Carcinogenesis PDF Lecture Notes

Carcinogenesis DSPR 139: Neoplasia and Genetics Daria Vasilyeva, DDS  1. Basic concepts in genetics 2. Cell cycle and signal transduction 3. Genetic basis of neoplasia 4. Targets of gene mutation in At a glance carcinogenesis:...

Carcinogenesis DSPR 139: Neoplasia and Genetics Daria Vasilyeva, DDS  1. Basic concepts in genetics 2. Cell cycle and signal transduction 3. Genetic basis of neoplasia 4. Targets of gene mutation in At a glance carcinogenesis: a) Proto-oncogenes b) Tumor suppressors c) Apoptosis d) DNA repair Gene Basic functional unit of heredity ○ Every person has two copies of each gene ○ One from each parent Most genes: the same in all people < 1% of genes: slightly different between people Alleles: variations of a single gene Contribute to each person’s unique physical features (= phenotype) Gene A distinct DNA sequence of nucleotides within a chromosome Acts as instructions to make molecules called proteins Genes vary in size from a few hundred DNA bases to > 2 million bases Allele: variations of single gene with small differences in sequence of DNA bases Humans have 20,000-25,000 genes  Information in DNA (deoxyribonucleic acid) is stored as code of four chemical bases on a phosphate-deoxyribose backbone (nucleotides) Sequence of bases determines the information ○ This information determines the proteins that will be built (translation)  Chromosome Very efficiently condensed packaging of DNA 46 chromosomes (23 pairs) in the nucleus of most human cells ○ 23 come from mother’s egg ○ 23 come from father’s sperm What chromosomes look like most of the time In preparation for cell division DNA replication occurs, and for a brief period of time there are two identical copies of DNA per chromosome Chromosome Anatomy Chromosome Anatomy: Address of a Gene Chromosome arms: further divided into regions, bands, sub-bands (counting away from the centromere) Every gene has an ‘address:’ e.g. APC lives at 5q21 Match definition to concept: Version of a gene A: Gene, B: Allele, C: Chromosome, D: Chromatid, E: Nucleotide Match definition to concept: Tightly packed DNA A: Gene, B: Allele, C: Chromosome, D: Chromatid, E: Nucleotide Match definition to concept: Building block of DNA A: Gene, B: Allele, C: Chromosome, D: Chromatid, E: Nucleotide  Match definition to concept: Contains instructions to make particular protein A: Gene, B: Allele, C: Chromosome, D: Chromatid, E: Nucleotide Match definition to concept: Has two arms A: Gene, B: Allele, C: Chromosome, D: Chromatid, E: Nucleotide How many chromosomes do most human cells have? A: 23, B: 46, C: 92, D: 5, E: 11 How many chromatids does a human chromosome usually have? A: 1, B: 2, C: 3, D: 4  Cell cycle G0 (resting) phase: where the cell spends most of its time Cell cycle: series of events that take place in a cell as it prepares for division (grows) and then divides into two daughter cells. 1. Cyclins (proteins) turn on kinases 2. Cyclin-dependent kinases (CDKs) (enzymes) increase in activity in response to elevated cyclin levels → drive progression through cell cycle 3. CDK inhibitors (different kinds of chemicals) inhibit CDK function The cell cycle is a highly orchestrated balance of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors  From Gene to Signaling Pathway Genes encode proteins Many proteins participate in signaling pathways ○ Chemical or physical signal is transmitted through a cell as a series of molecular events (e.g. protein phosphorylation catalyzed by protein kinases) → results in a cellular response (e.g. negative feedback, regulation of cell cycle, regulation of transcription) Many genes that are mutated in neoplasia encode proteins that are part of signal transduction pathways  Signal Transduction Pathways Basic components: Transmembrane receptor: binds extracellular signaling molecule ○ e.g., receptor tyrosine kinase, G protein-coupled receptor Cascading interaction of intracellular/cytoplasmic proteins downstream of receptor Most pathways end in nucleus Pathways modulate many different cellular functions ○ (e.g. cell division, gene