Read this essay to learn about how oncogenes arise and proteins produced by oncogenes contribute to the development of cancer.
1. Essay on How Cellular Oncogenes Arise:
Oncogenes can arise inside cells in two fundamentally different ways. One mechanism involves the participation of cancer viruses that introduce oncogenes into the cells they infect. The alternative, is based on a series of mechanisms that convert normal cellular genes into oncogenes, often as a result of exposure to carcinogenic agents.
Cellular Oncogenes Arise from Proto-Oncogenes:
Those normal cellular genes that can be converted into oncogenes are referred to as proto-oncogenes. Despite their harmful-sounding name, proto-oncogenes are not bad genes simply lying in wait for an opportunity to foster the development of cancer. Rather they are normal genes that make essential contributions to the regulation of cell proliferation and survival.
The term proto-oncogene simply implies that if and when the structure or activity of a proto-oncogene is disrupted by certain kinds of mutations, the altered form of the gene can cause cancer. In genetic terms, such mutations are considered to be “gain-of-function” mutations because they create a new function, namely the ability to induce tumor formation that is not originally present in proto-oncogenes.
Thus proto-oncogenes are normal genes that contribute to normal cell function, but they can also be converted into oncogenes, which are dysfunctional genes that produce proteins that perform a decidedly abnormal function—that is, inducing the development of cancer.
Detection of Cellular Oncogenes from Gene Transfer Experiments:
Oncogenes are not present in normal cells, so how do they arise in cancer cells? In some cancers, an infecting virus simply brings one or more viral oncogenes into the cells it infects. But what about cancers that are not caused by viruses, which is the situation with most human cancers? Do they also possess oncogenes, and if so, where do they come from?
Evidence that oncogenes are indeed present in non- viral cancers first emerged from gene transfer experiments in which DNA isolated from tumor cells was introduced into normal cells and tested for its ability to transform them into cancer cells. The initial studies of this type, carried out in the laboratories of Robert Weinberg and Geoffrey Cooper in the early 1980s, focused on the behavior of DNA isolated from human bladder cancers.
DNA was extracted from bladder cancer cells and applied to cultures of normal mouse cells under experimental conditions that stimulate DNA transfection—that is, uptake of foreign DNA into the cells and its incorporation into their chromosomes. Upon being transfected with tumor cell DNA, some of the mouse cells started to proliferate, and when the proliferating cells were injected back into mice, the animals developed cancer.
In contrast, transfection with DNA extracted from normal human cells did not transform mouse cells into cancer cells. It was therefore concluded that the DNA of bladder cancer cells contains genetic information, not present in normal DNA that is capable of causing cancer.
Because these bladder cancer transfection experiments employed total cellular DNA samples containing thousands of different genes, further work was needed to identify the cancer-causing gene(s) contained in such complex DNA mixtures. This goal was pursued using gene cloning techniques, a set of procedures that allow DNA molecules to be broken into gene-sized fragments that can be reproduced in large amounts.
When gene cloning was applied to bladder cancer DNA, it led to the isolation and identification of a single mutant gene that is responsible for the ability of the transfected DNA to cause cancer.
This mutant gene, which was the first human oncogene to be identified, is called a cellular oncogene to distinguish it from the viral oncogenes that are brought into cells by cancer viruses.
Although it is not a viral gene, the oncogene isolated from bladder cancer DNA did turn out to be related in base sequence to a viral oncogene, namely the v-ras oncogene found in retroviruses that cause sarcomas in rats. (The name ras was originally derived from “rat sarcoma”.) By convention, human versions of the v-ras oncogene are named using capital letters and italics—that is, they are called RAS oncogenes.
RAS oncogenes, like retroviral oncogenes, are altered forms of normal cellular proto-oncogenes. In the case of RAS oncogenes, we will see shortly that the corresponding RAS proto-oncogene is a normal gene that produces a protein involved in a normal pathway for stimulating cell proliferation.
So genes that produce this protein are linked to cancer in two different ways:
(1) V-ras oncogenes are present in certain retroviruses that cause sarcomas in rats, and
(2) Closely related RAS oncogenes appear in some human cancers that are not caused by viruses.
Following the discovery of the RAS oncogene—the first example of a human cellular oncogene—subsequent transfection experiments using DNA from other human tumor types revealed the existence of more than a dozen additional oncogenes. However, only about 20% of human cancers turn out to have cellular oncogenes when tested in this way.
Such results do not mean that other human cancers lack oncogenes, but simply that oncogenes cannot always be detected using DNA transfection techniques. As we will see shortly, the reason is related to differences in the mechanisms by which cellular oncogenes arise.
These mechanisms can be grouped into five basic categories:
(1) Point mutation,
(2) Gene amplification,
(3) Chromosomal translocation,
(4) DNA rearrangement, and
(5) Insertional mutagenesis.
