Cancer Genetics and Genomics I PDF

Cancer Genetics and Genomics [1] Julie W. Hirschhorn, Ph.D., HCLD Office: (843) 792-1181 Email: [email protected] A. Malignant Transformation 1. Alterations in Genotype a. Proto-oncogenes b. Tumor Suppressor Genes c. DNA Repair Genes d. Apoptosis Genes 2. DNA Alterations and Variant Classification...

Cancer Genetics and Genomics [1] Julie W. Hirschhorn, Ph.D., HCLD Office: (843) 792-1181 Email: [email protected] A. Malignant Transformation 1. Alterations in Genotype a. Proto-oncogenes b. Tumor Suppressor Genes c. DNA Repair Genes d. Apoptosis Genes 2. DNA Alterations and Variant Classification 3. Phenotypic Changes in the Cell 4. Neoplastic Transformation B. Trademarks of Cancer 1. Driver and Passenger Mutations 2. Clonal Selection and Expansion 3. Oncogene activation 4. Oncogene addiction 5. DNA Damage Repair C. Human Genome Project 1. 2. 3. 4. The Project itself Project Outcomes Impact on Human Genetics and Genomics Genetics versus Genomics Recommended Reading: 1. Mark’s Basic Medical Biochemistry, 6th Ed. 2022. Chapter 18, Sections: Introduction, Causes of Cancer, Damage to DNA Leading to Mutations, Oncogenes, TumorSuppressor Genes, Cancer and Apoptosis, Cancer Requires Multiple Mutations, At the Molecular Level, Cancer is Many Different Diseases. MUSC Link: https://pascal- musc.primo.exlibrisgroup.com/permalink/01PASCAL_MUSC/advtp6/alma991000451854 205641 2. Genetics in Medicine, 8th Ed, 2016, Thompson and Thompson: Chapter 15, Section: Genetic Basis of Cancer. MUSC Library Online Link: https://pascal- musc.primo.exlibrisgroup.com/permalink/01PASCAL_MUSC/6ubsj8/alma991000286805 105641 “The foundation [of cancer] has been set in the discovery of mutations that produce oncogenes with dominant gain of function and tumor suppressor genes with recessive loss of function; both classes of cancer genes have been identified through their alteration in human and animal cancer cells and by their elicitation of cancer phenotypes in experimental models.” Bishop and Weinberg, 1996. Page 1 of 17 OBJECTIVES 1. Describe why all cancers are genetic at the cellular level. 2. Distinguish between the four functional classes of genes to which mutations can cause malignant transformation. 3. Describe a proto-oncogene and an oncogene. 4. Define a tumor suppressor gene. 5. Describe how cancer development can happen because of malfunctions in DNA repair. 6. Describe how gene mutations, chromosome translocations, and gene amplifications can generate a gain of function mutation. 7. Explain how a loss of function mutation can result in malignant transformation or progression. 8. Describe the concepts of oncogene activation and addiction. 9. Describe the importance of the Human Genome Project. Illustrations adapted from: • • • • Henry’s Clinical Diagnosis and Management by Clinical Laboratory Methods, 23rd Edition, © 2017, Elsevier Inc. Molecular Pathology in Clinical Practice, 2nd Edition, © 2016, Springer International Publishing Genetics in Medicine, 8th Edition, © 2016, Elsevier Inc. Mark’s Basic Medical Biochemistry, 5th Edition, © 2018, Wolters Kluwer Other References: Hanahan and Weinberg. The Hallmarks of Cancer. 2000. Cell. 100(7):57-70. Hanahan and Weinberg. Hallmarks of Cancer: The Next Generation. 2011. Cell. 144:646-674. Li et al, Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer. 2017. J Mol Diag. 19(1):4-23. Richards S, et al. Standards and Guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. 2015. Genetics in Medicine. 17(5):405-424. Page 2 of 17 NOTES A. MALIGNANT TRANSFORMATION 1. Alterations in Genotype Cancer is a result of accumulation of gene mutations. Cancer is the second most common cause of death in the United States (the first is heart disease). The frequency of cancer increases with age with most cancer deaths occurring between the ages of 55 and 75 years old. The death rate declines after 75 years of age. Cancer is an abnormal growth and malignant transformation often involves the invasion or metastasis of those cancer cells to other sites within the body. These mutations occur in genes involved in regulation of proliferation, differentiation, DNA repair, and cell survival. Page 3 of 17 These cancer-related genes can be sorted into 4 functional classes: a. Proto-oncogenes and Oncogenes b. Tumor Suppressor Genes c. DNA Repair Genes d. Apoptosis Genes Mutations in cancer-related genes can be somatic or germline: a. Acquired or Somatic: An alteration in DNA that occurs after conception and is not present in the germline. These mutations occur spontaneously or induced by a mutagen b. Germline: A gene change in a reproductive or germ cell that becomes incorporated into the DNA of every cell in the body of an offspring. 