Function

1.Introduction

2.Overview

Introduction

Oncogenes, the mutated forms of proto-oncogenes, code for proteins that can transform cells (1) and energize a growth-stimulatory pathway that causes the cell to proliferate excessively (2). The primary function of oncogenes is to disrupt the cell's growth control systems. They cause continuous division by affecting the gene products that participate in growth control:

• growth factors
• growth factor receptors
• intracellular signal transducers
• nuclear transcription factors

In addition, metastasis and invasion contribute to the lethality of cancer cells. In order for successful metastasis to occur, oncogenes must escape the controls that keep normal cells in place:

• adhesion
• anchorage dependence
• basement membranes

How Cancer Cells Spread Return to top of page

Metastasis starts with the cancerous cell detaching from its origin and invading a blood or lymph vessel. It then establishes a new cellular colony at a distant site after traveling in the circulation system (3)

Cell-cell and cell-matrix interactions are essential for the maintenance of tissue homeostasis and the regulation of cell growth and cell differentiation. In normal tissues, cells adhere to one another and to the extracellular matrix. They survive and proliferate through anchorage dependence, a phenomenon that requires cells to attach to a surface in order to reproduce (3). Cadherins and selectins, two groups of Ca²⁺ dependent cell-cell adhesion molecules, and immunoglobulin (Ig) superfamily are the principle protein classes that mediate cell-cell adhesion (1); whereas integrins, cell-surface receptors, mediate surface cell attachment (3). If the mediation of E-cadhedrin, an adhesive protein that holds most epithelial sheets together by linking the cells' plasma membranes is blocked, noninvasive cells become invasive through the removal of Ca²⁺(3). This removal not only disrupts cell-cell adhesion but also does not generate the junctions between epithelial cells (i.e., gap junctions)(1).

Gap junctions participate in signal transductions by allowing cells to exchange ions and small water soluble molecules (i.e., secons messengers such as cAMP and Ca²⁺ ) and are believed to play a role in the regulation of cell proliferation and cell division(9). With the growth control mechanism impaired in carcinogenesis, researchers believe that the loss of gap junctional intercellular communication (GJIC) may contribute to the cell's lack of control. The accumulation of replication signals in the cell due to the inhibition of GJIC leads researchers to this conclusion(9). For example, the src oncogene inhibits GJIC when it phosphorylates a gap junctional protein (Cx 43) with a src tyrosine kinase. This phosphorylation changes the junction channels from open to closed thereby disabling GJIC(9).

In addition, the cyclin E-CDK2 (E- cyclin dependent kinase) complex, which regulates cell growth and division (FIG 1), becomes less active thereby stopping cell growth. Therefore, cells denied anchorage eventually undergo apoptosis --- induced cell suicide. However, oncogenes overcome anchorage dependence and apoptosis by messages conveying that the cell is properly attached when in reality it is not. This false message stops the cells from terminating its own growth and from dying (3). Whether attached or not, cyclin E-CDK2 complex remain active in cancerous cells. Hence, oncogenes are anchorage independent.

FIGURE 1: How Cyclin E-CDK2 regulates cell growth (2).

The third constraint cancer cells must overcome is the basement membranes that normal cells, with the exception of white blood cells, cannot penetrate. Normal cells cannot breach extracellular matrices because they do not contain high concentrations of mtalloproteinases, enzymes which dissolves basement membranes and other extracellular matrices. Instead, normal cells contain higher concentrations of enzymes that inhibit membrane penetration (3). With cancer cells releasing mtalloproteinase, the transformed cells can gain access to the blood stream and relocate elsewhere.

Affecting the Cell's Growth Control Systems Return to top of page

By affecting the genes products that participate in growth control, oncogenes disrupt the cells' growth control systems and proliferate excessively. The effects of these oncogenes on cells are dominant. Examples of how oncogenes affect these gene products are as follows.