Mechanism 1: Point Mutations can Convert Proto-Oncogenes into Oncogenes:
When the RAS oncogene was first isolated from human bladder cancer cells, the question immediately arose as to how this abnormal RAS gene (an oncogene) differs from the normal RAS gene (a proto-oncogene). Analyzing the base sequences of the two genes provided the answer- RAS oncogenes typically differ from the normal RAS proto- oncogene in a single nucleotide base. In other words, changing just a single base in the nucleotide sequence of a normal gene is sufficient to convert it into a gene that can cause cancer.
Figure 1 illustrates an example of such a mutation that is frequently encountered in RAS. In this particular case, changing a single base in the RAS proto- oncogene causes the resulting RAS oncogene to produce an abnormal Ras protein in which a single amino acid is converted from glycine to valine.
It is common practice to name human oncogenes using italicized capital letters (e.g., RAS gene) and to designate the protein encoded by the same gene without italics and often with only the initial letter capitalized (e.g., Ras protein).
The preceding example represents the simplest mechanism for creating an oncogene from a proto-oncogene- A single nucleotide in a proto-oncogene simply undergoes mutation, thereby creating an oncogene that codes for a protein that differs in a single amino acid from the normal protein encoded by the gene. Such mutations in a single nucleotide base are called point mutations.
RAS oncogenes produced by point mutation have been detected in many types of cancer, including cancers of the bladder (where they were originally discovered), lung, colon, pancreas, and thyroid. A point mutation can occur at any of several different sites within a RAS oncogene, and the particular site involved appears to be influenced by the carcinogen that caused it.
For example, asbestos, vinyl chloride, and dimethylbenzanthracene each trigger mutations at different locations within the RAS gene. The ability of these carcinogens to cause cancer may therefore stem from their ability to mutate different nucleotides in the same gene.
Point mutations that convert proto-oncogenes into oncogenes are not restricted to genes coding for Ras proteins. The RET gene can be converted by various point mutations into RET oncogenes that code for altered Ret receptor proteins. Individuals inheriting a single copy of such a mutant gene exhibit a roughly 70% risk of developing cancer within their lifetime, usually thyroid cancer.
In addition to playing a role in hereditary cancers, RET oncogenes containing point mutations also appear in some sporadic thyroid cancers, where they are thought to be caused by exposure to environmental carcinogens.
Mechanism 2: Gene Amplification can Convert Proto-Oncogenes into Oncogenes:
A second, fundamentally different mechanism for creating cellular oncogenes uses the process of gene amplification to create multiple, duplicate copies of the same gene. Gene amplification is accomplished by replicating the DNA located in a specific chromosomal region numerous times in succession, thereby creating dozens, hundreds, or even thousands of copies of the same stretch of DNA.
Chromosome regions containing amplified genes often exhibit a distinctive, abnormal appearance that can be recognized when chromosomes are examined by light microscopy (Figure 2).
The main types of abnormalities are homogeneously staining regions (HSRs) and double minutes (DMs). HSRs are chromosome regions that stain homogeneously rather than exhibiting the alternating pattern of light and dark bands that is typical of normal chromosomes.
In contrast, DMs are independent, chromosome-like bodies that are much smaller than typical chromosomes, often appearing as spherical, paired structures. Both types of chromosomal abnormalities represent regions of amplified DNA containing from several dozen to several hundred copies of one or more genes.
When a gene is amplified, most (if not all) of the multiple copies are actively expressed. The total amount of protein produced by an amplified gene is therefore much greater than when the gene is present in only a single copy per chromosome.
However, the base sequence of each copy of an amplified gene is usually normal. So unlike point mutations, which create oncogenes that produce an abnormal protein, gene amplification typically yields oncogenes that produce normal proteins but in excessive quantities.
Members of the MYC gene family—individually called MYC, MYCL, and MYCN—are among the most commonly encountered oncogenes to arise by gene amplification in human cancers. Some cancer viruses also introduce abnormal versions or trigger abnormal behavior of MYC genes.
Amplified MYC genes have been detected in a diverse array of tumors, including cancers of the breast, ovary, uterine cervix, lung, and esophagus. These amplified genes produce excessive amounts of normal Myc protein.
The extent of MYC gene amplification—that is, the number of gene copies per cell— varies among people with the same type of cancer, but tends to remain constant for a given person’s tumor. In some cases, a connection has been detected between the number of gene copies and tumor behavior.
For example, neuroblastomas with extensively amplified MYCN genes are more likely to invade and metastasize, and have lower survival rates, than neuroblastomas in which the MYCN gene is less amplified (Figure 3).
In addition to MYC genes, more than a dozen other examples of amplified oncogenes have been detected in human cancers. Depending on the particular type of cancer involved, amplified oncogenes are present in anywhere from 5% to 50% of cancer cases and exhibit repetition frequencies as high as 500 copies or more.
An amplified oncogene of significant clinical importance is the ERBB2 gene, which appears in amplified form in about 25% of all breast and ovarian cancers. As was the case for MYCN gene amplification in neuroblastomas, individuals whose tumors exhibit higher copy numbers of the ERBB2 gene tend to exhibit poorer survival rates.
A specific anticancer drug, called Herceptin, is designed to counteract the effects of the overactive ERBB2 gene in such individuals.
