Functions Of Human Oncogenes

The oncogenes of acutely transforming retroviruses (Table 4.1) as well as human oncogenes (Table 4.2) can be categorized according to their biochemical function or to the localization of their products. A sketch of these localizations (Figure 4.6) suggests that this spatial distribution may be more than incidential. Indeed, many proven or suspected human oncogenes belong to a functional network which transmits signals for proliferation and survival from the exterior of the cell to the nucleus.

Figure 4.6 Cellular localisation of oncogene proteins See text for further discussion.

A normal cell proliferates in response to extracellular signals that are conferred by soluble growth factors and are modulated by signals elicited as a result of cell adhesion to the extracellular matrix and neighboring cells. The first group of presumed human oncogene products accordingly comprises peptide growth factors like TGFa (transforming growth factor alpha), FGF1 (fibroblast growth factor) or WNT1 (wingless/int-1) which are mitogens for epithelial and/or mesenchymal cells. These or related factors are overproduced by many carcinomas.

Peptide growth factors bind to and activate receptors at the cell membrane such as the EGFR (epidermal growth factor receptor, Fig. 4.4), the product of the ERBB1 gene, or one of several FGF receptors. ERBB1 is overexpressed in different carcinomas, often as a consequence of gene amplification. FGFR expression is also altered in many human tumors and the FGFR3 in particular is activated by point mutations in cancers of the bladder (^-14.3) and the cervix. Further growth factor receptors such as ERBB2 (^-18.4), MET (^-15.3), IGFR1 (^-16.2), KIT (^-22.4), and RET (^-2.2) are also crucial oncogenes in human cancers. These receptors share structures and the mechanism of signaling and are summarized as receptor tyrosine kinases (RTKs, also: TRKs). RTKs constitute one of the biggest classes of oncogenes. However, some oncogene products are receptors belonging to different classes, e.g. cytokine receptors (^6.8).

Binding of a growth factor to the extracellular domain of a RTK leads to formation of receptor dimers or heterodimers, e.g. between ERBB1 and ERBB2. It also causes a conformational change by which a pseudosubstrate peptide loop in the intracellular domain of the receptor is removed from the active center of the intracellular tyrosine kinase domain. The tyrosine kinase becomes active and the receptor subunits phosphorylate each other in trans. Overexpression of RTKs in tumor cells favors dimer formation. Thereby, it sensitizes the cells to lower concentrations of ligand growth factors or even leading to growth factor-independent activation. Oncogenic point mutations typically occur in the inhibitory loop, thereby causing constitutive activation of the tyrosine kinase activity. Assembly of several receptor dimers to larger complexes in the cell membrane may occur followed by internalization into endosomes.

Cross- and auto-phosphorylation of RTKs provide phosphotyrosine for recognition by adaptor proteins containing SH2 domains which dock to the activated receptor. The SH2 domains in different proteins all bind phosphotyrosines, but recognize them in different peptide contexts. To various extents, RTKs also phosphorylate proteins other than themselves.

Usually, multiple proteins bind to one receptor by recognizing different phosphotyrosines. In this fashion, one receptor can activate several different signaling pathways. For instance, activated EGFR binds the adaptor proteins GRB2 and SHC (which again binds GRB2), PLCy (phospholipase Cy), the regulatory subunit of PI3K (phosphatidylinositol-3'-kinase), and GAP (GTPase activator protein).

Phosphotyrosines are substrates for tyrosine phosphatases and also serve as recognition sites for proteins which lead to internalization and degradation of the receptor, such as CBL proteins. Together, these help to limit the strength of growth factor signals in normal cells and ensure that the signals are transient.

The adaptor protein GRB2 in turn assembles a further protein named SOS to the complex, which interacts with and activates RAS proteins (Figure 4.7). All RAS proteins, HRAS, KRAS, or NRAS, are «21 kDa proteins and are linked to the inside of the cell membrane through their C-terminus which is posttranslationally modified by myristylation, farnesylation and methylation (^Figure 22.8). They belong to a larger superfamily of small monomeric G proteins that can bind alternatively GTP or GDP. In the active state, GTP is bound. Hydrolysis of GTP to GDP by the combined action of RAS and a GTPase activator protein (i.e., GAP) restores the basal inactive state. Normally, activation of RAS depends on interaction with SOS which acts as a guanine nucleotide exchange factor (GEF), loading the RAS protein with GTP instead of GDP. The activated state of normal RAS proteins is short-lived, since

Figure 4.7 The main MAPKpathway See text for further details

RTKs in parallel to the SOS GEF activate GAPs that stimulate GTP hydrolysis. However, mutations in RAS amino acids 12, 13, or 61, which surround the GTP binding site, obstruct the access for the GAP protein and prolong the active state.

RAS is the next branching point in the signaling network described here, since activated RAS acts on several pathways (^Fig. 6.2) which affect protein synthesis, the cytoskeleton and cell survival, notably the PI3K pathway (^6.3).

