Cancerrelated Genes 41 Oncogenes

Oncogenes are derived from normal host genes, also called protooncogenes, that become dysregulated as a consequence of mutation. Oncogenes contribute to the transformation process by driving cell proliferation or reducing sensitivity to cell death. Historically, oncogenes were identified in four major ways: chromosomal translocation, gene amplification, RNA tumor viruses, and gene transfer experiments. Gene transfer experiments consist of transfecting DNA isolated from tumor cells into normal rodent cells (usually NIH-3T3 cells) and observing any morphological changes. These morphological changes became the hallmarks for cell transformation, the process of becoming tumorigenic. As previously discussed, the characteristics of transformed cells are as follows: (1) the ability tot form foci instead of a monolayer in tissue culture; (2)the ability to grow without adherence to a matrix, or "anchorage-independent growth"; and (3) the ability to form tumors when injected into immunologically compromised animals.

There are seven classes of oncogenes, classified by their location in the cell and their biochemical activity (Table 1.3). All of these oncogenes have different properties that can lead to cancer. The classes of oncogenes are growth factors, growth factor receptors, membrane-associated guanine nucleotide-binding proteins, serine-threonine protein kinases, cytoplasmic tyrosine kinases, nuclear proteins, and cytoplasmic proteins that affect cell survival.

4.1.1 Growth Factors and Growth Factor Receptors. Cell growth and proliferation are subject to regulation by external signals that are typically transmitted to the cell in the

Table 1.3 Oncogenes

Oncogenes

Growth Factors sis int-2 trk

Growth Factor Receptors erb-Bl erb-B2/HER2/neu fms ros

Tyrosine kinases bcr-abl src lck

Serine-Threonine protein kinases raf mos

Guanine nucleotide binding proteins H-ras

K-ras

N-ras

Cytoplasmic proteins bcl-2

Nuclear proteins myc jun fos

Protein Function

Platelet-derived growth factor Fibroblast growth factor Nerve growth factor

Epidermal growth factor receptor

Heregulin

Hematopoietic colony stimulating factor Insulin receptor kinase

Tyrosine kinase Tyrosine kinase

Serine-threonine kinase Serine-threonine kinase

GTPase

GTPase

GTPase

Anti-apoptotic protein

Transcription factor Transcription factor (AP-1) Transcription factor (AP-1)

Neoplasm(s)

fibrosarcoma breast neuroblastoma squamous cell carcinoma breast carcinoma sarcoma astrocytoma chronic myelogenous leukemia colon colon sarcoma sarcoma melanoma; lung, pancreas leukemias; colon, lung, pancreas carcinoma of the genitourinary tract and thyroid; melanoma

B-cell lymphoma .

lymphoma osteosarcoma sarcoma form of growth factors that bind to and activate specific growth factor receptors. Predictably, one class of oncogenes consists of growth factors that can stimulate tumor cell growth. In normal cells and tissues, growth factors are produced by one cell type that then act on another cell type. This is termed paracrine stimulation. However, many cancer cells secrete their own growth factors as well as express the cognate receptors that are stimulated by those factors. Because of this autocrine stimulation, cancer cells are less dependent on external sources of growth factors for proliferation and their growth is unregulated. Examples of on-cogenic growth factors include v-sis, which is the viral homolog of the platelet-derived growth factor (PDGF) gene. PDGF stimulates the proliferation of cells derived from connective tissue such as fibroblasts, smooth muscle cells, and glial cells. Thus, tumors caused by excess stimulation by v-sis include fibrosarco-mas and gliomas.

The receptors that interact with growth factors are also another large family of onco-genes. Growth factor receptors are composed of three domains: an extracellular domain that contains the ligand binding domain that interacts with the appropriate growth factor, a hydrophobic transmembrane domain, and a cytoplasmic domain that typically contains a kinase domain that can phosphorylate tyrosine residues in other proteins. Hence, these receptors are frequently referred to as receptor tyrosine kinases (RTK). It is this kinase

Figure 1.9. Ras signaling pathway. Growth factor (GF) binds to its receptor and initiates dimeriza-tion and autophosphorylation. Grb2 interacts with SOS, which activates ras by promoting the GTP-bound form. Ras recruits Raf to the plasma membrane and initiates the Raf/MAPK signaling cascade. Protein kinase C also stimulates this pathway as well as another cascade of stress-activated kinases (SEK/JNK). Both of these signalingpathways promote cell proliferation by stimulatingthe transcription of genes like cyclooxygenase-2, activator protein-1, and nuclear factor-KB. Ras also signals phosphoinositol-3-kinase and Akt/protein kinase B for cell survival.

