Neoplasia

Benign Tumors

In general, benign tumors are designated by attaching the suffix -oma to the cell type from which the tumor arises. For example, a benign tumor arising in fibrous tissue is a fibroma; a benign cartilaginous tumor is a chondroma. More varied and complex nomenclature is applied to benign epithelial tumors. The term adenoma is generally applied not only to benign epithelial neoplasms that produce glandlike structures, but also to benign epithelial neoplasms that are derived from glands but lack a glandular growth pattern. Thus, a benign epithelial neoplasm arising from renal tubule cells and growing in a glandlike pattern is termed an adenoma, as is a mass of benign epithelial cells that produces no glandular patterns but has its origin in the adrenal cortex. Papillomas are benign epithelial neoplasms, growing on any surface, that produce microscopic or macroscopic fingerlike fronds. A polyp is a mass that projects above a mucosal surface, as in the gut, to form a macroscopically visible structure (Fig. 6.1). Although this term commonly is used for benign tumors, some malignant tumors also may grow as polyps, whereas other polyps (such as nasal polyps) are not neoplastic but inflammatory in origin. Cystadenomas are hollow cystic masses that typically arise in the ovary.

Malignant Tumors

The nomenclature of malignant tumors essentially follows that of benign tumors, with certain additions and exceptions.

The transformed cells in a neoplasm, whether benign or malignant, usually resemble each other, consistent with their origin from a single transformed progenitor cell. In some unusual instances, however, the tumor cells undergo divergent differentiation, creating so-called “mixed tumors”. Mixed tumors are still of monoclonal origin, but the progenitor cell in such tumors has the capacity to differentiate down more than one lineage. The best example is mixed tumor of salivary gland. These tumors have obvious epithelial components dispersed throughout a fibromyxoid stroma, sometimes harboring islands of cartilage or bone (Fig. 6.2). All of these diverse elements are thought to derive from a single transformed epithelial progenitor cell, and the preferred designation for these neoplasms is pleomorphic adenoma. Fibroadenoma of the female breast is another common mixed tumor. This benign tumor contains a mixture of proliferating ductal elements (adenoma) embedded in loose fibrous tissue (fibroma). Unlike pleomorphic adenoma, only the fibrous component is neoplastic, but the term fibroadenoma remains in common usage.

Teratoma is a special type of mixed tumor that contains recognizable mature or immature cells or tissues derived from more than one germ cell layer, and sometimes all three. Teratomas originate from totipotential germ cells such as those that normally reside in the ovary and testis and that are sometimes abnormally present in midline embryonic rests. Germ cells have the capacity to differentiate into any of the cell types found in the adult body; not surprisingly, therefore, they may give rise to neoplasms that contain elements resembling bone, epithelium, muscle, fat, nerve, and other tissues, all thrown together in a helter-skelter fashion.

The specific names of the more common neoplasms are presented in Table 6.1. Some glaring inconsistencies may be noted. For example, the terms lymphoma, mesothelioma, melanoma, and seminoma are used for malignant neoplasms. Unfortunately for students, these exceptions are firmly entrenched in medical terminology.

There also are other instances of confusing terminology:

Although the terminology of neoplasms is regrettably complex, an understanding of the nomenclature is important because it is the language by which a tumor's nature and significance is communicated among physicians in different disciplines involved in cancer care.

Differentiation and Anaplasia

Differentiation refers to the extent to which neoplasms resemble their parenchymal cells of origin, both morphologically and functionally; lack of differentiation is called anaplasia. In general, benign neoplasms are composed of well-differentiated cells that closely resemble their normal counterparts. A lipoma is made up of mature fat cells laden with cytoplasmic lipid vacuoles, and a chondroma is made up of mature cartilage cells that synthesize their usual cartilaginous matrix—evidence of morphologic and functional differentiation. In well-differentiated benign tumors, mitoses are usually rare and are of normal configuration.

By contrast, while malignant neoplasms exhibit a wide range of parenchymal cell differentiation, most exhibit morphologic alterations that betray their malignant nature. In well-differentiated cancers, these features may be quite subtle (Fig. 6.3). For example, well-differentiated adenocarcinoma of the thyroid gland may contain normal-appearing follicles, its malignant potential being only revealed by invasion into adjacent tissues or metastasis. The stroma carrying the blood supply is crucial to the growth of tumors but does not aid in the separation of benign from malignant ones. The amount of stromal connective tissue does, however, determine the consistency of a neoplasm. Certain cancers induce a dense, abundant fibrous stroma (desmoplasia), making them hard, so-called “scirrhous tumors”.

Tumors composed of undifferentiated cells are said to be anaplastic, a feature that is a reliable indicator of malignancy. The term anaplasia literally means “backward formation”—implying dedifferentiation, or loss of the structural and functional differentiation of normal cells. It is now known, however, that at least some cancers arise from stem cells in tissues; in these tumors, failure of differentiation of transformed stem cells, rather than dedifferentiation of specialized cells, accounts for their anaplastic appearance. Recent studies also indicate that in some cases, dedifferentiation of apparently mature cells occurs during carcinogenesis. Anaplastic cells often display the following morphologic features:

• Pleomorphism (i.e., variation in size and shape) (Fig. 6.4)

• Nuclear abnormalities, consisting of extreme hyperchromatism (dark-staining), variation in nuclear size and shape, or unusually prominent single or multiple nucleoli. Enlargement of nuclei may result in an increased nuclear-to-cytoplasmic ratio that approaches 1 : 1 instead of the normal 1 : 4 or 1 : 6. Nucleoli may attain astounding sizes, sometimes approaching the diameter of normal lymphocytes.

• Tumor giant cells may be formed. These are considerably larger than neighboring cells and may possess either one enormous nucleus or several nuclei.

• Atypical mitoses, which may be numerous. Anarchic multiple spindles may produce tripolar or quadripolar mitotic figures (Fig. 6.5).

• Loss of polarity, such that anaplastic cells lack recognizable patterns of orientation to one another. Such cells may grow in sheets, with total loss of communal structures, such as glands or stratified squamous architecture.

Well-differentiated tumor cells are likely to retain the functional capabilities of their normal counterparts, whereas anaplastic tumor cells are much less likely to have specialized functional activities. For example, benign neoplasms and even well-differentiated cancers of endocrine glands frequently elaborate the hormones characteristic of their cell of origin. Similarly, well-differentiated squamous cell carcinomas produce keratin (see Fig. 6.3), just as well-differentiated hepatocellular carcinomas secrete bile. In other instances, unanticipated functions emerge. Some cancers may express fetal proteins not produced by comparable cells in the adult. Cancers of nonendocrine origin may produce so-called “ectopic hormones.” For example, certain lung carcinomas may produce adrenocorticotropic hormone (ACTH), parathyroid hormone–like hormone, insulin, glucagon, and others. More is said about these so-called “paraneoplastic” phenomena later.

