critical role in directing normal tissues to become cancerous (Aaronson, 1991). In many types of cancers, growth factors or their receptors are aberrantly expressed. This chapter focuses primarily on EGF, PDGF, VEGF, TGFs, FGF, IGF, and their receptors and role in human malignancies. These growth factors are involved in cell proliferation and differentiation in various cell types. The growth factors that stimulate hematopoietic cells and lymphocytes will not be covered. Numerous molecular therapies targeted at aberrant growth factor signaling are being investigated, with some agents in the late stages of clinical testing. Targeted therapies may provide an important therapeutic option for patients with tumors that are often considered incurable using traditional cytotoxic approaches. (Schiller et al., 2002)

1.1. Growth Factor Receptor Molecular Structure

Growth factor receptors are categorized based on their primary signal transduction mechanisms. Types of receptors include ligand-gated ion-channels, GTPase (G-protein)-linked receptors, and protein kinase-linked receptors. Protein kinase-linked receptors have intrinsic tyrosine kinase or serine/threonine kinase activity and are linked to cytosolic kinases (reviewed in Mendelsohn, Baird, Fan, & Markowitz, 2001).

The majority of growth factors exert their effects through binding to receptors with an intrinsic tyrosine kinase (RTKs). These transmembrane receptors are composed of extracellular ligand binding, transmembrane, and cytoplasmic tyrosine kinase domains.

The extracellular domains of these receptors contain cysteine moieties that fold into tertiary structures and form either immunoglobulin (Ig)-like domains (loop structures) created by disulfide bonds between cysteine moieties, or cysteine-rich domains that exist as a complex structure resulting from formation of closely positioned disulfide bonds. Both of these structures create pockets that allow growth factors to bind to the receptor with high affinity (Ullrich & Schlessinger, 1990).

Tyrosine kinase transmembrane receptors have been divided into several classes or families based on their extracellular domain structure. Examples of these receptors include the following: the EGF receptors (EGFR), PDGF receptors (PDGFR), VEGF receptors (VEGFR), FGF receptors (FGFR), and the IGF receptors (IGFR). Most transmembrane tyrosine kinase receptors are monomeric. Upon ligand binding, the monomeric receptors undergo dimerization. However, the members of the IGFR exist as homodimers of cysteine-rich peptides that are linked by disulfide bounds. Following ligand binding, transphosphorylation of specific tyrosine residues occurs on the cytoplasmic portions of the receptors. PDGFR and FGFR have Ig-like structures as their extracellular domains, while the EGF receptor family members have a cysteine-rich extracellular domain (Ullrich & Schlessinger, 1990).

The TGF-p receptors possess serine/threonine kinase activity (Massague, 1998). Like RTKs, these receptors are transmembrane proteins consisting of extracellular (ligand-binding), transmembrane, and intracellular kinase domains. When the heterodimeric TGF-p receptor complex is activated via ligand binding, it induces a potent antiproliferative activity in many cell types (Massague, 1990). The mechanisms of cellular responses to the receptor serine/threonine kinases are not as well understood as those of the RTKs.

Growth factor binding to the extracellular domain of the receptors leads to activation of transcription factors and, eventually, production of protein molecules that control cell functions.

1.2. Ligands of Tyrosine Kinase Receptors and Signal Transduction Pathways

Tyrosine kinase receptors and their cognate ligands contain complementary domains created by particular amino acid sequences that render their receptor-ligand relationship unique. Binding of the ligand to the receptor and subsequent receptor oligomerization brings the kinase domains of the 2 receptor chains closer to each other, activating the intrinsic tyrosine kinase, which transphosphorylates tyrosine residues on the receptors and on signaling molecules in the cytosol. (Figure 1) Substrates in the cytosol containing Scr-homology-2 (SH2) domains bind to the activated receptors at docking sites that consist of a phosphotyrosine residue and a specific sequence of amino acids in close proximity on the receptor. Thus, receptor phosphorylation both stimulates kinase activity and allows binding of downstream-

signaling molecules. SH2 domains are present in a diverse range of eukaryotic proteins, including many proteins involved in signal transduction (Pawson, 1995).

Figure 1. Using the epidermal growth factor receptor as an example, this figure illustrates the mechanisms by which growth factors and their receptors transduce extracellular signals into intracellular processes, in particular, migration, angiogenesis, gene transcription, cell cycle progression, survival, and proliferation. In addition, some mechanisms by which signal transduction can be blocked are depicted.

