The Washington Manual of Oncology, 3 Ed.

Cancer Biology—Basics of Molecular Oncogenesis

Elizabeth C. Chastain • John D. Pfeifer

 I. SOURCES OF DNA DAMAGE

1. Endogenous sources of DNA damage. There are several constant, unavoidable sources of background DNA damage.
2. Reactive oxygen species. Reactive oxygen species (ROS) are byproducts of normal cellular metabolism and play important roles in cell signaling and homeostasis. The most common ROS include OH, NO, and peroxides. Increased environmental stress may dramatically increase ROS production, resulting in DNA damage. Collectively, these changes are known as oxidative damage and include sugar and base modifications, DNA–DNA and DNA–protein cross-links, and DNA strand breaks. ROS can also be generated by exogenous sources such as ionizing radiation, pollutants, and tobacco.
3. Spontaneous chemical reactions. The most common spontaneous chemical changes that alter the structure of DNA are deamination and depurination reactions, although spontaneous hydrolysis, alkylation, and adduction reactions may also occur. The mutagenic potential of the different types of reactions varies.
4. Metal ions. Although the evidence for DNA damage by endogenous metals is especially substantial for iron and copper, nickel, chromium, magnesium, and cadmium are also well-established human carcinogens. Metal-catalyzed reactions produce DNA adducts, resulting in a wide variety of organic compound metabolites. Metals such as arsenic, cadmium, lead, and nickel also directly inhibit DNA repair, which augments the mutagenic potential of the DNA damage they induce.
5. Exogenous sources of DNA damage
6. Chemicals. Although a virtually infinite number of chemicals can damage DNA, a few families of environmental and therapeutic compounds illustrate the general mechanisms involved.
7. Polycyclic aromatic hydrocarbons (PAHs) and related compounds. These molecules are converted into reactive intermediate metabolites by the normal physiologic action of cytochrome P-450, a process termed metabolic activation. These reactive intermediates are responsible for DNA damage through the formation of DNA adducts. Variation in the balance between metabolic activation and detoxification influences cancer rates.
8. Antineoplastic agents. Cell cycle–specific therapies include antimetabolites (i.e., 5-fluorouracil, 6-mercaptopurine, and methotrexate) that interfere with nucleotide production, and taxanes (i.e., docetaxel, paclitaxel) and vinca alkaloids, both of which disrupt microtubule formation. Cell cycle–nonspecific drugs include cytotoxic alkylating agents (i.e., cyclophosphamide, busulfan, nitrogen mustard, and thiotepa) that result in DNA damage through the formation of covalent linkages producing alkylated nucleotides, DNA–DNA cross-links, DNA–protein cross-links, and DNA strand breaks; anthracyclines (i.e., daunorubicin, doxorubicin) that inhibit topoisomerase II; and platinum-based therapies (i.e., carboplatin, cisplatin) that act primarily by causing intrastrand and interstrand cross-links.
9. Radiation. DNA damage caused by radiation can be classified into damage caused by ultraviolet radiation (UV light) and damage caused by ionizing radiation.
10. UV light. UV-B radiation from sunlight (wavelength 280 to 315 nm) produces cyclobutane pyrimidine dimers (due to covalent bonds between adjacent thymine residues within the same strand of DNA) as well as pyrimidine (6-4) pyrimidone photoproducts (due to covalent bonds between TC or CC dimers within the same strand of DNA). Damage caused by UV-A radiation (wavelength 315 to 400 nm) is usually due to ROS-mediated mechanisms.
11. Ionizing radiation. A broad spectrum of DNA damage is caused by ionizing radiation, including individual base lesions, cross-links, and single- and double-strand breaks. Low linear energy transfer (LET) radiation (x-rays, γ-rays, electrons, and β-particles have a typical LET of less than 10 keV/µm) and high-LET radiation (protons and neutrons have a typical LET of 10 to 100 keV/µm, whereas α-particles have an LET greater than 175 keV/µm) each produce a characteristic pattern of damage.
12. TYPES OF DNA ALTERATIONS
13. Single base pair substitutions. Single base pair (bp) substitutions are among the most commonly observed mutations and may be a consequence of numerous processes, including errors in DNA replication, spontaneous chemical reactions, ROS damage, chemical mutagenesis, ionizing radiation, and failure of DNA repair mechanisms. Coding and noncoding regions are almost equally susceptible to this type of alteration.