Also of relevance in the discussion of differentiation and anaplasia is dysplasia, referring to disorderly proliferation. Dysplastic epithelium is recognized by a loss in the uniformity of individual cells and in their architectural orientation. Dysplastic cells exhibit considerable pleomorphism and often possess abnormally large, hyperchromatic nuclei. Mitotic figures are more abundant than usual and frequently appear in abnormal locations within the epithelium. In dysplastic stratified squamous epithelium, mitoses are not confined to the basal layers, where they normally occur, but may be seen throughout the epithelium. In addition, there is considerable architectural anarchy. For example, the usual progressive maturation of tall cells in the basal layer to flattened squames on the surface may be lost and replaced by a disordered hodgepodge of dark basal-appearing cells. When dysplastic changes are severe and involve the entire thickness of the epithelium, the lesion is referred to as carcinoma in situ, a preinvasive stage of cancer (Fig. 6.6).

It is important to appreciate that dysplasia is not synonymous with cancer. Mild to moderate dysplasias that do not involve the entire thickness of the epithelium sometimes regress completely, particularly if inciting causes are removed. However, dysplasia is often noted adjacent to frankly malignant neoplasms (e.g., in cigarette smokers with lung cancer), and in general the presence of dysplasia marks a tissue as being at increased risk for developing an invasive cancer.

Local Invasion

The growth of cancers is accompanied by progressive infiltration, invasion, and destruction of surrounding tissues, whereas most benign tumors grow as cohesive expansile masses that remain localized to their sites of origin. Because benign tumors grow and expand slowly, they usually develop a rim of compressed fibrous tissue (Figs. 6.7 and 6.8). This capsule consists largely of extracellular matrix that is deposited by stromal cells such as fibroblasts, which are activated by hypoxic damage to parenchymal cells resulting from compression by the expanding tumor. Encapsulation creates a tissue plane that makes the tumor discrete, moveable (non-fixed), and readily excisable by surgical enucleation. However, it is important to recognize that not all benign neoplasms are encapsulated. For example, the leiomyoma of the uterus is discretely demarcated from the surrounding smooth muscle by a zone of compressed and attenuated normal myometrium, but lacks a capsule. A few benign tumors are neither encapsulated nor discretely defined; lack of demarcation is particularly likely in benign vascular neoplasms such as hemangiomas, which understandably may be difficult to excise. These exceptions are pointed out only to emphasize that although encapsulation is the rule in benign tumors, the lack of a capsule does not mean that a tumor is malignant. Sadly, because of their uncivilized nature, tumor cells sometimes do not follow the rules set by humans. We will see such deviations many times in this chapter.

Next to the development of metastases, invasiveness is the feature that most reliably distinguishes cancers from benign tumors (Figs. 6.9 and 6.10). Cancers lack well-defined capsules. There are instances in which a slowly growing malignant tumor deceptively appears to be encased by the stroma of the surrounding host tissue, but microscopic examination reveals tiny crablike feet penetrating the margin and infiltrating adjacent structures. This infiltrative mode of growth makes it necessary to remove a wide margin of surrounding normal tissue when surgical excision of a malignant tumor is attempted. Surgical pathologists carefully examine the margins of resected tumors to ensure that they are devoid of cancer cells (clean margins).

Metastasis

Metastasis is defined by the spread of a tumor to sites that are physically discontinuous with the primary tumor and unequivocally marks a tumor as malignant, as by definition benign neoplasms do not metastasize. The invasiveness of cancers permits them to penetrate into blood vessels, lymphatics, and body cavities, providing opportunities for spread (Fig. 6.11). Overall, approximately 30% of patients with newly diagnosed solid tumors (excluding skin cancers other than melanomas) present with clinically evident metastases. An additional 20% have occult (hidden) metastases at the time of diagnosis.

In general, the more anaplastic and the larger the primary neoplasm, the more likely is metastatic spread, but as with most rules there are exceptions. Extremely small cancers have been known to metastasize; conversely, some large and ominous-looking lesions may not. While all malignant tumors can metastasize, some do so very infrequently. For example, basal cell carcinomas of the skin and most primary tumors of the central nervous system are highly locally invasive but rarely metastasize. It is evident then that the properties of local invasion and metastasis are sometimes separable.

A special circumstance involves so-called “blood cancers”, the leukemias and lymphomas. These tumors are derived from blood-forming cells that normally have the capacity to enter the bloodstream and travel to distant sites; as a result, with only rare exceptions, leukemias and lymphomas are taken to be disseminated diseases at diagnosis and are always considered to be malignant.

Malignant neoplasms disseminate by one of three pathways: (1) seeding within body cavities, (2) lymphatic spread, or (3) hematogenous spread. Spread by seeding occurs when neoplasms invade a natural body cavity. This mode of dissemination is particularly characteristic of cancers of the ovary, which often cover the peritoneal surfaces widely. The implants literally may glaze all peritoneal surfaces and yet not invade the underlying tissues. Here is an instance where the ability to reimplant and grow at sites distant from the primary tumor seems to be separable from the capacity to invade. Neoplasms of the central nervous system, such as a medulloblastoma or ependymoma, may penetrate the cerebral ventricles and be carried by the cerebrospinal fluid to reimplant on the meningeal surfaces, either within the brain or in the spinal cord.

Lymphatic spread is more typical of carcinomas, whereas hematogenous spread is favored by sarcomas. There are numerous interconnections, however, between the lymphatic and vascular systems, so all forms of cancer may disseminate through either or both systems. The pattern of lymph node involvement depends principally on the site of the primary neoplasm and the natural pathways of local lymphatic drainage. Lung carcinomas arising in the respiratory passages metastasize first to the regional bronchial lymph nodes and then to the tracheobronchial and hilar nodes. Carcinoma of the breast usually arises in the upper outer quadrant and first spreads to the axillary nodes. However, medial breast lesions may drain through the chest wall to the nodes along the internal mammary artery. Thereafter, in both instances, the supraclavicular and infraclavicular nodes may be seeded. In some cases, the cancer cells seem to travel in lymphatic channels within the immediately proximate nodes to be trapped in subsequent lymph nodes, producing so-called “skip metastases.” The cells may traverse all of the lymph nodes ultimately to reach the vascular compartment by way of the thoracic duct.

A “sentinel lymph node” is the first regional lymph node that receives lymph flow from a primary tumor. It can be identified by injection of blue dyes or radiolabeled tracers near the primary tumor. Biopsy of sentinel lymph nodes allows determination of the extent of spread of tumor and can be used to plan treatment.

Of note, although enlargement of nodes near a primary neoplasm should arouse concern for metastatic spread, it does not always imply cancerous involvement. The necrotic products of the neoplasm and tumor antigens often evoke immunologic responses in the nodes, such as hyperplasia of the follicles (lymphadenitis) and proliferation of macrophages in the subcapsular sinuses (sinus histiocytosis). Thus, histopathologic verification of tumor within an enlarged lymph node is required.