Mechanism 3: Chromosomal Translocations can Convert Proto-Oncogenes into Oncogenes:
The third mechanism for creating oncogenes is by chromosomal translocation, a process in which a piece of one chromosome is broken off and moved to another chromosome. For example, the Philadelphia chromosome is an abnormal version of chromosome 22 that is associated with 90% of all cases of chronic myelogenous leukemia.
The Philadelphia chromosome is created by DNA breakage near the ends of chromosomes 9 and 22, followed by reciprocal exchange of DNA between the two chromosomes. This translocation creates a new oncogene comprised of portions of two normal genes that were initially located on chromosome 9 and chromosome 22, respectively.
One of the two genes, called ABL, resides near the end of chromosome 9; the other gene, called BCR, resides near the end of chromosome 22. During chromosomal translocation, a break occurs within the ABL gene on chromosome 9 and within the BCR gene on chromosome 22, and the two broken ends of the chromosomes are then switched (Figure 4).
As a result, the translocated versions of chromosomes 9 and 22 each acquire a new fusion gene—that is, a gene containing sequences derived from two different genes spliced together. The BCR-ABL fusion gene created on chromosome 22 behaves as an oncogene, coding for an abnormal protein that contributes to the development of cancer. This abnormal protein is called a fusion protein because it contains amino acid sequences encoded by both the BCR and the ABL genes.
Creating genes that code for fusion proteins is not the only way in which chromosomal translocations can produce oncogenes. Another mechanism is illustrated by the chromosomal translocation involving chromosomes 8 and 14 that occurs in Burkitt’s lymphoma.
In this case, the entire MYC proto-oncogene is moved from chromosome 8 to 14, where it becomes situated next to a highly active region of chromosome 14 that contains genes coding for antibody molecules. Moving the MYC gene so close to the highly active antibody genes causes MYC to become activated also, thereby leading to an overproduction of Myc protein that in turn stimulates cell proliferation.
The translocated MYC gene retains its normal structure and codes for a normal Myc protein, but it still behaves as an oncogene because its new location on chromosome 14 causes the gene to be overexpressed.
Thus chromosomal translocations contribute to cancer development through two distinct mechanisms, either by fusing two genes together to form an oncogene coding for a fusion protein or by activating the expression of a proto-oncogene by placing it near a highly active gene.
Chromosomal translocations acting in these ways have been detected in a variety of human cancers, especially leukemias and lymphomas. Besides the two examples described thus far (chronic myelogenous leukemia and Burkitt’s lymphoma), acute myelogenous leukemias have been found to exhibit a diverse array of chromosomal translocations, including translocations between chromosomes 3 and 5, chromosomes 6 and 9, chromosomes 7 and 11, chromosomes 8 and 16, chromosomes 9 and 12, chromosomes 12 and 22, and chromosomes 16 and 21. In each case, researchers have identified the two genes whose juxtaposition or fusion contributes to the development of cancer.
Mechanism 4: Local DNA Rearrangements can Convert Proto-Oncogenes into Oncogenes:
The fourth mechanism for creating oncogenes involves localized DNA rearrangements in which the movement of DNA base sequences in a particular region of a chromosome disrupts the expression or structure of a proto-oncogene located in that region. As shown in Figure 5, DNA rearrangements can be grouped into four basic categories.
The simplest types involve short stretches of DNA that are either lost or added during normal DNA replication, thereby causing mutations known as deletions or insertions, respectively. Rearrangements in which DNA segments are moved from one location to another are called transpositions, and DNA segments that have been excised and then reinserted backwards in the same location are termed inversions.
DNA rearrangements are frequently detected in human tumor cells, especially in certain types of cancer. In thyroid cancers, for example, rearrangements are present in nearly 50% of the tumors examined. Some of these rearrangements illustrate how a simple chromosomal inversion can create an oncogene from two perfectly normal genes. One well-studied example involves two genes, named NTRK1 and TPM3, that both reside on the same chromosome.
NTRK1 codes for a growth factor receptor and TPM3 codes for tropomyosin, a totally unrelated protein involved in muscle contraction and cell motility. In some thyroid cancers, a DNA inversion occurs that causes one end of the TPM3 gene to become fused to the opposite end of the NTRK1 gene, thereby creating a fusion gene called the TRK oncogene (Figure 6). The TRK oncogene produces a Trk fusion protein whose amino acid sequence is determined partly by the NTRK1 receptor gene and partly by the TPM3 gene.
The Trk fusion protein contains the part of the normal receptor molecule that exhibits tyrosine kinase activity, which means that it catalyzes the phosphorylation of the amino acid tyrosine in target proteins. The tyrosine kinase site of a normal receptor only becomes active after a growth factor binds to the receptor and causes adjacent receptor molecules to cluster together as dimers (two molecules joined together).
The Trk fusion protein, however, contains a portion of the tropomyosin molecule that forms coiled coils, which are structures that cause protein chains to join together as dimers. As a result, the fusion protein forms a permanent dimer and its tyrosine kinase is permanently activated (see Figure 6, step ④). The Trk fusion protein therefore contributes to cancer development by acting as a permanently activated receptor that continually stimulates cell proliferation, regardless of whether its growth factor is present.