A single bp substitution occurring within the coding sequence of a gene can lead to a significant change in the encoded amino acid, but does not cause a shift in the translational reading frame. These substitutions are classified into synonymous mutations and nonsynonymous mutations. Synonymous mutations are those in which a different codon still specifies the same amino acid and the vast majority of these changes are neutral. There are two types of nonsynonymous mutations, consisting of missense mutations (resulting in a codon that specifies a different amino acid) and nonsense mutations (production of a stop codon). The extent of disruption caused by missense mutations depends on the chemical similarities or differences between the two amino acids. Depending on the location, nonsense mutations may lead to premature termination and reduced protein function.

Single bp substitutions that occur outside the coding region of a gene can still be deleterious. Substitutions in the 5’ regulatory region of a gene can alter the pattern of gene expression, and substitutions in introns, exons, or untranslated regions of a gene can affect RNA processing.

1. Gross gene deletions. There are two types of recombination events that give rise to gross gene deletions. Homologous unequal recombination occurs at homologous sequences that are not paired precisely, generally at related gene sequences or repetitive sequence elements, resulting in rejoining of homologous but nonallelic DNA sequences. Regions of repetitive sequences are especially prone to unequal crossovers and are the site of many large-scale deletions, as well as insertions, duplications, amplifications, inversions, and translocations. Nonhomologous recombination (illegitimate recombination) occurs between DNA loci that have minimal or no sequence homology.
2. Gene conversion. This type of mutation is a nonreciprocal transfer of sequence information between two loci. One of the interacting sequences (the donor or source) remains unchanged, whereas the alternative sequence (the acceptor or target) is altered by partial or total replacement by the donor sequence. Gene families with tandem repeats and clusters are particularly prone to this type of alteration.
3. Short gene deletions. The mutation rate at some microsatellites is remarkably high. Because the mutant alleles differ from the wild type by a single repeat unit without exchange of flanking markers, the proposed mechanism involves misalignment of the short direct repeats during DNA replication (due to so-called polymerase slippage).
4. Insertions. Polymerase slippage can account for short insertions just as it can for short deletions. Homologous unequal recombination between repetitive sequence elements provides a mechanism for the generation of larger insertions (which may actually be gene duplications).
5. Expansion of unstable repeat sequences. A small subset of tandem trinucleotide repeats, as well as a very limited number of longer repeats, shows anomalous behavior that causes abnormal gene expression. Repeats above a certain threshold length become extremely unstable and are virtually never transmitted unchanged from parent to child. Genes containing unstable expanding trinucleotide repeats can be grouped into two major classes: one includes genes that show very large expansions outside coding sequences, while the other class consists of genes that show modest expansion within coding sequences.
6. Inversions. A high degree of sequence similarity between repeats on the same chromosome may predispose to inversions by a mechanism that involves bending back of the chromatid upon itself in a process that is essentially homologous intrachromosomal recombination. Inversions not associated with significant sequence homology are apparently the result of nonhomologous recombination.
7. Illegitimate recombination. The RAG enzyme, responsible for the double-stranded DNA breaks that underlie the V(D)J recombination of antigen receptor genes, occasionally cuts DNA at unrelated loci that have complementary recombination signals, producing a translocation.
8.  Mitochondrial DNA damage and mutations. Mitochondrial DNA (mtDNA) is susceptible to damage by the same processes responsible for nuclear DNA damage. ROS are an especially important source of damage, given the proximity of mtDNA to the reactive intermediates produced by the electron transport/oxidative phosphorylation of the respiratory chain. Although the types of mtDNA mutation are similar to those of nuclear DNA, the unique features of mitochondrial genetics result in an entirely different pattern of phenotype–genotype correlations.

 III. DNA REPAIR. Only rarely does DNA repair occur through simple chemical reversal of the damage; usually, it entails excision of altered DNA followed by resynthesis. However, it is important to emphasize that many DNA alterations, including insertions, deletions, duplications, inversions, and translocations, are not targets of DNA repair pathways and are therefore highly mutagenic.