While hematogenous spread is the favored pathway for sarcomas, carcinomas use it as well. As might be expected, arteries are penetrated less readily than are veins. With venous invasion, the bloodborne cells follow the venous flow draining the site of the neoplasm, with tumor cells often stopping in the first capillary bed they encounter. Since all portal area drainage flows to the liver, and all caval blood flows to the lungs, the liver and lungs are the most frequently involved secondary sites in hematogenous dissemination. Cancers arising near the vertebral column often embolize through the paravertebral plexus; this pathway probably is involved in the frequent vertebral metastases of carcinomas of the thyroid and prostate glands.

Certain carcinomas have a propensity to grow within veins. Renal cell carcinoma often invades the renal vein to grow in a snakelike fashion up the inferior vena cava, sometimes reaching the right side of the heart. Hepatocellular carcinomas often penetrate and grow within the radicles of portal and hepatic veins, eventually reaching the main venous channels. Remarkably, such intravenous growth may not be accompanied by widespread dissemination.

Many observations suggest that the anatomic localization of a neoplasm and its venous drainage cannot wholly explain the systemic distributions of metastases. For example, prostatic carcinoma preferentially spreads to bone, bronchogenic carcinoma tends to involve the adrenal glands and the brain, and neuroblastoma spreads to the liver and bones. Conversely, skeletal muscles, although rich in capillaries, are rarely sites of tumor metastases. The molecular basis of such tissue-specific homing of tumor cells is discussed later.

Thus, numerous features of tumors (Fig. 6.12) usually permit the differentiation of benign and malignant neoplasms.

Cancer Incidence

For the year 2012, the World Health Organization (WHO) estimated that there were about 14.1 million new cancer cases worldwide, leading to 8.2 million deaths (approximately 22,500 deaths per day). Moreover, due to increasing population size, by the year 2035 the WHO projects that the numbers of cancer cases and deaths worldwide will increase to 24 million and 14.6 million, respectively (based on current mortality rates). Additional perspective on the likelihood of developing a specific form of cancer can be gained from national incidence and mortality data. In the United States, it is estimated that the year 2016 will be marked by approximately 1.69 million new cases of cancer and 595,000 cancer deaths. Incidence data for the most common forms of cancer, with the major killers identified, are presented in Fig. 6.13.

Over several decades, the death rates for many forms of cancer have changed. Since 1995, the incidence of cancer in men and women in the United States has been roughly stable, but the cancer death rate has decreased by roughly 20% in men and 10% in women. Among men, 80% of the decrease is accounted for by lower death rates for cancers of the lung, prostate, and colon; among women, nearly 60% of the decrease is due to reductions in death rates from breast and colorectal cancers. Decreased use of tobacco products is responsible for the reduction in lung cancer deaths, while improved detection and treatment are responsible for the decrease in death rates for colorectal, female breast, and prostate cancers.

The last half-century has also seen a sharp decline in death rates from cervical cancer and gastric cancer in the United States. The decrease in cervical cancer is directly attributable to widespread use of the Papanicolaou (PAP) smear test for early detection of this tumor and its precursor lesions. The deployment of the human papillomavirus (HPV) vaccine may nearly eliminate this cancer in coming years. The cause of the decline in death rates for cancers of the stomach is obscure; it may be related to decreasing exposure to unknown dietary carcinogens.

Environmental Factors

Environmental exposures appear to be the dominant risk factors for many common cancers, suggesting that a high fraction of cancers are potentially preventable. This notion is supported by the geographic variation in death rates from specific forms of cancer, which is thought to stem mainly from differences in environmental exposures. For instance, death rates from breast cancer are about four to five times higher in the United States and Europe than in Japan. Conversely, the death rate for stomach carcinoma in men and women is about seven times higher in Japan than in the United States. Liver cell carcinoma is relatively infrequent in the United States but is the most lethal cancer among many African populations. Nearly all the evidence indicates that these geographic differences have environmental rather than genetic origins. For example, Nisei (second-generation Japanese living in the United States) have mortality rates for certain forms of cancer that are intermediate between those in natives of Japan and in Americans who have lived in the United States for many generations. The two rates come closer with each passing generation.

There is no paucity of environmental factors that contribute to cancer. They lurk in the ambient environment, in the workplace, in food, and in personal practices. They can be as universal as sunlight or be largely restricted to urban settings (e.g., asbestos) or particular occupations (Table 6.2). The most important environmental exposures linked to cancer include the following:

Thus, there is no escape: it seems that everything people do to earn a livelihood, to subsist, or to enjoy life turns out to be illegal, immoral, or fattening, or—most disturbing—possibly carcinogenic!

Age and Cancer

In general, the frequency of cancer increases with age. Most cancer deaths occur between 55 and 75 years of age; the rate declines, along with the population base, after 75 years of age. The rising incidence with age may be explained by the accumulation of somatic mutations that drive the emergence of malignant neoplasms (discussed later). The decline in immune competence that accompanies aging also may be a factor.

Although cancer preferentially affects older adults, it also is responsible for slightly more than 10% of all deaths among children younger than 15 years of age (Chapter 7). The major lethal cancers in children are leukemias, tumors of the central nervous system, lymphomas, and soft-tissue and bone sarcomas. As discussed later, study of several childhood tumors, such as retinoblastoma, has provided fundamental insights into the pathogenesis of malignant transformation.

Acquired Predisposing Conditions

Acquired conditions that predispose to cancer include disorders associated with chronic inflammation, immunodeficiency states, and precursor lesions. Many chronic inflammatory conditions create a fertile “soil” for the development of malignant tumors (Table 6.3). Tumors arising in the context of chronic inflammation are mostly carcinomas, but also include mesothelioma and several kinds of lymphoma. By contrast, immunodeficiency states mainly predispose to virus-induced cancers, including specific types of lymphoma and carcinoma and some sarcoma-like proliferations.

Precursor lesions are localized disturbances of epithelial differentiation that are associated with an elevated risk for developing carcinoma. They may arise secondary to chronic inflammation or hormonal disturbances (in endocrine-sensitive tissues), or may occur spontaneously. Molecular analyses have shown that precursor lesions often possess some of the genetic lesions found in their associated cancers (discussed later). However, progression to cancer is not inevitable, and it is important to recognize precursor lesions because their removal or reversal lowers cancer risk.

Many different precursor lesions have been described; among the most common are the following:

In this context it also may be asked, “What is the risk for malignant change in a benign neoplasm?”—or, stated differently, “Are benign tumors precancers?” In general the answer is no, but inevitably there are exceptions, and perhaps it is better to say that each type of benign tumor is associated with a particular level of risk, ranging from high to virtually nonexistent. As cited earlier, adenomas of the colon as they enlarge can undergo malignant transformation in up to 50% of cases; by contrast, malignant change is extremely rare in leiomyomas of the uterus.

Interactions Between Environmental and Genetic Factors

Cancer behaves like an inherited trait in some families, usually due to germ line mutations that affect the function of a gene that suppresses cancer (a so-called “tumor suppres­sor gene,” discussed later). What then can be said about the influence of heredity on sporadic malignant neoplasms, which constitute roughly 95% of the cancers in the United States?