Mechanism 5: Insertional Mutagenesis can Convert Proto-Oncogenes into Oncogenes:
Elucidation of the final mechanism for creating oncogenes emerged from the discovery that some cancer viruses possess no oncogenes of their own, but instead convert a cell’s own genes into oncogenes. This phenomenon is called insertional mutagenesis. The underlying mechanism involves the presence of special viral sequences called long terminal repeats (LTRs), which are located at both ends of the genome of retroviruses.
LTRs contain sequences that promote gene transcription so efficiently that they activate the transcription of cellular genes that lie near the inserted viral genes. Therefore, if a retrovirus happens to randomly insert its genes near a proto-oncogene, the LTRs may stimulate transcription of the proto-oncogene and trigger overproduction of a normal cellular protein that can contribute to cancer development.
Because virally induced cancers are more common in animals than they are in humans, the best understood examples of insertional mutagenesis have come from the study of animal viruses. Nonetheless, there are reasons for believing that a similar phenomenon applies to humans as well.
Insertional mutagenesis can inadvertently take place when retroviruses are used in gene therapy experiments to ferry healthy genes into cells exhibiting genetic defects. At least one retrovirus employed for such purposes occasionally integrates itself next to a proto-oncogene whose abnormal expression has been found to trigger leukemia.
Cellular Oncogenes Arise from Proto-Oncogenes by Mechanisms that Alter Gene Structure or Expression:
The general features of the five preceding mechanisms for converting proto-oncogenes into cellular oncogenes are summarized in Figure 7. In some cases, the structure of a proto-oncogene is altered in a way that causes it to produce an abnormal protein. In other cases, the expression of a proto-oncogene is enhanced, thereby leading to excessive production of a normal protein.
The existence of these alternative strategies helps explain why the oncogenes present in human cancers cannot always be detected using the DNA transfection technique. Only about 20% of human cancers have oncogenes that can be detected by DNA transfection.
The DNA transfection approach—in which DNA isolated from cancer cells is introduced into normal cells and tested for its ability to transform them into cancer cells— is best suited for detecting oncogenes containing mutations that cause them to produce an abnormal protein.
In the case of oncogenes that simply produce too much of a normal protein, this altered expression is not likely to persist after chromosome structure has been disrupted and DNA fragments are isolated, which is how DNA is prepared for use in transfection studies.
2. Essay on How Proteins are Produced by Oncogenes:
Thus far, we have seen that five distinct mechanisms exist for converting a proto-oncogene into an oncogene, which then produces either a structurally abnormal protein or a normal protein in excessive amounts. In either case, the question arises as to how these proteins cause cancer.
Addressing such a question is a complex task because more than 100 oncogenes have been identified to date and the proteins they produce fall into a variety of categories, including growth factors, receptors, enzymes that catalyze protein phosphorylation and proteins that bind to and regulate the activity of DNA or other proteins (Table 1).
Yet despite this diversity, a unifying theme can be found. Most of the proteins produced by oncogenes are components of signaling pathways that promote cell proliferation and survival. By producing abnormal versions or excessive quantities of proteins involved in these pathways, oncogenes disrupt normal signaling mechanisms and foster the excessive proliferation and inappropriate survival of cancer cells.
Oncogenes Typically Code for Components of Signaling Pathways that Activate Cell Proliferation:
Normally a cell will not grow and divide unless it is stimulated by an appropriate growth factor, which triggers proliferation by activating signaling pathways involving dozens of molecules within the targeted cell.
Oncogenes code for proteins that are participants in such signaling pathways, but rather than producing the proper amount of a correct protein, an oncogene produces either an abnormal protein or an excessive amount of a normal one. In either case, the net result is the unregulated activation of a signaling pathway and hence uncontrolled cell proliferation, even in the absence of growth factors.
In most cells, proliferation is controlled by multiple signaling pathways that function in overlapping networks. Yet despite the overall complexity, these pathways tend to share some features in common.
In general, the binding of a growth factor to a receptor located on the outer surface of a cell leads to receptor activation; the activated receptor then stimulates a series of molecules that relay information to various compartments within the cell, including the nucleus; and some of the relay molecules reaching the nucleus trigger changes in gene expression that stimulate cell proliferation or promote cell survival.
A good example of these principles is provided by the Ras-MAPK pathway, which plays a central role in stimulating normal cell proliferation (and which often behaves abnormally in cancer cells).
As shown in Figure 8, the Ras- MAPK pathway involves six main steps:
(1) A growth factor binds to a cell-surface receptor.
(2) The activated receptor becomes phosphorylated.
(3) The phosphorylated receptor binds to adaptor proteins that relay the signal to Ras proteins associated with the inner surface of the plasma membrane.
(4) Activated Ras triggers a cascade of intracellular protein phosphorylation reactions that lead to activation of a protein kinase called MAPK.
(5) Activated MAPK enters the nucleus and phosphorylates proteins called transcription factors, which bind to DNA and activate the transcription of specific genes.
