INTRODUCTION
Since the discovery of oncogenes in the 1970s, our understanding of cancer biology has expanded exponentially. Cancer is a product that evolves from cumulative genetic or epigenetic changes that progressively drive the transformation of normal cells into malignant derivatives.1 These changes result in alterations of various signaling pathways that are now being intensively studied and increasingly used in aiding diagnosis and guiding cancer therapy. Our current understanding of cancer biology is too broad to be review comprehensively here. In this chapter, we try to highlight the general mechanisms of tumorigenesis and metastasis.
HALLMARKS OF CANCER
Proliferation of normal cells is constantly held in check and intricately regulated both by the cellular intrinsic signaling mechanisms and the extrinsic microenvironment. In contrast, failure of these regulatory mechanisms is the general basis for uncontrolled cellular proliferation and tumorigenesis. Cancer cells acquire several intrinsic pathologic traits that not only liberate them from the homeostatic signaling from neighboring cells, but that endow them with the ability to subvert the surrounding tissues into supporting their proliferation. It is now widely accepted that several hallmarks, each originating from aberrations of distinct signaling pathways, are acquired during tumorigenesis. 2,3 Theseare:
Self-sufficiency in growth signals. Tumor cells can generate their own growth factors, stimulate the release of growth factors bound to the extracellular matrix, enhance their sensitivity to growth signals by overexpression of receptors, or proliferate independently of growth signals by constitutive activation of signaling pathways.
Insensitivity to antigrowth signals. Aberrant cell proliferation is normally constrained by growth inhibitors such as transforming growth factor beta (TGFβ)that is secreted by other cells in the microenvironment. These factors help maintain cells in the quiescent phase (G0) or inhibit uncontrolled progression from the G1 to S phase. Tumor cells can evade TGFβ inhibition by downregulating TGFβ receptors, displaying mutant receptors, or inactivating downstream signaling proteins.
Evasion of apoptosis. Signals evoked by oncogenes, DNA damage, detachment from basement membrane or hypoxic tumor environment often triggers apoptosis, a form of programmed cell death that involves p53 and the Bcl-2 family proteins such as Bad and Bax. Such control mechanisms are usually impaired in cancer cells by loss of p53 or overexpression of antiapoptotic proteins such as Bcl-2.
Limitless replication potential. Telomere is a structure consisting of highly repetitive DNA sequence and specialized proteins at the ends of chromosomes that protects the chromosomes from degradation. During each cycle of DNA replication, a small section of telomere is normally lost. When the length of the telomere reaches a critically low threshold, cells undergo senescence or apoptosis. On the other hand, cancer cells acquire the ability to maintain telomere length, either by reactivation of telomerase, an enzyme that elongates telomere or by another mechanism called ALT (alternative-lengthening of telomeres). This allows cancer cells to replicate indefinitely.
Angiogenesis. Tumors cannot grow larger than 0.2 mm in diameter, the diffusion limit of oxygen, without access to blood vessels. Tumor cells are capable of inducing formation of new blood vessels, termed angiogenesis to support their growth. Angiogenesis is often induced by hypoxia and requires upregulation of proangiogenic factors, such as vascular-endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-1 and -2, with simultaneous inhibition of antiangiogenic factors such as angiopoietin-1 and thrombospondin-1. Targeting angiogenesis by anti-VEGF agents, such as bevacizumab, is now a common strategy in cancer treatment. 4
Tissue invasion and metastasis. A defining feature of malignant as opposed to benign tumor is the ability to invade its surrounding tissue and metastasize. Cancer cells acquire the ability to disrupt the basement membrane by upregulating matrix metalloproteases (MMPs) and invade after undergoing epithelial–mesenchymal transition (EMT). An extremely few number of cancer cells which acquire the ability to intravasate, survive in bloodstream/lymphatic system, evade immune destruction and extravasate into distal organs will subsequently form metastatic clones.
Evasion of the immune system. In order to survive, cancer cells must escape the immune response, particularly cytotoxic T lymphocytes and natural killer cells, by either preventing immune recognition or inducing immune tolerance. Escape from immune recognition is mediated by downregulating mechanisms necessary for antigen presentation. Examples include downregulation of MHC molecules and inhibition of costimulatory molecules on tumor cells. Generation of immune tolerance involves altering the complex cellular and cytokine network of antigen presentation. Examples include production of inhibitory cytokines, suppression of stimulatory cytokines, and induction of formation of T-regulatory cells.