While the evidence suggests that sporadic cancers can largely be attributed to environmental factors or acquired predisposing conditions, lack of family history does not preclude an inherited component. It may in fact be difficult to tease out hereditary and genetic contributions because these factors often interact. Such interactions may be particularly complex when tumor development is affected by small contributions from multiple genes. Furthermore, genetic factors may alter the risk for developing environmentally induced cancers. Instances where this holds true often involve inherited variation in enzymes such as components of the cytochrome P-450 system that metabolize procarcinogens to active carcinogens. Conversely, environmental factors can influence the risk for developing cancer, even in individuals who inherit well-defined “cancer genes.” For instance, breast cancer risk in females who inherit mutated copies of the BRCA1 or BRCA2 tumor suppressor genes (discussed later) is almost three-fold higher for women born after 1940 than for women born before that year, perhaps because of changes in reproductive behavior or increases in obesity in more recent times.

Driver and Passenger Mutations

In the following sections, we briefly review the types of mutations that are commonly found in cancers. Before doing so, however, we must first touch on the important concept of driver mutations and passenger mutations. Driver mutations are mutations that alter the function of cancer genes and thereby directly contribute to the development or progression of a given cancer. They are usually acquired, but as mentioned earlier, occasionally inherited. By contrast, passenger mutations are acquired mutations that are neutral in terms of fitness and do not affect cellular behavior; they just come along for the proverbial ride. Because they occur at random, passenger mutations are sprinkled throughout the genome, whereas driver mutations tend to be tightly clustered within cancer genes. It is now appreciated that particularly in cancers caused by carcinogen exposure, such as melanoma and smoking-related lung cancer, passenger mutations greatly outnumber driver mutations.

Despite their apparently innocuous nature, passenger mutations have nevertheless proven to be important in several ways:

Gene Rearrangements

Gene rearrangements may be produced by chromosomal translocations or inversions. Specific chromosomal trans­locations and inversions are highly associated with certain malignancies, particularly neoplasms derived from hematopoietic cells and other kinds of mesenchymal cells. These rearrangements can activate proto-oncogenes in two ways:

• Some gene rearrangements result in overexpression of proto-oncogenes by removing them from their normal regulatory elements and placing them under control of an inappropriate, highly active promoter or enhancer. Two different kinds of B cell lymphoma provide illustrative examples of this mechanism. In more than 90% of cases of Burkitt lymphoma, the cells have a translocation, usually between chromosomes 8 and 14, that leads to overexpression of the MYC gene on chromosome 8 by juxtaposition with immunoglobulin heavy chain gene regulatory elements on chromosome 14 (Fig. 6.14). In follicular lymphoma, a reciprocal translocation between chromosomes 14 and 18 leads to overexpression of the anti-apoptotic gene, BCL2, on chromosome 18, also driven by immunoglobulin gene regulatory elements.

• Other oncogenic gene rearrangements create fusion genes encoding novel chimeric proteins. Most notable is the Philadelphia (Ph) chromosome in chronic myeloid leukemia, consisting of a balanced reciprocal translocation between chromosomes 9 and 22 (see Fig. 6.14). As a consequence, the derivative chromosome 22 (the Philadelphia chromosome) appears smaller than normal. This cytogenetic change is seen in more than 90% of cases of chronic myeloid leukemia and results in the fusion of portions of the BCR gene on chromosome 22 and the ABL gene on chromosome 9. The few Philadelphia chromosome–negative cases harbor a cryptic (cytogenetically silent) BCR-ABL fusion gene, the presence of which is the sine qua non of chronic myeloid leukemia. As discussed later, the BCR-ABL fusion gene encodes a novel tyrosine kinase with potent transforming activity.

Lymphoid tumors are most commonly associated with recurrent gene rearrangements. This relationship exists because normal lymphocytes express special enzymes that purposefully introduce DNA breaks during the processes of immunoglobulin or T cell receptor gene recombination. Repair of these DNA breaks is error-prone, and the resulting mistakes sometimes result in gene rearrangements that activate proto-oncogenes. Two other types of mesenchymal tumors, myeloid neoplasms (acute myeloid leukemias and myeloproliferative disorders) and sarcomas, also frequently possess gene rearrangements. Unlike lymphoid neoplasms, the cause of the DNA breaks that lead to gene rearrangements in myeloid neoplasms and sarcomas is unknown. In general, the rearrangements that are seen in myeloid neoplasms and sarcomas create fusion genes that encode either hyperactive tyrosine kinases (akin to BCR-ABL) or novel oncogenic transcription factors. A well-characterized example of the latter is the (11;22)(q24;q12) translocation in Ewing sarcoma. This rearrangement creates a fusion gene encoding a chimeric oncoprotein composed of portions of two different transcription factors called EWS and FLI1.

Identification of pathogenic gene rearrangements in carcinomas has lagged because karyotypically evident translocations and inversions (which point to the location of important oncogenes) are rare in carcinomas. However, advances in DNA sequencing have revealed recurrent cryptic pathogenic gene rearrangements in carcinomas as well. As with hematologic malignancies and sarcomas, gene rearrangements in solid tumors can contribute to carcinogenesis either by increasing expression of an oncogene or by generation of a novel fusion gene. Examples will be discussed along with specific cancers in other chapters. As with a fusion gene such as BCR-ABL, some of the fusion genes in solid tumors also provide drug targets (e.g., EML-ALK in lung cancer; Chapter 13).

Gene Amplifications

Proto-oncogenes may be converted to oncogenes by gene amplification, with consequent overexpression and hyperactivity of otherwise normal proteins. Such amplification may produce several hundred copies of the gene, a change in copy number that can be readily detected by molecular hybridization with appropriate DNA probes. In some cases, the amplified genes produce chromosomal changes that can be identified microscopically. Two mutually exclusive patterns are seen: multiple small, extrachromosomal structures called double minutes; and homogeneously staining regions. The latter derive from the insertion of the amplified genes into new chromosomal locations, which may be distant from the normal location of the involved genes; because regions containing amplified genes lack a normal banding pattern, they appear homogeneous in a G-banded karyotype. Two clinically important examples of amplification involve the NMYC gene in neuroblastoma and the HER2 gene in breast cancers. NMYC is amplified in 25% to 30% of neuroblastomas, and the amplification is associated with poor prognosis (Fig. 6.15). HER2 (also known as ERBB2) amplification occurs in about 20% of breast cancers, and antibody therapy directed against the receptor encoded by the HER2 gene has proved effective in this subset of tumors.

Epigenetic Modifications and Cancer

You will recall from Chapter 1 that epigenetics refers to reversible, heritable changes in gene expression that occur without mutation. Such changes involve posttranslational modifications of histones and DNA methylation, both of which affect gene expression. In normal, differentiated cells, the major portion of the genome is not expressed. These regions of the genome are silenced by DNA methylation and histone modifications. On the other hand, cancer cells are characterized by a global DNA hypomethylation and selective promoter-localized hypermethylation. Indeed, it has become evident during the past several years that tumor suppressor genes are sometimes silenced by hypermethylation of promoter sequences, rather than by mutation. In addition, genome-wide hypomethylation has been shown to cause chromosomal instability and can induce tumors in mice. Thus, epigenetic changes may influence carcinogenesis in many ways. As an added wrinkle, deep sequencing of cancer genomes has identified mutations in genes that regulate epigenetic modifications in many cancers. Thus, certain genetic changes in cancers may be selected because they lead to alterations of the “epigenome” that favor cancer growth and survival.