1. Direct repair. Human cells produce very few enzymes capable of directly reversing DNA damage. One such enzyme is O6-alkyguanine DNA alkyltransferase, produced by the O6-methylguanine methyltransferase (MGMT) gene. It removes the naturally occurring mutagenic nonnative methyl group from O6-methylguanine, restoring the base to guanine. This dealkylation reaction is significant because the altered base incorrectly pairs with thymine and hence is highly mutagenic. More recently, a set of enzymes has been discovered to catalyze the oxidative demethylation of 1-methyladenine and 3-methycytosine.
2. Base excision repair. A major source of DNA damage includes small chemical alterations. Base excision repair (BER) is responsible for repair of oxidized bases and alkylated bases, and is also responsible for correction of spontaneous depurination events, single-strand breaks, and some mismatched bases. While these lesions do not typically impede transcription or DNA replication, they are especially prone to produce mutations, and BER plays a crucial role in maintaining the integrity of the genome.
3. Nucleotide excision repair. This is the most versatile DNA repair pathway, in which the damaged or incorrect portion of a DNA strand is excised and the resulting gap is filled by repair replication using the complementary strand as a template. Lesions repaired by nucleotide excision repair (NER) typically serve as structural inhibitors to transcription and replication due to distortion of the helical conformation secondary to interference of normal base pairing.
4. Transcription-coupled repair. NER and BER are inefficient at repairing damage to the transcribed DNA strand of the actively expressed loci secondary to RNA polymerase II stalling at the site of the damage and sterically hindering repair. Because the stalled RNA polymerase effectively inactivates the gene, independent of whether the DNA damage would cause a mutation affecting gene function, transcription-coupled repair not only corrects DNA damage but also restores gene expression.
5. Mismatch repair. The mismatch repair (MMR) system corrects nucleotides mispaired by DNA polymerases within an otherwise complementary paired DNA strand. This repair mechanism can also excise small insertion/deletion loops of single-stranded DNA that result from polymerase slippage during replication of repetitive sequences or arise during recombination. The importance of this repair mechanism in maintaining genetic stability is illustrated by the observation that its absence results in mutation rates up to 1,000 times higher than normal, with a particular tendency for errors within short tandem repeats or homopolymeric stretches.
6. Translesion synthesis. The polymerases primarily responsible for replication of nuclear DNA are hampered when they encounter a chemically altered base. Translesion synthesis is a DNA damage tolerance process occurring in the vicinity of the DNA lesion that proceeds via replacement of the conventional polymerase by one or several specialized polymerases that have the ability to replicate damaged DNA. This process manages to effectively replicate damaged nuclear DNA templates despite the almost limitless diversity of DNA lesions. However, translesion synthesis polymerases display low fidelity replication of nondamaged templates, lack a proofreading function, and have flexible base-pairing properties. Hence, the capability of replicating damaged DNA through this mechanism comes at the expense of a high error rate.
7. Recombinatorial repair. Unrepaired double-strand breaks (DSBs) are highly disruptive events that interfere with proper chromosome segregation during cell division and often induce various chromosomal aberrations including aneuploidy, deletions, and chromosomal translocations. The two main DSB repair mechanisms are homologous recombination and nonhomologous end joining. Both initiate a cascade of kinase reactions that not only recruit repair factors to the site of the break, but also delay or terminate the cell cycle through DNA damage checkpoint control. The exact mechanism of cross-link repair in human cells remains unknown.
8. Defective DNA damage repair. Some of the most striking examples of hereditary cancer syndromes are due to mutations affecting genes involved in DNA repair, which emphasizes the fundamental role of repair pathways in oncogenesis.
9. Defective BER. The most direct evidence linking the role of BER in cancer comes from germline mutations in the MYH gene, which produces a glycosylase responsible for removing adenine mispaired with 8-oxoguanine or guanine. Mutations lead to recessive inheritance of multiple colorectal adenomas.
10. Defective NER. At least four photosensitivity-related syndromes have been attributed to inborn errors in NER including the autosomal recessive xeroderma pigmentosum, the brittle hair disorder trichothiodystrophy, UV sensitivity syndrome (UVSS), and a clinical disorder that combines features of both xeroderma pigmentosum and Cockayne syndrome.
11. Defective transcription-coupled repair. Inborn defects in transcription-coupled repair are associated with Cockayne syndrome.
12. Defective MMR. Defective MMR facilitates malignant transformation through the production of mutations in genes that harbor microsatellites in their coding regions, some of which have critical roles in the regulation of cell growth and apoptosis (e.g., TGFBR2 gene, which encodes the transforming growth factor β receptor II, and the BAX gene, which encodes a proapoptotic protein). Defects in MMR genes are responsible for hereditary nonpolyposis colorectal carcinoma (HNPCC). However, it is important to recognize that although microsatellite instability can be demonstrated in a variety of malignancies, in most cases the phenotype is due to somatic rather than germline mutations at MMR loci.
13. Defective translesion synthesis. Defective replication of damaged DNA due to inherited mutations in one of the translesion polymerases is responsible for xeroderma pigmentosum variant.
14. Defective double-strand break and cross-link repair. Diseases associated with impaired double-strand break repair by homologous recombination include ataxia telangiectasia, ataxia telangiectasia–like disorder, Nijmegen breakage syndrome, Fanconi anemia, familial breast–ovarian cancer syndrome, Werner’s syndrome, Bloom’s syndrome, and Rothmund–Thomson syndrome. Prominent clinical features shared by these diseases include radiosensitivity, genomic instability, cancer susceptibility, and immunodeficiency.

