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Introduction to the cell membrane and organelles Konstantinos Voskarides Learning objectives 1. Describe the basic structure of the human cell 2. Outline the structure and function of the cell membrane 3. Outline the basic structure and key functions of cellular organelles 4. D...

Introduction to the cell membrane and organelles Konstantinos Voskarides Learning objectives 1. Describe the basic structure of the human cell 2. Outline the structure and function of the cell membrane 3. Outline the basic structure and key functions of cellular organelles 4. Discuss, with examples, the implications of dysfunction of cellular organelles Light microscope, Electron microscope All Eukaryotic Cells Have the Same Basic Set of Membrane-enclosed Organelles All Eukaryotic Cells Have the Same Basic Set of Membrane-enclosed Organelles All Eukaryotic Cells Have the Same Basic Set of Membrane-enclosed Organelles All Eukaryotic Cells Have the Same Basic Set of Membrane-enclosed Organelles  The cell membrane is a mosaic lipid bilayer Figure 10-1 Molecular Biology of the Cell (© Garland Science 2008) Phosphoglycerides, Sphingolipids, and Sterols Are the Major Lipids in Cell Membranes Two different modes of transport through the cell membrane Evolutionary Origins May Help Explain the Topological Relationships of Organelles A simplified “road map” of protein traffic within a eukaryotic cell Vesicle budding and fusion during vesicular transport Examples of signal sequences that direct proteins to different intracellular locations NUCLEUS Nuclear Pore Complexes (NPC) Perforate the Nuclear Envelope Nuclear pores control export and import of molecules The Ran GTPase Imposes Directionality on Transport Through NPCs MITOCHONDRION THE TRANSPORT OF PROTEINS INTO MITOCHONDRIA PEROXISOMES Peroxisomes Use Molecular Oxygen and Hydrogen Peroxide to Perform Oxidation Reactions Detoxify blood from several toxic substances, alcohol and reactive oxygen species (ROS)  ENDOPLASMIC RETICULUM (ER) n Endoplasmic reticulum has two major regions: smooth endoplasmic reticulum and rough endoplasmic reticulum. Rough ER contains attached ribosomes, smooth not n Via the attached ribosomes, rough endoplasmic reticulum synthesizes proteins. Rough ER also manufactures membranes n Smooth endoplasmic reticulum serves as a transitional area for transport vesicles. It also functions in carbohydrate and lipid synthesis, like cholesterol and phospholipids, and detoxification of metabolic wastes and drugs n Rough and smooth ER are typically connected to one another so that the proteins and membranes made by the rough ER can freely move into the smooth ER for transport to other parts of the cell The ER Is Structurally and Functionally Diverse A Signal-Recognition Particle (SRP) Directs the ER Signal Sequence to a Specific Receptor in the Rough ER Membrane A Signal-Recognition Particle (SRP) Directs the ER Signal Sequence to a Specific Receptor in the Rough ER Membrane In Single-Pass Transmembrane Proteins, a Single Internal ER Signal Sequence Remains in the Lipid Bilayer as a Membrane-spanning a Helix Combinations of Start-Transfer and Stop-Transfer Signals Determine the Topology of Multipass Transmembrane Proteins Rhodopsin Most Proteins Synthesized in the Rough ER Are Glycosylated by the Addition of a Common N-Linked Oligosaccharide Oligosaccharides Are Used as Tags to Mark the State of Protein Folding The unfolded protein response (UPR, or ER stress)  Dufey et al, 2014 Ozawa et al, 2009 Role of the UPR in physiology and diseases. Genetic manipulation of major UPR components has revealed its relevance in the function of diverse organs and cell types, in addition to its contribution to a variety of diseases using preclinical mouse models. Important functions have been reported in brain, bone marrow, heart, liver, pancreas, intestine, and gastric system (blue boxes). Pathologies where abnormal ER stress levels play a relevant role in disease include diabetes, neurodegeneration, ischemia, cancer, and other diseases (red boxes) GOLGI apparatus n The Golgi apparatus modifies proteins and lipids that it receives from the endoplasmic reticulum. n These molecules leave the Golgi to be delivered to different intracellular or extracellular targets. Protein processing – carbohydrate regions of glycoproteins are altered by addition, removal or modification of carbohydrates. Lipid processing – adds phosphate groups and glycoproteins to lipids from the endoplasmic reticulum (such as cholesterol) to create the phospholipids that make up the cell membrane The Golgi Apparatus Consists of an Ordered Series of Compartments The Golgi Apparatus Consists of an Ordered Series of Compartments Oligosaccharide Chains Are Processed in the Golgi Apparatus Proteoglycans Are Assembled in the Golgi Apparatus Significance of glycosylation