Figure 1. Using the epidermal growth factor receptor as an example, this figure illustrates the mechanisms by which growth factors and their receptors transduce extracellular signals into intracellular processes, in particular, migration, angiogenesis, gene transcription, cell cycle progression, survival, and proliferation. In addition, some mechanisms by which signal transduction can be blocked are depicted.

SH2 domains are stretches of 100 amino acids with highly conserved residues that create binding compartments for molecules containing phosphotyrosine residues. The molecules containing SH2 domains are involved in tyrosine phosphorylation and dephosphorylation, phospholipid metabolism, activation of Ras-like GTPases, gene expression, protein trafficking, and cytoskeletal architecture (Pawson, 1995). SH2 is also a component of adapter proteins, for instance Grb-2, which serve to link activated receptors to specific enzymes.

Many adapter proteins also contain SH3 domains, composed of up to 75 amino acids, which are responsible for protein-protein interactions. The SH3 segments of adapter molecules that also contain SH2 domains are able to connect tyrosine-phosphorylated receptors to downstream effector proteins, achieving signal transduction (Pawson, 1995). Important signal transduction pathways and proteins involved in growth factor signaling, including phosphatidylinositol 3-kinase (PI3K)

dependent pathways, Ras proteins, and Janus kinase (Jak) signaling molecules, are described in detail elsewhere in this volume.

1.3. Receptor and Ligand Modulation

Receptor activation is achieved by very low concentrations of growth factors and is short lived. Once the growth factor binds to its receptor, the growth factor-receptor complex is internalized into endosomes within less than 1 hour (Carpenter, 1987). While in the endosomes, the binding equilibrium is shifted due to decreased pH leading to ligand release. The separated ligand and receptor are transported to lysosomes and are catabolized. Some of the free receptors may be recycled and transported to the cell surface, where they are used for further activation before they are catabolized in the lysosomes.

Activation of a particular signal pathway is achieved within seconds of the binding of the growth factor to its receptor. Shortly after activation by a growth factor, receptor internalization and degradation occurs, and signal transduction terminates. In order to achieve a prolonged receptor-mediated signal, new receptors and growth factors must be produced (Carpenter, 1987).

1.4. The Role of Growth Factors in Cell Cycle Progression

One of the most important roles of growth factors is stimulating quiescent (G0 cells into active traversal of the cell cycle and cell division. In this regard, growth factors are divided into 2 groups: competence factors, such as EGF, FGF, and PDGF; and progression factors, including insulin and IGF. Quiescent cells are initially advanced into the G( phase under the influence of competence factors and then become committed to DNA synthesis (S phase) by progression factors (Pledger, Stiles, Antoniades, & Scher, 1977). The stimulatory effects of growth factors must be present throughout this transitional process, which takes several hours, and if the signal is disrupted before the cell becomes committed to DNA synthesis, the cell will retreat to the G0 phase (Figure 2).

At a critical point in the cell cycle, the restriction point, the cell is committed to progress into the S phase (Pardee, 1989). This point of the cell cycle occurs at the G, checkpoint, which is controlled by the level of phosphorylation of the retinoblastoma (Rb) protein. There are several factors affecting the level of phosphorylation of Rb proteins, including cyclins, cyclin-dependent kinases (CDKs), and the CDK inhibitors. For instance, elevated cyclin D levels shorten the duration of the G, phase and reduce the dependency of the cell on exogenous growth factors (Sherr, 1996; Sherr & Roberts, 1995).

Figure 2. This diagram illustrates the various points at which growth factors regulate cell cycle progression. The competence factors, EGF, PDGF, and FGF are important early in G,, while the progression factors are more important in late G,. In addition, the inhibitory effects of TGF-fi are illustrated.

Figure 2. This diagram illustrates the various points at which growth factors regulate cell cycle progression. The competence factors, EGF, PDGF, and FGF are important early in G,, while the progression factors are more important in late G,. In addition, the inhibitory effects of TGF-fi are illustrated.

1.5. Growth Factors and Their Receptors 1.5.1. The Epidermal-related, Growth Factors

There are several ligands that can bind to the EGFR: EGF, TGF-a, amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), cripto, vaccinia virus growth factor (VVGF), betacellulin (BTC), tomoregulin, neuregulin, and epiregulin (EPR). (Ciccodicola et al., 1989; Derynck, 1988; Higashiyama, Abraham, Miller, Fiddes, & Klagsbrun, 1991; Laurence & Gusterson, 1990; Normanno, Bianco, De Luca, & Salomon, 2001; Reisner, 1985; Shoyab, Plowman, McDonald, Bradley, & Todaro, 1989; Toyoda et al., 1995) EGF and TGF-a are believed to be the most important and widely expressed endogenous ligands.

