Lecture 7 Cancer Genetics and Genomics Cancer is not a single disease, it is used to describe the more virulent forms of neoplasia, a disease process characterized by uncontrolled cellular proliferation leading to a mass or tumor (neoplasm). For a neoplasm to be a cancer, however, it must also be malignant, which means its growth is no longer controlled and the tumor is capable of progression by (1) invading neighboring tissues, (2) spreading (metastasizing) to more distant sites, or both. Tumors that do not invade or metastasize are not cancerous but are referred to as benign tumors. There are three main forms of cancer: – sarcomas, in which the tumor has arisen in mesenchymal tissue, such as bone, muscle, or connective tissue, or in nervous system tissue; – carcinomas, which originate in epithelial tissue, such as the cells lining the intestine, bronchi, or mammary ducts; and – hematopoietic and lymphoid malignant neoplasms, such as leukemia and lymphoma, which spread throughout the bone marrow, lymphatic system, and peripheral blood. Within each of the major groups, tumors are classified by site, tissue type, histological appearance, and degree of malignancy. GENETIC BASIS OF CANCER Neoplasia is an abnormal accumulation of cells that occurs because of an imbalance between cellular proliferation and cellular attrition. Cells proliferate as they pass through the cell cycle and undergo mitosis. Attrition, due to programmed cell death, removes cells from a tissue. The Genetic Basis of Cancer Regardless of whether a cancer occurs sporadically in an individual, as a result of somatic mutation, or repeatedly in many individuals in a family as a hereditary trait, cancer is a genetic disease. Genes in which mutations cause cancer fall into two distinct categories: oncogenes and tumor-suppressor genes (TSGs). TSGs in turn are either "gatekeepers" or "caretakers". An oncogene is a mutant allele of a proto- oncogene, a class of normal cellular protein- coding genes that promote growth and survival of cells. Oncogenes facilitate malignant transformation by stimulating proliferation or inhibiting apoptosis. Oncogenes encode proteins such as: – proteins in signaling pathways for cell proliferation – transcription factors that control the expression of growth-promoting genes – inhibitors of programmed cell death machinery Gatekeeper TSGs control cell growth. Gatekeeper genes block tumor development by regulating the transition of cells through checkpoints ("gates") in the cell cycle or by promoting programmed cell death and, thereby, controlling cell division and survival. Loss-of-function mutations of gatekeeper genes lead to uncontrolled cell accumulation. Gatekeeper TSGs encode: – regulators of various cell-cycle checkpoints – mediators of programmed cell death Caretaker TSGs protect the integrity of the genome. Loss of function of caretaker genes permits mutations to accumulate in oncogenes and gatekeeper genes, which, in concert, go on to initiate and promote cancer. Caretaker TSGs encode: – proteins responsible for detecting and repairing mutations – proteins involved in normal chromosome disjunction during mitosis – components of programmed cell death machinery Tumor initiation. Different types of genetic alterations are responsible for initiating cancer. These include mutations such as: – activating or gain-of-function mutations, including gene amplification, point mutations, and promoter mutations, that turn one allele of a proto-oncogene into an oncogene – ectopic and heterochronic mutations of proto- oncogenes – chromosome translocations that cause misexpression of genes or create chimeric genes encoding proteins with novel functional properties – loss of function of both alleles, or a dominant negative mutation of one allele, of TSGs. Tumor progression. Once initiated, a cancer progresses by accumulating additional genetic damage, through mutations or epigenetic silencing, of caretaker genes that encode the machinery that repairs damaged DNA and maintains cytogenetic normality. A further consequence of genetic damage is altered expression of genes that promote vascularization and the spread of the tumor through local invasion and distant metastasis. Some tumor-suppressor genes directly regulate proto-oncogene function (gatekeepers); others act more indirectly by maintaining genome integrity and correcting mutations during DNA replication and cell division (caretakers). Activation of an antiapoptotic gene allows excessive accumulation of cells, whereas loss of function of apoptotic genes has the same effect. Activation of oncogenes or antiapoptotic genes is