23.1 Tumor Cells and the Onset of Cancer
23.2 The Genetic Basis of Cancer
23.3 Oncogenic Mutations in Growth-Promoting Proteins
23.4 Mutations Causing Loss of Growth-Inhibiting and Cell-Cycle Controls
23.5 The Role of Carcinogens and DNA Repair in Cancer
▲ FIGURE 23-1 Overview of changes in cells that cause cancer. During carcinogenesis, six fundamental cellular properties are altered, as shown here, to give rise to the complete, most destructive cancer phenotype. Less dangerous tumors arise when only some of these changes occur. In this chapter we examine the genetic changes that result in these altered cellular properties. [Adapted from D. Hanahan and R. A. Weinberg, 2000, Cell 100:57.]
Metastasis is a complex process with many steps. Invasion of new tissues is nonrandom, depending on the nature of both the metastasizing cell and the invaded tissue. Metastasis is facilitated if the tumor cells produce growth and an-giogenesis factors (blood vessel growth inducers). Motile, invasive, aggregating, deformable cells are most dangerous. Tissues under attack are most vulnerable if they produce growth factors and readily grow new vasculature. They are more resistant if they produce anti-proliferative factors, inhibitors of proteolytic enzymes, and anti-angiogenesis factors.
Research on the genetic foundations of a particular type of cancer often begins by identifying one or more genes that are mutationally altered in tumor cells. Subsequently it is important to learn whether an altered gene is a contributing cause for the tumor, or an irrelevant side event. Such investigations usually employ multiple approaches: epidemiolog-ical comparisons of the frequency with which the genetic change is associated with a type of tumor, tests of the growth properties of cells in culture that have the particular mutation, and the testing of mouse models of the disease to see if the mutation can be causally implicated. A more sophisticated analysis is possible when the altered gene is known to encode a component of a particular molecular pathway (e.g., an intracellular signaling pathway). In this case it is possible to alter other components of the same pathway and see whether the same type of cancer arises.
Because the multiple mutations that lead to formation of a tumor may require many years to accumulate, most cancers develop later in life. The occurrence of cancer after the age of reproduction may be one reason that evolutionary restraints have not done more to suppress cancer. The requirement for multiple mutations also lowers the frequency of cancer compared with what it would be if tumorigenesis were triggered by a single mutation. However, huge numbers of cells are, in essence, mutagenized and tested for altered growth during our lifetimes, a sort of evolutionary selection for cells that proliferate. Fortunately the tumor itself is not inherited.
Before examining in detail the genetic basis of cancer, we consider the properties of tumor cells that distinguish them from normal cells and the general process of oncogenesis. The genetic changes that underlie oncogenesis alter several fundamental properties of cells, allowing cells to evade normal growth controls and ultimately conferring the full cancer phenotype (see Figure 23-1). Cancer cells acquire a drive to proliferate that does not require an external inducing signal. They fail to sense signals that restrict cell division and continue to live when they should die. They often change their attachment to surrounding cells or the extracellular matrix, breaking loose to divide more rapidly. A cancer cell may, up to a point, resemble a particular type of normal, rapidly dividing cell, but the cancer cell and its progeny will exhibit inappropriate immortality. To grow to more than a small size, tumors must obtain a blood supply, and they often do so by signaling to induce the growth of blood vessels into the tumor. As cancer progresses, tumors become an abnormal organ, increasingly well adapted to growth and invasion of surrounding tissues.
Tumors arise with great frequency, especially in older individuals, but most pose little risk to their host because they are localized and of small size. We call such tumors benign; an example is warts, a benign skin tumor. The cells composing benign tumors closely resemble, and may function like, normal cells. The cell-adhesion molecules that hold tissues together keep benign tumor cells, like normal cells, localized to the tissues where they originate. A fibrous capsule usually delineates the extent of a benign tumor and makes it an easy target for a surgeon. Benign tumors become serious medical problems only if their sheer bulk interferes with normal functions or if they secrete excess amounts of biologically active substances like hormones. Acromegaly, the overgrowth of head, hands, and feet, for example, can occur when a benign pituitary tumor causes overproduction of growth hormone.
In contrast, cells composing a malignant tumor, or cancer, usually grow and divide more rapidly than normal, fail to die at the normal rate (e.g., chronic lymphocytic leukemia, a tumor of white blood cells), or invade nearby tissue without a significant change in their proliferation rate (e.g., less
▲ FIGURE 23-2 Gross and microscopic views of a tumor invading normal liver tissue. (a) The gross morphology of a human liver in which a metastatic lung tumor is growing. The white protrusions on the surface of the liver are the tumor masses. (b) A light micrograph of a section of the tumor in (a) showing areas of small, dark-staining tumor cells invading a region of larger, light-staining, normal liver cells. [Courtesy of J. Braun.]
harmful tumors of glial cells). Some malignant tumors, such as those in the ovary or breast, remain localized and encapsulated, at least for a time. When these tumors progress, the cells invade surrounding tissues, get into the body's circulatory system, and establish secondary areas of proliferation, a process called metastasis. Most malignant cells eventually acquire the ability to metastasize. Thus the major characteristics that differentiate metastatic (or malignant) tumors from benign ones are their invasiveness and spread.
