Cancer Arises from Genetic Dysfunction in the Regulation of the Cell Cycle, Cell Life Span, and Cell Suicide

Cancer is a genetic disease (but not usually a hereditary disease) and a uniquely cellular disease. As shown in Figure 2-1, tumors and other cancers arise from the division of a single mutant cell whose descendants accumulate several additional mutations to become increasingly damaged with respect to control of cellular proliferation, life span, and cell death. That is, cancer is a genetic disease caused by the accumulation of mutations in body cells, such as those of the epithelia lining the lungs or the secretory epithelia of the mammary glands.

All the cells of a tumor can trace their ancestry back to a single cell that developed an initial deleterious mutation. This first mutation usually occurs in a gene controlling proliferation, such that the cell produces a mutant protein¹ that is a dysfunctional, more permissive regulator of the cell cycle. This greater “permissiveness” provides the mutant cell with more opportunity to proliferate, and it thus has a selective advantage compared with its normal neighbors. Perhaps because of this selective advantage, or because of continued exposure to mutagens (e.g., cigarette smoke, agricultural chemicals), a descendant of this cell accumulates another mutation that also affects some aspect of the cell cycle or cell death. This increases the doubly mutant cell’s selective advantage further still, and the downward spiral of increasingly abnormal, dividing cells begins to spin out of control. Scientists agree that this accumulation of mutations in individual genes is necessary for cancer to develop, but some think it is not sufficient. Rather, they argue that cancer only results when the accumulation of mutations eventually leads to large-scale genetic instability, such that whole chromosomes are gained and lost. The majority of spontaneous tumors do have cells with abnormal sets of chromosomes, a phenomenon called aneuploidy. Whether aneuploidy is necessary for cancer remains to be seen, but there is no disagreement that cancer cells are in some way badly damaged with respect to genes controlling growth.

The mutations leading to cancer are the same type as those that underlie Mendel’s familiar laws of heredity. These include base-pair changes, deletions or additions of nucleotides in the gene, and translocation of one piece of a chromosome to another. However, it is important to understand that the cells in which the mutations are occurring are different than those underlying Mendel’s laws of inheritance. Mendelian inheritance results from mutations occurring in the germ line of the organism. These are the cells that will become gametes, either sperm or eggs, and whose deoxyribonucleic acid (DNA) will be passed down to every cell of the offspring. The mutations leading to cancer are occurring in nonreproductive cells throughout the body, called somatic cells. These are passed down only to a limited number of other somatic cells by cell division, not to offspring through sexual reproduction. Thus, although cancer is a genetic disease, only about 10% of the time is it a “hereditary disease,” that is, the result of mutation inherited from a parent. In general, cancer appears to be the result of the accumulation of mutations leading to genetic instability in a particular lineage of somatic cells.

Traditionally, cancers are divided into categories based on the cell type involved. Carcinomas are cancers of epithelial cells; sarcomas are derived from connective tissue or muscle; and leukemias are cancers of blood-forming cells. There are many subdivisions based on specific cell types and location of the tumors. However, these names are traditional only; they do not reflect any fundamental differences in the biology of the cancer. Rather, it is now clear that cancers of all types share broadly similar types of dysfunctions controlling cell proliferation, cell suicide, and cell life span.

Cyclin-Dependent Kinases Are the “Engines” Driving the Cell Cycle

Recall from Chapter 1 that protein kinases, which are enzymes that phosphorylate other proteins, are important as elements of signaling pathways. For example, the second messenger cyclic adenosine monophosphate (cAMP) acts by activating protein kinase A (see Figure 1-18), and diacylglycerol as a second messenger activates protein kinase C (see Figure 1-19). Protein kinases play a major role in many aspects of control of the cell cycle; most importantly, CDKs, when activated, can directly cause a cell to enter either S phase or mitosis, whether the cell is ready or not.