The epigenetic state of particular cell types—a feature described as the epigenetic context—also dictates their response to signals that control growth and differentiation. As mentioned earlier, epigenetic modifications regulate gene expression, allowing cells with the same genetic makeup (e.g., a neuron and a keratinocyte) to have completely different appearances and functions. In some instances, the epigenetic state of a cell dramatically affects its response to otherwise identical signals. For example, the NOTCH1 gene has an oncogenic role in T-cell leukemia, yet acts as a tumor suppressor in squamous cell carcinomas. As would be expected, this dichotomy exists because activated NOTCH1 turns on pro-growth genes in T-cell progenitors and tumor suppressor genes in keratinocytes.

Self-Sufficiency in Growth Signals

The self-sufficiency in growth that characterizes cancer cells generally stems from gain-of-function mutations that convert proto-oncogenes to oncogenes. Oncogenes encode proteins called oncoproteins that promote cell growth, even in the absence of normal growth-promoting signals. To appreciate how oncogenes drive inappropriate cell growth, it is helpful to review briefly the sequence of events that characterize normal cell proliferation (introduced in Chapter 1). Under physiologic conditions, cell proliferation can be readily resolved into the following steps:

The mechanisms that endow cancer cells with the ability to proliferate can be grouped according to their role in the growth factor–induced signal transduction cascade and cell cycle regulation. Indeed, each one of the listed steps is susceptible to corruption in cancer cells.

Downstream Signal-Transducing Proteins

Cancer cells often acquire growth autonomy as a result of mutations in genes that encode components of signaling pathways downstream of growth factor receptors. The signaling proteins that couple growth factor receptors to their nuclear targets are activated by ligand binding to growth factor receptors. The signals are trasnmitted to the nucleus through various signal transduction molecules. Two important oncoproteins in the category of signaling molecules are RAS and ABL. Each of these is discussed briefly next.

RAS

RAS is the most commonly mutated oncogene in human tumors. Approximately 30% of all human tumors contain mutated RAS genes, and the frequency is even higher in some specific cancers (e.g., pancreatic adenocarcinoma). RAS is a member of a family of small G proteins that bind guanosine nucleotides (guanosine triphosphate [GTP] and guanosine diphosphate [GDP]). Signaling by RAS involves the following sequential steps:

• Normally, RAS flips back and forth between an excited signal-transmitting state and a quiescent state. RAS is inactive when bound to GDP; stimulation of cells by growth factors such as EGF and PDGF leads to exchange of GDP for GTP and subsequent conformational changes that generate active RAS (Fig. 6.18). This excited signal-emitting state is short-lived, however, because the intrinsic guanosine triphosphatase (GTPase) activity of RAS hydrolyzes GTP to GDP, releasing a phosphate group and returning the protein to its quiescent GDP-bound state. The GTPase activity of activated RAS is magnified dramatically by a family of GTPase-activating proteins (GAPs), which act as molecular brakes that prevent uncontrolled RAS activation by favoring hydrolysis of GTP to GDP.

• Activated RAS stimulates downstream regulators of proliferation by several interconnected pathways that converge on the nucleus and alter the expression of genes that regulate growth, such as MYC. While details of the signaling cascades (some of which are illustrated in Fig. 6.18) downstream of RAS are not discussed here, an important point is that mutational activation of these signaling intermediates mimics the growth promoting effects of activated RAS. For example, BRAF, which lies in the so-called “RAF/ERK/MAP kinase pathway” is mutated in more than 60% of melanomas and is associated with unregulated cell proliferation. Mutations of phosphatidly inositol-3 kinase (PI3 kinase) in the PI3K/AKT pathway also occur with high frequency in some tumor types, with similary consequences.

RAS most commonly is activated by point mutations in amino acid residues that are either within the GTP-binding pocket or in the enzymatic region that carries out GTP hydrolysis. Both kinds of mutations interfere with breakdown of GTP, which is essential to inactivate RAS. RAS is thus trapped in its activated, GTP-bound form, and the cell is forced into a continuously proliferating state. It follows from this scenario that the consequences of activating mutations in RAS should be mimicked by loss-of-function mutations in GAPs, which would lead to a failure to simulate GTP hydrolysis and thereby restrain RAS. Indeed, the GAP neurofibromin-1 (NF1) is mutated in the cancer-prone familial disorder neurofibromatosis type 1 (Chapter 22) and is a bona fide tumor suppressor. Similarly, another important tumor suppressor called PTEN is a negative inhibitor of PI3 kinase and is frequently mutated in carcinomas, certain leukemias, and other cancers as well.

ABL

Several non–receptor tyrosine kinases function as signal transduction molecules. In this group, ABL is the best defined with respect to carcinogenesis.

The ABL proto-oncoprotein has tyrosine kinase activity that is dampened by internal negative regulatory domains. As discussed earlier (see Fig. 6.14), in chronic myeloid leukemia and certain acute leukemias, a part of the ABL gene is translocated from its normal abode on chromosome 9 to chromosome 22, where it fuses with part of the breakpoint cluster region (BCR) gene. This fusion gene encodes as BCR-ABL hybrid protein that contains the ABL tyrosine kinase domain and a BCR domain that self-associates, an event that unleashes a constitutive tyrosine kinase activity. Of interest, the BCR-ABL protein activates all of the signals that are downstream of RAS, making it a potent stimulator of cell growth.

The crucial role of BCR-ABL in cancer has been confirmed by the dramatic clinical response of patients with chronic myeloid leukemia to BCR-ABL kinase inhibitors. The prototype of this kind of drug, imatinib mesylate (Gleevec), galvanized interest in design of drugs that target specific molecular lesions found in various cancers (so-called “targeted therapy”). BCR-ABL also is an example of the concept of oncogene addiction, wherein a tumor is profoundly dependent on a single signaling molecule. BCR-ABL fusion gene formation is an early, perhaps initiating, event that drives leukemogenesis. Development of leukemia probably requires other collaborating mutations, but the transformed cell continues to depend on BCR-ABL for signals that mediate growth and survival. BCR-ABL signaling can be seen as the central lodgepole around which the transformed state is “built”. If the lodgepole is removed by inhibition of the BCR-ABL kinase, the structure collapses. In view of this level of dependency, it is not surprising that acquired resistance of tumors to BCR-ABL inhibitors often is due to the outgrowth of a subclone with a mutation in BCR-ABL that prevents binding of the drug to the BCR-ABL protein.

