CHAPTER 101 Growth Factors in Glial Tumors
This last part of surgery, namely, operations, is a reflection on the healing art; it is a tacit acknowledgement of the insufficiency of surgery. It is like an armed savage who attempts to get that by force which a civilized man would get by stratagem.1
Progressive loss of cellular control through accumulation of multiple genetic defects leads to tumor formation and malignant progression.2 These changes can involve activation of growth-promoting oncogenes or inactivation of tumor suppressor genes.3 Polypeptide growth factors play a major role in tumor growth and progression.
Growth Factors
Growth factors are defined by Tannock and colleagues as “a polypeptide produced by cells that stimulates or inhibits proliferation by either the same cell or other cells.”4 Most growth factors are secreted soluble polypeptides that activate specific membrane-bound receptors. They usually act via binding to receptors situated on the cell surface. Through complex molecular cascades this signal is transduced and passed on in an alternative format to ultimately bring about changes in growth or function of the cell. Growth factors play a role in regulating diverse cellular processes, including cell growth and division, cell survival and apoptosis, cellular differentiation, and motility. Abnormalities in these functions are seen in virtually all brain tumors. Astrocytomas have been the most intensely studied. The malignant phenotype is associated with genomic instability, immortality, increased cell survival and division, enhanced migration and invasion, neoangiogenesis, and the ability to avoid detection by the immune surveillance system.5
There are three mains ways that growth factors are involved in the genesis of gliomas.6 The first involves concurrent expression of both a growth factor and its corresponding receptor, which allows autocrine stimulation of cellular growth. The second scenario is growth factor expression without receptor expression. Through paracrine actions on tissues such as vascular endothelium, an environment supportive of tumor growth is created. Finally, tumor cells may express receptors for growth-promoting ligands that are produced elsewhere in the body.
Intracellular Signaling
Abnormalities in intracellular signaling are almost universally involved in the transition from normal cells to malignant cells, which results in increased cell division and abnormal survival.7
Phosphorylation of proteins by kinases is a major mechanism by which the function of growth factors is modulated. Cellular signaling will usually involve the following steps, as illustrated in Figure 101-1.7 First, a ligand molecule binds to a receptor situated on the cell surface. This leads to activation of the receptor, which may involve conformational structural changes in the receptor itself or recruitment of other proteins called adaptor proteins, which in turn activate downstream signaling pathways. This process often involves changes in phosphorylation of target proteins and subsequent activation of transcription factors, which alters gene expression in the cell.
Disruption of a growth factor signal transduction pathway for therapeutic purposes can occur at any level along the signaling pathway. There is considerable overlap and intercommunication among the different pathways. Examples of common systems are given in the subsequent text, but interested readers are referred to the more detailed material cited at the end of this chapter (see, for example, Kapoor and O’Rourke8).
Receptor Tyrosine Kinases
The receptor tyrosine kinases (RTKs) are a very important, evolutionarily conserved family of signaling molecules.9 These receptors are made up of an extracellular ligand-binding domain, a transmembrane region, and a domain contained within the cytoplasm that has the ability to enzymatically phosphorylate target proteins,7 thereby altering their interactions with other molecules. RTKs contain a single hydrophobic transmembrane domain, as opposed to the seven–transmembrane receptor family, which lacks intrinsic protein kinase activity and is coupled to G proteins. There are approximately 60 genes in this family of receptors,10,11 with wide-ranging effects on the cell. They are subdivided into 20 subfamilies.9 Mutations in RTKs are found in approximately 30% of human cancers.9
The enzymatically active intracellular portions are normally locked into an inactive conformation.9 Ligand binding permits oligomerization of the receptor components. This brings the catalytic and cytoplasmic domains into juxtaposition and permits transphosphorylation of a tyrosine residue in a region called the activation loop, which leads to the activation of kinase. Next, phosphorylation of segments in the cytoplasmic segment by the activated enzyme permits interaction with intracellular docking proteins. These docking proteins contain sites, such as SH2 sites,12 that bind specifically to the phosphorylated tyrosine–containing segments of the receptor complex. Downregulation of receptor activity occurs via internalization into endosomes or dephosphorylation.7
Cytokine Receptor Superfamily
Interleukins, prolactin, growth hormone, and interferons are examples of ligands that bind to cytokine receptors.7,13 Ligand binding leads to substrate tyrosine phosphorylation in a process dependent on members of the Janus protein tyrosine kinase (JAK) family.14 There are four members in this protein family: JAK1, JAK2, JAK3, and Tyk2. If a specific JAK molecule is absent, the function of all cytokine receptors with which it normally associates is lost. Protein recruitment to the receptor complex after tyrosine phosphorylation results in activation of mitogen-activated protein kinases (MAPKs) and frequently phosphatidylinositol-3′-kinase (PI3K), which gives rise to further downstream cellular changes, including activation of members of the signal transducer and activator of transcription (STAT) family of transcription factors.7 A further level of complexity is added by the action of the suppressor of cytokine signaling (SOCS) family of proteins, which target the phosphorylated tyrosine–containing proteins for degradation, thereby blocking STAT signaling.13,15
Integrin Signaling
There are at least 24 different integrin receptors, which consist of 1 of 8 core β subunits and 1 of 18 α subunits.16 These receptors are linked to the cellular microfilament system and influence cell adhesion and movement. Intracellular signaling can alter the binding ability of the extracellular domain.15,16 Integrin receptor complexes regulate the activity of the Rho family of guanosine triphosphatases (GTPases) and interact with PI3K, phospholipase C-γ (PLC-γ), Shc, and Grb family adaptor proteins.7
Signal Transducers and Activators of Transcription
The STAT family of proteins acts downstream from the tyrosine phosphorylation step.13 They contain SH2 domains in their carboxyl regions. Phosphorylation of tyrosine residues within this SH2 domain allows STAT dimerization.16 These dimers then undergo translocation to the nucleus, where they activate gene transcription by binding to specific DNA sequences.