dominant. Mutations in tumor-suppressor genes are recessive; when both alleles are mutated or inactivated, cell growth is unregulated or genomic integrity is compromised. Loss of pro-apoptotic genes may occur through loss of both alleles or through a dominant negative mutation in one allele. The development of cancer (oncogenesis) results from mutations in one or more of the vast array of genes that regulate cell growth and programmed cell death. When cancer occurs as part of a hereditary cancer syndrome, the initial cancer-causing mutation is inherited through the germline and is therefore already present in every cell of the body. Most cancers, however, are sporadic because the mutations occur in a single somatic cell, which then divides and proceeds to develop into the cancer. It is not surprising that somatic mutations can cause cancer. Large numbers of cell divisions are required to produce an adult organism of an estimated 1014 cells from a single-cell zygote. Given a frequency of 10-10 replication errors per base of DNA per cell division, and an estimated 1015 cell divisions during the lifetime of an adult, replication errors alone result in thousands of new DNA mutations in the genome in every cell of the organism. Genome and chromosome mutations add to the mutational burden. The genes mutated in cancer are not inherently more mutable than other genes. Many mutations doubtlessly occur in somatic cells and cause one cell among many to lose function or die, but they have no phenotypic effects because the loss of one cell is masked by the vast majority of healthy cells in an organ or tissue. What distinguishes oncogenic mutations is that, by their very nature, they allow even one mutant cell to develop into a life-threatening disease. Micro-RNA genes The catalogue of genes involved in cancer also includes genes that are transcribed into noncoding RNAs from which regulatory microRNAs (miRNAs) are generated. There are at least 250 miRNAs in the human genome that carry out RNA-mediated inhibition of the expression of their target protein-coding genes, either by inducing the degradation of their targets' mRNAs or by blocking their translation. Approximately 10% of miRNAs have been found to be either greatly overexpressed or down-regulated in various tumors, and are referred to as oncomirs. One example is the 100-fold overexpression of the miRNA miR-21 in glioblastoma multiforme, a highly malignant form of brain cancer. Overexpression of some miRNAs can suppress the expression of tumor-suppressor gene targets, whereas loss of function of other miRNAs may allow overexpression of the oncogenes they regulate. Since each miRNA may regulate as many as 200 different gene targets, overexpression or loss of function of miRNAs may have widespread oncogenic effects because many genes will be dysregulated. Initiation and Progression of Cancer Once it is initiated, a cancer progresses by accumulating additional genetic damage through mutations in caretaker genes encoding the cellular machinery that repairs damaged DNA and maintains cytogenetic normality. Damage to these genes produces an ever- widening cascade of mutations in an increasing assortment of the genes that control cellular proliferation and repair DNA damage. In this way, the original clone of neoplastic cells serves as a reservoir of genetically unstable cells, referred to as cancer stem cells. These give rise to multiple sublineages of varying degrees of malignancy, each carrying a set of mutations that are different from but overlap with mutations carried in other sublineages. In this sense, cancer is fundamentally a "genetic" disease, and mutations are central to its etiology and progression. A paradigm for the development of cancer, as illustrated in Figure 16-2, provides a useful conceptual framework for considering the role of genetic changes in cancer. It is a general model that probably applies to many if not most cancers, although it is best elucidated in the case of colon cancer. Figure 16-2. Stages in the evolution of cancer. Increasing degrees of abnormality are associated with sequential loss of tumor- suppressor genes from several chromosomes and activation of proto-oncogenes, with or without a concomitant defect in DNA repair. Multiple lineages carrying somewhat different mutational spectra and epigenetic changes are likely, particularly once metastatic disease appears. Cancer in Families Many forms of cancer have a higher incidence in relatives of patients than in the general population. Most prominent among these familial forms of cancer are the nearly 50 mendelian hereditary cancer syndromes in which the