The main route by which RAS relays a proliferation signal is via interaction with RAF proteins. The three RAF proteins in human cells are Ser/Thr protein kinases. Like many protein kinases, they contain a regulatory domain and a C-terminal catalytic domain. In the inactive state, the protein resides in the cytosol and the kinase activity is blocked by the regulatory domain. Activated RAS translocates RAF to the cell membrane and relieves the inhibition of the regulatory domain. Interestingly, RAF activity is also modulated by phosphorylation of the CR2

segment in its regulatory domain. A tyrosine in this domain is phosphorylated by SRC family kinases, and serine and threonine residues are phosphorylated by protein kinase C isozymes which can be activated indirectly by PLC (^6.5). So, here are further links in the network. In some human cancers, BRAF is altered by specific point mutations to become overactive (^12.4). Mutations in BRAF occur in the same tumor types as mutations in RAS genes, but alternately to them. Since RAS proteins act on several pathways, this finding is important in showing that signaling via RAF is indeed significant for transformation. Tellingly, the main modification in the retroviral v-raf oncogene is the inactivation of the regulatory domain of the cellular protein.

Activated RAF is the first one in a cascade of protein kinases that further comprises MEK and ERK proteins. Alternative names for these are MAPK (mitogen-activated protein kinase) for ERK and MAPKK for MEK; RAF proteins can therefore be counted as MAPKKK (or MEKK). There are several additional MEKKs in parallel pathways in human cells (^6.2). Most human cells contain two MEK and two ERK protein kinases. MEK are highly specific and phosphorylate predominantly ERK proteins at a tyrosine and threonine each in a TEY sequence. Activated ERKs phosphorylate a variety of substrates to activate protein synthesis, alter the structure of the cytoskeleton, and induce gene expression. Interaction of MEKs, ERKs and MAPKKs is supported and its specificity is enhanced by scaffold proteins. Phosphorylation of MAPKKs, MEKs, and ERKs is removed by several specific and less specific protein phosphatases in order to terminate the signal. Some of these phospatases, like MKP1 (MAP kinase phosphatase 1), are themselves activated by MAPK signaling, while others may be constitutively active.

Increased protein synthesis and cytoskeletal changes elicited by MAPK signalling in the cytoplasm are important for cell growth and also necessary for cell migration. Stimulation of cell proliferation in addition requires an altered pattern of gene expression and activation of the cell cycle in the nucleus. The mitogenic signal from growth factors for altered transcription is to a large extent relayed by the MAPK signaling cascade. Activated ERKs phosphorylate several transcription factors (Figure 4.7) directly or stimulate other protein kinases like p90RSK1 to do so. This induces the transcription of a larger set of genes, which again are organized as a cascaded network in the nucleus.

The first set of genes induced upon activation of the MAPK pathway by growth factors in previously resting cells are the 'early-response' genes. They are, e.g. induced by treatment of quiescent cells in cell culture with growth-factors or serum. Among them are several from the oncogene lists, such as FOS, JUN, MYB, and MYC. The most important one of these in the context of human cancers is MYC which is frequently activated by overexpression or deregulation and thereby becomes active independent of growth factor signaling. As a rule, FOS, JUN and MYB are necessary for the growth of human cancers, but do not usually seem to act as oncogenes, strictly spoken. It is not entirely clear, why this is so. Certainly, one obvious reason is that the viral counterparts of these genes are severely altered and over-expressed. Moreover, cells from long-lived humans may have better checks against tumor formation than cells of many animals. Indeed, overexpression of proteins like FOS or JUN can induce apoptosis in some human cells.

Following their induction, the products of the 'early response' genes induce the expression of genes required for cell cycle progression, directly or indirectly. In normal cells, one of the most important proteins that links the mitogenic signal to cell cycle regulation is Cyclin D1 (^6.4), the product of the CCND1 gene (^4.3). Overexpression of Cyclin D1 caused by gene amplification or translocation of CCDN1 is important in several different human cancers. Moreover, Cyclin D1 is often overexpressed without alterations in the gene itself, likely as a consequence of 'upstream' mutations in the MAPK pathway. Its overexpression may (partly) mediate the oncogenic effect of these alterations. The related gene CCDN2 is expressed in a more restricted range of tissues than CCDN1. Accordingly, overexpression and amplification of this gene occur in a more limited range of human tumors, e.g. prominently in testicular cancers. D-Cyclins activate the cyclin-dependent kinase 4 to promote progression through the cell cycle (^5.2, ^6.4). Amplification and overexpression of CDK4 are also observed in human cancers, e.g. in gliomas and hepatomas.

Finally, physiological signaling for cell proliferation requires a parallel signal for cell survival, by the same growth factor and its receptor or by a complementing pathway (^6.4). For instance, insulin-like growth factors also stimulate the MAPK cascade, but more strongly confer a survival signal, which is mediated through PI3K (^■6.3). Therefore, the overexpression of the insulin-like growth factors IGF1 and IGF2 as well as PI3K and its downstream kinase AKT must be considered as an oncogenic event in many human cancers. Alternatively, cell death by apoptosis as a consequence of inappropriate stimulation of cell proliferation can be prevented by oncogenic overexpression of proteins like BCL2 (^7.3). This is the crucial event in follicular lymphoma, but this change or equivalent ones are also found in other hematological cancers as well as many carcinomas. The synergism between oncogenes that stimulate cell proliferation and oncogenes that prevent apoptosis induced by this stimulation is evident in many human cancers, most transparently in certain lymphomas (^10.2). This is another example of oncogene cooperation.