2. DNA Alterations and Variant Classification There are many types of variation that can occur in the human genome. Not all variants contribute to cancer or an increased cancer risk. Page 4 of 17 • A synonymous variant does not lead to a change in the amino acid. Note that a silent variant is a type of synonymous variant that leads to no phenotypic change. Synonymous variants, however, may not always be silent: they can potentially affect RNA degradation or translational efficiency. • Missense variants may or may not have an impact on protein function based on whether the amino acid change leads to properties like the wildtype amino acid or a more significant change. • Frameshift variants lead to a change in the coding frame of the sequence that will result in all downstream amino acids being altered. • Polymorphisms exist in a population at greater than 1% allele frequency. Variant Classification: Type of Variant Pathogenic Likely Pathogenic Description Directly contributes to the development of disease. Additional evidence is not expected to alter the classification of this variant. [Note: not all pathogenic variants are fully penetrant] Very likely to contribute to the development of disease, but scientific evidence is currently insufficient to prove this conclusively. Uncertain Significance There is not enough information currently to support a more definitive classification of this variant. Likely Benign Not expected to have a major effect on disease, but the scientific evidence is currently insufficient to prove this conclusively. Benign Does not cause disease. Additional evidence is not expected to alter the classification of this variant. Adapted from Richards et al. 2015. Determining the functional change in a protein from a variant change at the amino acid level is not always straight forward. • In some cases, there are pre-clinical studies in cell or animal models that describe the impact of the variant on protein expression that may or may not translate directly to humans. • For sites not described in the literature, the use of in silico (computational) prediction algorithms can be used to predict if a nucleotide change in a Page 5 of 17 gene will change the structure and function of the protein (Li et al 2017). The most common characteristics considered to come up with a “label” for the variant change by these in silico models include items such as degree of evolutionary conservation of the nucleotide or amino acid, the biochemical impact of the substituted amino acid, location of the variant in the functional domains of the translated protein. • Some sites have been described and biochemically characterized in human clinical trials. There are different labeling conventions for germline and somatic variants. Below is the germline labeling system. For somatic variants, the classifications are damaging, likely damaging, uncertain, likely benign and benign. Functional Mutations Classifications: a. Gain of Function: Protein product has abnormal function(s) caused by overexpression or by acquisition of a new function; disorders are usually dominant. b. Loss of Function: Protein product has reduced or abolished function; disorders are usually recessive. Heterozygotes with 50% function are either asymptomatic or exhibit mild phenotype. 3. Phenotypic Changes in the Cell a. Alterations in cell cancer genotypes were summarized into six categories by Hanahan and Weinberg in their formative Hallmarks of Cancer paper (Cell, 2000). This figure describes that alterations in genes related to these 6 categories of cell physiology can promote malignant growth. For example, the three acquired capabilities of growth signal autonomy, insensitivity to growth signals, and resistance to apoptosis lead to an uncoupling of the cell’s ability to read growth signals from its environment and promote its own proliferation program separate from the normal cells around the tumor cell. 4. Neoplastic Transformation Neoplastic transformation is the conversion of normal cells into tumor cells and is the result of genetic changes. Malignant neoplasms do not tend to arise from benign neoplasms. In some