risk of cancer is very high and the approximately 100 additional mendelian disorders listed in Online Inheritance in Man that predispose to cancer. Extensive epidemiological studies have shown, however, that some families have an above-average risk of cancer even in the absence of an obvious mendelian inheritance pattern. For example, an increased incidence of cancer, in the range of 2- to 3-fold, has been observed in first- degree relatives of probands with most forms of cancer, which suggests that many cancers are complex traits resulting from both genetic and environmental factors. Thus, a family history of cancer in multiple first- degree or second-degree relatives of a patient should arouse the physician's suspicion of increased cancer risk in the patient. Although individuals with a hereditary cancer syndrome represent probably less than 5% of all patients with cancer, identification of a genetic basis for their disease has great importance both for clinical management of these families and for understanding cancer in general. First, the relatives of individuals with strong hereditary predispositions, which are most often due to mutations in a single gene, can be offered testing and counseling to provide appropriate reassurance or more intensive monitoring and therapy, depending on the results of testing. Second, as is the case with many common diseases, understanding the hereditary forms of the disease provides crucial insights into disease mechanisms that go far beyond the rare hereditary forms themselves. ONCOGENES An oncogene is a mutant gene whose altered function or expression results in abnormal stimulation of cell division and proliferation. The mutation can be an activating gain-of-function mutation in the coding sequence of the oncogene itself, a mutation in its regulatory elements, or an increase in its genomic copy number, leading to unregulated heterochronic or ectopic function of the oncogene product. Oncogenes have a dominant effect at the cellular level; that is, when it is activated or overexpressed, a single mutant allele is sufficient to initiate the change in phenotype of a cell from normal to malignant. Activated oncogenes encode proteins that act in many steps in the pathway that controls cell growth, including growth factors that stimulate cell division, the receptors and cytoplasmic proteins that transduce these signals, the transcription factors that respond to the transduced signals, and the proteins that counteract programmed cell death (apoptosis). Fig 16-3. Mechanisms of tumorigenesis by oncogenes of various classes. Unregulated growth factor signaling may be due to mutations in genes encoding growth factors themselves (1), their receptors (2), or intracellular signaling pathways (3). Downstream targets of growth factors include transcription factors (4), whose expression may become unregulated. Both telomerase (5) and antiapoptotic proteins that act at the mitochondria (6) may interfere with cell death and lead to tumorigenesis. Table 16-1. Mechanisms of Activation of Proto-oncogenes Mechanism Type of Gene Activated Result Regulatory mutation Growth factor genes Increased expression Structural mutation Growth factor receptors, Allows autonomy of signal-transducing proteins expression Translocation, retroviral Transcription factors Overexpression insertion, gene amplification Regulatory mutation, Oncomirs Overexpression, down- translocation, retroviral insertion regulates tumor-suppressor genes Deletion, inactivating mutation Oncomirs Loss of expression, up- regulates oncogenes TUMOR-SUPPRESSOR GENES Whereas the proteins encoded by oncogenes promote cancer, mutations in tumor-suppressor genes (TSGs) contribute to malignancy by a different mechanism, that is, through loss of function of both alleles of the gene. TSGs are highly heterogeneous. Some truly suppress tumors by regulating the cell cycle or causing growth inhibition by cell-cell contact; TSGs of this type are gatekeepers because they regulate cell growth directly. Other TSGs, the caretakers, are involved in repairing DNA damage and maintaining genomic integrity. Loss of both alleles of genes that are involved in repairing DNA damage or chromosome breakage leads to cancer indirectly by allowing additional secondary mutations to accumulate either in proto-oncogenes or in other TSGs. Table 16-3. Selected Tumor-Suppressor Genes Gene Gene product and possible DISORDERS IN WHICH THE GENE IS function sporadic AFFECTED Gatekeepers Familial Sporadic RB1 p110 Retinoblastoma Retinoblastoma, small Cell cycle regulation cell lung carcinomas, breast cancer TP53 p53 Li-Fraumeni Lung