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The Genetic Basis of Cancer

As we have seen, mutations in two broad classes of genes— proto-oncogenes (e.g., ras) and tumor-suppressor genes (e.g., APC)—play key roles in cancer induction. These genes encode many kinds of proteins that help control cell growth and proliferation (Figure 23-8). Virtually all human tumors have inactivating mutations in genes that normally act at various cell-cycle checkpoints to stop a cell's progress through the cell cycle if a previous step has occurred incorrectly or if DNA has been damaged. For example, most cancers have inactivating mutations in the genes coding for one or more proteins that normally restrict progression through the G1 stage of the cell cycle. Likewise, a constitutively active Ras or other activated signal-transduction protein is found in several kinds of human tumor that have different origins. Thus malignancy and the intricate processes for controlling the cell cycle discussed in Chapter 21 are two faces of the same coin. In the series of events leading to growth of a tumor, oncogenes combine with tumor-suppressor mutations to give rise to the full spectrum of tumor cell properties described in the previous section (see Figure 23-7).

In this section, we consider the general types of mutations that are oncogenic and see how certain viruses can cause cancer. We also explain why some inherited mutations increase the risk for particular cancers and consider the relation between cancer and developmentally important genes. We conclude this section with a brief discussion of how genomics methods are being used to characterize and classify tumors.

► FIGURE 23-8 Seven types of proteins that participate in controlling cell growth and proliferation. Cancer can result from expression of mutant forms of these proteins. Mutations changing the structure or expression of proteins that normally promote cell growth generally give rise to dominantly active oncogenes. Many, but not all, extracellular signaling molecules (I), signal receptors (II), signal-transduction proteins (III), and transcription factors (IV) are in this category. Cell-cycle control proteins (VI) that function to restrain cell proliferation and DNA-repair proteins (VII) are encoded by tumor-suppressor genes. Mutations in these genes act recessively, greatly increasing the probability that the mutant cells will become tumor cells or that mutations will occur in other classes. Apoptotic proteins (V) include tumor suppressors that promote apoptosis and oncoproteins that promote cell survival. Virus-encoded proteins that activate signal receptors (Ia) also can induce cancer.

Virus-encoded activators of signal receptors (Ia)

<q -Signaling molecule (I)

O / Signal receptor (II)

Virus-encoded activators of signal receptors (Ia)

<q -Signaling molecule (I)

O / Signal receptor (II)

Gain-of-Function Mutations Convert Proto-oncogenes into Oncogenes

Recall that an oncogene is any gene that encodes a protein able to transform cells in culture or to induce cancer in animals. Of the many known oncogenes, all but a few are derived from normal cellular genes (i.e., proto-oncogenes) whose products promote cell proliferation. For example, the ras gene discussed previously is a proto-oncogene that encodes an intracellular signal-transduction protein; the mutant rasD gene derived from ras is an oncogene, whose encoded protein provides an excessive or uncontrolled growth-promoting signal. Other proto-oncogenes encode growth-promoting signal molecules and their receptors, anti-apoptotic (cell-survival) proteins, and some transcription factors.

Conversion, or activation, of a proto-oncogene into an oncogene generally involves a gain-of-function mutation. At least four mechanisms can produce oncogenes from the corresponding proto-oncogenes:

• Point mutation (i.e., change in a single base pair) in a proto-oncogene that results in a constitutively active protein product

• Chromosomal translocation that fuses two genes together to produce a hybrid gene encoding a chimeric protein whose activity, unlike that of the parent proteins, often is constitutive

• Chromosomal translocation that brings a growth-regulatory gene under the control of a different promoter that causes inappropriate expression of the gene

• Amplification (i.e., abnormal DNA replication) of a DNA segment including a proto-oncogene, so that numerous copies exist, leading to overproduction of the encoded protein

An oncogene formed by either of the first two mechanisms encodes an "oncoprotein" that differs from the normal protein encoded by the corresponding proto-oncogene. In contrast, the other two mechanisms generate oncogenes whose protein products are identical with the normal proteins; their oncogenic effect is due to production at higher-than-normal levels or in cells where they normally are not produced.