Role of Tyrosine Phosphatases
Tyrosine phosphatases contain a catalytic domain that dephosphorylates previously activated tyrosine residues. They can be either cytoplasmic or membrane bound16 and cause both upregulation and downregulation of intracellular signaling processes. SHP-2 is an example of a tyrosine kinase that positively influences cell signaling.16 It seems particularly important in regulation of the MAPK pathway.
Serine/Threonine Phosphorylation Systems
Two major families of receptors, the Toll/interleukin-1 (IL-1) receptor family and the transforming growth factor-β (TGF-β) receptor family, induce their effects via phosphorylation of serine and threonine residues7 rather than tyrosine.
The Toll/IL-1 receptor family includes six Toll-like receptors (TLR1 to TLR6), the IL-1 receptor, and the IL-18 receptor.17 Binding results in activation of the nuclear factor NF-κB and c-Jun N-terminal kinases (JNKs).7
Role of G Proteins in Intracellular Signaling
There are two broad types of G proteins: heterotrimeric G proteins and small G proteins.18 Both types are active when bound to adenosine triphosphate (ATP) and inactive when bound to adenosine diphosphate (ADP). There are more than 1000 G protein–coupled receptors, which represents 2% of the human genome.19 They mediate a wide variety of cellular functions, including hormonal signaling, neurotransmitter signaling, light perception, and chemokine functioning.20 This variety of intracellular effects is mediated in part by molecular differences in the intracellular adaptor molecules created by combinatorial construction of trimers from a diverse pool of subunits, as outlined in the following text.
Structurally, all receptors in this family have seven transmembrane α helices, an extracellular ligand-binding site, and an intracellular cytoplasmic domain that interacts with the so-called heterotrimeric G-protein complex.7,19,20
Various combinations of α, β, and γ subunits form heterotrimers. The guanine nucleotide–binding portion is known as the α subunit.7,20 There are 16 known α subunits.7,20 The α subunit has an inhibitory effect on the function of the combined βγ complex. The β and γ subunits cooperate to activate intracellular protein targets. There are 4 different β and 7 different γ subunits.7,20 In the quiescent nonactivated state, one molecule of guanosine diphosphate (GDP) is bound to the α subunit, which in turn is bound to the βγ complex.21 This trimeric complex is recruited to the receptor upon ligand binding and results in activation of the G-protein complex by exchange of a molecule of guanosine triphosphate (GTP) for the GDP already bound to the α subunit. The activated GTP-α portion then dissociates from the βγ complex, which leaves it free to modulate the function of intracellular targets. Cleavage of a phosphate group from the GTP-α group permits reassociation with the βγ complex and terminates intracellular propagation of the signal.21 Figure 101-2 illustrates the cycle of G-protein activation.
In addition, the GTP-bound α subunit can activate the enzyme adenylyl cyclase, which produces the intracellular second messenger cyclic adenosine monophosphate (cAMP).7 Another common target of G protein–associated receptors is phospholipase C-β (PLC-β), which can interact with either α or βγ complexes. Members of the PLC-β family generate the intracellular messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in the cellular membrane.7 IP3 acts to transiently increase intracellular calcium concentrations, which has a variety of effects, including protein kinase activation.
Finally, ion channel function can be modulated by G proteins via coupling to cyclic guanosine monophosphate (cGMP) phosphodiesterase, as in the case with the light-sensing molecule rhodopsin.7 Free GTP-coupled α subunit lowers cGMP levels by activation of the phosphodiesterase.