Growth factors and growth receptors Return to Introduction

Growth factors and growth inhibitors control cell proliferation. Growth factors stimulate the cells to divide. In the case of growth factor oncogenes, cell production is a continuous process. Since oncogenes rarely arise from genes encoding growth factors, only one naturally occurring growth factor oncogene has been discovered --- the sis oncogene (1). Since the sis oncogene is derived from the gene for the platelet-derived growth factor (PDGF), it encodes a protein similar to it. The sis oncogene transform cells that naturally have the PDGF receptor. When a ligand binds to this mutated receptor, cell proliferation is induced (1).

The receptors for growth factors involve an external ligand binding to a specific receptor that activates a tyrosine kinase activity (4). The receptors transmit growth signals by phosphorylating tyrosine residues. The transfer of phosphate groups from ATP to the side chains of the amino acid tyrosine creates sites on the protein that activates other target proteins and results in a cascade of biochemical events(10). Hence, the genes for such receptors, like the Erb-B and neu gene, become oncogenic when mutated in such a way that the receptors remain active even in the absence of a bound ligand (1). The mutation occurs with the neu proto-oncogene undergoing a point mutation (valine to glutamine) and the Erb-B proto-oncogene undergoing a deletion of its receptor binding domain. The receptors lose ligand control and grow independent of the factors that regulate growth. Uncontrollable growth ensues.

Intracellular Signal Transducers Return to introduction

Interactions between protein growth factors and their receptors result in the production of signals that cause biochemical events within the cell. These biochemical cascades are called second messenger pathways. They transduce ligand-receptor signals into the cell's interior (1). The best understood transducers are the G proteins, guanine nucleotide proteins, that activate cells and are involved in the control of a variety of metabolic processes as an on-off (GTP bound-GDP bound) molecular switches (5). Normal G proteins, such as G_s, control cAMP synthesis. When a ligand occupies a cell surface receptor, a G_s protein recognizes it and binds to GTP. Once converted to its activated state, the G_s protein, with the bound GTP, binds to and activates an effector enzyme, adenyl cyclase. This enzyme synthesizes cAMP. The G_s protein, in turn, returns to its inactive state by hydrolyzing the bound GTP (1).

Mutations in the Gs protein results in an oncogene with the GTPase activity eliminated. With the absence of a termination signal, the concentration of cAMP increases and unregulated cell proliferation occurs (1). Src, ras, and crk proto-oncogenes are examples of intracellular signal transducers involving GTPase activity that work as molecular switches for the relay of extracellular signals from the exterior of the cell to the nucleus.

Like the G proteins, normal ras proteins function as intracellular on-off molecular switches with an active state bound to GTP and an inactive state bound to GDP (1). The activation and deactivation [Fig 2] of the Ras protein follow a similar pattern to the G proteins. A guanine nucleotide-exchange factor (GEF) binds to the Ras*GDP complex thereby causing the disassociation of the bound GDP (1). With the release of the GEF, GTP binds spontaneously to the Ras molecule. The binding of a GTPase-activating protein (GAP) to the Ras*GTP complex begins the process of ras deactivation (1). GTP is hydrolyzed and P_i and GAP is released. Hence, GAP promotes the hydrolysis of bound GTP to return the Ras to its inactive state (GDP bound) (7).

Figure 2: Cycling of the Ras protein between inactive (GDP-bound) and active (GTP-bound) forms(1)

Normal Ras genes direct extracellular signals to the nucleus where specific genes are activated for cell growth, division, and cell differentiation (7). It behaves as an on-off switch in cell division (6). RasGAP, a RAS GTPase activating protein, controls the activation/deactivation of the Ras gene by limiting the lifetime of a signaling Ras protein through the increase of GTPase activity. However, when valine substitutes glycine at position 12 of the sequence, the proto-oncogene mutates (1). This mutation results in the inefficient hydrolosis of GTP. By blocking the Ras GTPase activity, the signalling path is kept on (7). The cell is flooded with growth stimulatory signals. The mutated ras gene encodes proteins that transmit these signals continuously even in the absence of growth factor receptors (11).