Correct folding of proteins. More than 50% of proteins are glycosylated Quality control in ER Cell-to-cell adhesion (e.g. in lectins, proteins that recruit immune cells) ABO blood group proteins Envelopes of viruses may have glycans to shield from immune recognition  Examples of congenital disorders of glycosylation Reily et al, 2019 Protein trafficking Konstantinos Voskarides Learning objectives 1. Outline the basic routes of protein trafficking inside the cell 2. Outline the basic structure and key functions of cellular organelles 3. Discuss, with examples, the implications of dysfunction of cellular organelles VESICLES TRANSPORT Intracellular transport is the movement of vesicles and substances within a cell. Intracellular transport is required for maintaining homeostasis within the cell by responding to physiological signals. Proteins synthesized in the cytosol are distributed to their respective organelles, according to a special signal peptide, in the N-terminus of the protein Intracellular vesicles main routes  Vesicle are formed by budding, from the membrane Vesicles are coated with special proteins Clathrin-coated vesicles, are responsible for the uptake of extracellular molecules from the plasma membrane by endocytosis as well as the transport of molecules from the trans Golgi network to lysosomes COPII-coated vesicles bud from the ER and carry their cargo forward along the secretory pathway, to the Golgi apparatus COPI-coated vesicles transport resident ER proteins marked by the KDEL or KKXX retrieval signals back to the ER Electron micrographs of clathrin-coated, COPI-coated, and COPII-coated vesicles There Are Various Types of Coated Vesicles The Assembly of a Clathrin Coat Drives Vesicle Formation The assembly and disassembly of a clathrin coat  Vesicle fusion is mediated by interactions between specific pairs of proteins, called SNAREs, on the vesicle and target membranes (v-SNAREs and t- SNAREs, respectively) Specific SNAREs are required for different vesicle fusions in different locations The formation of complexes between v-SNAREs on the vesicle and t-SNAREs on the target membranes that leads to membrane fusion, is not fully understood Tethering of a transport vesicle to a target membrane Endocytosis of viruses has similarities with vesicle fusion TRANSPORT FROM THE ENDOPLASMIC RETICULUM THROUGH THE GOLGI APPARATUS n Proteins Leave the ER in COPII-coated Transport Vesicles n Only Proteins That Are Properly Folded and Assembled Can Leave the ER n Vesicular Tubular Clusters Mediate Transport from the ER to the Golgi Apparatus n The Retrieval Pathway to the ER Uses Sorting Signals n Many Proteins Are Selectively Retained in the Compartments in Which They Function Proteins Leave the ER in COPII-Coated Transport Vesicles Vesicular Tubular Clusters Mediate Transport from the ER to the Golgi Apparatus The Retrieval Pathway to the ER Uses Sorting Signals EXOCYTOSIS The three best-understood pathways of protein sorting in the trans Golgi network The formation of secretory vesicles The process is illustrated schematically (top) and in an electron micrograph that shows the release of insulin from a secretory vesicle of a pancreatic β cell Some proteins are produced as pro-peptides and mature by proteolysis inside the vesicles Four examples of regulated exocytosis leading to plasma membrane enlargement Two ways of sorting plasma membrane proteins in a polarized epithelial cell ENDOCYTOSIS n Phagocytosis n Pinocytosis n Lysosomes are very important for the endocytotic pathways Endosome maturation: the endocytic pathway from the plasma membrane to lysosomes A low-density lipoprotein (LDL) particle The receptor-mediated endocytosis of LDL Storage of plasma membrane proteins in recycling endosomes: The example of glucose transporters  Impaired recycling of glucose transporters can couse insulin resistance. This is the first step for the development of diabetes mellitus. Fazakerley et al, 2019 Pathways synopsis n Endocytosis -> early endosome -> Recycling endosome -> Plasma membrane n Endocytosis -> early endosome -> late endosome -> Lysosome Many neurotoxins directly affect SNARE complexes. Such toxins as the botulinum and tetanus toxins work by targeting the SNARE components. These toxins prevent proper vesicle recycling and result in poor muscle control, spasms, paralysis, and even death. 