(6) The activated genes produce proteins that stimulate cell proliferation.
Among these proteins are cell-cycle regulators such as Cdk and cyclin, which stimulate progression through the restriction point and into S phase.
Although the Ras-MAPK pathway is only one of several signaling mechanisms used by cells for controlling cell proliferation, it is a good starting point for discussing how oncogenes work because it illustrates the main types of proteins produced by oncogenes, and the pathway often functions abnormally in human cancers.
In the following six sections we will look at each step of the Ras-MAPK pathway in more depth, examining how it operates normally and providing examples of oncogenes that cause it to act in a hyperactive or uncontrolled fashion. Other signaling pathways that tend to behave abnormally in cancer cells will also be described, as we discuss the roles played by oncogenes in the development of cancer.
Some Oncogenes Produce Growth Factors:
The first step in the Ras-MAPK pathway involves the binding of a growth factor to a target cell (see Figure 8, step ①). That normal cell proliferation requires the presence of an appropriate growth factor can be demonstrated by placing cells in a culture medium containing nutrients and vitamins but no growth factors.
Under these conditions, progression through the cell cycle is halted during G1 (at the restriction point) and cell proliferation ceases. Progression through the cell cycle can be restarted by adding small amounts of blood serum, which contains several growth factors. One is platelet-derived growth factor (PDGF), a protein produced by blood platelets that stimulates the proliferation of connective tissue cells.
Another is epidermal growth factor (EGF), a protein widely distributed in normal tissues that acts on a variety of cell types, mainly (but not exclusively) of epithelial origin. The normal function of growth factors is to stimulate the cell proliferation that is required during events such as embryonic development, tissue regeneration, and wound repair.
For example, blood platelets accumulate at sites of tissue injury and release the growth factor PDGF, which is instrumental in stimulating the cell proliferation needed for wound healing.
Proliferation of a target cell population normally depends on growth factors produced by other cells that detect the need for tissue growth and produce the appropriate growth factors. But what would happen if a cell were to produce a growth factor that stimulates its own proliferation?
This scenario would create an uncontrolled situation in which cells proliferate to create more cells of the same type, which then produce more of the growth factor that continues to stimulate proliferation of the same cells. One of the first oncogenes found to trigger such a scenario was v-sis, a gene from the simian sarcoma virus that causes sarcomas in monkeys. The v-sis oncogene codes for a mutant form of the growth factor PDGF.
When the virus infects a monkey cell whose proliferation is normally stimulated by PDGF, the mutant PDGF produced by the v-sis oncogene continually stimulates the cell’s own proliferation (in contrast to the normal situation in which cells are only exposed to PDGF when it is released from surrounding blood platelets). The net result is that PDGF produced by the v-sis oncogene causes the infected cells to constantly stimulate their own uncontrolled proliferation.
This type of mechanism, originally discovered in an animal retrovirus, is now known to apply to human cancers as well. In certain human sarcomas, a chromosomal translocation creates a fusion gene in which part of the PDGF gene is joined to part of an unrelated gene that codes for collagen, a protein component of the extracellular matrix.
The fused gene (COL1A1-PDGFB) behaves as an oncogene because it produces PDGF in an uncontrolled fashion, thereby causing cells containing the gene to continually stimulate their own proliferation.
Some Oncogenes Produce Receptor Proteins:
The next step in the Ras-MAPK pathway involves the plasma membrane receptors that transmit signals from growth factors to the cell interior (see Figure 8, step ②). Growth factor receptors are typically trans-membrane proteins, which mean that one end of the receptor is exposed outside the cell and the other end is exposed inside the cell.
The exterior portion of the receptor contains a binding site for its corresponding growth factor, and the end protruding inside the cell transmits signals to the cell interior, usually by acting as a protein kinase. A protein kinase is an enzyme that catalyzes protein phosphorylation (the attachment of phosphate groups to protein molecules). Receptors involved in the Ras-MAPK pathway specifically phosphorylate the amino acid tyrosine in target proteins, so such receptors are called receptor tyrosine kinases.
Binding of a growth factor to its receptor site exposed at the outer cell surface leads to activation of the receptor’s tyrosine kinase site protruding inside the cell. This process of receptor activation is accomplished through the ability of growth factors to promote or stabilize the clustering of two or more receptor molecules into configurations that activate their tyrosine kinase sites.
For example, the growth factor EGF is a single protein chain that binds to two receptor molecules simultaneously, thereby joining two receptors together to form a dimer (Figure 9a).
After receptor molecules have become clustered and activated by the binding of growth factor, the tyrosine kinase activity of each receptor catalyzes the phosphorylation of the adjacent receptor at multiple sites. Since receptors are phosphorylating other receptor molecules of the same type, the process is referred to as autophosphorylation.
Several dozen oncogenes are known to code for receptor tyrosine kinases. Many of these oncogenes produce mutant receptors whose tyrosine kinase activity is permanently activated rather than being dependent on a growth factor to trigger activation. An example is the v-erb-b oncogene, found in the avian erythroblastic leukemia virus that causes a cancer of red blood cells in chickens.