Reprogramming of metabolism. Unlike normal cells in which energy is generated mainly from oxidative phosphorylation in the mitochondria, cancer cells are often reprogrammed to derive their energy from glycolysis in the cytoplasm (Warburg effect). Such transformation not only helps the cancer cells to adapt to the hypoxic microenvironment, but also provides the essential substrates for synthesis of macromolecules such as amino acids and nucleosides required for rapid proliferation. 5 Such reprogramming is usually driven by oncoproteins such as AKT1, mTOR, and myc. Clinically, the high dependence of cancer cells on glucose metabolism provides the basis for [18F] fluorodeoxyglucose positron emission tomography (FDG–PET) imaging, a powerful tool to detect andmonitortumorigenicgrowth.
ROOT CAUSE OF CANCER: GENETIC AND EPIGENETIC ALTERATIONS
Mechanisms underlying the above-mentioned phenotypes typically originate from sequential gain or loss of gene functions that can occur within the gene (genetic alterations) or the regulatory process that control gene expression (epigenetic alterations).6
Etiologies of Genetic Alterations Include the Following
Inherited defects. Germline mutation in certain genes can predispose one to developing cancer over time, and is the cause of most familial cancer syndromes.
Exogenous damage. Chemicals, especially aromatic hydrocarbons, heavy metals, and substances such as asbestos fibers can damage the DNA are all potential carcinogens. UVA induces production of reactive oxygen species (ROS), while UVB causes cyclobutane pyrimidine dimers and pyrimidine pyrimidone photoproducts. Platinum and alkylating chemotherapeutic agents are known to cause DNA crosslinks.
Oncogenic viruses. Cells infected with certain DNA viruses and retroviruses can ultimately become tumorigenic. This can be caused by aberrant expression of oncogenes (transduction) or through enhanced expression of cellular protooncogenes (proviral insertion). Examples include HPV in cervical cancer, EBV in Burkitt’s lymphoma and nasopharyngeal carcinoma, HBV and HCV in hepatocellular carcinoma, and HTLV-1 and -2 in T-cell leukemia.
Genomic instability. DNA replication is not 100% fail-safe. Replication errors such as point mutation, chromosomal translocation, amplification or rearrangement can occur and are normally rectified through specified DNA repair mechanisms. If the error cannot be repaired, DNA damage response is triggered and cells are programmed to undergo apoptosis. However, certain errors such as point mutations, small insertions/deletions or reciprocal translocation can sometimes escape DNA repair mechanisms and be passed onto daughter cells. Importantly, cells with defects, either inherited or acquired, in DNA repair mechanism (such as BRCA1/2 mutation) or DNA damage response (such as ataxia-telangiectasia, germline p53 mutation or inhibition by viral proteins such as HPV E6 and E7) are much more likely to accumulate and propagate replication errors. The result is genomic instability and increased chance of cancer formation.
Epigenetic Changes
Other than changes in primary nucleotide sequence, gene expression is also regulated by epigenetic mechanisms such as methylation/demethylation of gene promoters/enhancers and histone modifications.7 In the last decade, control of gene expression at posttranscriptional level by micro-RNAs was discovered and will also be discussed in this section.
DNA Methylation
Methylation of cytosines in CpG dinucleotides by DNA methyltransferases generally leads to transcriptional silencing. Repetitive CpG dinucleotides (or CpG islands) are often found within the upstream promoters of most genes. When methylated, these regions become inaccessible to transcription factors and can attract histone modifying proteins that lead to formation of compact, inactive chromatin. DNA methylation is essential in normal developmental processes such as genomic imprinting and cellular differentiation. However, when the promoter of a tumor suppressor gene is hyper-methylated, transcription is shut down and cells are propelled toward malignant transformation. For example, hypermethylation of the promoter for MLH1, a gene involved in DNA mismatch repair, has been implicated in the pathogenesis of hereditary nonpolyposis colorectal cancer. Two DNA methyltransferase inhibitors, 5-azacytadine and decitabine (2′-deoxy-5-azacytidine), are approved therapy for the treatment of myelodysplastic syndrome and also have activity in acute myeloid leukemia.