Active CDKs are composed of two different types of protein subunits (Figure 2-3). The catalytic subunits (numbered CDK1, CDK2, etc.) are the subunits that have enzymatic activity for hydrolyzing adenosine triphosphate (ATP) and transferring the phosphate group to a protein substrate. The other subunit is an activator of the catalytic subunit and is called a cyclin; the abundance of this protein increases and decreases during the cell cycle (i.e., the protein concentration cycles up and down during the cell cycle). Different cyclins are specific for various CDKs and for the different phases of the cell cycle. The various cyclins are identified by letters, such as cyclin A and cyclin B. Cyclins must reach a threshold concentration to activate the catalytic subunit, and the threshold is achieved as a result of protein accumulation from new synthesis during the G phases.

When the cyclins have bound to their appropriate catalytic subunit, the cyclin-CDK complex as a whole is activated by achieving a particular state of phosphorylation. There are inhibitory sites of phosphorylation around amino acid 15 of the catalytic subunit, and these must be dephosphorylated. There is also a stimulatory phosphorylation site at amino acid 167, and this must be phosphorylated for cyclin-CDK activity. When activated, the CDK phosphorylates various substrates associated with either S phase or M phase. For example, the cyclin-CDK complex responsible for mitosis directly phosphorylates the protein filaments that strengthen the nuclear membrane (lamins). This phosphorylation causes the filaments to disassemble, in turn allowing the nuclear membrane to dissolve, which is an early event of mitosis.

The different phases of the cell cycle are controlled by different cyclin-CDK pairs, as shown in Figure 2-4. Thus the complex of CDK1 with either cyclin B or cyclin A is the particular CDK pair responsible for driving the cell into mitosis. Cyclins E and A interacting with CDK2 play important roles in initiating and maintaining DNA synthesis in S phase. Cyclin D interacting with either CDK4 or CDK6 functions in late G1 in a “decision” by the cell to commit to DNA synthesis. This decision is called the restriction (R) point and is discussed in the later section on tumor suppressors.

Given the importance of cyclins and CDKs in driving the cell cycle, one would expect they would have some connection to cancer. Overexpression of cyclin D is associated with human and mouse breast cancer, and ablation of cyclin D provides some protection against breast cancer in mice. Virtually all multiple myelomas, a type of leukemia, show overexpression of cyclin D. Overexpression of cyclin A is strongly associated with some lung cancers and with testicular cancer of humans, and overexpression of cyclin E is associated with certain human leukemias. Curiously, in contrast to the cyclin subunit, the CDK enzymatic subunit is not known to be mutant in any common cancer.

The CDK “Engines” Are Controlled by Both Throttle (Oncogene) and Brake (Tumor Suppressor) Controls

The CDK-cyclin pairs are controlled by both stimulatory and inhibitory pathways, analogous to an automobile engine controlled by throttle and brake mechanisms. The throttle mechanisms are largely the result of the cell’s environmental inputs. That is, various environmental cues, both soluble signal molecules and insoluble molecules found in tissue, are required for cells to divide. However, the pathways sending inhibitory signals to the cell cycle, the “brakes” for cell division, are largely internal and are activated by damage or stress to the cell. In general, these inhibitory signals are like the safety interlocks on an automobile. Just as one cannot start a car in gear, so the cell should not divide if DNA synthesis has not exactly duplicated all the genes and chromosomes, or if something is wrong with the mitotic spindle.

The environmental stimulatory signals for cell division can be as simple and nonspecific as availability of nutrients, to the extent that cells only divide when they have approximately doubled in size through synthetic growth. However, two more specific stimulators of the cell cycle are primarily implicated in cancer. One is the response to soluble growth factors found in the circulation and in the extracellular fluid surrounding cells (see Chapter 1). Growth factors are proteins secreted by a variety of other cell types that are required for the division, and indeed survival, of normal, noncancerous cells. Cancer cells, however, can divide and survive with little or no stimulation from growth factors because of the acquired ability to synthesize growth factors of their own, or the activation of downstream elements in the signaling pathway.