33 THE DEGRADATION AND RECYCLING OF MACROMOLECULES IN LYSOSOMES n Lysosomes Are the Principal Sites of Intracellular Digestion n Lysosomes Are Heterogeneous n Plant and Fungal Vacuoles Are Remarkably Versatile Lysosomes n Multiple Pathways Deliver Materials to Lysosomes n Cells Can Acquire Nutrients from the Extracellular Fluid by Macropinocytosis n Specialized Phagocytic Cells Can Ingest Large Particles n Cargo Recognition by Cell-surface Receptors Initiates Phagocytosis n Autophagy Degrades Unwanted Proteins and Organelles Lysosome acid hydrolases are hydrolytic enzymes that are active under acidic conditions Four pathways to degradation in lysosomes (A) Scanning electron micrograph of a mouse macrophage phagocytosing two chemically altered red blood cells. The red arrows point to edges of thin processes (pseudopods) of the macrophage that are extending as collars to engulf the red cells. (B) An electron micrograph of a neutrophil phagocytosing a bacterium, which is in the process of dividing Autophagy is mediated by receptors that recruit cargo to the autophagosome membrane Lysosome storage diseases Developmental delay, movement disorders, seizures, dementia, deafness, and/or blindness Some people with lysosomal storage diseases have enlarged livers or spleens, pulmonary and cardiac problems, and bones that grow abnormally Bellettato and Scarpa, 2010 https://www.ucl.ac.uk/immunity-transplantation/clinical-services/diseases- treatments/inherited-diseases/lysosomal-storage-disease Organization of the human genome Konstantinos Voskarides Learning objectives 1. Describe the structure of DNA and chromatin and compare intragenic and extragenic DNA 2. Describe the basic structure of the human gene 3. Describe the basic structure of the human chromosome THE STRUCTURE AND FUNCTION OF DNA n A DNA Molecule Consists of Two Complementary Chains of Nucleotides n The Structure of DNA Provides a Mechanism for Heredity n In Eukaryotes, DNA Is Enclosed in a Cell Nucleus  A cross-sectional view of a typical cell nucleus Chromatin: The complex of DNA with proteins Heterochromatin: More condensed, inactive genes Euchromatin: Less tightly compacted, active genes Comparison of our DNA with other species CHROMOSOMAL DNA AND ITS PACKAGING IN THE CHROMATIN FIBER n Eukaryotic DNA Is Packaged into a Set of Chromosomes n Chromosomes Contain Long Strings of Genes n The Nucleotide Sequence of the Human Genome Shows How Our Genes Are Arranged n Each DNA Molecule That Forms a Linear Chromosome Must Contain a Centromere, Two Telomeres, and Replication Origins n Nucleosomes Are a Basic Unit of Eukaryotic Chromosome Structure n Procaryotes (bacteria) don’t have nucleosomes CHROMOSOMAL DNA AND ITS PACKAGING IN THE CHROMATIN FIBER n The Structure of the Nucleosome Core Particle Reveals How DNA Is Packaged n Nucleosomes Have a Dynamic Structure and Are Frequently Subjected to Changes Catalyzed by ATP-dependent Chromatin-remodeling Complexes n Attractions Between Nucleosomes Compact the Chromatin Fiber A mitotic chromosome The human karyotype The organization of genes on a human chromosome Nucleosome Nucleosomes as seen in the electron microscope A model for the role played by histone tails in the compaction of chromatin The structure of a nucleosome core particle, as determined by x-ray diffraction analyses of crystals Chromosome organization THE GLOBAL STRUCTURE OF CHROMOSOMES n Chromosomes Are Folded into Large Loops of Chromatin n Chromosome Loops Decondense When the Genes Within Them Are Expressed n Mammalian Interphase Chromosomes Occupy Discrete Territories in the Nucleus, with Their Heterochromatin and Euchromatin Distributed Differently  TYPICAL STRUCTURE OF A EUCARYOTIC GENE Procaryotes (bacteria) don’t have introns and mRNA modifications The beta- globin gene  The Sequence of the Human Genome, Gene categorization Science, 2001 mtDNA DNA methylation can suppress gene expression Genomic imprinting: A standard set of genes are inherited methylated and inactivated. Only one allele is transcriptionally active for those genes. For some of them, methylation pattern has been inherited form mothers and for some others from fathers. Methylation pattern is erased and established again during gamete production. Falls et al, 1999 Transcriptomics and Proteomics techniques Konstantinos Voskarides Learning objectives 1. Briefly outline techniques to study the transcriptome and proteome ANALYZING PROTEINS Proteins Can Be Separated by SDS Polyacrylamide- Gel Electrophoresis according their molecular weight (SDS-PAGE)  Big proteins The most common method for in-gel protein detection is staining with Coomassie blue dye. This method stains all the proteins in the gel Small proteins Two-dimensional (2D) Gel Electrophoresis Provides Greater Protein Separation Separation of protein molecules by isoelectric focusing 2D polyacrylamide-gel electrophoresis Biomarker discovery Functional analysis Total protein from homogenates of tumor or normal breast tissues from the same patient were loaded onto each gel and separated by 2D gel electrophoresis. Carcoforo et al, 2013 Mass Spectrometry Provides a Highly Sensitive Method for Identifying Unknown Proteins 2D protein electrophoresis can be used in combination with mass spectometry Conrotto and Souchelnytskyi, 2008 Specific Proteins Can Be Detected by Blotting with Antibodies: Western blot Qualitative and quantitative