 IV. VIRUSES. Several RNA and DNA viruses are associated with the development of malignancies.

1. RNA viruses. Three RNA viruses associated with malignancies include two retroviruses, human T-lymphotropic virus (HTLV-1) and human immunodeficiency virus (HIV), and the flavivirus hepatitis C virus (HCV).
2. Retroviruses. In general, oncogenic retroviruses can be classified into two main groups on the basis of disease-causing mechanisms. Acute transforming retroviruses are typically replication defective and cause rapid induction of tumors because they carry viral oncogenes. On the other hand, nonacute retroviruses (of which HTLV-1 is an example) are replication competent and do not carry oncogenes but rather exert their oncogenic effects by integrating within or adjacent to a cellular proto-oncogene. Retroviruses that cause immunodeficiency (of which HIV is an example) apparently promote oncogenesis only indirectly, most likely as a consequence of the immunosuppression associated with infection.
3. Flaviviruses. HCV infection alone is not sufficient to give rise to hepatocellular carcinoma (HCC), and oncogenesis is thought to develop as a result of the increased cellular turnover caused by long-term hepatocyte necrosis and regeneration, fibrosis, and inflammation associated with infection.
4. DNA viruses. DNA viruses from several different families have oncogenic properties.
5. Hepadnaviruses. Hepatitis B virus (HBV) encodes a gene (known as X) that is involved in transcriptional activation and signal transduction and has been implicated to contribute directly to tumorigenesis. Additionally, the virus can contribute to oncogenesis through direct insertional mutagenesis. However, HBV primarily promotes hepatocellular carcinogenesis indirectly, occurring most likely through increased cellular turnover caused by long-term hepatocyte necrosis and regeneration, fibrosis, and inflammation associated with infection.
6. Herpesviruses
7. Epstein–Barr virus (EBV) is associated with the oncogenesis of lymphoid malignancies (including Burkitt lymphoma and classical Hodgkin lymphoma), gastric cancer, and nasopharyngeal carcinoma. The viral genome encodes more than 100 genes (including several that play a direct role in cellular transformation) and numerous microRNAs (miRNA).
8. Kaposi sarcoma–associated herpesvirus (KSHV), also known as human herpes virus-8 (HHV-8), is associated with the development of Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV encodes many genes related to tumor development, including cyclins, inhibitors of apoptosis, and cytokines, all of which are candidates for contributing to viral oncogenesis.
9. Papillomaviruses. Human papillomavirus (HPV) is associated with cervical cancer, other anogenital cancers, and a subset of squamous carcinomas of the head and neck, particularly those of the oropharynx. The high-risk, cancer-associated serotypes (most commonly, HPV-16 and HPV-18) harbor two principal transforming genes, E6 and E7. The E6 protein inactivates p53, and the E7 protein interacts with the retinoblastoma (RB1) gene product. However, E6 and E7 alone are insufficient for carcinogenesis. The development of a fully transformed cellular phenotype requires alterations in a number of cellular pathways. It remains unclear why only a subset of patients infected with high-risk HPV serotypes eventually develop malignant disease.