expression, cell metabolism)  From Gene to Signaling Pathway… to Gene Mutations in genes that encode proteins participating in signal transduction pathways can change gene expression or activate cell signaling/ division independent of external stimuli Questions  Neoplasia is Genetic Carcinogenesis is a result of non-lethal genetic damage ○ Tumor is formed by clonal expansion of a single precursor cell that experienced genetic damage ○ Tumors are clonal Genomic alterations may be inherited, arise from environmental influences, or occur stochastically Driver alterations: contribute to development of malignant phenotype Passenger alterations: much more frequent than driver alterations but not relevant to development of malignant phenotype Carcinogenesis results from step-wise accumulation of complementary driver mutations over time  Neoplasia is Genetic Tumors continue to evolve genomically during outgrowth and progression  Genetic Mutations Genetic mutations are a type of genomic alteration ○ Permanent changes in DNA sequence Mutations that arise in germ cells are transmitted to offspring and can give rise to inherited diseases ○ Germ cells: egg, sperm Mutations arising in somatic cells are important in giving rise to cancers and some congenital malformations ○ Do not cause hereditary disease Mutations interfere with gene expression How many chromosomes do germ cells have? A: 23, B: 46, C: 68, D: 2  Neoplasia is Genetic There are four classes of normal regulatory genes that are the main targets of cancer-causing mutations: Proto-oncogenes (growth-promoting) Tumor suppressor genes (growth-inhibiting) Genes that regulate programmed cell death/apoptosis Genes involved in DNA repair 1. Oncogenes: Self-sufficiency in growth signaling Genes that promote autonomous cell growth in cancer cells Participate in signaling pathways that drive cell proliferation Oncogenes are mutated proto-oncogenes (normal version of a gene) ○ gain-of-function mutations Encode mutant proteins called oncoproteins Oncoproteins have gained function to promote cell growth independently of normal extracellular growth-promoting signals  MAPK (mitogen activated protein kinase) pathway alterations present in 78-88% of ameloblastomas BRAF mutations most common Mutation in BRAF gene produces mutant BRAF protein that allows ‘downstream’ signaling of MAPK pathway independent of external stimulus  BRAF and BRAFV600E mutation BRAFV600E mutation: 90% of all BRAF mutations seen in neoplasia Amino acid valine (V) is replaced by glutamate (E) at codon 600 ○ Codon: nucleotide triplet encoding specific amino acid BRAFV600E mutation results from BRAF c.1799T>A: ○ Thymine (T) replaced by adenine (A) at coding DNA sequence (nucleotide) 1799 GTG: encodes valine GAG: encodes glutamate This is a point mutation This is an activating mutation BRAF protein consists of 766 amino acids All of the following can be oncogenic: Growth factors Growth factor receptors Proteins involved in signaling cascade Nuclear regulatory proteins Cell cycle regulators  RAS and MYC mutations RAS - most commonly mutated oncogene MYC Oncogene that activates expression of many genes involved in cell growth Can reprogram plain somatic cells into pluripotent stem cells Upregulates telomerase in some contexts Contributes to stem-cell like immortalization of tumor cells Telomerase: Enzyme that lengthens telomeres → limitless replicative potential Contributes to ‘cell immortality’ Describe oncogene in one word  2. Tumor suppressor genes: Insensitivity to growth inhibition Tumor suppressor genes normally apply brakes to cell proliferation Abnormalities lead to failure of growth inhibition ○ loss-of function mutations: proteins not able to function properly Mutated genes encode proteins that have lost function to regulate signal transduction pathways and other critical cellular functions  Knudson ‘two-hit’ hypothesis Humans have two alleles (copies) of a gene With tumor suppressor genes, both alleles must be mutated prior to tumor development One allele can still ‘pick up the slack’ for its mutated counterpart Retinoblastoma: may occur in familial and sporadic settings  RB and the Cell Cycle Named after tumor in which its role was first discovered (retinoblastoma) Directly or indirectly inactivated in most human cancers Regulates expression of genes necessary for dividing cells to pass through G1/S cell cycle checkpoint  Which retinoblastoma is more likely occur at a younger age?  