cancer, breast Cell cycle regulation syndrome cancer, many others DCC Dcc-receptor None known Colorectal cancer Decreases cell survival in the absence of survival signal from its netrin ligands VHL Vhl von Hippel- Clear cell renal Forms part of a cytoplasmic Lindau syndrome carcinoma destruction complex with APC that normally inhibits induction of blood vessel growth when oxygen is present Gene Gene product and DISORDERS IN WHICH THE GENE IS possible function AFFECTED sporadic Caretakers Familial Sporadic BRCA1, Brca1, Brca2 Familial breast Breast cancer, ovarian BRCA2 Chromosome repair in and ovarian cancer response to double- cancer stranded DNA breaks MLH1, Mlh1, Msh2 Hereditary Colorectal cancer MSH2 Repair nucleotide nonpolyposis mismatches between colon cancer strands of DNA The Two-Hit Origin of Cancer The existence of TSG mutations leading to cancer was originally proposed in the 1960s to explain why certain tumors can occur in both hereditary and sporadic forms. For example, it was suggested that the hereditary form of the childhood cancer retinoblastoma might be initiated when a cell in a person heterozygous for a germline mutation in a tumor- suppressor retinoblastoma gene, required to prevent the development of the cancer, undergoes a second, somatic event that inactivates the other allele. As a consequence of this second somatic event, the cell loses function of both alleles, giving rise to a tumor. The second hit is most often a somatic mutation, although loss of function without mutation, such as occurs with transcriptional silencing, has also been observed in some cancer cells. In the sporadic form of retinoblastoma, both alleles are also inactivated, but in this case, the inactivation results from two somatic events occurring in the same cell. The "two-hit" model is now widely accepted as the explanation for many familial cancers besides retinoblastoma, including familial polyposis coli, familial breast cancer, neurofibromatosis type 1 (NF1), hereditary nonpolyposis colon carcinoma, and a rare form of familial cancer known as Li- Fraumeni syndrome. In all of these syndromes, the second hit is often but not always a mutation. Silencing due to epigenetic changes such as DNA methylation, associated with a closed chromatin configuration and loss of accessibility of the DNA to transcription factors, is another important, alternative molecular mechanism for loss of function of a TSG. Because an alteration in gene function due to methylation is stably transmitted through mitosis, it behaves like a mutation; because there is no change in the DNA itself, however, the alteration is referred to as an epigenetic rather than a genetic change. Epigenetic silencing of gene expression is a normal phenomenon that explains such widely diverse phenomena as X inactivation, genomic imprinting, and regulation of a specialized repertoire of gene expression in the development and maintenance of differentiation of specific tissues. Gatekeeper Tumor-Suppressor Genes in Autosomal Dominant Cancer Syndromes Retinoblastoma Retinoblastoma, the prototype of diseases caused by mutation in a TSG, is a rare malignant tumor of the retina in infants, with an incidence of about 1 in 20,000 births. Diagnosis of a retinoblastoma must usually be followed by removal of the affected eye, although smaller tumors, diagnosed at an early stage, can be treated by local therapy so that vision can be preserved. About 40% of cases of retinoblastoma are of the heritable form, in which the child inherits one mutant allele at the retinoblastoma locus (RB1) through the germline. A somatic mutation or other alteration in a single retinal cell leads to loss of function of the remaining normal allele, thus initiating development of a tumor. The disorder is inherited as a dominant trait because the large number of primordial retinoblasts and their rapid rate of proliferation make it very likely that a somatic mutation will occur in one or more of the more than 106 retinoblasts. Since the chance of the second hit in the heritable form is so great, the hit occurs frequently in more than one cell, and thus heterozygotes for the disorder are often affected with multiple tumors, often affecting both eyes. On the other hand, the occurrence of the second hit is a matter of chance and does not occur 100% of the time; the penetrance of retinoblastoma, therefore, although high, is not complete. The other 60% of cases of retinoblastoma are nonheritable (sporadic); in these cases, both RB1 alleles in a single retinal cell have been inactivated independently. Because two hits in the same cell is a rare event, there is