Highly signifficant in Biomedical research Signifficant in disease diagnosis, e.g. infectious diseases SDS-PAGE followed by Western blot Medical application example: Western blot is required for the diagnosis of Lyme disease  ELISA method Qualitative and Quantitative protein assay ELISA applications Indirect ELISA: Autoimmune disease diagnosis (lupus, rheumatoid arthritis, Sjögren’s syndrome etc), antibodies detection after vaccination, HIV infection etc Direct ELISA: hormones (e.g. pregnancy test), cytokines, cancer biomarkers etc PCR PCR: Polymerase Chain Reaction. An artificial DNA replication process. A laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail. PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment. A pair of primers directs the synthesis of a desired segment of DNA in a test tube PCR uses repeated rounds of strand separation, hybridization, and synthesis to amplify DNA  DNA electrophoresis. A way to visualise DNA DNA is loaded and electrophorised in agarose gels https://www.khanacademy.org/ Once the DNA fragments have been separated, we can examine the gel and see what sizes of bands are found on it. When a gel is stained with a DNA-binding dye and placed under UV light, the DNA fragments will glow, allowing us to see the DNA present at different locations along the length of the gel. https://www.khanacademy.org/ PCR can be used to detect the presence of a viral genome in a nasal sample PCR is used in forensic science to distinguish one individual from another The DNA sequences analyzed are short tandem repeats (STRs), known also as microsatellite markers, composed of sequences such as CACACA... or GTGTGT.... STRs are found in various positions (loci) in the human genome. The number of repeats in each STR locus is highly variable in the population, ranging from 4 to 40 in different individuals. Because of the variability in these sequences, an individual will usually inherit a different number of repeats at each STR locus from his mother and from his father; two unrelated individuals, therefore, rarely contain the same pair of sequences at a given STR locus. TRANSCRIPTOMICS Expression of Individual Genes Can Be Measured Using Quantitative RT-PCR (real-time PCR) Global Analysis of mRNAs by RNA-sequencing Provides a Snapshot of Gene Expression  Global Analysis of mRNAs by RNA-seq Provides a Snapshot of Gene Expression Human fibroblasts were deprived of serum for 48 hours; serum was then added back to the cultures at time 0, and the cells were harvested for mRNA measurements at different time points. red indicates an increase in expression; green is a decrease in expression. Gene control and Epigenetics Konstantinos Voskarides Learning objectives 1. Define epigenetic regulation and outline the different mechanisms 2. Outline the basic principles of gene control Epigenetic changes n Epigenetic changes affect DNA expression n Epigenetic changes are triggered by environmental factors n Cytosine methylation n Histones modifications n Patterns of DNA Methylation Can Be Inherited When Vertebrate Cells Divide n When promoter of genes are methylated (CG islands), transcription is suppressed The Core Histones Are Covalently Modified at Many Different Sites The Core Histones Are Covalently Modified at Many Different Sites Covalent Modifications and Histone Variants Act in Concert to Control Chromosome Functions Gene control n Procaryotic cells (bacteria) have operons: Control of expression of multiple genes through a common promoter n Example: The Tryptophan Repressor Switches Genes Off The Different Cell Types of a Multicellular Organism Contain the Same DNA n Gene Expression Can Be Regulated at Many of the Steps in the Pathway from DNA to RNA to Protein n Transcription Regulators (transcription factors) Contain Structural Motifs That Can Read DNA Sequences n Dimerization of Transcription Regulators Increases Their Affinity and Specificity for DNA n Nucleosome Structure Promotes Cooperative Binding of Transcription Regulators n A Eukaryotic Gene Control Region Includes Many cis-Regulatory Sequences n Eukaryotic Transcription Regulators Work in Groups n Activator Proteins Promote the Assembly of RNA Polymerase at the Start Point of Transcription n Eukaryotic Transcription Activators Direct the Modification of Local Chromatin Structure Transcriptional synergy n Eukaryotic Transcription Repressors Can Inhibit Transcription in Several Ways A single transcription regulator can coordinate the expression of many different genes Genomic imprinting and X- chromosome inactivation are epigenetic phenomena  DNA replication Konstantinos Voskarides Learning objectives 1. Outline the process of DNA replication 2. List the potential errors during DNA replication and discuss their potential implication if they remain uncorrected 3. Describe the structure and function of telomeres and their role in the