The second stimulatory pathway of general importance in cancer is cell attachment. The cells of multicellular organisms must be tightly attached to one another and to their surrounding matrix, similar to tendon; otherwise we would be jelly, juice, and bubbles on the floor. Also, however, attachment of cells to their surroundings is a source of specific and complex information to the physiology of the cell. One of the most important such messages is a “permissive” signal to divide. Normal cells must be anchored to some substrate in order to respond to other signals to divide. That is, most normal animal cells show anchorage dependence of growth. For this reason, vertebrate cells in culture are grown on the surface of a dish or flask, not in suspension the way bacteria are cultured. Again, cancer cells have lost this normal restriction on proliferation, and many cancer cells can divide and survive in suspension. The common test for the absence of anchorage dependence is growth in soft agar: cancer cells will, but normal cells will not, divide and form colonies when suspended in soft agar. Thus, cancer cells can survive unattached while riding the circulation to relocate in a different tissue than that of the original tumor. In this way, cancer is able to spread through the body, a process called metastasis, which is ultimately the cause of death in most cases of cancer.

The Rube Goldberg pathways that underlie the proliferative signals of growth factors and adhesion are similar and intersect. These “throttle” contraptions begin with a soluble signal binding to a growth factor receptor and a “solid-state” signal about attachment to the surrounding tissue. However, both pathways quickly converge on the same stimulation pathway for conserved cell division. These stimulatory pathways are driven by proteins that were originally identified as being encoded by genes in viruses that caused cancer in animals. Thus these were named oncogenes, literally “cancer genes.” A major breakthrough came with the discovery that these oncogenes were actually derived from the host genome, not genes normally encoded in the virus. That is, viruses had stolen cell cycle control genes from their animal host cell. Being viruses, they did not take good care of the animal cell cycle genes they stole. The stolen genes mutated into deranged cell cycle regulators. Subsequently, the same mutant genes that were found in viruses were found to explain many spontaneous cancers in humans and in the long-used experimental tumors of mice. The finding that cancer was caused by abnormal host genes helped confirm that cancer was a somatic genetic disease due to mutations in the tumor cells.

Further analysis revealed that these oncogenes often encode normal stimulators of the cell cycle, and the mutations involved had the effect of permanently activating an element in the cell cycle pathway. You can see how this would work based on the Rube Goldberg cartoon of Figure 1-13. Note that all the elements in the garage door opener are stimulatory; if any one turns “on,” a signal is sent “downstream” to cause the garage door to open. If the fish tank of the cartoon were to “mutate” by developing a leak, an “on” signal would be sent downstream of the fish tank, regardless of whether a car had pulled into the driveway. So it is with the oncogene elements controlling the cell cycle. If one of the elements mutates to turn itself “on,” that is, acquired a gain-of-function mutation, it will stimulate cell division and contribute to cancer. To return to the automobile analogy, oncogenes represent a stuck throttle or accelerator pedal. The normal, well-behaved versions of the oncogene (a watertight fish tank before the bullet, Figure 1-13) are called proto-oncogenes. Thus, strictly speaking, oncogenes have their normal equivalent as proto-oncogenes. However, given this awkward usage, increasingly the normal versions are also informally called oncogenes, and it is usually clear from the context whether the mutant or normal version is being discussed. The molecules and molecular events of the oncogene pathway (also called the growth factor or MAP kinase pathway) are discussed later.