Cancer cells can often be distinguished from normal cells by microscopic examination. They are usually less well differentiated than normal cells or benign tumor cells. In a specific tissue, malignant cells usually exhibit the characteristics of rapidly growing cells, that is, a high nucleus-to-cytoplasm ratio, prominent nucleoli, and relatively little specialized structure. The presence of invading cells in an otherwise normal tissue section is used to diagnose a malignancy (Figure 23-2).
Normal cells are restricted to their place in an organ or tissue by cell-cell adhesion and by physical barriers such as the basal lamina, which underlies layers of epithelial cells and also surrounds the endothelial cells of blood vessels (Chapter 6). Cancer cells have a complex relation to the extracellular matrix and basal lamina. The cells must degrade the basal lamina to penetrate it and metastasize, but in some cases cells may migrate along the lamina. Many tumor cells secrete a protein (plasminogen activator) that converts the serum protein plas-minogen to the active protease plasmin. Increased plasmin activity promotes metastasis by digesting the basal lamina, thus allowing its penetration by tumor cells. As the basal lamina disintegrates, some tumor cells will enter the blood, but fewer than 1 in 10,000 cells that escape the primary tumor survive to colonize another tissue and form a secondary, metastatic tumor. In addition to escaping the original tumor and entering the blood, cells that will seed new tumors must then adhere to an endothelial cell lining a capillary and migrate across or through it into the underlying tissue. The multiple crossings of tissue layers that underlie malignancy often involve new or variant surface proteins made by malignant cells.
In addition to important changes in cell-surface proteins, drastic changes occur in the cytoskeleton during tumor-cell formation and metastasis. These alterations can result from changes in the expression of genes encoding Rho and other small GTPases that regulate the actin cytoskeleton (Chapter 19). For instance, tumor cells have been found to over-express the RhoC gene, and this increased activity stimulates metastasis.
In order for most oncogenic mutations to induce cancer, they must occur in dividing cells so that the mutation is passed on to many progeny cells. When such mutations occur in nondividing cells (e.g., neurons and muscle cells), they generally do not induce cancer, which is why tumors of muscle and nerve cells are rare in adults. Nonetheless, cancer can occur in tissues composed mainly of nondividing differentiated cells such as erythrocytes and most white blood cells, absorptive cells that line the small intestine, and keratinized cells that form the skin. The cells that initiate the tumors are not the differentiated cells, but rather their precursor cells. Fully differentiated cells usually do not divide. As they die or wear out, they are continually replaced by proliferation and differentiation of stem cells, and these cells are capable of transforming into tumor cells.
In Chapter 22, we learned that stem cells both perpetuate themselves and give rise to differentiating cells that can regenerate a particular tissue for the life of an organism (see Figure 22-2). For instance, many differentiated blood cells have short life spans and are continually replenished from hematopoietic (blood-forming) stem cells in the bone marrow (see Figure 22-5). Populations of stem cells in the intestine, liver, skin, bone, and other tissues likewise give rise to all or many of the cell types in these tissues, replacing aged and dead cells, by pathways analogous to hematopoiesis in bone marrow. Similarly within a tumor there may be only certain cells with the ability to divide uncontrollably and generate new tumors; such cells are tumor stem cells.
Because stem cells can divide continually over the life of an organism, oncogenic mutations in their DNA can accumulate, eventually transforming them into cancer cells. Cells that have acquired these mutations have an abnormal pro-liferative capacity and generally cannot undergo normal processes of differentiation. Many oncogenic mutations, such as ones that prevent apoptosis or generate an inappropriate growth-promoting signal, also can occur in more differentiated, but still replicating, progenitor cells. Such mutations in hematopoietic progenitor cells can lead to various types of leukemia.
Normal animal cells are often classified according to their embryonic tissue of origin, and the naming of tumors has followed suit. Malignant tumors are classified as carcinomas if they derive from endoderm (gut epithelium) or ectoderm (skin and neural epithelia) and sarcomas if they derive from mesoderm (muscle, blood, and connective tissue precursors). The leukemias, a class of sarcomas, grow as individual cells in the blood, whereas most other tumors are solid masses. (The name leukemia is derived from the Latin for "white blood": the massive proliferation of leukemic cells can cause a patient's blood to appear milky.)
Tumor Growth Requires Formation of New Blood Vessels
Tumors, whether primary or secondary, require recruitment of new blood vessels in order to grow to a large mass. In the
▲ EXPERIMENTAL FIGURE 23-3 Scanning electron micrographs reveal the organizational and morphological differences between normal and transformed 3T3 cells.