Insensitivity to Growth Inhibitory Signals: Tumor Suppressor Genes

Isaac Newton theorized that every action has an equal and opposite reaction. Although Newton was not a cancer biologist, his formulation holds true for cell growth. Whereas oncogenes encode proteins that promote cell growth, the products of tumor suppressor genes apply brakes to cell proliferation. Disruption of such genes renders cells refractory to growth inhibition and mimics the growth-promoting effects of oncogenes. The following discussion describes tumor suppressor genes, their products, and possible mechanisms by which loss of their function contributes to unregulated cell growth.

In principle, anti-growth signals can prevent cell proliferation by several complementary mechanisms. The signal may cause dividing cells to enter G₀ (quiescence), where they remain until external cues prod their reentry into the proliferative pool. Alternatively, the cells may enter a postmitotic, differentiated pool and lose replicative potential. Nonreplicative senescence, alluded to earlier, is another mechanism of escape from sustained cell growth. And, as a last-ditch effort, the cells may be programmed for death by apoptosis. As we will see, tumor suppressor genes have all these “tricks” in their toolbox designed to halt wayward cells from becoming malignant.

RB: Governor of the Cell Cycle

RB, a key negative regulator of the cell cycle, is directly or indirectly inactivated in most human cancers. The retinoblastoma gene (RB) was the first tumor suppressor gene to be discovered and is now considered the prototype of this family of cancer genes. As with many advances in medicine, the discovery of tumor suppressor genes was accomplished by the study of a rare disease—in this case, retinoblastoma, an uncommon childhood tumor. Approximately 60% of retinoblastomas are sporadic, while the remaining ones are familial, the predisposition to develop the tumor being transmitted as an autosomal dominant trait. To account for the sporadic and familial occurrence of an identical tumor, Knudson, in 1974, proposed his now famous two-hit hypothesis, which in molecular terms can be stated as follows:

• Two mutations (hits) are required to produce retinoblastoma. These involve the RB gene, which has been mapped to chromosomal locus 13q14. Both of the normal alleles of the RB locus must be inactivated (hence the two hits) for the development of retinoblastoma (Fig. 6.19).

• In familial cases, children inherit one defective copy of the RB gene in the germ line; the other copy is normal. Retinoblastoma develops when the normal RB gene is lost in retinoblasts as a result of somatic mutation. Because in retinoblastoma families a single germ line mutation is sufficient to transmit disease risk, the trait has an autosomal dominant inheritance pattern.

• In sporadic cases, both normal RB alleles are lost by somatic mutation in one of the retinoblasts. The end result is the same: a retinal cell that has lost both of the normal copies of the RB gene becomes cancerous.

From the above, it is evident that although the risk for developing retinoblastoma in retinoblastoma families is inherited as a dominant trait, at the level of the cell, one intact RB gene is all that is needed for normal function.

Although the loss of normal RB genes initially was discovered in retinoblastomas, it is now evident that biallelic loss of this gene is a fairly common feature of several tumors, including breast cancer, small cell cancer of the lung, and bladder cancer. Patients with familial retinoblastoma also are at greatly increased risk for developing osteosarcomas and some soft-tissue sarcomas.

The function of the RB protein is to regulate the G1/S checkpoint, the portal through which cells must pass before DNA replication commences. Although each phase of the cell cycle circuitry is monitored carefully, the transition from G₁ to S is an extremely important checkpoint in the cell cycle “clock.” In the G₁ phase, diverse signals are integrated to determine whether the cell should progress through the cell cycle, or exit the cell cycle and differentiate. The RB gene product, RB, is a DNA-binding protein that serves as a point of integration for these diverse signals, which ultimately act by altering the phosphorylation state of RB. Specifically, signals that promote cell cycle progression lead to the phosphorylation and inactivation of RB, while those that block cell cycle progression act by maintaining RB in an active hypophosphorylated state.

To appreciate this crucial role of RB in the cell cycle, it is helpful to review the mechanisms that enforce the G₁/S transition.

• The initiation of DNA replication (S phase) requires the activity of cyclin E/CDK2 complexes, and expression of cyclin E is dependent on the E2F family of transcription factors. Early in G₁, RB is in its hypophosphorylated active form, and it binds to and inhibits the E2F family of transcription factors, preventing transcription of cyclin E. Hypophosphorylated RB blocks E2F-mediated transcription in at least two ways (Fig. 6.20). First, it sequesters E2F, preventing it from interacting with other transcriptional activators. Second, RB recruits chromatin remodeling proteins, such as histone deacetylases and histone methyltransferases, which bind to the promoters of E2F-responsive genes such as cyclin E. These enzymes modify chromatin at the promoters to make DNA insensitive to transcription factors.

• This situation is changed on mitogenic signaling. Growth factor signaling leads to cyclin D expression and activation of cyclin D–CDK4/6 complexes. The level of cyclin D-CDK4/6 activity is tempered by antagonists such as p16, which is itself subject to regulation by growth inhibitors such as TGFβ that serve to set a threshold for mitogenic responses. If the stimulus is sufficently strong, cyclin D-CDK4/6 complexes phosphorylate RB, inactivating the protein and releasing E2F to induce target genes such as cyclin E. Cyclin E/CDK complexes then stimulate DNA replication and progression through the cell cycle. When the cells enter S phase, they are committed to divide without additional growth factor stimulation. During the ensuing M phase, the phosphate groups are removed from RB by cellular phosphatases, regenerating the hypophosphorylated form of RB.

• E2F is not the sole target of RB. The versatile RB protein binds to a variety of other transcription factors that regulate cell differentiation. For example, RB stimulates myocyte-, adipocyte-, melanocyte-, and macrophage-specific transcription factors. Thus, the RB pathway couples control of cell cycle progression at G₁ with differentiation, which may explain how differentiation is associated with exit from the cell cycle.

In view of the centrality of RB to the control of the cell cycle, an interesting question is why RB is not mutated in every cancer. In fact, mutations in other genes that control RB phosphorylation can mimic the effect of RB loss and are commonly found in many cancers that have normal RB genes. For example, mutational activation of CDK4 and overexpression of cyclin D favor cell proliferation by facilitating RB phosphorylation and inactivation. Indeed, cyclin D is overexpressed in many tumors because of amplification or translocation of the cyclin D1 gene. Mutational inactivation of genes encoding CDKIs also can drive the cell cycle by removing important brakes on cyclin/CDK activity. As mentioned earlier, the CDKN2A gene, which encodes the CDK inhibitor p16, is an extremely common target of deletion or mutational inactivation in human tumors.

It is now accepted that loss of normal cell cycle control is central to malignant transformation and that at least one of the four key regulators of the cell cycle (p16, cyclin D, CDK4, RB) is mutated in most human cancers. Notably, in cancers caused by certain oncogenic viruses (discussed later), this is achieved through direct targeting of RB by viral proteins. For example, the human papillomavirus (HPV) E7 protein binds to the hypophosphorylated form of RB, preventing it from inhibiting the E2F transcription factors. Thus, RB is functionally deleted, leading to uncontrolled growth.

Summary

RB: Governor of the Cell Cycle

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• Like other tumor suppressor genes, both copies of RB must be dysfunctional for tumor development to occur.