Figure 3: A growth factor binds to a receptor tyrosine kinase and is phosphorylated. The phosphorylated growth factor receptors interact with protein exchange factors which activate Ras (stimulates the GDP-GTP exchange). Ras activates a series of mitogen activated protein kinases which eventually phosphorylate specific transcription factors. These activated transcription factors bind to specific DNA sites thereby activating the transcription of a particular gene (7).

On the other hand, the src genes encode protein tyrosine kinase (PTK). The src gene product phosphorylates tyrosine instead of the amino acids serine and threonine like other protein kinases (4). It also cooperates with growth control factor receptors that lack their own PTK (4). As nuclear proteins lacking extracellular domains, they contain myristate, a long chain of fatty acids, bound to its N-terminal glycine (1). This hydrophobic anchor allows normal src proteins to bind partially to the plasma membrane. (Fig. 4) However, if the proto-oncogene is transformed, the N-terminal glycine is removed and the proteins encoded by the src oncogenes can no longer bind to the plasma membrane (1). In addition, phosphatidylinositol 3-kinase (PI 3-kinase), a cytoplasmic enzyme, binds to pp60^src. PI 3-kinase phosphorylates PI, phosphatidylinsitol. This fatty molecule, once phosphorylated, stimultes the cell to perform several functions, one of which may be cell division. Hence, the cell undergoes abnormal cell division.

Figure 4: Src protein with myristrate anchor (1)

In conjunction, crk oncogenes, which are Src homologies, have SH2 and SH3 domains. These structures are found in many signaling proteins(1). The SH2 domains interact with protein sites that have phosphorylated tyrosine; hence, they bind to PTK target proteins. The SH3 domains interact with the proline rich regions of the target (1). Crk oncogenes engage in highly specific interactions and cause cancer without a catalytic domain. The SH domains, on the other hand, selectively bind to the sequences around the phosphotyrosine or prolines (1). It is this specific aggregation to areas that normally serve as signaling units for cellular events that cause oncogenesis (1). Therefore, mutations of the src and crk proto-oncogenes transform the cells to affect the cell's growth control system.

Nuclear transcription factors Return to introduction

Oncogenes exert direct effects on transcription rates and cause changes in the proportion of different mRNAs in cells as they bind to DNA and modify the rate of transcription initiation (1). The Fos, FBJ osteosarcoma, and jun, Avian sarcoma virus 17, genes are examples of oncogenes affecting normal nuclear transcription factors.

The jun oncogene act as a transcription factor. It plays a role in cellular growth and differentiation. It is comprised of a set of related genes (jun C, jun B, and jun D) that are each expressed independently in different tissues (8). The jun oncogene codes for proteins which regulate gene expression directly or indirectly. By binding to a common cellular and viral DNA sequence motif, TGAGTCA, jun oncogenes encode proteins directly (8). Indirect gene expression includes its dimerization and cooperativity with the fos oncogenes (8).

The v-jun oncoprotein is a mutant form of the AP-1 transcription factor that binds to the TGACTCA sequence and is implicated in such processes as proliferation and differentiation. The fos oncogene encodesmembers of AP-1 transcription factors. The expression for the c-fos gene is necessary for the cell to progress through the cell cycle and for oncogenic cell transformation along the growth factor signal transduction pathway. Since Fos and Jun join to form a fully active transcription factor, their phosphorylation cause them to bind to specific DNA sequences near the myc gene (10). Myc proteins are transcription factors which activate genes that force cells forward --- they activate genes that initiate the prgression of cells through G1 to the S phase of the cell cycle(1). It binds to and activates transcription through specific CACGTG binding sites in collaboration with Max, a protein similar to myc. When mutated, myc is present in high levels even in the absence of growth factors(10).

In normal cells, the fos gene controls the cell growth and differentiation of hematopolectic, embryonal, and neuronal cells (5). Its expression is also associated with nerve cell adaptation, cell proliferation and cellular transformation (5). Fos proteins interact with different DNA sequences and transcription factors; hence, it participates in the transduction of cytoplasmic information into the nucleus (5). Both the fos and jun oncogenes activate the transcription of key genes that encode growth promoting proteins. They can inhibit transcription of growth repressing genes (1).