EGF is synthesized as a large 1217-amino acid transmembrane precursor, which initially is anchored to the cell surface. It has biological activity, since it can stimulate EGFR located on the same cell or nearby cells. Subsequently, an extracellular portion of this protein is cleaved to release a 53-amino acid molecule, which has biological activity (Carpenter & Cohen, 1990; Normanno et al., 2001). EGF has important effects on cell growth, differentiation, and survival. EGF is produced primarily in the submandibular salivary glands, Brunner's glands in the small intestine, and the kidney (Kajikawa et al., 1991).

TGF-a is synthesized in many cell types and was first isolated from the culture medium of an oncogenically transformed cell line (de Larco & Todaro, 1978). TGF-is synthesized as a precursor protein and, after 2 sequential endoproteolytic cleavages, forms a 50-amino acid single polypeptide chain that shares 42% homology with EGF (Dunn, Hesse, & Black, 2000; Massague & Pandiella, 1993).

is present in regenerating epithelial cells and cells of many other normal and malignant adult tissues as well (Yasui et al., 1992).

1.5.2. Epidermal Growth Factor (erb-B) Receptor Family

The EGFR family of RTKs consists of 4 related receptors: EGFR/HER1 (c-erb-Bl), HER2 (c-erb-B2), HER3 (c-erb-B3), and HER4 (c-erb-B4). (Figure 3) These receptors are functional as homoditners or heterodimers of combinations of receptors and have different affinities to various growth factors.

Figure 3. EGF receptors are commonly expressed on most epithelial and mesenchymal cells and are responsible for cell proliferation and differentiation. EGF binds with higher affinity to heterodimers of receptors that contain HER2 than to any other EGF pairs. Similarly, one of the HER3 and HER4 ligands, heregulin, binds preferentially to heterodimers of HER3 and HER2 than to homodimers of HER3 (Pinkas-Kramarski et al., 1996; Sliwkowski, 1994) (Larger arrows indicate higher affinity).

Figure 3. EGF receptors are commonly expressed on most epithelial and mesenchymal cells and are responsible for cell proliferation and differentiation. EGF binds with higher affinity to heterodimers of receptors that contain HER2 than to any other EGF pairs. Similarly, one of the HER3 and HER4 ligands, heregulin, binds preferentially to heterodimers of HER3 and HER2 than to homodimers of HER3 (Pinkas-Kramarski et al., 1996; Sliwkowski, 1994) (Larger arrows indicate higher affinity).

1.5.3. Vascular Endothelial Growth Factor

VEGF, also known as vascular permeability factor (VPF), is a heparin-binding glycoprotein that is secreted as a homodimeric protein. Currently, there are 6 related proteins identified as vascular endothelial growth factors: VEGF-A, placenta growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D, and VEGF-E. VEGF-A (generally known as VEGF) is the most potent angiogenic isoform. Alternative splicing of mRNA in the process of VEGF production creates various isoforms composed of 205-, 189-, 165-, 145-, and 121-amino acid residues (Gerwins, Sköldenberg, & Claesson-Welsh, 2000; Kerbel, 2000). Several studies have indicated that the different VEGF isoforms have distinct functions. For instance, in animal models lacking certain VEGF isoforms (VEGFiäs and VEGFm), myocardial angiogenesis is impaired and ischemic cardiomyopathy has resulted. (Carmeliet 1999 VEGF-A has potent mitogenic effects, specifically on vascular endothelial cells (angiogenesis), and is one of the most important growth and survival factors for the endothelium (Ferrara, 1999; Houck, Leung, Rowland, Winer, & Ferrara, 1992; Park, Keller, & Ferrara, 1993). Additionally, VEGF causes vasodilatation partly through stimulation of nitric oxide synthase in endothelial cells (Yang et al., 1996) VEGF can also stimulate cell migration and inhibit apoptosis (programmed cell death) (Alon et al., 1995).