Knudson ‘two-hit’ hypothesis and cancer predisposition syndromes MANY cancer predisposition syndromes are characterized by germline mutation in one allele of the tumor suppressor gene Germline mutation: mutated gene within egg or sperm (germ cells) Can be passed on to subsequent generations Present in every cell in the body - Somatic mutation: Autosomal dominant mutated gene within any ○ APC: familial adenomatous polyposis cell of the body other than ○ NF1: neurofibromatosis 1 germ cells - Cannot be passed on to ○ PTCH: nevoid basal cell carcinoma syndrome subsequent generations ○ PTEN: Cowden syndrome ○ RB: familial retinoblastoma syndrome ○ TP53: Li-Fraumeni syndrome Patients with germline mutations in tumor suppressor genes are at markedly elevated risk for tumor development One allele of a given tumor suppressor gene is already inactivated in every single cell in the body Nevoid basal cell carcinoma (Gorlin) syndrome: germline mutation in PTCH1 (9q22.3) tumor suppressor gene  PTCH regulates Hedgehog signaling pathway Patched 1 protein binds/inactivates SMO (a proto-oncogene) Provides negative regulation of hedgehog signaling pathway PTCH1 is mutated in: A: Odontogenic keratocyst, B: Ameloblastoma, C: Melanoma  APC and Colonic Neoplasia Tumor suppressor that downregulates Wnt/ β-catenin signaling pathway  APC and Colonic Neoplasia Familial adenomatous polyposis (FAP) Cancer predisposition syndrome with germline APC mutations Thousands of adenomatous colonic polyps by age 30, with risk for transformation to colorectal carcinoma approaching 100% APC mutations also present in 70-80% of sporadic colorectal carcinoma Gardner syndrome (mutation of APC gene on 5q21-22) - Subgroup of familial adenomatous polyposis syndrome - Multiple epidermoid cysts + other skin lesions - Osteomas and supernumerary teeth (seen on pano X-ray) - craniofacial bony and dental signs often develop in puberty and precede gastrointestinal symptoms - Multiple premalignant colorectal polyps (100% become malignant if untreated; median age 40yo) - -  TP53: Guardian of Genomic Stability Regulates cell cycle progression, DNA repair, cellular senescence, apoptosis Monitors DNA damage and cell stress p53 (protein encoded by TP53) induces: ○ Growth arrest and DNA repair ○ Apoptosis if DNA cannot be repaired Most frequently mutated gene in human cancers  Gene Deletions: Meningioma Meningioma: most common brain tumor Deletions in 22q as a result of errors in DNA replication Deleted region contains NF2 tumor suppressor gene Gene deletion is another mechanism of tumor suppressor gene inactivation Gene amplification and p53: Liposarcoma Amplification: abnormal generation of multiple copies of a specific sequence of DNA as a result of errors during DNA replication 12q13-15 amplification in liposarcoma ○ Multiple copies of the same genes within amplified region ○ Protein products of these genes are not mutated but there is a lot more of them MDM2 amplification by FISH  MDM2 is a negative regulator of p53 Overexpression of MDM2 may have the same effect as TP53 mutation  CDK4 overexpression promotes progression through the cell cycle Most frequently mutated gene in human cancers is A: RB, B: BRAF, C: TP53, D: CDK4 3. Evasion of programmed cell death (apoptosis) Apoptosis Programmed cell death in response to pathologic conditions (such as carcinogenic mutations) if cell remained viable Bcl-2 family: 25 pro-apoptotic or anti-apoptotic genes Bcl-2 (anti-apoptotic), Bcl-XL, Bax, BAD, etc. Relative expression of these proteins determines cell fate  Follicular Lymphoma evades Cell Death Overexpression of Bcl-2 via a chromosomal translocation ○ Chromosomal translocation: a segment of a chromosome becomes interchanged with or attached to