10. Polyomavirus. The most recently described viral-associated malignancy is Merkel cell carcinoma, caused by Merkel cell polyomavirus (MCPyV). This rare but highly aggressive neuroendocrine skin cancer arises in elderly, immunosuppressed, and immunodeficient patients. MCPyV integrates into the host genome and may mediate oncogenesis through inactivation of the tumor suppressor pRB.
11. INDIVIDUAL GENES THAT ARE TARGETS OF ONCOGENIC MUTATIONS. According to the clonal model of carcinogenesis, a malignant tumor arises from a single cell. This founder cell acquires an initial mutation that provides its progeny with a selective growth advantage, and from within this expanded population, another single cell acquires a second mutation that provides an additional growth advantage, and so on, until a fully transformed malignant tumor emerges. Tumor suppressor genes and oncogenes are frequent targets of mutation in this multistep process of tumor evolution.
12. Tumor suppressor genes. Normal cellular functions of tumor suppressor gene products are highly diverse, including regulation of the cell cycle, cell differentiation, apoptosis, and maintenance of genomic integrity. Dozens of tumor suppressor genes have been identified, and numerous potential candidates have been described. Many of these genes were identified by virtue of the fact that they are mutated in the germline of persons who are affected by cancer syndromes; nevertheless, for the vast majority of tumor suppressor genes, somatic mutations play a far more significant role in cancer development than do germline mutations. Tumor suppressor genes have been broadly divided into two classes, gatekeepers and caretakers.
13. Gatekeepers. This group directly regulates cell growth by inhibiting cellular proliferation or promoting apoptosis; familiar examples include the products of RB1, TP53, APC, NF1, NF2, WT1, MEN1, and VHL genes. Because the functions of the proteins encoded by gatekeeper genes are rate-limiting for tumor growth, tumor development occurs only when both copies of the gene are inactivated. Individuals with an autosomal dominant cancer susceptibility syndrome inherit one damaged copy, and so require only one somatic mutation to inactivate the remaining wild-type allele and initiate tumor formation. Nonetheless, inactivating mutations of both alleles of a gatekeeper are still insufficient for acquisition of a fully transformed malignant phenotype. Gatekeepers vary with tissue type, and so germline inactivation of a particular gatekeeper gene leads to only specific forms of cancer predisposition.
14. Caretakers. These genes are responsible for DNA repair and genomic maintenance; therefore, when inactivated by mutations, these genes indirectly facilitate oncogenesis by promoting an increased rate of mutation. Genes that encode proteins involved in DNA repair are the classic examples of caretakers, including MSH2, MLH1, PMS1, ATM, XPA through XPG, and FANCA through FNACL.
15. Oncogenes. The normally functioning cellular counterparts, termed proto-oncogenes, are important regulators of many aspects of cell physiology, including cell growth and differentiation. More than 75 proto-oncogenes have been identified, and their products include extracellular cytokines and growth factors (e.g., int-1 and int-2), transmembrane growth factor receptors (e.g., c-erb2/EGFR, HER2/neu, src, c-abl, c-ret, H-ras, K-ras, and N-ras), cytoplasmic kinases (e.g., BRAF), and nuclear proteins involved in the control of DNA replication (e.g., c-myc, N-myc, L-myc, c-myb).