Inactivation of any of 4 of these growth factors (VEGF-A, PIGF, VEGF-B, and VEGF-E) influences vascular endothelial cells, while others, VEGF-C and -D, act on lymphatic endothelial cells. (Gerwins et al., 2000) VEGF-B is likely to be involved in vasculogenesis and activation of invasive enzymes on endothelial cells (Aase, 1999; Olofsson et al., 1998). VEGF-C has been linked to lymph angiogenesis and, recently, to tumor angiogenesis (Lymboussaki et al., 1998; Salven et al., 1998; Tsurusaki et al., 1999; Veikkola & Alitalo, 1999).

The family of VEGFR includes 3 structurally related tyrosine kinase receptors, VEGFR-1 (flt-1), VEGFR-2 (KDR/flk-1), and VEGFR-3 (flt-4). These receptors are almost exclusively expressed on endothelial cells. Like VEGF isoforms, the receptor types have different roles in the angiogenesis process. VEGFR-2 expression is crucial in the differentiation of angioblasts into endothelial cells (Shalaby et al., 1995); VEGFR-1 gene disruption results in abnormal vessel morphogenesis (Fong, Rossant, Gertsenstein, & Breitman, 1995); and inactivation of VEGFR-3 leads to defective lumen formation in large vessels (Dumont et al., 1998).

1.5.4. Platelet-derived Growth Factor (PDGF)

PDGF is a potent mitogenic growth factor for cells of mesenchymal origin that express high levels of PDGFR. PDGFs are dimers of disulfide-bound polypeptide chains, A and B, creating 3 biologically active isomers of PDGF (AA, AB, and BB) (Inui, Kitami, Tani, Kondo, & Inagami, 1994). Additional two novel members of this growth factor family were recently identified, i.e., PDGF-C and

PDGF-D (Heldin, Eriksson, & Ostman, 2002). PDGF isoforms stimulate and inhibit cellular activities in diverse ways, depending on the cell type, thus generating a tremendous range of possibilities for biological responses. In vascular smooth muscle cells, PDGF-AA increases protein synthesis (hypertrophy) while PDGF-BB initiates mitosis (hyperplasia) (Inui et al., 1994). In fibroblasts, however, PDGF-AA inhibits chemotaxis, while PDGF-BB stimulates chemotactic activities (Siegbahn, Hammacher, Westermark, & Heldin, 1990). In general, the biological activities of PDGF include migration, proliferation, contraction, inhibition of gap junctional communications, cytokine production, and lipoprotein uptake. Collectively, these activities promote wound healing in adults and the formation of blood vessels, kidney glomeruli, and lung alveoli in embryos. PDGFs are ligands for two receptors, PDGFR a and P, which are primarily localized on connective tissue, smooth muscle, and vascular endothelial cells and are not normally expressed on epithelial cells (Beitz, Kim, Calabresi, & Frackelton, 1991; Westermark & Sorg, 1993).

1.5.5. Fibroblast Growth Factor (FGF)

FGFs and their signaling pathways play significant roles in the normal development of embryonic cells and in wound healing by regulating cell growth and differentiation. Twenty FGFs have been identified (FGF1-FGF20). This family ofpolypeptide growth factors stimulates proliferative, chemotactic, and angiogenic activities primarily in cells of mesodermal origin; however, they also have effects on cells derived from the ectoderm and endoderm (Basilico & Moscatelli, 1992). The members of this family of growth factors are classified as FGFs solely on the basis of their structural similarities and not on their biological activities. For instance, FGF-7 does not stimulate fibroblasts (Powers, McLeskey, Wellstein, 2000).

FGFs exert their mitogenic and angiogenic effects in target cells by signaling through cell-surface tyrosine kinase receptors. In order to affect differing cells as extensively as FGFs do, the signaling system requires a variety of receptors. There are 4 distinct genes that encode for FGFR, designated as FGFR1 (Flg), FGFR2 (Bek), FGFR3, and FGFR4. The diverse collection of FGFRs is produced through alternate splicing of the same gene or analogous splicing of different genes (Powers et al., 2000). The various isoforms of FGFRs bind to FGF ligands with differing affinities. (Ornitz 1992) Similarly, different FGFRs may have different signaling roles, evident by the involvement of FGFR1, more than the other receptors in the FGFR family, in malignancies and cell transformation (reviewed in Mendelsohn et al., 2001).

1.5.6. Transforming Growth Factor-f)

The TGF group includes TGF-a and -P, which are structurally unrelated and bind to a completely different family of receptors (Dunn, Heese, & Black, 2000). TGF-a binds to EGF receptors and acts similarly to EGF, as described above.