The localized amplification of DNA to produce as many as 100 copies of a given region (usually a region spanning hundreds of kilobases) is a common genetic change seen in tumors. This anomaly may take either of two forms: the duplicated DNA may be tandemly organized at a single site on a chromosome, or it may exist as small, independent

▲ EXPERIMENTAL FIGURE 23-9 DNA amplifications in stained chromosomes take two forms, visible under the light microscope. (a) Homogeneously staining regions (HSRs) In a human chromosome from a neuroblastoma cell. The chromosomes are uniformly stained with a blue dye so that all can be seen. Specific DNA sequences were detected using fluorescent in situ hybridization (FISH) in which fluorescently labeled DNA clones are hybridized to denatured DNA in the chromosomes. The chromosome 4 pair is marked (red) by in situ hybridization with a large DNA cosmid clone containing mini-chromosome-like structures. The former case leads to a homogeneously staining region (HSR) that is visible in the light microscope at the site of the amplification; the latter case causes extra "minute" chromosomes, separate from the normal chromosomes that pepper a stained chromosomal preparation (Figure 23-9).

I Gene amplification may involve a small number Kç jB of genes, such as the N-myc gene and its neighbor lillilllUll DDX1 that are amplified in neuroblastoma, or a chromosome region containing many genes. It can be difficult to determine which genes are amplified, a first step in determining which gene caused the tumor. DNA microarrays offer a powerful approach for finding amplified regions of chromosomes. Rather than look at gene expression, the application of microarrays we described earlier, these experiments involve looking for abnormally abundant DNA sequences. Genomic DNA from cancer cells is used to probe arrays containing fragments of genomic DNA and spots with amplified DNA give stronger signals than control spots. Among the amplified genes, the strongest candidates for the relevant ones can be identified by also measuring gene expression. A breast carcinoma cell line, with four known amplified chromosome regions, was screened for amplified genes, and the expression levels of those genes were also studied on microarrays. Fifty genes were found to be amplified, but only five were also highly expressed. These five are new candidates as oncogenes. I

the N-myc oncogene. On one of the chromosome 4's an HSR is visible (green) after staining for a sequence enriched in the HSR. (b) Optical sections through nuclei from a human neuroblastoma cell that contain double minute chromosomes. The normal chromosomes are the green and blue structures; the double minute chromosomes are the many small red dots. Arrows indicate double minutes associated with the surface or interior of the normal chromosomes. [Parts (a) and (b) from I. Solovei et al., 2000, Genes Chromosomes Cancer 29:297-308, Figures 4 and 17.]

However they arise, the gain-of-function mutations that convert proto-oncogenes to oncogenes are genetically dominant; that is, mutation in only one of the two alleles is sufficient for induction of cancer.

Cancer-Causing Viruses Contain Oncogenes or Activate Cellular Proto-oncogenes

Pioneering studies by Peyton Rous beginning in 1911 led to the initial recognition that a virus could cause cancer when injected into a suitable host animal. Many years later molecular biologists showed that Rous sarcoma virus (RSV) is a retrovirus whose RNA genome is reverse-transcribed into DNA, which is incorporated into the host-cell genome (see Figure 4-43). In addition to the "normal" genes present in all retroviruses, oncogenic transforming viruses like RSV contain the v-src gene. Subsequent studies with mutant forms of RSV demonstrated that only the v-src gene, not the other viral genes, was required for cancer induction.

In the late 1970s, scientists were surprised to find that normal cells from chickens and other species contain a gene that is closely related to the RSV v-src gene. This normal cellular gene, a proto-oncogene, commonly is distinguished from the viral gene by the prefix "c" (c-src). RSV and other oncogene-carrying viruses are thought to have arisen by incorporating, or transducing, a normal cellular proto-oncogene into their genome. Subsequent mutation in the transduced gene then converted it into a dominantly acting oncogene, which can induce cell transformation in the presence of the normal c-src proto-oncogene. Such viruses are called transducing retroviruses because their genomes contain an oncogene derived from a transduced cellular proto-oncogene.

Because its genome carries the potent v-src oncogene, the transducing RSV induces tumors within days. In contrast, most oncogenic retroviruses induce cancer only after a period of months or years. The genomes of these slow-acting retroviruses differ from those of transducing viruses in one crucial respect: they lack an oncogene. All slow-acting, or "long latency," retroviruses appear to cause cancer by integrating into the host-cell DNA near a cellular proto-oncogene and activating its expression. The long terminal repeat (LTR) sequences in integrated retroviral DNA can act as an enhancer or promoter of a nearby cellular gene, thereby stimulating its transcription. For example, in the cells from tumors caused by avian leukosis virus (ALV), the retroviral DNA is inserted near the c-myc gene. These cells overproduce c-Myc protein; as noted earlier, overproduction of c-Myc causes abnormally rapid proliferation of cells. Slow-acting viruses act slowly for two reasons: integration near a cellular proto-oncogene (e.g., c-myc) is a random, rare event, and additional mutations have to occur before a full-fledged tumor becomes evident.