conditions, such as liver cirrhosis and chronic Page 6 of 17 ulcerative colitis, malignant transformation may occur, and this is due to the ongoing proliferation leading to a greater likelihood for mutation to occur. Neoplasms have a greater likelihood for karyotypic abnormalities, such as translocations, deletions, and gene amplifications. Some neoplastic growth can be influenced by host factors, such as hormones. The major characteristics of neoplastic cells include: a. Growth is not inhibited by contact with surrounding cells. The tumor cells can behave independently from their surroundings. b. Tumor cells are transplantable and can adapt to other locations within the body, which favors invasion and metastasis. c. Tumor cells can bind to laminin and fibronectin in connective tissues, and then secrete collagenases and proteases to invade. d. Neoplastic cells can avoid cell death and/or can divide indefinitely. Genetics in Medicine. Figure 15-1 B. TRADEMARKS OF CANCER 1. Driver and Passenger Mutations The number of mutations present in a cancer can range from just a few to many tens of thousands. One way to describe mutations in cancer is as drivers and passengers: a. Driver Mutations: These are mutations presumed to be involved in the development or progression of the cancer. Often occur in cancer-related genes. b. Passenger Mutations: These mutations probably occurred as the cancer developed and probably did not cause the neoplasia. Most mutations are passengers and occur randomly. Driver mutations can be further classified into oncogenes/proto-oncogenes and tumor suppressor genes. Page 7 of 17 • A proto-oncogene is a class of genes that under normal cellular circumstances promote the normal survival and growth of cells. o A proto-oncogene with a gain-of-function mutation is referred to as an activated oncogene. The gain-of-function mutation will cause transformation to a malignant phenotype. This type of mutation is considered dominant because just one copy of a proto-oncogene turned oncogene can produce a transformed phenotype. • An oncogene is a gene that has the potential to cause cancer. Oncogenes can also be activated because of gene fusions or changes in expression levels. • Tumor suppressor genes are activated because of a loss-of-function mutation. o Normally, these genes function by inhibiting cellular proliferation in response to stimuli such as DNA damage o They are protective in the normal cell situation, but when there is a loss-of-function mutation then the protective function of these proteins is inactivated o A tumor suppressor gene mutation must be recessive - meaning that both alleles of the tumor suppressor gene must be inactivated Examples of Driver Mutations: A. The BRAF gene (proto-oncogene) a. Constitutively activating mutations in BRAF have been identified in approximately 5-10% of all human cancers; most variants are single Page 8 of 17 (6) nucleotide changes (most prevalent is V600E), but also fusions • Melanoma, colorectal cancer, ovarian tumor, thyroid carcinoma, hairy cell leukemia, and lung adenocarcinoma b. BRAF is normally involved in the activation of the RAS/MAPK signaling pathway, which relates growth and proliferation of cells, differentiation, migration, and apoptosis • The MAPK pathway is only activated in response to ligand binding to the cell surface receptor tyrosine kinases or to cytokine receptors • When activated these RTKs or cytokine receptors will activate monomeric G protein family members to promote the formation of signal-transduction complexes and activate downstream signal cascades finally activating ERK • ERK is a serine/threonine kinase that can phosphorylate various targets, including transcription factors c. When an activating variant is present constitutive activation of the MAP kinase/ERK signaling pathways causes the cell undergoes oncogenic transformation • V600E activates the kinase domain • Other variants in exons 11 and 15 will occur in a glycine-rich loop and activation segment of the kinase domain • Variants in these regions disrupt the normal interactions that hold BRAF in an inactive conformation B. The CDKN2A gene (Tumor Suppressor) a. This gene contains 4 exons and alternative splicing results in two tumor suppressors: the p14ARF protein or the p16INK4a protein • Both share exon 2 b. Normally, p14 acts to sequester the p53-specific ubiquitin ligase HDM2. c. If p14 is lost, HDM2 targets p53 for ubiquitination