• In cases of familial retinoblastoma, one defective copy of the RB gene is present in the germ line, so that only one additional somatic mutation is needed to completely eliminate RB function.

• RB exerts anti-proliferative effects by controlling the G₁-to-S transition of the cell cycle. In its active form, RB is hypophosphorylated and binds to E2F transcription factors. This interaction prevents transcription of genes like cyclin E that are needed for DNA replication, and so the cells are arrested in G₁.

• Growth factor signaling leads to cyclin D expression, activation of cyclin D–CDK4/6 complexes, inactivation of RB by phosphorylation, and thus release of E2F.

• Loss of cell cycle control is fundamental to malignant transformation. Almost all cancers have a disabled G₁ checkpoint due to mutation of either RB or genes that affect RB function, such as cyclin D, CDK4, and CDKIs.

• Many oncogenic DNA viruses, like HPV, encode proteins (e.g., E7) that bind RB and render it nonfunctional.

TP53: Guardian of the Genome

The p53-encoding tumor suppressor gene, TP53, is the most commonly mutated gene in human cancer. The p53 protein is a transcription factor that thwarts neoplastic transformation by three interlocking mechanisms: activation of temporary cell cycle arrest (termed quiescence), induction of permanent cell cycle arrest (termed senescence), or triggering of programmed cell death (termed apoptosis). If RB is a “sensor” of external signals, p53 can be viewed as a central monitor of internal stress, directing the stressed cells toward one of these pathways.

A variety of stresses trigger the p53 response pathways, including anoxia, inappropriate pro-growth stimuli (e.g., unbridled MYC or RAS activity), and DNA damage. By managing the DNA damage response, p53 plays a central role in maintaining the integrity of the genome, as discussed next.

In nonstressed, healthy cells, p53 has a short half-life (20 minutes) because of its association with MDM2, a protein that targets p53 for destruction. When the cell is stressed, for example, by an assault on its DNA, “sensors” that include protein kinases such as ATM (ataxia telangiectasia mutated) are activated. These activated sensors catalyze posttranslational modifications in p53 that release it from MDM2, increasing its half-life and enhancing its ability to drive the transcription of target genes. Hundreds of genes whose transcription is triggered by p53 have been found. These genes suppress neoplastic transformation by three mechanisms:

• p53-mediated cell cycle arrest may be considered the primordial response to DNA damage (Fig. 6.21). It occurs late in the G₁ phase and is caused mainly by p53-dependent transcription of the CDKI gene CDKN1A (p21). The p21 protein inhibits cyclin–CDK complexes and prevents phosphorylation of RB, thereby arresting cells in the G₁ phase. Such a pause in cell cycling is welcome, because it gives the cells “breathing time” to repair DNA damage. The p53 protein also induces expression of DNA damage repair genes. If DNA damage is repaired successfully, p53 upregulates transcription of MDM2, leading to its own destruction and relief of the cell cycle block. If the damage cannot be repaired, the cell may enter p53-induced senescence or undergo p53-directed apoptosis.

• p53-induced senescence is a form of permanent cell cycle arrest characterized by specific changes in morphology and gene expression that differentiate it from quiescence or reversible cell cycle arrest. Senescence requires activation of p53 and/or Rb and expression of their mediators, such as the CDKIs. The mechanisms of senescence are unclear but seem to involve global chromatin changes, which drastically and permanently alter gene expression.

• p53-induced apoptosis of cells with irreversible DNA damage is the ultimate protective mechanism against neoplastic transformation. It is mediated by upregulation of several pro-apoptotic genes, including BAX and PUMA (discussed later).

To summarize, p53 is activated by stresses such as DNA damage and assists in DNA repair by causing G₁ arrest and inducing the expression of DNA repair genes. A cell with damaged DNA that cannot be repaired is directed by p53 to either enter senescence or undergo apoptosis (see Fig. 6.21). In view of these activities, p53 has been rightfully called the “guardian of the genome.” With loss of normal p53 function, DNA damage goes unrepaired, mutations become fixed in dividing cells, and the cell turns onto a one-way street leading to malignant transformation.

Confirming the importance of TP53 in controlling carcinogenesis, more than 70% of human cancers have a defect in this gene, and the remaining malignant neoplasms often have defects in genes upstream or downstream of TP53. Biallelic abnormalities of the TP53 gene are found in virtually every type of cancer, including carcinomas of the lung, colon, and breast—the three leading causes of cancer deaths. In most cases, mutations affecting both TP53 alleles are acquired in somatic cells. In other tumors, such as certain sarcomas, the TP53 gene is intact but p53 function is lost because of amplification and overexpression of the MDM2 gene, which encodes a potent inhibitor of p53. Less commonly, patients inherit a mutant TP53 allele; the resulting disorder is called the Li-Fraumeni syndrome. As in the case with familial retinoblastoma, inheritance of one mutant TP53 allele predisposes affected individuals to develop malignant tumors because only one additional hit is needed to inactivate the second, normal allele. Patients with the Li-Fraumeni syndrome have a 25-fold greater chance of developing a malignant tumor by 50 years of age compared with the general population. In contrast to tumors developing in patients who inherit a mutant RB allele, the spectrum of tumors that develop in patients with the Li-Fraumeni syndrome is much more varied; the most common types are sarcomas, breast cancer, leukemia, brain tumors, and carcinomas of the adrenal cortex. Compared with individuals diagnosed with sporadic tumors, patients with Li-Fraumeni syndrome develop tumors at a younger age and may develop multiple primary tumors.

As with RB, normal p53 also can be rendered nonfunctional by certain oncogenic DNA viruses. Proteins encoded by oncogenic HPVs, certain polyoma viruses, and hepatitis B virus bind to p53 and nullify its protective function. Thus, transforming DNA viruses subvert two of the best-understood tumor suppressors, RB and p53.

Summary

TP53: Guardian of the Genome

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• TP53 encodes p53, the central monitor of stress in the cell, which can be activated by anoxia, inappropriate oncogene signaling, or DNA damage. Activated p53 controls the expression and activity of genes involved in cell cycle arrest, DNA repair, cellular senescence, and apoptosis.

• DNA damage leads to activation of p53 by phosphorylation. Activated p53 drives transcription of CDKN1A (p21), which prevents RB phosphorylation, thereby causing a G₁-S block in the cell cycle. This pause allows the cells to repair DNA damage.

• If DNA damage cannot be repaired, p53 induces cellular senescence or apoptosis.

• Of human tumors, 70% demonstrate biallelic mutations in TP53. Patients with the rare Li-Fraumeni syndrome inherit one defective copy of TP53 in the germ line, such that only one additional mutation is required to lose normal p53 function. Li-Fraumeni syndrome patients are prone to develop a wide variety of tumors.

• As with RB, p53 can be incapacitated when bound by proteins encoded by oncogenic DNA viruses such as HPV.