aging cell The Maintenance of DNA Sequences n Mutation Rates Are Extremely Low n Low Mutation Rates Are Necessary for Life as We Know It DNA Replication Mechanisms n Base-pairing Underlies DNA Replication and DNA Repair n The DNA Replication Fork Is Asymmetrical n The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms n DNA Replication in the 5'-to-3' Direction Allows Efficient Error Correction n A Special Nucleotide-polymerizing Enzyme Synthesizes Short RNA Primer Molecules n Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork DNA Replication Mechanisms n A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA n The Proteins at a Replication Fork Cooperate to Form a Replication Machine n DNA Replication Is Fundamentally Similar in Eukaryotes and Bacteria n A Strand-directed Mismatch Repair System Removes Replication Errors That Remain in the Wake of the Replication Machine n The Accidental Incorporation of Ribonucleotides During DNA Replication Is Corrected n DNA Topoisomerases Prevent DNA Tangling During Replication DNA acts as a template for its own replication The chemistry of DNA synthesis How DNA polymerase adds a deoxyribonucleotide to the end of a growing DNA strand DNA replication is semi-conservative Two replication forks moving in opposite directions on the E. coli chromosome, a large circular DNA molecule At each replication fork, the lagging DNA strand is synthesized in pieces During DNA synthesis, DNA polymerase proofreads its own work RNA primers are synthesized by an RNA polymerase called DNA primase, which uses a DNA strand as a template Different enzymes act in series to synthesize DNA on the lagging strand DNA ligase joins together Okazaki fragments on the lagging strand during DNA synthesis How DNA helicase enzymes can separate strands as they move along a DNA single strand The effect of single-strand DNA-binding proteins (SSB proteins) on the structure of single-stranded DNA The sliding clamp that holds DNA polymerase on the DNA Bacteria Eucaryotes DNA mismatch repair (MMR) DNA polymerization errors can create base mismatches between the paternal and the new DNA strand (e.g. A:C or G:T) Special MMR proteins can correct the errors according the paternal DNA strand Consrved mechanism, from bacteria to mammals Mutations in MMR genes cause cancer Topisomerases Relieve stress caused by DNA supercoling Topoisomerase inhibitors have cytotoxic effects since un-repaired single- and double stranded DNA breaks, that topoisomerases cause, can lead to cell death. Useful are anticancer agents The Initiation and Completion of DNA Replication in Chromosomes n DNA Synthesis Begins at Replication Origins n Bacterial Chromosomes Typically Have a Single Origin of DNA Replication n Eukaryotic Chromosomes Contain Multiple Origins of Replication n In Eukaryotes, DNA Replication Takes Place During Only One Part of the Cell Cycle n Eukaryotic Origins of Replication Are “Licensed” for Replication by the Assembly of an Origin Recognition Complex (ORC) n Features of the Human Genome That Specify Origins of Replication Remain to Be Fully Understood The Initiation and Completion of DNA Replication in Chromosomes n Properties of the ORC Ensure That Each Region of the DNA Is Replicated Once and Only Once in Each S Phase n New Nucleosomes Are Assembled Behind the Replication Fork n Termination of DNA Replication Occurs Through the Ordered Disassembly of the Replication Fork n Telomerase Replicates the Ends of Chromosomes n Telomeres Are Packaged Into Specialized Structures That Protect the Ends of Chromosomes n Telomere Length Is Regulated by Cells and Organisms Bacterial Chromosomes Have a Single Origin of DNA Replication Nucleosomes are rapidly re-assembled behind the replication fork The human telomerase structure Telomere replication A t-loop protects the ends of the mammalian chromosome Yeast cells control the length of their telomeres It is believed that the same is happening in germ- line cells and stem cells of mammals Biological ageing Konstantinos Voskarides Learning objectives 1. Describe the main aging theories  Genetics Biochemistry Cell biology Evolution Endocrinology theory Ageing Comparative Bioethics biology Ageing-related disease Regenerative cardiovascular medicine Demography disease, Alzheimer’s, stem cells cancer, diabetes, etc  Life expectancies as calculated for year (US) Life expectancy Life expectancy at birth at age 65 Year Men Women Men Women ---------------------------------------------------------------------------------------- 1900 47.9 50.9 11.3 12.0 1930 58.0 61.3 11.8 12.9 1950 65.6 71.1 12.8 15.1 1970 67.1 74.9 13.1 17.1 1980 69.9 77.5 14.0 18.4 1990 71.4 78.3 14.9 18.8 ---------------------------------------------------------------------------------------- Increase 23.5 27.4 3.6 6.8 ----------------------------------------------------------------------------------------  