The v-erb-b oncogene produces an altered version of the receptor for EGF that retains tyrosine kinase activity but lacks the EGF binding site. As a consequence, the receptor is constitutively active—that is, it exhibits tyrosine kinase activity even in the absence of EGF (see Figure 9b), whereas the normal form of the receptor only exhibits tyrosine kinase activity when bound to EGF. Because its tyrosine kinase is always active, the receptor produced by the v-erb-b oncogene permanently stimulates the Ras-MAPK pathway and thereby triggers excessive cell proliferation.
Comparable oncogenes have been detected in some human cancers. For example, thyroid cancers frequently possess RET or TRK oncogenes, which code for mutant receptor tyrosine kinases whose uncontrolled activity stimulates cell proliferation.
Another group of oncogenes produces normal receptors but in excessive quantities, which can also lead to hyperactive signaling (see Figure 9c). An example is provided by the ERBB2 gene, which codes for a member of the EGF receptor family. The ERBB2 gene is amplified in about 25% of human breast and ovarian cancers, where the multiple copies of the ERBB2 gene produce excessive amounts of a normal receptor protein. The presence of so many receptor molecules at the cell surface leads to a magnified response to growth factor binding and hence excessive cell proliferation.
Receptors do not always possess their own tyrosine kinase activity. In the case of some receptors, binding of growth factor instead causes the activated receptor to stimulate the activity of an independent tyrosine kinase molecule. One such tyrosine kinase is Jak, a central component of a signaling mechanism called the Jak-STAT pathway.
As shown in Figure 10, binding of growth factors to receptors involved in this pathway causes the receptors to activate Jak molecules, which in turn catalyze the phosphorylation of cytoplasmic proteins called STATs (an abbreviation for Signal Transducers and Activators of Transcription).
The phosphorylated STAT molecules then join together and move from the cytoplasm to the nucleus, where they trigger changes in gene expression that stimulate cell proliferation. One oncogene that codes for a receptor involved in the Jak-STAT pathway has been detected in the myeloproliferative leukemia virus, which causes leukemia in mice. The oncogene, called v-mpl, codes for a mutant version of the receptor for thrombopoietin, which is a growth factor that uses the Jak-STAT pathway to stimulate the production of blood platelets.
Some Oncogenes Produce Plasma Membrane G Proteins:
After a growth factor has bound to and activated its receptor, a number of different signaling pathways can be triggered by the activated receptor. In the case of Ras-MAPK signaling, the phosphorylated tyrosines created on the receptor by autophosphorylation serve as binding sites for adaptor proteins that relay the signal to Ras, a protein associated with the inner surface of the plasma membrane.
The Ras protein is a member of a class of molecules called G proteins because their activity is regulated by the two small nucleotides GTP (guanosine triphosphate) and GDP (guanosine diphosphate).
G proteins are molecular switches whose “on” or “off” state depends on whether they are bound to GTP or to GDP (Figure 11). In the absence of receptor stimulation, Ras is normally bound to GDP and is inactive. To become active, it must release GDP and acquire GTP in a reaction that requires the help of another protein called a guanine- nucleotide exchange factor (GEF). This role is played by one of the adaptor proteins that relays the signal from the activated receptor to Ras (see Figure 8, step ③).
The central role played by Ras proteins in the control of cell proliferation has been demonstrated by experiments involving cells that have stopped dividing after removal of growth factors. Injecting mutant, hyperactive forms of the Ras protein into such cells can cause them to begin dividing again, even in the absence of growth factor. Conversely, injecting cells with antibodies that inactivate the Ras protein prevents cells from dividing when growth factors are subsequently added.
Oncogenes coding for Ras proteins arise in two different ways: They may be brought into cells by a retrovirus (as occurs mainly in animal cancers), or they may be created by point mutations in proto-oncogenes (as is common in human cancers).
Human cells possess three closely related RAS proto-oncogenes known as HRAS, KRAS, and NRAS; each can incur point mutations that produce oncogenes coding for abnormal Ras proteins. Such mutations are detected in roughly 30% of all human cancers, making RAS oncogenes the most commonly encountered type of human oncogene.
Point mutations in RAS oncogenes cause a single incorrect amino acid to be inserted at one of several possible locations within the Ras protein. The net result is often a hyperactive Ras protein that retains bound GTP instead of degrading it to GDP, thereby maintaining the protein in a permanently activated state.
In this hyperactive state, the Ras protein continually sends a stimulatory signal to the rest of the Ras-MAPK pathway, regardless of whether an appropriate growth factor is bound to the cell’s growth factor receptors.
Of the three types of RAS genes, KRAS is the most frequently mutated in human cancers. Point mutations in KRAS are present in about 30% of lung cancers, 50% of colon cancers, and up to 90% of pancreatic cancers. Mutations in NRAS are less frequent in epithelial cancers but are detected in about 25% of acute leukemias. Finally, HRAS mutations are encountered primarily in bladder cancers, where they appear in about 10% of cases.