Histone Deacetylation
Acetylation/deacetylation of lysine residues on histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively, is another mode of epigenetic modification which can alter gene expression. Deacetylation causes histones to wrap more tightly around DNA, which blocks access of transcription factors to DNA. In cancer, tumor suppressor genes that are silenced by histone deacetylation can theoretically be reactivated by HDAC inhibitors. Currently, HDAC inhibitors such as Vorinostat and Romidepsin are approved for treatment of cutaneous T-cell lymphoma.
Posttranscriptional Regulation: Micro-RNAs
Micro-RNAs, an important discovery of the last decade, are a species of short, single-stranded, noncoding RNAs that can bind to the complementary sequences within target mRNAs and generally lead to their degradation. More than half of human genes are now believed to be regulated by micro-RNAs. Aberrant expression of micro-RNAs is found in many human cancers. 8 For example, miR-15a and miR-16–1, which negatively regulate Bcl-2, an antiapoptotic gene, are often deleted or down-regulated in B-cell CLL. On the other hand, miR-9, which is overexpressed in breast cancer, promotes metastasis by downregulating E-cadherin. Therapies targeting micro-RNAs are still under investigation.
Outcome of Genetic/Epigenetic Alterations: Oncogenes and Tumor Suppressor Genes
The two major classes of genes are that are subject to these changes are the oncogenes and tumor suppressor genes. In principle, tumorigenic changes involve gain of oncogene function and loss of tumor suppressor function.
Oncogenes are altered versions of normal genes (termed proto-oncogenes) that have acquired gain-of-function point mutations, gene copy amplifications or translocations. The results are cellular proliferation, growth, survival, invasion, and angiogenesis. Several categories of oncogenes have been described and these include:
1. Growth factors: sis, trk
2. Growth-factor receptors: EGFR, HER2/neu, c-kit, PDGFR, VEGFR
3. Tyrosine kinases: bcr-abl, src, Syk-ZAP-70, BTK
4. Serine-threonine kinases: Raf, Akt, MAPK, cyclin-dependent kinases, Aurora kinases
5. GTPases: K-Ras, N-Ras, H-Ras, Cdc42, Rac, Ral
6. Cytoplasmic proteins: Bcl-2, survivin
7. Transcription factors: c-myc, jun, fos, Nf-kB
Tumor suppressor genes or gate-keeper genes function to restrain cellular proliferation and/or maintain cellular homeostasis. Classic examples include PTEN, NF1, NF2, and APC which are negative regulators of proliferation; INK4a-ARF which controls cell cycle, p53 which is the master switch of apoptosis, BRCA1/ BRCA2 which are key players in DNA damage response and VHJ which negatively regulates angiogenesis. In cancer cells, expression of tumor suppressor genes is usually lost by biallelic gene deletion, loss-of-function mutations or transcriptionally silenced by epigenetic mechanisms. While loss of tumor suppressor genes alone is not sufficient for malignant transformation, it does predispose cells to accumulate further genetic changes and cooperates with oncogenes to complete cellular transformation. Clinically, germline mutations/deletions of tumor suppressor genes are associated with several familial cancer syndromes (See Table15-1).
p53 mutations.
The tumor suppressor p53 is the most frequently mutated gene in human cancer and therefore warrants further discussion. p53 is normally activated in response to stresses such as DNA damage, hypoxia, and oncogene activation. It encodes a transcription factor that primarily activates genes responsible for cell cycle arrest and apoptosis by several mechanisms. It activates cell cycle inhibitors such as p21 and upregulation of proapoptotic genes such as Bax. Inactivating mutations of p53 are found in more than 50% of all cancers. The majority of p53 mutations (~75%) are missense mutations, usually within the DNA-binding domain that results in an abnormal tertiary structure with impaired ability to regulate transcription of target genes. Hypermethylation of the promoter for p53 and upregulation of its inhibitors, such as HDM-2 are also found in human cancers. Patients with Li–Fraumeni syndrome have germline mutations in the p53 gene and are at high risk for early-onset cancers of multiple tissues types, including breast, bone, soft tissue, head and neck, and brain and, less commonly, lung, stomach, colon, and blood (leukemia). In sporadic cancers, the timing of p53 loss can differ. p53 alterations are usually early events in lung, esophageal, head and neck, breast, cervical, bladder, and stomach cancers, but are late events in brain, thyroid, prostate, and ovarian cancers. There is no consistent pattern in p53 alterations in colon, bladder, and liver cancers. Delivery of p53 by gene therapy is currently in clinical trials. 9
TUMOR METASTASIS
Tumor metastasis warrants more detailed discussion because it accounts for more than 90% of cancer deaths. In most cancers, metastatic trait is acquired late in the multistep oncogenic process. However, clinically the natural history, route and site of metastasis vary with different cancers, which underscore the complex signaling mechanisms behind metastasis. In fact, metastasis in itself is a consummation of multiple distinct properties that an extremely small number of cancer cells acquire during evolution from a genetically heterogeneous population. 10 These properties include:
Detachment from the primary tumor. Adherence of tumor cells to adjacent cells and extracellular matrix is almost always altered. Intercellular adhesion is greatly diminished by downregulation of normal adhesion proteins such as E-cadherin and interactions with extracellular matrix is augmented by expression of specific integrins such as α;6β4. These changes facilitate detachment of tumor cells from their primary locus.