In natural bird and mouse populations, slow-acting retro-viruses are much more common than oncogene-containing retroviruses such as Rous sarcoma virus. Thus, insertional proto-oncogene activation is probably the major mechanism by which retroviruses cause cancer. Although few human tumors have been associated with any retrovirus, the huge investment in studying retroviruses as a model for human cancer paid off both in the discovery of cellular oncogenes and in the sophisticated understanding of retroviruses, which later accelerated progress on the HIV virus that causes AIDS.

A few DNA viruses also are oncogenic. Unlike most DNA viruses that infect animal cells, oncogenic DNA viruses integrate their viral DNA into the host-cell genome. The viral DNA contains one or more oncogenes, which permanently transform infected cells. For example, many warts and other benign tumors of epithelial cells are caused by the DNA-containing papillomaviruses. Unlike retroviral oncogenes, which are derived from normal cellular genes and have no function for the virus except to allow their proliferation in tumors, the known oncogenes of DNA viruses are integral parts of the viral genome and are required for viral replication. As discussed later, the oncoproteins expressed from integrated viral DNA in infected cells act in various ways to stimulate cell growth and proliferation.

Loss-of-Function Mutations in Tumor-Suppressor Genes Are Oncogenic

Tumor-suppressor genes generally encode proteins that in one way or another inhibit cell proliferation. Loss-of-

function mutations in one or more of these "brakes" contribute to the development of many cancers. Five broad classes of proteins are generally recognized as being encoded by tumor-suppressor genes:

• Intracellular proteins that regulate or inhibit progression through a specific stage of the cell cycle (e.g., p16 and Rb)

• Receptors or signal transducers for secreted hormones or developmental signals that inhibit cell proliferation (e.g., TGFp, the hedgehog receptor patched)

• Checkpoint-control proteins that arrest the cell cycle if DNA is damaged or chromosomes are abnormal (e.g., p53)

• Proteins that promote apoptosis

• Enzymes that participate in DNA repair

Although DNA-repair enzymes do not directly inhibit cell proliferation, cells that have lost the ability to repair errors, gaps, or broken ends in DNA accumulate mutations in many genes, including those that are critical in controlling cell growth and proliferation. Thus loss-of-function mutations in the genes encoding DNA-repair enzymes prevent cells from correcting mutations that inactivate tumor-suppressor genes or activate oncogenes.

Since generally one copy of a tumor-suppressor gene suffices to control cell proliferation, both alleles of a tumor-suppressor gene must be lost or inactivated in order to promote tumor development. Thus oncogenic loss-of-function mutations in tumor-suppressor genes are genetically recessive. In many cancers, tumor-suppressor genes have deletions or point mutations that prevent production of any protein or lead to production of a nonfunctional protein. Another mechanism for inactivating tumor-suppressor genes is methyl-ation of cytosine residues in the promoter or other control elements. Such methylation is commonly found in nontran-scribed regions of DNA.

Inherited Mutations in Tumor-Suppressor Genes Increase Cancer Risk

Individuals with inherited mutations in tumor-suppressor genes have a hereditary predisposition for certain cancers. Such individuals generally inherit a germ-line mutation in one allele of the gene; somatic mutation of the second allele facilitates tumor progression. A classic case is retinoblas-toma, which is caused by loss of function of RB, the first tumor-suppressor gene to be identified. As we discuss later, the protein encoded by RB helps regulate progress through the cell cycle.