Maximum human longevity Mme Jeanne Calment, died 1998, aged 122 Sunflowers, Vincent van Gogh, 1888 Mutation accumulation hypothesis Haldane, Medawar, Williams Suggested that mutation accumulation is related with ageing Natural selection cannot discard deleterious mutations from our genome Somatic mutation rates are probably related with ageing A multi-species study Cagan et al, 2022, Nature  The Rate-of-Living Theory (1928) “…the duration of life varies inversely as the rate of energy expenditure … the length of life depends on the rate of living” Loeb and Northrop (1916, 1917): increasing temperature Raymond Pearl reduces Drosophila lifespan Effect of temperature on 30˚C 27˚C 21˚C 18˚C Drosophila lifespan Coefficient relating lifespan to ambient temperature = 2-3, like that of chemical reactions  The free radical theory of ageing Denham Harman (1956) “A free radical is any species capable of independent existence (hence the term ‘free’) that contains one or more unpaired electron” Barry Halliwell & John Gutteridge Y + -> Y e. - -. X -> e + X + - O2 + e - -> O2.- Superoxide Stefamatos and Sabz, 2017  Cellular Theories The Hayflick Limit (1961) Pre-1961: “All metazoan cells are potentially immortal. Ageing not cell autonomous” Leonard Hayflick Fibroblasts: connective tissue cells, e.g. from skin Hayflick and Moorhead (1961) Isolate cells from human tissue, place in culture vessel with nutrient medium Cells divide and form confluent layer on vessel surface Discard half the cells, allow remainder to grow to confluency = one passage Continue to passage the cells Cell replication slows and stops after 50 ± 10 passages: cells have reached the Hayflick limit and undergone replicative senescence Is replicative senescence the cause of ageing? Can telomeres explain ageing?? Chromosome Ends are specialized structures called Telomeres Blue = DNA White = Telomere protein  Telomeres Repeated G rich sequence on one strand in humans: (TTAGGG)n Repeats can be several thousand basepairs long. In humans, telomeric repeats average 5-15 kilobases. Telomere specific proteins, eg. TRF1 & TRF2 bind to the repeat sequence and protect the ends. Chromosomes with short telomeres inhibit cell devision  Telomerase Telomerase is a ribonucleoprotein enzyme complex (a cellular reverse transcriptase). TERT - RNA directed DNA polymerase. TERC - RNA template. It stabilizes telomere length by adding hexameric (TTAGGG) repeats onto the telomeric ends of the chromosomes, thus compensating for the erosion of telomeres that occurs in its absence.  The telomere theory of ageing Potentially immortal cells (germ cells, cancer cells) maintain telomerase activity Can divide indefinitely. Cells with a limited replicative lifespan Should have no telomerase activity. Progressively shortening telomeres. Cell division serves as a mitotic clock for replicative senescence. Provides a mechanistic explanation for the Hayflick limit. The IGF-1/insulin pathway  C. elegans age-1(hx546) mutation 65% increase in mean lifespan Nematode worm 110% increase in maximum lifespan Remains youthful for longer Mutations in daf-2 greatly increase lifespan  daf-2 (-) and age-1 (-) switch on the dauer longevity programme in the adult Normal adult Short lived daf-2 (-) age-1 (-) Mutant Long adult lived Dauer longevity programme switched on age-1 Catalytic subunit of phosphatidyl inositol 3-kinase daf-2 Insulin or IGF-1 receptor  Insulin/IGF-1 signalling modulates ageing in insects as well as nematodes Drosophila melanogaster + chico1 *Mutations in INR (fly daf-2): mean female lifespan increased by up to 85% *Mutation of chico (insulin receptor substrate), increases lifespan by up to 48% Implications *Wide evolutionary conservation of the role of insulin/IGF-signalling in the modulation of ageing: a universal mechanism of ageing? The insulin-like pathway P P P dFOXO dFOXO Increased longevity? Does it control ageing?  Extended longevity in mice lacking the insulin receptor in adipose tissue Ron Kahn (2003): fat-specific insulin receptor knockout (FIRKO) mouse Protected against age-related obesity 18% increase in mean lifespan in both sexes Worms, flies: One insulin/IGF-1 receptor Mammals: insulin receptor, IGF-1 receptor, insulin like-receptor…. (Blüher et al. Science 2003)  IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice Holzenberger (2002): Mice heterozygous for a deletion of the IGF-1 receptor gene Resistant to oxidative stress Increased mean lifespan (33% females, males not long lived) (Holzenberger et al. Nature 2002) Caloric restriction and sirtuins McKay’s experiment: Feed rats a reduced diet rich in vitamins and minerals Results: Caloric restricted male rats lived 75% longer than controls. Maximum lifespan +1 yr., 35% longer. No difference for female rats In later experiments using a reformulated diet, found lifespan extension in both male and female rats  McCay CM et al. J. Nutrition 1935, 10: 63-79 McCay, C. M., and M. F. Crowell. 