Oncogenes represent mutated forms of proto-oncogenes, resulting in neoplastic transformation. Despite their variety, oncogenes can be divided into two general groups on the basis of the mechanism of their action. One group induces continuous or unregulated cell proliferation by inactivation of growth inhibitory signals, or by activation of growth-promoting genes, growth factors, receptors, intracellular signaling pathways, or nuclear oncoproteins. The other group immortalizes cells by rescuing them from senescence and apoptosis. Accumulated evidence from human malignancies and transgenic animal models indicates that mutation of a single oncogene is insufficient for acquisition of a fully transformed, malignant phenotype.

Only a few inherited cancer syndromes are due to germline mutations in oncogenes. Instead, most oncogene mutations are somatic and therefore associated with sporadic malignancies. For many tumor types, the involved oncogene and the type of mutation are characteristic.

 VI. INTRACELLULAR PATHWAYS THAT ARE TARGETS OF ONCOGENIC MUTATIONS

1. Signal transduction. In the context of oncology, important signal transduction pathways are those that regulate cellular proliferation, differentiation, and death, often through regulation of gene transcription. Signal transduction pathways have evolved to respond to an enormous variety of stimuli and are highly interconnected to permit dynamic regulation of the strength, duration, and timing of cell responses. Many of the genes involved in signal transduction are classified as tumor suppressor genes and oncogenes.
2. Ligands. The various types of ligands include proteins (soluble, cell bound, and matrix), individual amino acids, lipids, gases, and soluble and polymerized nucleotides.
3. Receptors. Examples of cell surface receptors include receptor tyrosine kinases, serine kinases, and phosphatases; members of the Notch family; and G protein-coupled receptors. The guanylate cyclase family of receptors provides an example of related receptors that can be either membrane bound or soluble. The transcription factors that bind glucocorticoids, thyroxine, and vitamin D are examples of receptors that are located in the nucleus.
4. Signal propagation. Many ligand–receptor interactions transmit signals through small molecule second messengers, which then bind noncovalently to protein targets and affect their function. Examples of small molecule second messengers include cyclic adenosine monophosphate (cAMP), phospholipases (that generate inositol triphosphate and diacylglycerol), Ca²⁺, and eicosanoids.
5. Regulation of the cell cycle. Cell proliferation is rigorously regulated to preserve function and integrity throughout development and adult life. Many signal transduction pathways specifically regulate promitogenic and antimitogenic proteins that control the cell cycle including the A-, B-, D-, and E-type cyclins, cyclin-dependent kinases (CDKs), and the skp, cullin, F-box containing (SCF) and anaphase-promoting complex or cyclosome (APC/C) families of protein-ubiquitin ligases. Not surprisingly, many of the genes involved in cell cycle regulation are commonly mutated or show an altered pattern of expression in a variety of human malignancies.
6. Cell cycle checkpoints. Progression through the cell cycle prior to repair of DNA damage is potentially harmful; therefore, a number of cell cycle checkpoints are employed to allow for proper DNA repair. These DNA damage checkpoints occur before S phase entry, during S phase, and before M phase entry. Additional cell cycle checkpoints include a replication checkpoint (that ensures DNA replication is complete prior to initiation of M phase), a spindle integrity checkpoint (that ensures appropriate partitioning of the chromosomes occurs before initiation of anaphase during mitosis), and a restriction checkpoint (that blocks cell cycle progression at mid-G1 phase in the absence of essential growth factors or nutrients). Loss of function of one or more of these checkpoints is a characteristic feature of many malignancies.
7. Apoptosis. Signal transduction pathways control not only cell proliferation but also programmed cell death. Apoptosis is a genetic pathway for rapid and efficient killing of unnecessary or damaged cells, which can be divided into two distinct but not mutually exclusive signaling pathways known as extrinsic and intrinsic apoptosis. Extrinsic apoptosis is elicited by the ligand-induced activation of plasma membrane proteins of the death receptor family, such as CD95/FAS and the tumor necrosis factor-α 1. Provocation of this pathway leads to downstream activation of proteolytic caspases (final effectors of the apoptotic pathway). Several cellular inhibitor of apoptosis proteins (IAPs), including cIAP1, cIAP2, and XIAP, are able to inhibit apoptosis as a result of their ability to interfere with caspase activation. Intrinsic apoptosis (also known as mitochondrial apoptosis) is regulated by the integrity of mitochondrial structure and function. If proapoptotic signals are predominant, mitochondrial membranes become permeable and release proteins such as cytochrome C, endonuclease G, Smac/DIABLO, and HTRA2 that lead to activation of caspases. In both pathways, control of apoptosis is achieved through regulation of the proapoptotic proteins BAX and BAK and the antiapoptotic proteins BCL-2, BCL-XL, and MCL-1.

Not surprisingly, there is substantial cross-talk between cellular proliferation and apoptosis pathways, often focused at various cell cycle checkpoints. Mutations or alterations in the level of expression of pro- and antiapoptotic proteins are characteristic of many human malignancies.

1. Telomere metabolism. The telomere is the nucleoprotein complex present at the end of each chromatid that functions to protect the chromosome. The enzyme telomerase catalyzes the unique reaction by which the long TTAGGG tandem repeat arrays of the telomere are synthesized. Telomerase activity is tightly controlled, although the details of the different regulatory mechanisms are not fully understood. Dysfunctions of telomerase maintenance have an important role in tumorigenesis as well as in genomic instability, since chromosomes lacking a telomere are unstable and tend to fuse with other broken chromosomes, undergo recombination, or get degraded.

 VII. EXTRACELLULAR PATHWAYS THAT ARE TARGETS OF ONCOGENIC MUTATIONS

1. Angiogenesis. Tumor growth requires the development of an adequate blood supply, known as angiogenesis. Two types of angiogenesis have been described. Sprouting angiogenesis occurs through branching of new capillaries from preexisting capillaries, a process that includes degradation of the basement membrane, migration of endothelial cells in the direction of the angiogenic stimulus, endothelial cell proliferation, and capillary tube formation. Nonsprouting angiogenesis occurs through proliferation of endothelial cells within preexisting vessels, with subsequent lumen enlargement, splitting, or fusion. It has been calculated that any increase in tumor size beyond a diameter of 0.5 mm requires angiogenesis.