Hereditary versus Sporadic Retinoblastoma Children with hereditary retinoblastoma inherit a single defective copy of the RB gene, sometimes seen as a small deletion on one of the copies of chromosome 13. The children develop retinal tumors early in life and generally in both eyes. One essential event in tumor development is the deletion or

▲ FIGURE 23-10 Role of spontaneous somatic mutation in retinoblastoma. This disease is marked by retinal tumors that arise from cells carrying two mutant RB~ alleles. (a) In hereditary (familial) retinoblastoma, a child inherits a normal RB+ allele from one parent and a mutant RB~ allele from the other parent. A single mutation in a heterozygous somatic retinal cell that inactivates the normal allele will produce a cell homozygous for two mutant alleles. (b) In sporadic retinoblastoma, a child inherits two normal RB+ alleles. Two separate somatic mutations in a particular retinal cell or its progeny are required to produce a homozygous RB~/RB~ cell.

mutation of the normal RB gene on the other chromosome, giving rise to a cell that produces no functional Rb protein (Figure 23-10). Individuals with sporadic retinoblastoma, in contrast, inherit two normal RB alleles, each of which has undergone a loss-of-function somatic mutation in a single retinal cell. Because losing two copies of the RB gene is far less likely than losing one, sporadic retinoblastoma is rare, develops late in life, and usually affects only one eye.

If retinal tumors are removed before they become malignant, children with hereditary retinoblastoma often survive until adulthood and produce children. Because their germ cells contain one normal and one mutant RB allele, these individuals will, on average, pass on the mutant allele to half their children and the normal allele to the other half. Children who inherit the normal allele are normal if their other parent has two normal RB alleles. However, those who inherit the mutant allele have the same enhanced predisposition to develop retinal tumors as their affected parent, even though they inherit a normal RB allele from their other, normal parent. Thus the tendency to develop retinoblastoma is inherited as a dominant trait. As discussed below, many human tumors (not just retinal tumors) con tain mutant RB alleles; most of these arise as the result of somatic mutations.

Inherited Forms of Colon and Breast Cancer Similar hereditary predisposition for other cancers has been associated with inherited mutations in other tumor-suppressor genes. For example, individuals who inherit a germ-line mutation in one APC allele develop thousands of precancerous intestinal polyps (see Figure 23-7). Since there is a high probability that one or more of these polyps will progress to malignancy, such individuals have a greatly increased risk for developing colon cancer before the age of 50. Likewise, women who inherit one mutant allele of BRCA1, another tumor-suppressor gene, have a 60 percent probability of developing breast cancer by age 50, whereas those who inherit two normal BRCA1 alleles have a 2 percent probability of doing so. In women with hereditary breast cancer, loss of the second BRCA1 allele, together with other mutations, is required for a normal breast duct cell to become malignant. However, BRCA1 generally is not mutated in sporadic, noninherited breast cancer.

Loss of Heterozygosity Clearly, then, we can inherit a propensity to cancer by receiving a damaged allele of a tumor-suppressor gene from one of our parents; that is, we are heterozygous for the mutation. That in itself will not cause cancer, since the remaining normal allele prevents aberrant growth; the cancer is recessive. Subsequent loss or inactiva-tion of the normal allele in a somatic cell, referred to as loss of heterozygosity (LOH), is a prerequisite for cancer to develop. One common mechanism for LOH involves mis-segregation during mitosis of the chromosomes bearing the affected tumor-suppressor gene (Figure 23-11a). This process, also referred to as nondisjunction, is caused by failure of the spindle-assembly checkpoint, which normally prevents a metaphase cell with an abnormal mitotic spindle from completing mitosis (see Figure 21-32, 2). Another possible mechanism for LOH is mitotic recombination between a chromatid bearing the wild-type allele and a homologous chromatid bearing a mutant allele. As illustrated in Figure 23-11b, subsequent chromosome segregation can generate a daughter cell that is homozygous for the mutant tumor-suppressor allele. A third mechanism is the deletion or mutation of the normal copy of the tumor-suppressor gene; such a deletion can encompass a large chromosomal region and need not be a precise deletion of just the tumor-suppressor gene.

Hereditary cancers constitute about 10 percent of human cancers. It is important to remember, however, that the inherited, germ-line mutation alone is not sufficient to cause tumor development. In all cases, not only must the inherited normal tumor-suppressor allele be lost or inactivated, but mutations affecting other genes also are necessary for cancer to develop. Thus a person with a recessive tumor-suppressor-gene mutation can be exceptionally susceptible to environmental mutagens such as radiation.

(a) Mis-segregation

Normal allele

Normal allele

- Mutant allele

Heterozygous for mutant allele

- Mutant allele

Heterozygous for mutant allele

Chromosome duplication

Chromosome duplication

Aberrant 3:1 segregation

Dies

Aberrant 3:1 segregation

Dies

Heterozygous for mutant allele

Random loss of extra chromosome

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