another chromosomal segment 2/3 of chromosomal translocations arise from erroneous repair of DNA double-strand breaks Most famous translocation: Philadelphia chromosome t(9;22): characteristic chromosomal translocation in chronic myelogenous leukemia, known as Philadelphia chromosome Chromosomal translocation: ‘shortcut’ to cancer Mucoepidermoid carcinoma of hard palate with (11;19) CRTC1-MAML2 translocation Both CRTC1 and MAML2 are transcription co-activators Common mechanisms of genetic damage Point mutations Gene deletions Gene amplifications Chromosomal translocations  4. Genomic instability: Defects in genes involved in DNA repair Genetic aberrations that increase mutation rates are common in cancer → faster acquisition of driver mutations necessary for tumor progression For example, TP53 tumor suppressor gene, when mutated: ○ Cannot arrest cell division to repair DNA damage commonly occurring in each replication cycle ○ Cannot initiate apoptosis in irreversibly damaged cells  Humans are surrounded by mutagenic environmental agents ○ Sunlight, radiation, chemicals Cancer is actually a relatively rare outcome of these exposures due to: ○ Ability of normal cells to repair DNA damage ○ Apoptosis as protective response ○ Normal surveillance mechanisms of immune system  Xeroderma pigmentosum Cancer predisposition syndrome Impaired ability to repair UV-induced DNA damage ○ UV radiation frequently causes abnormal cross-linking of pyrimidine bases Normal, error-free DNA replication cannot occur Pyrimidine bases: thymine and cytosine Purine bases: adenine and guanine  UV-induced DNA damage is normally repaired by nucleotide excision repair (NER) system ○ Group of proteins involved in excision and replacement of damaged DNA NER does not work properly in XP  Xeroderma pigmentosum Autosomal recessive syndrome Inherited mutations in genes that encode NER proteins (XPA, XPB, XPC, XPD, XPE, XPF, XPG, XPV) ○ Results in accelerated rate of (unrepaired) DNA damage 1000-fold increased risk of skin cancers Median age of tumor development 8 yo Increased risk of squamous cell carcinoma of lower lip and tip of tongue  Hereditary nonpolyposis colon cancer syndrome (Lynch syndrome) During DNA replication, DNA mismatch repair (MMR) proteins act as ‘spell-checkers’ ○ Detect, excise, and repair defects such as erroneous base pair insertions and deletions If ‘proof-reading’ function is lost, genetic mutations are more likely to accumulate Increase in mutational load → accumulation of complementary driver mutations  Hereditary nonpolyposis colon cancer syndrome (Lynch syndrome) MMR proteins are encoded by MSH2, MLH1, MSH6, PMS2, EPCAM genes Germline mutation in any one of these genes: ○ Increases risk of colorectal cancer ○ Increases risk of other cancers Endometrium, stomach, brain, skin, etc. 3% of all colorectal cancers Most common syndrome associated with colon cancer  MMR and microsatellite instability Lynch syndrome colorectal carcinoma is microsatellite instability-high (MSI-H) ○ Microsatellite: tract of repetitive DNA ○ Short DNA sequences (2-6 base pairs) repeated up to 50 times ○ Have higher mutation rate than other areas of DNA → unstable if MMR deficient Microsatellite instability: ○ Can affect gene expression and protein shape/function ○ Often leads to frameshift mutations  BRCA and familial breast cancer 10-15% of breast cancers are hereditary BRCA1 and BRCA2 are mutated in 25% of familial cases Cells that don’t have functional BRCA1/BRCA2 proteins develop chromosomal breaks and aneuploidy (wrong number of chromosomes) BRCA proteins repair DNA breaks through a process called homologous recombination DNA break is repaired during replication using normal sister chromatid as template  Neoplasia is Genetic Four classes of normal regulatory genes are the main targets of cancer-causing mutations: Proto-oncogenes (growth-promoting) Tumor suppressor genes (growth-inhibiting) Genes that regulate programmed cell death/apoptosis Genes involved in DNA repair Carcinogenesis results from accumulation of complementary driver mutations in a stepwise fashion over time