Altered Cellular Metabolism

Even in the presence of ample oxygen, cancer cells demonstrate a distinctive form of cellular metabolism characterized by high levels of glucose uptake and increased conversion of glucose to lactose (fermentation) via the glycolytic pathway. This phenomenon, called the Warburg effect and also known as aerobic glycolysis, has been recognized for many years (indeed, Otto Warburg received the Nobel Prize in 1931 for its discovery). Clinically, the “glucose-hunger” of tumors is used to visualize tumors via positron emission tomography (PET) scanning, in which patients are injected with ¹⁸F-fluorodeoxyglucose, a glucose derivative that is preferentially taken up into tumor cells (as well as normal, actively dividing tissues such as the bone marrow). Most tumors are PET-positive, and rapidly growing ones are markedly so.

Warburg's discovery was largely neglected for many years, but over the past decade, metabolism has become one of the most active areas of cancer research. Metabolic pathways (like signaling pathways) in normal and cancer cells are still being elucidated, and the details are complex, but at the heart of the Warburg effect lies a simple question: why is it advantageous for a cancer cell to rely on seemingly inefficient glycolysis (which generates two molecules of ATP per molecule of glucose) instead of oxidative phosphorylation (which generates up to 36 molecules of ATP per molecule of glucose)? While pondering this question, it is important to recognize that rapidly proliferating normal cells, such as in embryonic tissues and lymphocytes during immune responses, also rely on aerobic fermentation. Thus, “Warburg metabolism” is not cancer specific, but instead is a general property of growing cells that becomes “fixed” in cancer cells.

The answer to this riddle is simple: Aerobic glycolysis provides rapidly dividing tumor cells with metabolic intermediates that are needed for the synthesis of cellular components, whereas mitochondrial oxidative phosphorylation does not. The reason growing cells rely on aerobic glycolysis becomes readily apparent when one considers that a growing cell has a strict biosynthetic requirement; it must duplicate all of its cellular components—DNA, RNA, proteins, lipid, and organelles—before it can divide and produce two daughter cells. While oxidative phosphorylation yields abundant ATP, it fails to produce any carbon moieties that can be used to build the cellular components needed for growth (proteins, lipids, and nucleic acids). Even cells that are not actively growing must shunt some metabolic intermediates away from oxidative phosphorylation in order to synthesize macromolecules that are needed for cellular maintenance.

By contrast, in actively growing cells only a small fraction of the cellular glucose is shunted through the oxidative phosphorylation pathway, such that on average each molecule of glucose metabolized produces approximately four molecules of ATP. Presumably, this balance (heavily biased toward aerobic fermentation, with a bit of oxidative phosphorylation) hits a metabolic “sweet spot” that is optimal for growth. It follows that growing cells do rely on mitochondrial metabolism. However, a major function of mitochondria in growing cells is not to generate ATP, but rather to carry out reactions that generate metabolic intermediates that can be shunted off and used as precursors in the synthesis of cellular building blocks. For example, lipid biosynthesis requires acetyl-CoA, and acetyl-CoA is largely synthesized in growing cells from intermediates such as citrate that are generated in mitochondria.

So how is this profound reprogramming of metabolism, the Warburg effect, triggered in growing normal and malignant cells? As might be guessed, metabolic reprogramming is produced by signaling cascades downstream of growth factor receptors, the very same pathways that are deregulated by mutations in oncogenes and tumors suppressor genes in cancers. Thus, whereas in rapidly dividing normal cells aerobic glycolysis ceases when the tissue is no longer growing, in cancer cells this reprogramming persists due to the action of oncogenes and the loss of tumor suppressor gene function. Some of the important points of cross-talk between pro–growth signaling factors and cellular metabolism are shown in Fig. 6.23 and include the following:

The flipside of the coin is that tumor suppressors often inhibit metabolic pathways that support growth. We have already discussed the “braking” effect of the tumor suppressors NF1 and PTEN on signals downstream of growth factor receptors and RAS, allowing them to oppose the Warburg effect. Indeed, it may be that many (and perhaps all) tumor suppressors that induce growth arrest suppress the Warburg effect. For example, p53, arguably the most important tumor suppressor, upregulates target genes that collectively inhibit glucose uptake, glycolysis, lipogenesis, and the generation of NADPH (a key cofactor needed for the biosynthesis of macromolecules). Thus, it is increasingly clear that the functions of many oncoproteins and tumor suppressors are inextricably intertwined with cellular metabolism.

Beyond the Warburg effect, there are two other links between metabolism and cancer that are of sufficient importance to merit brief mention, autophagy and an unusual set of oncogenic mutations that lead to the creation of oncometabolites, small molecules that appear to directly contribute to the transformed state.

Oncometabolism

Another surprising group of genetic alterations discovered through tumor genome sequencing studies are mutations in enzymes that participate in the Krebs cycle. Of these, mutations in isocitrate dehydrogenase (IDH) have garnered the most interest, as they have revealed a new mechanism of oncogenesis termed oncometabolism (Fig. 6.24).

The proposed steps in the oncogenic pathway involving IDH are as follows:

• IDH acquires a mutation that leads to a specific amino acid substitution involving residues in the active site of the enzyme. As a result, the mutated protein loses it ability to function as an isocitrate dehydrogenase and instead acquires a new enzymatic activity that catalyzes the production of 2-hydroxglutarate (2-HG).

• 2-HG in turn acts as an inhibitor of several other enzymes that are members of the TET family, including TET2.

• TET2 is one of several factors that regulate DNA methylation, which you will recall is an epigenetic modification that controls normal gene expression and often goes awry in cancer. According to the model, loss of TET2 activity leads to abnormal patterns of DNA methylation.

• Abnormal DNA methylation in turn leads to misexpression of currently unknown cancer genes, which drive cellular transformation and oncogenesis.

Thus, according to this scenario, mutated IDH acts as an oncoprotein by producing 2-HG, which is considered a prototypical oncometabolite. Oncogenic IDH mutations have now been described in a diverse collection of cancers, including a sizable fraction of cholangiocarcinomas, gliomas, acute myeloid leukemias, and sarcomas. Of clinical significance, because the mutated IDH proteins have an altered structure, it has been possible to develop drugs that inhibit mutated IDH and not the normal IDH enzyme. These drugs are now being tested in cancer patients and have produced encouraging therapeutic responses. This developing story is a remarkable example of how detailed understanding of oncogenic mechanisms can yield entirely new kinds of anti-cancer drugs.

Summary

Altered Cellular Metabolism

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• Warburg metabolism is a form of pro-growth metabolism favoring glycolysis over oxidative phosphorylation. It is induced in normal cells by exposure to growth factors and becomes fixed in cancer cells due to the action of certain driver mutations.

• Many oncoproteins (RAS, MYC, mutated growth factor receptors) induce or contribute to Warburg metabolism, and many tumor suppressors (PTEN, NF1, p53) oppose it.

• Stress may induce cells to consume their components in a process called autophagy. Cancer cells may accumulate mutations to avoid autophagy, or may corrupt the process to provide nutrients for continued growth and survival.

• Some oncoproteins such as mutated IDH act by causing the formation of high levels of “oncometabolites” that alter the epigenome, thereby leading to changes in gene expression that are oncogenic.