and proteasomal degradation. The loss of p53 impairs the normal targeting of damaged Page 9 of 17 cells for destruction (cell cycle arrest or apoptosis) and there is a proliferation of these damaged cells leading to genomic instability. d. Normally, p16 acts as a negative regulator of the proliferation of normal cells by interacting strongly with CDK4 and CDK6, keeping pRb in a hypo-phosphorylated active state, which will decelerate the cell’s progression from G1 to S phase. e. If p16 is lost, then CDK4/CDK6 can bind cyclin D and phosphorylate Rb. Phosphorylated pRb dissociates from the transcription factor E2F, which will translocate to the nuclease and promotes transcription of genes promoting entry into S phase and DNA synthesis. C. TP53 Gene (Tumor Suppressor) a. Normally in unstressed cells, TP53 is targeted for E3 ubiquitin ligase degradation by MDM2 (HDM2). During cellular stress, p53 can be activated leading to downstream responses such as apoptosis, G1 to S phase cell-cycle arrest, senescence, or modulation of autophagy. • Activation of p53 can be dependent on the type of stress. For example, when exposed to ionizing radiation, Chk2 (a DNAdamage induced kinase) will phosphorylate a site within the amino terminus of p53 that inhibits the interaction of p53 with MDM2, preventing p53 degradation. This pathway does not respond to UVmediated DNA damage. b. Most investigated mechanisms of p53-mediated tumor suppressor are focused on transcriptional activation of target genes. Page 10 of 17 c. The p53 gene is referred to as the “Guardian of the Genome” d. Loss of both p53 alleles is found in >50% of ALL human cancers e. There is complexity to the transcriptional regulation by p53 signaling. There is variability in the mechanism of response, and this can vary by cell type, stress type, and other microenvironment factors. f. In response to stress, p53 can work through multiple mechanisms. Two of those mechanisms are described here: • p53 can stimulate transcription of p21. This gene product will inhibit the formation of cyclin-CDK complexes and prevent RB phosphorylation and release of E2F proteins preventing the cell from entering S phase. • p53 can stimulate the transcription of DNA repair enzymes, like GADD45, if DNA repair is successful then p53 will downregulate itself through activation of the mdm2 gene. If DNA repair is not successful, then p53 will activate apoptosis genes (e.g. bax and IGF-BP3). Mark’s Basic Medical Biochemistry, Figure 18.10 Page 11 of 17 2. DNA Damage Repair Damage control checkpoints normally function as a mechanism to arrest cell cycle progression in response to either DNA damage or un-replicated DNA. All carcinogens ultimately damage DNA. DNA damage can result from: • Point mutations • Gene rearrangements • Gene amplifications (copy number changes) Page 12 of 17 3. Clonal Selection and Expansion Mutations and epigenetic changes accumulate in cell lineages by clonal selection and expansion. Mark’s Basic Medical Biochemistry, Figure 18.1 “Tumor progression” results from subclones that arise over time from the original malignant clone. These additional mutations may contribute to characteristics of invasiveness, metastatic potential, and response to therapy. Tumor growth is also dependent on the ability of the tumor to develop a blood supply to continue to feed the growing population of tumor cells. The tumor cells secrete factors that promote angiogenesis and fibroblast proliferation. 4. Oncogene activation A proto-oncogene with a gain-of-function mutation is referred to as an activated oncogene. A variety of different mutation types can cause protooncogene activation. Oncogenes activated by gene translocation and rearrangement can lead to the creation of a novel protein (e.g. Philadelphia Chromosome, t(9;22)(q34;q11)). The functional products of oncogene activation can lead to oncogene addiction and oncogene progression. Page 13 of 17 a. BRAF V600E • See more detail on page 8 of these notes • Caused by a single nucleotide change or two neighboring nucleotides that results in an amino acid change from Valine at codon 600 • Results in constitutive activation of the BRAF protein b. Exon 14 Skipping Mutation in Lung Cancer • Alternative splicing results in the exclusion of exon 14 of the MET gene leading to impaired receptor degradation and oncogenic transformation • • Prevalence of approximately 3% of metastatic NSCLC patients per year in the