Figure 1.9. Ras signaling pathway. Growth factor (GF) binds to its receptor and initiates dimeriza-tion and autophosphorylation. Grb2 interacts with SOS, which activates ras by promoting the GTP-bound form. Ras recruits Raf to the plasma membrane and initiates the Raf/MAPK signaling cascade. Protein kinase C also stimulates this pathway as well as another cascade of stress-activated kinases (SEK/JNK). Both of these signalingpathways promote cell proliferation by stimulatingthe transcription of genes like cyclooxygenase-2, activator protein-1, and nuclear factor-KB. Ras also signals phosphoinositol-3-kinase and Akt/protein kinase B for cell survival.

activity that is essential to the intracellular signaling that is stimulated by an activated receptor and in all oncogenic receptors mutations that lead to constitutive intracellular signaling promote unregulated cellular proliferation. RTKs can become oncogenically activated by mutations in each of the protein domains. Genetic mutations that result in the production of an epidermal growth factor receptor (EGFR) lacking the extracellular li-binding domain leads to constitutive signaling. This oncogenic EGFR is known as erb-Bl (Fig. 1.9).

Normally, EGF binds to the extracellular portion of the EGFR and causes dimeriza-tion of the intracellular part of the receptor and association with adaptor proteins, Son of Sevenless (SOS), and growth factor receptor binding protein 2 (Grb 2). These proteins interact through src-homology (SH) do mains SH2 and SH3, respectively. Through an unknown mechanism, the SOS-Grb 2 complex activates the oncogene ras. Ras induces an intracellular cascade of kinases to promote proliferation. These signaling cascades become constitutive when the extracellular portion of the EGFR becomes truncated, as in the case of erb-Bl. Oncogenic activation of a related RTK, erb-B2, occurs as a consequence of a single point mutation that falls within the transmembrane region of this receptor (72). This mutated receptor is frequently found in breast cancers. Finally, mutations in the cytoplasmic kinase domain can also cause constitutive activity leading to constitutive signaling.

4.1.2 G Proteins. In many cases, signaling that is initiated by growth factors activating their receptors passes next to membrane asso-

ciated guanine nucleotide-binding proteins, which when activated by mutation, constitute another class of oncogenes. The prototypical member of this family of oncogenes is the ras oncogene. There are three ras genes in this family of oncogenes, which include H-ras, K-ras, and N-ras. These genes differ in their expression patterns in different tissues. All have been found to have point mutations in human cancers including liver, colon, skin, pancreatic, and lung cancers, which lead to constitutive signaling of genes involved in proliferation, cell survival, and remodeling of the actin cytoskeleton. Ras is a small molecular weight protein that is post-translationally modified by attachment of a farnasyl fatty acid moiety to the C-terminus. Because this post-transla-tional modification is essential for activity of the ras oncogenes, this process has become a target for drug development aimed at interfering with ras activity (73).

Ras binds both guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) reversibly but is only in the activated state and capable of signaling when bound to GTP. The activated, GTP-bound form of ras signals a variety of mitogen-induced and stress-induced pathways, leading to transcription of genes necessary for cell growth and proliferation

(74). Mitogens such as growth factors can activate ras through the epidermal growth factor receptor, and stress factors affecting ras include ultraviolet light, heat, and genotoxins. Guanine nucleotide exchange factors (GEFs) foster ras activation by promoting the exchange of GDP for GTP. In contrast, GTPase activating proteins (GAPs) suppress ras activity by promoting GTP hydrolysis by ras, resulting in the GDP-bound inactive form of ras

(75). Importantly, because GAPs function to suppress cell proliferation, they can be thought of as tumor suppressors. Indeed, the neurofibromatosis gene, NF-1, is a GAP that acts as a tumor suppressor gene and can be inherited in a mutated and nonfunctional form giving rise to the Von Recklinghausen neurofibromatosis or neurofibromatosis type 1 cancer syndrome (76).

comprised of the serine/thereonine kinases.