1934. Prolonging the life span. Science Monthly 39:405–414. Escobar et al, 2018 Severe Diet Doesn’t Prolong Life in Rhesus Monkeys Is longevity related with sirtuins? Enzymes with ribosyltransferase or deacylase activity Lee 2019 Introduction to carcinogenesis Prof Constantina Constantinou Molecular Cancer Biologist [email protected] Monday October 21, 2024 Learning Objective Outline the key events in the neoplastic process and metastasis, and the main signalling pathways related with cancer  Cancer Image Credit: Cancer Research UK What is Cancer? Cancer is an abnormal growth of cells which tend to proliferate in an uncontrolled way and, in some cases, to metastasise (spread) to other parts of the body NIH (2024). Cancer as a microevolutionary process Most cancers derive from a single abnormal cell A single mutation is insufficient to transform a normal cell into a cancer cell Cancer cells contain somatic mutations Many cancers develop gradually through successive rounds of random inherited changes, followed by natural selection Cancers can also evolve abruptly due to genetic instability Some cancers may harbor a small population of stem cells Cancer cells bypass normal proliferation controls and colonize other tissues Cancer as a microevolutionary process Common characteristics of Cancerous Growth o Altered Control of Growth and Homeostasis: Cancer cells display an altered control of growth and homeostasis. o Escape from Proliferation Limits: Human cancer cells can bypass the natural limits on cell division, allowing them to divide uncontrollably. o Bypassing Death Signals Cancer cells have an abnormal ability to bypass death signals. o Altered Sugar Metabolism: Cancer cells have altered sugar metabolism. o Influence of Tumor Microenvironment: The tumor microenvironment influences cancer development. o Survival in Foreign Environments: Cancer cells must survive and proliferate in a foreign environment. Types of Cancer Based on Tissue Origin Carcinomas: Cancers derived from epithelial cells. Includes most common cancers (breast, prostate, lung, pancreas, colon). Sarcomas: Cancers arising from connective tissue tissue (i.e. bone, cartilage, fat, nerve), each of which develops from cells originating in mesenchymal cells outside the bone marrow Lymphomas and leukemias: Arise from hematopoietic (blood- forming) cells that leave the marrow and tend to mature in the lymph nodes and blood, respectively. Types of Cancer Based on Tissue Origin Foroly Farma (2024) Benign vs Malignant Tumors Benign tumors do not invade and do not spread. Malignant tumors invade and spread, giving rise to metastases, making the cancer hard to eradicate. Shown in this fusion image is a whole-body scan of a patient with metastatic non- Hodgkin’s lymphoma (NHL). Cancer Incidence and Mortality Cancer incidence and mortality in the United States Approximately 39.5% of men and women will be diagnosed with cancer at some point during their lifetimes (based on 2015–2017 data) (National Cancer Institute) 22% of deaths in the US in 2016 were from cancer, making it the second leading cause of death after heart disease in both men and women Mutations and Epigenetic changes More than 90% of cancer cases result from mutations or epigenetic changes that happened in our somatic cells, during our life In most cases, one mutation is not enough to cause cancer. Mutations are frequently caused by mutagens (radiation, chemicals). Hallmarks of cancer 1. Uncontrolled Cell Growth and Division: Cells grow and divide without receiving the appropriate signals. 2. Persistent Growth Despite Inhibitory Signals: Cells continue to grow and divide even when they receive signals to stop. 3. Evasion of Programmed Cell Death (Apoptosis): Cells avoid undergoing apoptosis, which is the process of programmed cell death. 4. Unlimited Replicative Potential: Cells can divide an unlimited number of times. 5. Induction of Angiogenesis: Cells promote the construction of new blood vessels to supply nutrients. 6. Tissue Invasion and Metastasis Formation: Cells invade surrounding tissues and form Hanahan, Douglas et al (2000). Cell, metastases, spreading to other parts of the 100(1), 57 – 70. body. Growth of human tumours The growth of a typical human tumor, such as a tumor of the breast Years may elapse before the tumor becomes noticeable. The doubling time for a typical breast tumor, for example, is about 100 days. However, particularly aggressive tumors may grow much more rapidly. Cancer prevalence as a function of age Cancer prevalence as a function of age Cancer and risk factors Smoking and lung cancer Cancer progression Stages of progression in the development of cancer of the epithelium of the uterine cervix Clonal evolution during tumor progression Most cancers are thought to originate from a