U.S. • • Exon 14 contains the juxtamembrane domain that is the binding site for the E3 ubiquitin ligase, CBL. FDA approved treatment for non-small cell lung cancer with these mutations The hypothesized mechanism of action for this splice variant is that the lack of the binding site for CBL there is a reduction in ubiquitination and degradation of MET. This leads to MET protein stability and increased downstream ligand-dependent signaling. Page 14 of 17 Drilon A. MET Exon 14 Alterations in Lung Cancer: Exon Skipping Extends HalfLife. Clin Cancer Res. 2016 Jun 15;22(12):2832-4. c. Philadelphia Chromosome, t(9;22), BCR-ABL1 • Oncogene activation can occur because of a translocation that results in the formation of a new protein • More than 40 oncogenic translocations have been described, mostly in leukemias and lymphomas (rarely in connective tissue sarcomas) • The Philadelphia chromosome is the most notable resulting in a reciprocal and balanced translocation between chromosomes 9 (ABL, protooncogene) and 22 (BCR gene, unknown function) • This translocation is observed in more than 90% of chronic myelogenous leukemia (CML) cases • The fusion gene encodes a chimeric protein (BCR-ABL1) is an activated tyrosine kinase and can be targeted with tyrosine kinase inhibitors (TKIs) Page 15 of 17 d. HER2 • Encoded by the Human ERBB2 gene • Amplification is observed in 15-30% of breast cancers; amplification also observed in ovarian, stomach, adenocarcinoma of the lung and some types of uterine cancer • FDA approved drug that targets HER2 e. t(8;14) translocation in Burkitt’s Lymphoma • Translocation results in overexpression of the MYC proto-oncogene (chromosome 8) by removing it from its normal location and placing it under the control of the highly active immunoglobulin heavy chain promoter (chromosome 14) • MYC encodes a nuclear phosphoprotein that is involved in cell cycle progression, apoptosis, and cellular transformation • MYC amplification is observed in numerous cancers. • More than 90% of cases of Burkitt Lymphoma have this translocation 5. Oncogene addiction This is a phenomenon where the survival and growth of a cancer cell is dependent on an activated oncogene or the inactivation of a tumor suppressor gene. This concept of oncogene addiction is the rationale behind targeted therapies. Also, tumor cells can “escape” from a given state of oncogene addiction through mutations in the same gene or other genes and pathways. C. HUMAN GENOME PROJECT a. The Project Just like the Rosetta stone was a huge archeological find necessary to unlock the mystery of ancient Egyptian hieroglyphics, the Human Genome Project was a necessary step to allow for rapid progress in the application and study of human genomics. The human genome project began in 1990 as an international public effort to sequence all 3 billion nucleotide base pairs in the human genome. The goal of the project was to provide researcher with powerful tools to understand the underlying genetic factors in human disease and allow for innovation in Page 16 of 17 diagnosis, treatment, and prevention. b. Project Outcomes From the beginning all of the data generated by the project was made freely and rapidly available. This allowed for the creation of additional publicly available databases that have worked to categorize the information in the human genome. There are approximately 23,000 genes in the human genome, which is considered the coding portion of the genome and only accounts for about 1% of the human genome. Much of the human genome is non-coding and what is known about non-coding regions of the human genome is considerably less than the coding regions. c. Impact on Human Genetics and Genomics Molecular profiling and the study of the genes involved in diseases would be more difficult without the completion of the human genome. Knowing the sequence of the human genome and mapping out the genes and non-coding regions have helped to accelerate research into the mechanisms underlying disease. Summary At this beginning of this discussion, we looked at an image from 2000 summarizing the hallmarks of cancer. With just 10 additional years of research, 4 additional hallmarks were added, including genomic instability and mutation, tumor-promoting inflammation, avoiding immune destruction, and de-regulating cellular energetics. You should know the main hallmarks, not the types of inhibitors used to treat these hallmarks. Page 17 of 17