(a) Normal 3T3 cells are elongated and are aligned and closely packed in an orderly fashion. (b) 3T3 cells transformed by an oncogene encoded by Rous sarcoma virus are rounded and covered with small hairlike processes and bulbous projections.
absence of a blood supply, a tumor can grow into a mass of about 106 cells, roughly a sphere 2 mm in diameter. At this point, division of cells on the outside of the tumor mass is balanced by death of those in the center due to an inadequate supply of nutrients. Such tumors, unless they secrete hormones, cause few problems. However, most tumors induce the formation of new blood vessels that invade the tumor and nourish it, a process called angiogenesis. This complex process requires several discrete steps: degradation of the basal lamina that surrounds a nearby capillary, migration of endothelial cells lining the capillary into the tumor, division of these endothelial cells, and formation of a new basement membrane around the newly elongated capillary.
Many tumors produce growth factors that stimulate angiogenesis; other tumors somehow induce surrounding normal cells to synthesize and secrete such factors. Basic fibroblast growth factor (bFGF), transforming growth factor a (TGFa), and vascular endothelial growth factor (VEGF), which are secreted by many tumors, all have angiogenic properties. New blood vessels nourish the growing tumor, allowing it to increase in size and thus increase the probability that additional harmful mutations will occur. The presence of an adjacent blood vessel also facilitates the process of metastasis.
Several natural proteins that inhibit angiogenesis (e.g., angiogenin and endostatin) or antagonists of the VEGF receptor have excited much interest as potential therapeutic agents. Although new blood vessels are constantly forming during embryonic development, few form normally in adults except after injury. Thus a specific inhibitor of angiogenesis not only might be effective against many kinds of tumors but also might have few adverse side effects. I
The transformed cells that grow have lost the side-by-side organization of the normal cells and grow one atop the other. These transformed cells have many of the same properties as malignant cells. Similar changes are seen in cells transfected with DNA from human cancers containing the rasD oncogene. [Courtesy of L.-B. Chen.]
Cultured Cells Can Be Transformed into Tumor Cells
The morphology and growth properties of tumor cells clearly differ from those of their normal counterparts; some of these differences are also evident when cells are cultured. That mutations cause these differences was conclusively established by transfection experiments with a line of cultured mouse fi-broblasts called 3T3 cells. These cells normally grow only when attached to the plastic surface of a culture dish and are maintained at a low cell density. Because 3T3 cells stop growing when they contact other cells, they eventually form a monolayer of well-ordered cells that have stopped proliferating and are in the quiescent G0 phase of the cell cycle (Figure 23-3a).
When DNA from human bladder cancer cells is trans-fected into cultured 3T3 cells, about one cell in a million incorporates a particular segment of the exogenous DNA that causes a distinctive phenotypic change. The progeny of the affected cell are more rounded and less adherent to one another and to the dish than are the normal surrounding cells, forming a three-dimensional cluster of cells (a focus) that can be recognized under the microscope (Figure 23-3b). Such cells, which continue to grow when the normal cells have become quiescent, have undergone onco-genic transformation. The transformed cells have properties similar to those of malignant tumor cells, including changes in cell morphology, ability to grow unattached to an extracellular matrix, reduced requirement for growth factors, secretion of plasminogen activator, and loss of actin microfilaments.
Figure 23-4 outlines the procedure for transforming 3T3 cells with DNA from a human bladder cancer and cloning the specific DNA segment that causes transformation. It was remarkable to find a small piece of DNA with this capability; had more than one piece been needed, the experiment would
► EXPERIMENTAL FIGURE 23-4 Transformation of mouse cells with DNA from a human cancer cell permits identification and molecular cloning of the rasP oncogene.
Addition of DNA from a human bladder cancer to a culture of mouse 3T3 cells causes about one cell in a million to divide abnormally and form a focus, or clone, of transformed cells. To clone the oncogene responsible for transformation, advantage is taken of the fact that most human genes have nearby repetitive DNA sequences called Alu sequences. DNA from the initial focus of transformed mouse cells is isolated, and the oncogene is separated from adventitious human DNA by secondary transfer to mouse cells. The total DNA from a secondary transfected mouse cell is then cloned into bacteriophage X; only the phage that receives human DNA hybridizes with an Alu probe. The hybridizing phage should contain part of or all the transforming oncogene. This expected result can be proved by showing either that the phage DNA can transform cells (if the oncogene has been completely cloned) or that the cloned piece of DNA is always present in cells transformed by DNA transfer from the original donor cell.
Extract DNA, transform new mouse cells
Extract DNA, transform new mouse cells
Extract genomic DNA
Surviving human DNA | Introduce into phage vector
Extract genomic DNA
Surviving human DNA | Introduce into phage vector
Phage library /
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