Invasion, migration, and intravasation. Expression of integrins such as α6β4 can greatly augment the invasion of cancer cells through stroma. Disruption of basement membrane is usually achieved via activation of metalloproteases. By undergoing epithelial-to-mesenchymal transition, cancer cells of epithelial origin acquire a mesenchymal cell phenotype that allows for motility and invasion.11 Migration is further directed by various growth factors such as EGF, FGF, HGF and insulin-like growth factor. Subsequently, cancer cells are able to penetrate vascular endothelial lining (intravasation) and enter the bloodstream or lymphatic system.
Survival in the vasculature or lymphatic system. Once in the circulation or lymphatic system, cancer cells must evade immune recognition as described before. Also, by abrogating their major apoptotic machinery, cancer cells are able to avoid anoikis, a type of apoptosis triggered by loss of contact with extra-cellular matrix. Nonetheless, most cancer cells succumb to the high-velocity shear stress within the circulation and only a tiny number of cells survive to reach target organs.
Arrest at the metastatic site and extravasation. The anatomy of blood and lymphatic vasculature, as well as presence of specific chemokines is an important determinant of the site of metastasis. For example, breast cancer cells have high expression of the chemokine receptor, CXCR4, while its ligand, CXCL12, is highly expressed in organs to which it most commonly metastasizes: lung, liver, bone, and regional lymph nodes. Once at the target organ site, cells must arrest in the vasculature and extravasate, which again involves alterations in cell adhesion molecules and proteases.
Survival in the metastatic microenvironment. Once at the target organ, tumor cells (seeds) must successfully interact with their microenvironment (soil) in order to initiate colonization. In addition, angiogenic “switch” must be triggered by secretion of various angiogenic factors to sustain metastatic growth.
FUTURE
The discovery of important signaling pathways propelling certain cancer has led to successful stories of targeted agents such as imatinib, and currently more targeted agents are being developed and tested. However, most cancers consist of highly heterogeneous subpopulations of transformed cells that are driven by complex network of signaling mechanisms. Therefore, it is highly unlikely that targeting one or two signaling pathways is sufficient in curbing cancer growth in most cases. The advent of new, high-throughput techniques such as microarray, genomic sequencing (termed oncogenomics) and recently functional proteomics, in conjunction with bioinformatics and system biology now allow broader view into the signaling lesions underlying every type of cancer, with the potential of allowing combinations of targeted agents. 12 Importantly, these techniques are beginning to redefine or reclassify many cancers based on their molecular profile rather than histology alone. Application of these techniques in aiding diagnosis and treatment decision will likely become more common in the future and become a crucial part in personalizing cancer management.
REFERENCES
1. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61: 759–767.
2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70.
3. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144: 646–674.
4. Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov. 2007;6:273–286.
5. Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat Rev Cancer. 2011;11:85–95.
6. Harris TJ, McCormick F. The molecular pathology of cancer. Nat Rev Clin Oncol. 2010; 7:251–265.
7. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:683–692.
8. Inui M, Martello G, Piccolo S. MicroRNA control of signal transduction. Nat Rev Mol Cell Biol. 2010;11:252–263.
9. Brown CJ, Lain S, Verma CS, et al. Awakening guardian angels: drugging the p53 pathway. Nat Rev Cancer. 2009;9:862–873.
10. Chiang AC, Massague J. Molecular basis of metastasis. N Engl J Med. 2008;359: 2814–2823.
11. Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer. 2009;9:265–273.
12. Bild AH, Potti A, Nevins JR. Linking oncogenic pathways with therapeutic opportunities. Nat Rev Cancer. 2006;6:735–741.