The mechanisms to stop the cell cycle, the “brakes,” are called checkpoints. Progress through the cell cycle depends on appropriate conditions being reached within the cell before a “decision” is made to go ahead with division. The first such checkpoint occurs before S phase. During G1, the cell checks itself over particularly with respect to DNA damage. The cell has sophisticated pathways to detect and repair DNA damage, such as mismatched bases detected in the double helix. For needed repairs to take place, however, DNA synthesis is delayed; the checkpoint is “engaged.” If the DNA is properly repaired, the checkpoint is disengaged, and after the delay, the cell goes ahead into S phase. However, if the DNA damage cannot be repaired, the checkpoint machinery is supposed to signal a more serious consequence. If the checkpoint is not disengaged after about a day, the cell “commits suicide.” Thus the checkpoint (or braking machinery) is tied into both the CDK engines and the processes of cell suicide, as described later. Similarly, the second checkpoint is in mitosis and checks for proper mitotic spindle assembly and correct chromosome alignment. Here again, if damage is detected, there are repair mechanisms, and a properly repaired cell will go into M phase after a delay for repair. If no repair can be made, the cell commits suicide.

The molecules and their interactions that underlie both oncogene (“throttle”) pathways and checkpoint (“brake”) pathways are now covered in greater detail, beginning with the role of growth factors.

The Ras Oncogene Contributes to Many Cancers and Serves as a Model for Understanding Small G Proteins

After activation of the RTK, the next major step in the growth factor/oncogene pathway in normal cells is activation of the protein product of the ras proto-oncogene. Investigations of how it worked revealed that the Ras protein was an important member of the small G-protein family of molecular regulators, all of which have intrinsic guanosinetriphosphatase (GTPase) activity and serve as molecular “on-off switches.” These proteins control many basic cellular functions, and the heterotrimeric G protein evolved from Ras-like ancestor proteins (see Chapter 1). Indeed, in yeast it is Ras, not a heterotrimeric G protein, that controls adenyl cyclase and phospholipase C (see Figure 1-18). Figure 2-6 illustrates the duty cycle of this on-off switch and its basic similarity to the alpha subunit (G_α) of the heterotrimeric G proteins. Ras, other small G proteins, and G_α all are in the “on” state when they have guanosine triphosphate (GTP) bound to them (because of receptor activation). All are in the “off” state when the G protein hydrolyzes its GTP so that guanosine diphosphate (GDP) is now bound. You can see how this gene could be discovered as an oncogene, that is, a gene in which a gain-of-function mutation contributes to the development of cancer. If the GTPase activity is lost by mutation, this simple, enzymatic on-off switch remains trapped in the “on” position (the accelerator pedal is stuck). It continues to send an activating signal to the downstream cell cycle machinery without the presence of growth factors or the activation of RTKs. In fact, such mutations in Ras underlie its oncogenic function, and it is estimated that 30% of human cancers have activating mutations in their ras gene.

Other small G proteins control a myriad of cellular functions, including others involved in cancer. Thus the Rho subfamily of small G proteins is directly involved in the spread of cancer because it helps regulate actin assembly and activity. As described later, the spread of cancer depends on the ability of cells to migrate through tissues. This “crawling” motility in turn depends on a musclelike mechanism based on actin and myosin (see Figure 1-4). Although the basic, on-off activity of Ras and Rho are the same as that shown in Figure 2-6, Rho is connected to actin, whereas active Ras activates the elements of the MAP kinase pathway.

The MAP Kinase Pathway Leads to the Expression of Cyclins and Other Stimulators of the Cell Cycle

GTP-bound Ras causes the sequential activation of a series of protein kinases, called Raf, Mek, and Erk. Raf phosphorylates and activates Mek, which in turn phosphorylates and activates Erk, as shown in Figure 2-5. This trio of kinases is called the mitogen-activated protein kinase, or MAP kinase, pathway (a mitogen is a stimulator of mitosis, e.g., a growth factor). If any of these three protein kinases should experience a gain-of-function mutation irreversibly activating the protein kinase, a stimulatory signal is sent down the remainder of the pathway. Thus, as with ras, these three kinase genes act as oncogenes.