Some Oncogenes Produce Intracellular Protein Kinases:
After the Ras protein has been activated, it triggers a cascade of intracellular protein phosphorylation reactions, beginning with the phosphorylation of a protein kinase called Raf kinase (Figure 8, step ④). Activated Raf kinase in turn catalyzes phosphorylation of another intracellular protein kinase called MEK, which phosphorylates another intracellular protein kinase called a MAP kinase or simply MAPK (an abbreviation for Mitogen-Activated Protein Kinase).
Unlike receptor tyrosine kinases, the intracellular kinases involved in this cascade of protein phosphorylation reactions attach phosphate groups mainly to the amino acids serine and threonine in target proteins, rather than to tyrosine.
Such enzymes are therefore referred to as serine/threonine kinases. Several oncogenes code for serine/threonine kinases involved in this cascade. An example is the BRAF oncogene, which codes for mutant forms of the Raf kinase in roughly two-thirds of human melanomas and at a lower frequency in a variety of other cancers.
In addition to the preceding types of serine/threonine kinases, several tyrosine kinases are likewise involved in intracellular signaling pathways. Unlike the receptor tyrosine kinases, which span the plasma membrane, these intracellular tyrosine kinases may be nuclear, cytoplasmic, or associated with the inner surface of the plasma membrane. Intracellular tyrosine kinases do not possess receptor sites and are therefore referred to as non-receptor tyrosine kinases.
Three examples are briefly described below:
1. Src kinase:
The Src kinase is an intracellular tyrosine kinase found in normal cells that can induce cancer when present in an abnormal form. For example, we have already seen that the v-src oncogene of the Rous sarcoma virus produces an abnormal Src kinase that triggers sarcoma development in chickens.
In addition, mutations in the human SRC gene are associated with certain forms of colon cancer. Src kinase and related members of the Src kinase family interact with a broad range of growth factor receptors. In some cases, these intracellular tyrosine kinases transmit signals from activated receptors that do not possess their own tyrosine kinase activity.
In other situations, they augment the activity of receptors that do possess tyrosine kinase activity. For example, Src kinase associates with receptors activated by the growth factors EGF and PDGF, catalyzing the phosphorylation of these receptors in a way that enhances the ability of the activated receptor to trigger the next steps in the signaling pathway.
2. Jak kinase:
In the Jak-STAT pathway, receptors transmit signals to the cell interior by activating intracellular tyrosine kinases that are members of the Jak family (see Figure 10). In certain leukemias, a translocation between chromosomes 9 and 12 has been identified that creates a fusion gene called TEL-JAK2.
This oncogene codes for a fusion protein in which the catalytic region of a Jak kinase is fused to a segment of an unrelated protein that causes the tyrosine kinase activity of Jak to become permanently activated. The resulting stimulation of the Jak-STAT signaling pathway leads to excessive cell proliferation.
3. Abl kinase:
The Abl tyrosine kinase, which is produced by the ABL proto-oncogene, functions in the cell nucleus as part of a normal signaling pathway that causes cells with damaged DNA to self-destruct by apoptosis. In chronic myelogenous leukemia, a chromosomal translocation event involving chromosomes 9 and 22 creates a Philadelphia chromosome in which segments of the BCR and ABL genes are fused together to form a BCR-ABL oncogene (see Figure 4).
The BCR-ABL oncogene codes for an abnormal version of the Abl tyrosine kinase that remains in the cytoplasm and therefore cannot trigger apoptosis. So in this particular case, an oncogene fosters an excessive accumulation of proliferating cells by enhancing their survival rather than by stimulating their proliferation.
The preceding examples represent just a few of the many intracellular protein kinases whose uncontrolled activity can contribute to cancer development by stimulating pathways that activate cell proliferation, promote cell survival, or both.
Some Oncogenes Produce Transcription Factors:
Some of the intracellular protein kinases that are activated in cells stimulated by growth factors will in turn trigger changes in transcription factors, which are proteins that bind to DNA and alter the expression of specific genes. Activation of transcription factors is a common feature of the signaling pathways that control cell proliferation and survival. It occurs in both the Ras-MAPK and Jak-STAT pathways.
In the case of the Ras- MAPK pathway, activated MAP kinases enter the nucleus and phosphorylate several different transcription factors, including Jun and members of the Ets family of proteins (see Figure 8, step ⑤). These activated transcription factors then stimulate the transcription of “early genes” that code for the production of other transcription factors, including the proteins Myc, Fos, and Jun, which then activate the transcription of a family of “delayed genes”.
Oncogenes that produce altered forms or excessive quantities of specific transcription factors have been detected in a broad range of human and animal cancers. Among the most common are oncogenes coding for Myc transcription factors, which control the expression of numerous genes involved in cell proliferation and survival.
Three examples of virally induced cancers in which an oncogene coding for a Myc protein plays a central role are as follows: The first example involves the avian leukosis virus, a retrovirus that causes leukemia in chickens by an insertional mutagenesis event in which the proviral DNA is integrated near the normal cellular gene coding for Myc.