The best studied of these serine-threonine protein kinases is the raf oncogene, which is activated when it is recruited to the plasma membrane by ras (77). Raf then initiates a cascade of mitogen-induced protein kinases which culminate in the nucleus with the activation of genes containing Elk-1 transcription factor binding sites. Raf can also directly activate protein kinase C, which signals another set of kinases that phosphorylate the c-jun transcription factor.

Another ras effector gene is phosphoinosi-tol 3-kinase (PI-3K), which initiates a signaling pathway for cell survival (78). PI-3K phosphorylates phosphatidalinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3)f an important intracellular second messenger, thus aiding in the transmission of signals for proliferation to the nucleus. PI-3K consists of a catalytic subunit, pi 10, and a regulatory subunit, p85, and there are five isoforms of each subunit. PI-3K phosphorylates protein kinase B (Akt/PKB) on serine and threonine residues, which in turn modulate cellular processes like glycoly-sis and translation initiation and elongation. Akt/PKB also phosphorylates Bad, a pro-apop-totic protein. When Bad is phosphorylated, it is sequestered by the 14-3-3 protein, rendering it incapable of binding to the anti-apoptotic protein, bcl-2, and thus, results in apoptosis. Akt's phosphorylation of Bad serves to inhibit apoptosis and promote cell survival. This has deleterious effects for the organism because tumor cells are not permitted to undergo apoptosis and will survive and divide.

PI-3K has been linked to the development of colon cancer by a study showing that genetic inactivation of the pllOgamma catalytic subunit of PI-3K leads to the development of invasive colorectal adenocarcinomas in mice (79). This pathway is not completely separate from the Raf/MAPK pathway, because Akt has been found to inhibit Raf activity. In fact, none of the aforementioned ras-mediated pathways operate completely independently; there are multiple examples of crosstalk between these signaling pathways.

4.1.3 Serine/Threonine Kinases. Once activated, ras then transmits the growth signal to a third class of signaling molecules that is

4.1.4 Nonreceptor Tyrosine Kinases. In addition to growth factor receptors, other nonre-ceptor kinases target protein tyrosines for

phosphorylation and can become activated as oncogenes. Indeed, one of the first oncogenes to be discovered, src, is the best characterized member of a family of proteins that have oncogenic potential. The src family of proteins are post-translationally modified by attachment of a myristate moiety to the N-terminus, which enables association with the plasma membrane. The members of the src family of proteins exhibit 75% homology at the amino acid level with the greatest degree of similarity found in three regions that have been labeled src homology domains 1, 2, and 3 (e.g., SHI, SH2, and SH3). The SHI domain encompases the domain that contains kinase activity. The SH2 and SH3 domains are located adjacent to and N-terminal to the kinase domain and function to promote protein/protein interactions. The SH2 domain binds with phosphor-ylated tyorsines, whereas the SH3 domain has affinity for the proline rich regions of proteins. Importantly,SH2 and SH3 domains are found in a large number of other proteins that are involved in intracellular signaling and that have oncogenic potential, and the structure of these domains are strongly conserved. Because SH2 and SH3 domains serve to potentiate signal transduction, they have also become targets for drug discovery programs aimed at disrupting the constitutive signaling generated by oncogenic activity (80).

A second oncogenic protein tyrosine kinase of considerable clinical importance is the Bcr-Abl oncogene. The Bcr-Abl protein is a chi-meric fusion protein formed by a reciprocal translocation involving chromosomes 9 and 22. This chromosomal rearrangement is diagnostic for the hematopoietic malignancy, chronic myelogenous leukemia (CML), and the rearranged chromosome is known as the Philadelphia chromosome (81). The c-Abl gene maps to chromosome 9 and is a tyrosine kinase, whereas the BCR gene is now known to be GTPase-activating protein (GAP), which when fused to Abl results in an unregulated tyrosine kinase that functions to promote cellular proliferation (82). The bcr-abl protein interacts with SH2 domains on Grb 2 and relocates to the cytoskeleton and initiates ras signaling, a primary mode of tumorigenic potential. Bcr-abl reduces growth factor dependence, alters adhesion properties, and en hances viability of CML cells. Consequently, the kinase activity of Bcr-Abl is a primary factor in stimulating the proliferation of CML cells, and therefore, has become the target for drug therapies aimed at combating this cancer. Indeed, the drugSTI571 has been spectacularly successful in the clinic at causing remission of this disease (83).

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