Angiogenesis requires induction or elevated expression of one or more proangiogenic growth factors, coinciding with the downregulation with one or more endogenous inhibitors within the local tissue environment. The proangiogenic and antiangiogenic factors are produced by tumor cells, inflammatory cells, and adjacent normal tissue cells, and their levels are regulated by interdependent stimuli such as tissue hypoxia (mediated through the hypoxia-inducible factor-1 [HIF-1] pathway), tissue pH, and soluble growth factors. Proangiogenic molecules bind to specific receptors on endothelial cells, smooth muscle cells, and pericytes, and include members of the vascular endothelial growth factor (VEGF) family, members of the angiopoietin (Ang) family, members of the fibroblast growth factor (FGF) family, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and various chemokines. Endogenous inhibitors of angiogenesis include extracellular matrix glycoproteins (e.g., thrombospondin-1 (TSP-1), endostatin, tumstatin, canstatin), members of clotting/coagulation cascade family (e.g., angiostatin), and vasostatin (which is a fragment of calreticulin). Another recently described regulator is the Notch receptor-DLL4 signaling pathway, a system that mediates embryonic angiogenesis but appears to function as an inhibitor of tumor angiogenesis in adults.

1. Invasion and metastasis. Metastasis is the spread of cancer from the organ of origin (primary site) to distant tissues and consists of a series of interrelated steps known as the metastatic cascade. The metastatic cascade includes detachment of a tumor cell (or cells) from surrounding cells; migration through the stroma and basement membrane into the lumen of a capillary, venule, or lymphatic; intravascular survival; arrest within a capillary bed with subsequent adherence to the wall; extravasation into adjacent tissue; and proliferation with associated angiogenesis and evasion of host defenses. This cascade is dependent on numerous alterations in tumor cells including motility, cell migration, protease expression, and autocrine and paracrine growth factor expression.
2. Cell motility. A number of stimuli increase cell motility, including tumor-secreted molecules, local tissue microenvironment-derived factors, and growth factors. Tumor-secreted molecules generally function in autocrine loops.
3. Cell migration. A process central to cell migration includes diminished cell surface adhesion to produce an altered pattern of tumor cell interaction with the intercellular matrix and nonneoplastic cells. Important adhesion molecules include members of the cadherin family, members of the Ig superfamily (especially VCAM-1 and NCAM), and integrins.
4. Proteases. Tumor cells must produce a number of proteases that are required in order to mediate cellular changes required for migration through the extracellular matrix and connective tissue barriers. The group of proteases that has been most extensively studied is the matrix metalloproteinases (MMPs), which are inhibited by tissue inhibitors of metalloproteinases (TIMPs).
5. Autocrine and paracrine growth factors. Growth factors produced by transformed cells as well as nonneoplastic cells are required for many steps in the metastatic cascade. The role of nonneoplastic cells in the local tissue microenvironment is increasingly being recognized as a significant source of various growth factors.

VIII. EPIGENETIC REGULATORS. Epigenetic processes not only play important roles during development, but are also involved in maintaining tissue-specific patterns of gene expression in differentiated cells. Almost all human cancers contain substantial epigenetic abnormalities that cooperate with genetic lesions to generate a transformed phenotype. Epigenetic changes tend to arise early in carcinogenesis, often preceding somatic mutations, and allow orchestration of activation and silencing pathways.

1. DNA methylation. The addition of methyl groups to cytosine residues of CpG dinucleotides to form 5-methylcytosine is catalyzed by DNA methyltransferases (DNMTs). Methylation typically occurs within CpG-rich regions (known as CpG islands) that are often distributed in the transcriptional start sites (TSS) of regulatory regions (i.e., promoter/enhancer regions) of genes. Increased methylation in these regions often (but not always) leads to epigenetic silencing, as often occurs for tumor suppressor genes. Additional gene expression defects linked to alterations in DNA methylation patterns involve the family of Tet methylcytosine dioxygenase proteins (TET1, TET2, and TET3) and isocitrate dehydrogenase genes (IDH1 and IDH2). Of note, hypomethylation caused by mutations in genes responsible for DNA methylation has also been suggested to play a role in several cancer types.
2. Chromatin modifiers. Active chromatin remodeling is essential for development and maintenance of normal physiologic functions, but becomes pathologically altered in cancer, leading to widespread mitotically heritable aberrations in gene expression that are characteristic of oncogenesis. The chromatin modifiers include proteins that transfer or remove acetyl or methyl modifications from histone tails, alter the position of nucleosomes along the DNA, remove nucleosomes from DNA entirely, or change the histone composition of nucleosomes in specific regions of the genome. Other proteins, known as readers, interpret these modifications and further alter local chromatin structure to either stimulate or repress gene expression.
3. RNA interference. Several classes of double- or single-stranded RNA molecules also have a role in regulation of gene expression, including short interfering RNA (siRNA), micro-RNA (miRNA), small modulatory RNA (smRNA), and long noncoding RNA (lncRNA). Also referred to as RNA interference (RNAi), RNA-mediated control of gene expression is abnormal in many malignancies.