Insertion of the proviral DNA enhances the rate at which the nearby Myc gene is transcribed, thereby leading to overproduction of the normal Myc protein. The second example involves the avian myelocytomatosis virus, a retrovirus possessing a v-myc oncogene that induces cancer by producing an abnormal version of the Myc protein.
The final example is Burkitt’s lymphoma, a human cancer triggered by the Epstein-Barr virus. Burkitt’s lymphoma is associated with a chromosomal translocation in which the MYC gene is translocated to chromosome 14, bringing it into close proximity to genes coding for antibody molecules. This event leads to excessive production of the Myc protein in cells where antibody genes are active—that is, in lymphocytes.
Burkitt’s lymphoma is only one of several human cancers in which the Myc protein is overproduced. In these other cancers, gene amplification rather than chromosomal translocation is usually responsible for the excessive production of Myc.
For example, amplification of the MYC gene is frequently observed in small cell lung cancers and, to a lesser extent, in a wide range of other carcinomas, including 20% to 30% of breast and ovarian cancers. Two other members of the MYC gene family, which code for slightly different versions of the Myc protein, have also been implicated in cancer development.
One is called MYCN (because it was first discovered in neuroblastomas), and the other is MYCL (first discovered in lung cancers). About 30% to 40% of small cell lung cancers exhibit amplification of the MYC, MYCN, or MYCL gene. MYCN is also amplified in other tumor types, including neuroblastomas and glioblastomas. In neuroblastomas, patients whose tumor cells exhibit MYCN gene amplification have poorer survival rates than patients whose tumors do not (see Figure 3).
Myc is just one of several transcription factors known to be produced by oncogenes. In animal retroviruses, for example, the oncogenes v-fos, v-jun, v-myb, v-ets, and v-erb-a each codes for a different transcription factor. Oncogenes coding for a variety of different transcription factors have also been reported in human cancers, although MYC family members are still the most prevalent.
Some Oncogenes Produce Cell Cycle or Cell Death Regulators:
In the final step of growth factor signaling pathways, transcription factors activate the expression of genes coding for proteins involved in cell proliferation (see step ⑥ in Figures 8 and 10). The activated genes include those coding for cyclin-dependent kinases (Cdks) and cyclins. Several human oncogenes produce proteins in this category.
For example, a cyclin-dependent kinase gene called CDK4 is amplified in certain sarcomas and glioblastomas, and the cyclin gene CYCD1 is often amplified in breast cancers and is altered by chromosomal translocation in some lymphomas. The presence of such oncogenes causes the production of excessive amounts or hyperactive versions of Cdk-cyclin complexes, which then stimulate progression through the cell cycle.
Stimulating progression through the cell cycle is not the only way of increasing the number of proliferating cells. The number of cells in a growing tumor is also influenced by the rate at which cells die. Normal tissues maintain a carefully regulated balance between cell proliferation and cell death, but in tumors this balance is disrupted in ways that lead to a progressive increase in dividing cells.
This increase can arise from enhanced cell proliferation, decreased cell death, or some combination of the two. Most of the oncogenes discussed in this essay act mainly by stimulating cell proliferation, but a few oncogenes act primarily or solely by inhibiting cell death. One example is the BCL2 gene, which codes for a protein called Bcl2. The Bcl2 protein resides on the outer surface of mitochondria and acts as a retraining influence on the pathway by which cells are destroyed by apoptosis.
In non-Hodgkin’s lymphomas, a common chromosomal translocation causes the BCL2 gene to produce too much Bcl2. The excessive amounts of Bcl2 block the pathway for apoptosis, thereby leading to a progressive accumulation of cells that would otherwise have been destroyed.
Another gene that affects cell death, called MDM2, is amplified in some human sarcomas and produces excessive amounts of a protein (Mdm2) that inhibits the ability of cells to self-destruct by apoptosis. Oncogenes such as BCL2 and MDM2 help cancer cells evade the apoptotic pathways that would otherwise trigger their destruction.
Oncogene-Induced Disruptions in Signaling Pathways Exhibit Some Common Themes:
In discussing how oncogenes work, this essay has focused on two signaling pathways- the Ras-MAPK pathway and the Jak-STAT pathway. In reality, these two signaling mechanisms are components of a larger network of pathways, involving numerous branches and shared components, that work together to determine whether cells will proliferate, stop proliferating, or die.
Yet despite the complexity of the branched interconnections, the various pathways involved in controlling cell proliferation and survival share some common features. First, binding of a growth factor to its receptor leads to receptor activation. Next, the activated receptor triggers a complex chain of events that includes a series of protein phosphorylation reactions. These protein phosphorylations then trigger changes in transcription factors that alter the expression of specific genes. Finally, the activated or inhibited genes produce proteins that influence cell proliferation and cell death.
Oncogenes exert their harmful effects by producing excessive quantities or hyperactive versions of proteins involved in these steps. The net result is that the presence of an oncogene leads to excessive cell proliferation and, in some cases, diminished cell death. The absence (or loss of function) of a tumor suppressor gene can likewise lead to excessive cell proliferation and diminished cell death.