Members of the TGF-P superfamily signal through a single common receptor complex that is a heteromeric serine/threonine kinase receptor. Mammalian TGF-p exists in 3 isoforms: TGF-pl, TGF-P2, and TGF-|33 (Massague, 1990). The various TGF-P isoforms share many biological activities and their actions on cells are qualitatively similar in most cases. These isoforms are involved in embryogenesis, cell proliferation, tissue repair, hematopoiesis, and regulation of the immune response (Kulkarni et al., 1993; Massague, 1990; Shull et al., 1992).

The TGF-P heteromeric receptor complex contains type I (RI) and type II (RII) subunits. Additionally, subunit RIII has been identified but has no signaling domain and serves as an auxiliary unit in presenting TGF-(32 to the RI and RII components, inhibits proliferation in a number of normal cell types, with several proposed mechanisms, and it antagonizes mitogenic effects of several growth factors, including PDGF, EGF, and FGF2 (Hunter, Sporn, & Davies, 1993). The chemical and structural changes in the receptor after binds to it, lead to phosphorylation of a number of downstream effectors, including Smad proteins. After phosphorylation by the TGF-P receptor, Smad2 and Smad3 form heterodimers with Smad4 and translocate to the nucleus to activate gene expression. Increased expression of several cyclin-dependent kinase inhibitors, including P21WAF1/CIP1, P27KIP1 and P15INK4B, has been observed after TGF-P receptor activation (Rich, Zhang, Datto, Bigner, & Wang, 1999). Expression of these proteins has been shown to be associated with decreased activity of Cdk2 followed by hypophosphorylation of Rb, and, therefore cell cycle arrest (Platten, Wick, & Weller, 2001).

1.5.7. Insulin-like Growth Factor

Insulin, IGF-I, and IGF-II are the members of this peptide-based family of growth-stimulating molecules, with a 50% similarity at the structural level (Daughaday & Rotwein, 1989). IGF-I and -II are produced mainly by the liver, the major source of endocrine IGFs (Werner & Rotwein, 2000), and insulin is produced by the P-cells in the islets of Langerhans. Six IGF binding proteins (IGFBPs), IGFBP-related proteins, and IGFBP-proteases modulate the activity of the IGFs by altering their available free fraction since IGFBPs have a higher affinity (2- to 50-fold) to IGFs than do IGF receptors (reviewed in Mendelsohn et al., 2001).

The circulating growth hormone level in the body controls IGF-I release patterns; therefore, IGF-I gene expression and blood levels are increased by 10- to 100-fold between birth and adulthood (Roberts et al., 1986). Increased levels of circulating IGF-II are detected in adults compared to those in children. Since multiple studies have demonstrated that many tissue cells are capable of producing IGFs regardless of development stage, it is believed that IGFs have local (autocrine and paracrine) activities in addition to their endocrine actions (Adamo, Ben-Hur, Roberts, & LeRoith, 1991; D'Ercole, Applewhite, & Underwood, 1980).

Insulin regulates metabolic functions primarily by affecting cells in liver, muscle, and adipose tissues (Kahn, 1985). In contrast, IGF-I and -II modulate the growth and differentiation of cells in almost every tissue in the body (Daughaday & Rotwein, 1989). IGF-I regulates several cellular activities, including cell proliferation, differentiation, and apoptosis; IGF-I acts as a potent mitogen for a variety of cell types stimulating cyclin D1 expression, which, in turn stimulates cell cycle progression from G, to S phase (Dufourny et al., 1997; Furlanetto, Harwell, & Frick, 1994). IGF-I also inhibits apoptosis by stimulating expression of Bcl protein and suppressing expression of Bax (Minshall et al., 1997; Parrizas & LeRoith, 1997; Wang, Ma, Markovich, Lee, & Wang, 1998). IGF-II also has mitogenic and antiapoptotic activities and regulates cellular proliferation and differentiation.

The IGF receptors include insulin, IGF-I, and IGF-II receptors. IGF-I and insulin receptors are structurally similar; however, the IGF-II receptor is a mannose 6-phosphate receptor, and its cellular actions are not understood. IGF-I receptors are expressed on actively proliferating cells, whereas insulin receptors are mainly present on highly differentiated, noncycling cells, including hepatocytes and adipocytes (O'Dell, 1998).

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