 IX. THE CANCER GENOME AND PERSONALIZED MEDICINE. Because cancer is essentially a genetic disease caused by accumulation of molecular alterations in the genome of somatic cells, advances in the knowledge of these alterations and the technologies to detect them are transforming the field of oncology. New paradigms are emerging including a shift in tumor taxonomy from histology to genetic-based, development of drugs that target specific molecular alterations, individualized treatments focused on matching patients with targeted therapy for their tumor, and the use of specific genetic alterations as highly sensitive biomarkers for disease monitoring.

1. Next-generation sequencing. Within the last decade, several next-generation sequencing (NGS) platforms have been developed that enable high-throughput DNA sequencing of large sets of genes, whole exomes, or even whole genomes from tissue samples at a reasonable cost and a short turnaround time. These NGS methods provide several advantages over traditional Sanger sequencing methods including increased sensitivity and simultaneous detection of numerous types of DNA alterations (single nucleotide variants, copy number alterations, insertions and deletions, and structural rearrangements). However, NGS approaches require very sophisticated bioinformatic analytic methods, since it is a complicated process to assemble hundreds of millions of short sequences and map them to a reference genome. With so-called deep sequence coverage, careful validation, and highly specialized bioinformatic pipelines, many groups have demonstrated that NGS can be used to guide the routine care of cancer patients.
2. “Passenger” and “Driver” mutations. At the time of diagnosis, cancer is comprised of billions of cells carrying a wide array of DNA alterations. Some of these acquired alterations have a functional role in malignant proliferation (“drivers”), but many have no functional role in tumorigenesis (“passengers”).

It is becoming clear that only a minor fraction of genetic alterations are likely responsible for driving cancer evolution by providing cells a selective advantage over their neighbors. However, in many cases, passenger and driver mutations occur at similar frequencies, and proper classification remains a challenge in cancer genetics. Approaches to distinguish drivers from passengers include identification of recurrent mutations within a specific amino acid or adjacent amino acids, in a small region of a protein, or in evolutionarily conserved residues.

1. Optimizing treatment. Many large-scale studies such as The Cancer Genome Project (TCGA) and the International Cancer Genome Consortium (ICGC) have identified novel genes as potential therapeutic targets in various types of cancer. Although these studies have demonstrated that the repertoire of oncogenic mutations in several types of cancer is extremely heterogeneous, they have made it possible to define clinically relevant tumor subtypes that can be best managed with therapies tailored to the underlying specific mutations.

SUGGESTED READINGS

Bozic I, Antal T, Ohtsuki H, et al. Accumulation of driver and passenger mutations during tumor progression. Proc Natl Acad Sci USA 2010;107:18545–18550.

Campos El, Reinberg D. Histones: annotating chromatin. Annu Rev Genet 2009;43:559–599.

Jinek M, Doudna JA. A three-dimensional view of the molecular machinery of RNA interference. Nature 2009;457:405–412.

Laird PW. Principles and challenges of genome wide DNA methylation analysis. Nat Rev Genet 2010;11:191–203.

López CA, Cleary JD, Pearson CE. Repeat instability and the basis for human diseases and as a potential target for therapy. Nat Rev Mol Cell Biol 2010;11:165–170.

Nguyen DX, Box PD, Massaqué J. Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer 2009;9:274–284.

Pfeifer JD. DNA damage, mutations, and repair. In: Molecular Genetic Testing in Surgical Pathology. Philadelphia, PA: Lippincott Williams & Wilkins, 2006:29–57.

Schultz DR, Harrington WJ Jr. Apoptosis: programmed cell death at a molecular level. Semin Arthritis Rheum 2003;32:345–369.

Weinberg RA. Biology of Cancer. 2nd ed. New York, NY: Garland Science Publishers, 2013.