Unlike their heterotrimeric counterparts, small G proteins possess intrinsic adenosine triphosphatase (ATPase) activity.19 Two categories of modulatory molecules act on small G proteins. Guanosine nucleotide exchange factors (GEFs) activate small G proteins by promoting the dissociation of GDP, thereby allowing a molecule of GTP to bind and activate the G protein. GTPase-activating proteins (GAPs), in contrast, promote inactivation of the G protein by fostering hydrolysis of GTP to GDP.18 There are five subfamilies of small G proteins, the most well characterized being the p21-ras subfamily.18
G proteins play an important role in tumors of the nervous system. The activated form of the small G protein p21-ras is present at elevated levels in astrocytomas,18 possibly because of overactivation by upstream activators such as the epidermal growth factor receptors (EGFRs). Suppression of p21-ras activity via the farnesyltransferase inhibitor L-744,832 has been shown to suppress astrocytoma growth.22 In the setting of neurofibromatosis type 1, loss of the NF1 gene, which is a downregulator of p21-ras activity, leads to upregulation of ras activity.18
Growth Factors Involved in Brain Tumors
Designing agents to inhibit glioma growth factors requires knowledge not only of the growth factor and its receptor or receptors but also of the downstream signaling chain. Instead of thinking about inhibiting the growth factor in isolation, it is useful to conceptualize intervention to disrupt a signaling cascade (see, for example, Wick and associates23).
Epidermal Growth Factor Receptor
EGFR (HER1, c-erbB1) is a 170-kD glycoprotein encoded by a gene on chromosome 17 (Table 101-1).24 It contains 1186 amino acid residues. Although epidermal growth factor (EGF) is not typically overexpressed in brain tumors, its receptor EGFR is. Microglia found in the vicinity of glioma cells have been shown to secrete EGF, thus suggesting a possible paracrine stimulatory pathway.25 EGFR belongs to the erbB receptor family and is also known as erbB1 receptor. Other members of this receptor family are erbB2 (HER2/Neu), erbB3, and erbB4.81 These transmembrane receptors have a single transmembrane-spanning domain, and ligand binding activates an intracellular tyrosine kinase that normally promotes cell growth and division. TGF-α is overexpressed in malignant gliomas and appears to be a prime ligand for binding to EGFR. Other ligands for EGFR include EGF, amphiregulin, heparin-binding EGF-like growth factor, and epiregulin.26
TABLE 101-1 Receptors and Ligands of the Epidermal Growth Factor Receptor Family
| RECEPTOR | LIGAND |
|---|---|
| EGFR (erbB1, HER1) | EGF, TGF-α, HB-EGF, amphiregulin, decorin, betacellulin, epiregulin |
| HER2 (erbB2, neu) | — |
| HER3 (erbB3) | NRG-1 |
| HER4 (erbB4) | NRG-1 to NRG-4 |
EGF, epidermal growth factor; EGFR, EGF receptor; HB-EGF, heparin-binding EGF; TGF, transforming growth factor.
Adapted from Nicholas MK, Lukas RV, Jafri NF, et al. Epidermal growth factor receptor–mediated signal transduction in the development and therapy of gliomas. Clin Cancer Res. 2006;12:7261.
A constitutively active form of EGFR (EGFRvIII) appears to be involved in the progression of gliomas rather than their initiation. EGFR is overexpressed in 40% to 50% of high-grade gliomas.26 In 50% of grade IV astrocytomas, EGFR gene rearrangement is present and commonly involves deletion of the ligand-binding domains. The resulting EGFRvIII has been shown to interfere with apoptosis by increasing Bcl-xl, which normally decreases apoptosis. In addition, mutant EGFR can increase drug resistance and suppress caspase activation. This EGFR mutation is commonly seen in the primary type of glioblastoma multiforme (GBM), which arises in older individuals and is associated with loss of the p16 tumor suppressor gene and expression of wild-type p53. It seems to be associated with a worse prognosis.27 EGFR-C958, resulting from genomic deletion of exons 23 to 25 of the cytoplasmic domain, is the second most common mutant and is found in 20% of GBMs.28 An autocrine loop involving EGFR and heparin EGF-like growth factor has been demonstrated in glioma cell lines.27
Another member of the EGFR family that is mutated in astrocytomas is neu (HER2/p185).30 The gene for this 185-kD protein is also found on chromosome 17, and it shares 50% sequence homology with EGFR. Its ligand is unknown, but mutations found in high-grade gliomas result in continuous ligand-independent growth signals.30
Therapies have focused mainly on antagonizing EGFR or interfering with tyrosine kinase activity.31 Attempts at therapy with monoclonal antibodies32 and vaccines33 have met with little success to date. A bispecific cytotoxin composed of recombinant IL-13 and EGF moieties bound to the diphtheria toxin is in development and has been shown to inhibit the growth of various cell lines,34 but no success in human trials has been reported.