Since human cancers accumulate many genetic and epigenetic alterations during their progression, in a typical cancer many genes are overexpressed and many of their products are overactive, and some may show mutations. Many of these genes may well be functionally important and promote tumor growth. It is tempting to regard every gene of this kind as an oncogene, particularly, if it shows a fitting biochemical property such as a protein kinase activity or DNA binding. In the context of human cancers, however, caution is warranted. This is because many cancers have developed over a long period, with many displaying genomic instability and/or an increased rate of point mutations (^3.4). As a consequence, there may be a larger number of genes with alterations in their sequence and dosage in the definitive tumor clone than absolutely required for its growth. To prove that a gene of this sort is an oncogene by a strict definition, one would have to show that the altered or overexpressed gene product indeed dominantly confers an essential property for the survival and sustained growth of the cancer, and that its overexpression and/or overactivity are caused by substantial changes in the gene itself, i.e. mutations or amplifications.

Further reading

Varmus HM, Weinberg RA (1993) Genes and the biology of cancer. Scientific American Publishing

Hesketh R (1997) The oncogene and tumor suppressor factbook. Academic Press.

Knipe DM, Howley PM (eds.) Fields Virology 4th ed. 2 vols. Lippincott Williams & Wilkins 2001

Lewis TS, Shapiro PS, Ahn NG (1998) Signal transduction through MAP kinase cascades. Adv. Cancer Res. 74, 49-139

Bar-Sagi D, Hall A (2000) Ras and Rho GTPases: a family reunion. Cell 103, 227-238

Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103, 211-225

Wilkinson MG, Millar JBA (2000) Control of the eukaryotic cell cycle by MAP kinase signaling pathways. FASEB J. 14, 2147-2157 Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411, 355-365 Kerkhoff E, Rapp UR (2001) The Ras-Raf relationship: an unfinished puzzle. Adv. Enzyme Regul. 41, 261-267

Savelyeva L, Schwab M (2001) Amplification of oncogenes revisited: from expression profiling to clinical application. Cancer Lett. 167, 115-123 Klein G (2002) Perspectives in studies of human tumor viruses. Front Biosci. 7, d268-274 Pelengaris S, Khan M, Evan G (2002) c-MYC: more that just a matter of life and death. Nat. Rev. Cancer 2, 764-776

Mikkers H, Berns A (2003) Retroviral insertional mutagenesis: tagging cancer pathways. Adv. Cancer Res. 88, 53-99

Nilson JA, Cleveland JL (2003) Myc pathways provoking cell suicide and cancer. Oncogene 22, 90079021

Mercer KE, Pritchard CA (2003) Raf proteins and cancer: B-Raf is identified as a mutational target. BBA 1653,25-40

Box 4.1 Carcinogenesis by HTLV-I

About 20 million people worldwide are estimated to be infected with HTLV-I. Up to 2% of them eventually develop a slow-growing, but obstinate and usually fatal malignancy of clonal CD3+ CD4+ cells, termed adult T-cell lymphoma/leukemia (ATLL). This cancer is clearly initiated by the retrovirus, but as it progresses, it develops chromosomal aberrations and may become completely independent of proteins expressed from the proviral genome it harbors. Through much of its development, it appears to also require stimulation by the antigen recognized by the particular T-cell clone.

In addition to the standard gag, pol, and env proteins of retroviruses(cf. Fig. 4.1), HTLV-I expresses several accessory proteins thought to be involved in the initial immortalization and clonal expansion of a T-cell infected by the virus. The most important one is tax, a transactivator protein with gross similarities to HIV tat.

Tax acts in a pleiotropic fashion, i.e. it influences the expression of many proviral and cellular genes. It binds to certain transcriptional activators and augments their interaction with transcriptional co-activators, in particular with CBP/p300. In this fashion, gene activation by NFKB (^6.9), AP1 and other factors activated by MAPK signaling (^-6.2), as well as CREB (^-12.4) is enhanced. In T-cells, this leads to the increased production of cytokines, e.g. IL2, of cytokine receptors, e.g. IL2R, and of anti-apoptotic proteins such as BCL-Xl and the IAP survivin (^6.9).

In addition to proliferation and apoptosis, Tax and other viral proteins also influence cell cycle regulation directly and the control of genomic stability by the TP53 protein (^5.3). Tax may sequester CBP/p300 from TP53, prohibiting gene activation by TP53 in response to chromosomal defects. Moreover, the provirus integration site can act as a hotspot for chromosomal breaks.

HTLV-I action in carcinogenesis might be schematically illustrated as follows:

Antigen stimulation cytokine receptor cytokine cytokine receptor


Franchini G, Nicot C, Johnson JM (2003) Seizing of T cells by HTLVI. Adv. Cancer Res. 89, 69-107.

Franchini G, Nicot C, Johnson JM (2003) Seizing of T cells by HTLVI. Adv. Cancer Res. 89, 69-107.

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