One important example of a gain-of-function mutation among the three MAP kinases involves the first of these MAP kinases, Raf. A single–amino-acid mutation in the kinase domain of Raf (a substitution of glutamate for normal valine at amino acid 600) causes permanent activation of Raf in approximately 50% of human melanomas, a very deadly cancer, and is also common in thyroid cancers. As described for mutations in Ras, activation of Raf sends an unregulated stimulatory signal downstream to the other MAP kinases, leading to unregulated proliferation of the cancer cells. Recent clinical progress involving melanoma illustrates the importance of understanding which particular mutations are involved in a given patient’s cancer. A newly developed drug, vemurafenib, targets the mutant Raf and significantly prolongs the life span of those melanoma patients harboring this raf mutation, but has no effect in cases of melanoma with normal Raf/raf.

Raf, Mek, and Erk are a specific example of yet another conserved but diverse general module of information transduction. There are MAP kinase trios other than Raf, Mek, and Erk. Although it is not worthwhile to give names to all the various specific pathways, it should be noted that these trios have a systematic set of names for their elements. Raf is a MAP kinase, kinase, kinase (a MAPKKK). Mek is a MAP kinase, kinase (MAPKK), and Erk protein is the MAP kinase (MAPK) itself. This jargon is awkward, but it is widely used and logical, as Figure 2-5 suggests.

When activated, Erk activates one or more transcription factors that control the transcription and translation of a key regulator of the cyclin-CDK engine. One of these transcription factors, Myc (“mick”), is encoded by another important oncogene/proto-oncogene. As with ras, the myc gene is mutated in a high frequency of human tumors, giving rise to an oncogenic form able to activate the cell cycle. As shown in Figure 2-5, Myc protein is involved in the transcription of a variety of cyclins and of the CDK2 catalytic subunit and plays a significant role in allowing the cell to pass from G1 to S phase. Myc is also involved in many other transcription events related to cell growth, differentiation, and cancer.

This completes the growth stimulatory pathway beginning with a growth factor binding to its RTK receptor that, through Ras, a MAP kinase cascade, and a transcription factor, eventually leads to a direct “throttling up” of a cyclin-CDK engine. This same pathway is used similarly to transduce the information about the other major stimulator of cell division, cell attachment.

The MAP Kinase Pathway also Mediates the Stimulation of the Cell Cycle by Cell Adhesion

As noted earlier, the other major throttle mechanism to regulate the cyclin-CDK engines of the cell cycle is cell adhesion. Cell adhesion, as with growth factor stimulation, ultimately stimulates cyclin-CDK pairs through the MAP kinase pathway. Two types of cell contact are involved in normal growth and proliferation. The most obvious is cell-cell adhesion; most cells are tightly attached to their neighboring cells. The second type is cell adhesion to an extracellular matrix (ECM) of fibrous proteins. Eighty percent of human and mouse cancers arise from epithelial cells (carcinomas), and all epithelial layers are attached to an ECM. The adhesion proteins that bind to other cells or to the ECM are adhesion receptors. Adhesion receptors are responsible for the mechanical aspect of attachment, but also act similar to other receptors in transducing information across the plasma membrane. In this case, adhesion receptors communicate the information that the cell is anchored and can divide.

Both cell-cell and cell-ECM adhesion activate the MAP kinase pathway, similar to growth factors, but the Ras intermediate is less important here. Figure 2-7 shows the activation of the MAP kinase pathway as a result of cell-ECM adhesion. The adhesion receptors that bind to ECM are called integrins and these activate the MAP kinase pathway via two important intermediates that are oncogenes. One is Src (“sark”), a protein tyrosine kinase and the first oncogene (src) to be discovered. Unlike the RTKs previously described, Src is not a receptor. However, Src is located on the inside face of the plasma membrane, where it can interact with adhesion receptors. Another important intermediate is also a protein tyrosine kinase, called Fak (focal adhesion kinase). As before, activation of Src and Fak activate the MAP kinase pathway, leading to increased cell division. Again, mutation or overexpression of src and fak sends inappropriate stimulation to the cell cycle machinery, which facilitates cancer. As mutant oncogenes, fak is associated with aggressive melanomas in humans. The src oncogene was named because of its ability to cause sarcomas in chickens.