The small-molecule inhibitors gefitinib (Iressa) and erlotinib (Tarceva)25 both bind to the receptor ATP-binding pocket, thereby preventing ATP binding and inhibiting signal transduction (Table 101-2).33 A phase II trial with gefitinib (Iressa/ZD1839—an EGFR tyrosine kinase inhibitor) in patients with recurrent glioblastoma75 failed to show any objective effects on tumor growth, although it was generally well tolerated.76 Combination with the mTOR (mammalian target of rapamycin) inhibitor sirolimus was more promising, with a 25% 6-month progression-free survival rate in patients with GBM.44 Despite previous studies showing good tolerability,41 a similar phase II study of erlotinib, which also inhibits the EGFRvIII mutant,77 likewise failed to show dramatic results.78 A phase II trial comparing erlotinib with temozolomide and other standard agents as first-line therapy failed to show any responses.79 It appears that cells expressing the constitutively active mutant form of EGFR (EGFRvIII) along with PTEN (phosphatase and tensin homologue from chromosome 10) are more responsive to EGFR kinase inhibitors.80 PTEN is a lipid phosphatase that acts to dephosphorylate the 3′ position of the intracellular signaling molecules inositol-3,4-bisphosphate31 and inositol-3-phosphate,81,82 thereby inhibiting the EGFR/PI3K/Akt/mTOR signaling pathway.33,83
Another approach has been to combine gefitinib with everolimus (RAD-001), an agent that interferes with the downstream mTOR protein kinase. This has resulted in a modest radiographic response, but no increase in survival.84 There is also new interest in combining this agent with radiation therapy,85 and timing of dose administration appears to be important.
Trials of the combined EGFR/erbB2 inhibitor lapatinib (GW-572016) and the combined EGFR/vascular endothelial growth factor receptor (VEGFR) inhibitor AEE788 are under way.86 There have been case reports of successful use of cetuximab87 and other monoclonal antibodies directed against the extracellular portion of EGFR, and clinical trials are also under way.52
Tumor Necrosis Factor
Another receptor family that contains about 25 receptors is the tumor necrosis factor receptor (TNFR) family.88 Common ligands include the tumor necrosis factor (TNF) family, Fas ligand, CD40, CD30, and CD27.89 Ligand binding leads to receptor aggregation and subsequent conformational changes in the receptors. Various receptor-specific adaptor proteins are then recruited to the receptor complex.
TNF-α exists in a membrane-associated and a freely soluble form. The former is a transmembrane protein, whereas the latter is a trimeric complex of 17-kD proteins.90 Expression of TNF-α is upregulated by the hypoxic environment often found in association with high-grade gliomas.91 There are two TNFRs: TNFRI is activated by the soluble form of TNF-α, and TNFRII binds to the membrane-associated form.90
Interestingly, activation of TNFRs, especially TNFRI, can facilitate two diametrically opposite responses. A common motif in this family of receptors is the death domain, which has the ability to induce cellular apoptosis. Examples of receptors with cytoplasmic death domains are TNFRI, Fas, and the nerve growth factor (NGF) receptor p75.7 This motif is also seen in the DR4 receptor for the ligand TRAIL (tumor necrosis factor–related apoptosis-inducing ligand),92 which is also the subject of study as an antitumor agent.93 This cytoplasmic domain interacts with death domains on intracellular effector molecules such as TNFR-associated death domain (TRADD) and Fas-associated death domain (FADD).18 In turn, these molecules activate caspase cascades leading to apoptosis.94
In contrast to this proapoptotic effect, a second effect of TNFRI activation is activation of the transcription factor NF-κB via AP-1, which has a multitude of intracellular effects, including protection of the cell from apoptosis.95 Knockout mice in which the TNF-α gene is lacking exhibit increased resistance to the development of experimental cancers.36 TNFRII has been less well characterized. It is known that activation of TNFRII leads to subsequent activation of several intracellular pathways, including MAPK, extracellular signal–regulated kinase (ERK), p38, NF-κB, and JNK.90,94
Monoclonal antibody–based inhibitors of TNF have seen widespread use in the treatment of chronic inflammatory conditions such as inflammatory bowel disease and arthritis.96 The much maligned drug thalidomide has shown some activity against gliomas and acts to inhibit RNA processing of TNF-α and other cytokines90; clinical trials are ongoing, although to date there have been few clear-cut benefits demonstrated.53–58