usually only a single clonal tumor and the retinoblastoma is found in one eye only. Although sporadic retinoblastoma usually occurs in one place in one eye only, 15% of patients with unilateral retinoblastoma have the heritable type but by chance develop a tumor in only one eye. Another difference between hereditary and sporadic tumors is that the average age at onset of the sporadic form is in early childhood, later than in infants with the heritable form. Figure 16-5 Retinoblastoma in a young girl, showing as a white reflex in the affected eye when light reflects directly off the tumor surface. Loss of Heterozygosity Geneticists studying DNA polymorphisms in the region close to the RB1 locus made an unusual but highly significant genetic discovery when they analyzed the alleles seen in tumor tissue from retinoblastoma patients. Individuals with retinoblastoma who were heterozygous at polymorphic loci near RB1 in normal tissues, such as in their white blood cells, had tumors that contained alleles from only one of their two chromosome 13 homologues, revealing a loss of heterozygosity (LOH) in the region of the gene. In familial cases, the retained chromosome 13 markers were the ones inherited from the affected parent, that is, the one with the abnormal RB1 allele. Thus, LOH represented the second hit of the remaining allele. LOH may occur by interstitial deletion, but there are other mechanisms, such as mitotic recombination or nondisjunction. LOH is the most common mutational mechanism by which the function of the remaining normal RB1 allele is disrupted in heterozygotes. When LOH is not seen, the second hit is usually a second somatic gene mutation or, occasionally, transcriptional inactivation of a nonmutated allele through methylation. LOH is a feature of a number of other tumors, both heritable and sporadic, and is often considered evidence for the existence of a TSG, even when that gene is unknown. Figure 16-6 Comparison of mendelian and sporadic forms of cancers such as retinoblastoma and familial polyposis of the colon. Figure 16-7 Chromosomal mechanisms that could lead to identical DNA markers at or near a tumor-suppressor gene in an individual heterozygous for an inherited germline mutation. The figure depicts the events that constitute the "second hit" that leads to retinoblastoma. Local events such as mutation, gene conversion, or transcriptional silencing, however, could cause loss of function of both RB1 genes without producing LOH. + is the normal allele, rb the mutant allele. The RB1 gene maps to chromosome 13, in band 13q14. In a small percentage of patients with retinoblastoma, the first mutation is a cytogenetically detectable deletion or translocation of this portion of chromosome 13. Such chromosomal changes, if they also disrupt genes adjacent to RB1, may lead to dysmorphic features in addition to retinoblastoma. Li-Fraumeni syndrome There are rare "cancer families" in which there is a striking history of many different forms of cancer (including several kinds of bone and soft tissue sarcoma, breast cancer, brain tumors, leukemia, and adrenocortical carcinoma), affecting a number of family members at an unusually early age, inherited in an autosomal dominant pattern. This highly variable phenotype is known as the Li-Fraumeni syndrome (LFS). Because the TSG TP53, encoding the protein p53, is inactivated in the sporadic forms of many of the cancers found in LFS, TP53 was considered a candidate for the gene defective in LFS. DNA analysis of several families with LFS has now confirmed this hypothesis; affected members in more than 70% of families with LFS carry a mutant form of the TP53 gene as a germline mutation. As seen also in retinoblastoma, one of the two mutations necessary to inactivate the TP53 gene is present in the germline in familial LFS, whereas in many sporadic cancers, both mutations are somatic events. Figure 16-8 A pedigree of the Li-Fraumeni syndrome, in which breast cancer, sarcomas, and other malignant tumors have occurred. Ages at diagnosis are shown. The p53 protein is a DNA-binding protein that appears to be an important component of the cellular response to DNA damage. In addition to being a transcription factor that activates the transcription of genes that stop cell division and allow repair of DNA damage, p53 also appears to be involved in inducing apoptosis in cells that have experienced irreparable DNA damage. Loss of p53 function, therefore, allows cells with damaged DNA to survive and divide, thereby propagating potentially oncogenic mutations. The TP53 gene can therefore be considered to also be a gatekeeper TSG.