single cell that has experienced an initial mutation, The progeny of this cell undergoes further changes, requiring numerous additional mutations, to become cancerous. Tumor progression, which usually takes many years, reflects the unfortunate operation of evolution by mutation and natural selection among somatic cells. Clonal evolution during tumor progression Cancer cells are genetically unstable. Chromomal abnormalities and other mutations increase through time, and that can make cancer cells more aggressive. Chromosome complements (karyotypes) of colon cancers showing different kinds of genetic instability. (A) The karyotype of a typical cancer shows many gross abnormalities in chromosome number and structure. Considerable variation can also exist from cell to cell (not shown). (B) The karyotype of a tumor that has a stable chromosome complement with few chromosomal anomalies; the genetic abnormalities in these tumors are mostly invisible, having been created by defects in DNA repair. Chromosome segregation defects Chromosome segregation defects can give rise to aneuploidy and/or chromothripsis Chromothrypsis Genomic phenomenon in which a chromosome or a segment of a chromosome shatters into many pieces, which are then stitched back together in a random order. It contributes to cancer development and progression by disrupting genes and regulatory regions, potentially activating oncogenes or inactivating tumor suppressor genes. Observed in various types of cancers. The exact mechanisms are still under investigation. Lifetime cancer risk and its association with division rate of the cell of origin of the cancer The lifetime cancer risk is correlated with the division rate of the cell of origin of the cancer Cancer Stem Cells Cancer Stem Cells (CSCs) are a subpopulation of cells within a tumor that can self-renew and differentiate into various cell types found in the cancer. CSCs are thought to contribute to tumor growth, metastasis, and recurrence. They have been identified in various cancers, including: Breast Cancer Brain Cancer Colon Cancer Leukemia Prostate Cancer While CSCs have been identified in these and other cancers, their presence and role can vary between different types of tumors and individual patients. Cancer stem cells Cancer stem cells can be responsible for a tumor’s growth and yet remain only a small part of the tumor-cell population Tumorigenesis= increased cell division and decreased cell death The Tumor Microenvironment The tumor microenvironment is crucial in tumorigenesis The Warburg effect in tumor cells The Warburg effect in tumor cells reflects a dramatic change in glucose uptake and sugar metabolism Non-Proliferating Cells o These cells use glucose from the blood to produce ATP. o ATP production occurs through oxidative phosphorylation in mitochondria. o When oxygen is low, they switch to glycolysis for ATP. o Glycolysis converts pyruvate to lactate to regenerate NAD+. Tumor Cells o Tumor cells have a high rate of glycolysis and glucose import. o Tumor cells produce a lot of lactate even with oxygen present. o This behavior is similar to rapidly growing embryonic cells. o Both need many small molecules from glucose for biosynthesis. Loss of contact inhibition Steps in the process of metastasis Studies in animals show that fewer than 1 in every 1000 malignant tumor cells introduced into the bloodstream is viable after 24 hours, and that less than 0.1% of these surviving circulating tumor cells (CTCs) will colonize a new tissue so as to produce a detectable tumor at a new site. Carcinogens = Mutagens Benzene in tobacco Genes implicated in cancer Cell cycle genes Apoptosis genes DNA repair genes Telomerase and related genes Genes implicated in cancer Tumor Suppresor Genes (TSGs) Oncogenes Types of mutations Driver Mutations: Genetic changes that contribute to cancer development by providing a growth advantage to cells. Passenger Mutations: Genetic changes that do not contribute to cancer progression and occur alongside driver mutations. Gain-of-Function Mutations: Mutations that enhance the activity of a gene or protein, often leading to increased cell growth. Loss-of-Function Mutations: Mutations that reduce or eliminate the activity of a gene or protein, potentially disrupting normal cellular functions  Disruptions in a Handful of Key Pathways Are Common to Many Cancers Cell signaling pathways and their role in cancer Mutation in the Epidermal Growth Factor receptor gene (protooncogene is converted to an oncogene→ Oncoprotein) Mutation of the epidermal growth factor (EGF) receptor can make it active even in the absence of EGF, and consequently oncogenic Mutation in the Ras gene (protooncogene is converted to an oncogene → Oncoprotein) Mutations in the tumor suppressor gene P53 Modes of action of the p53 tumor suppressor Mutations in the tumor suppressor gene APC In most sporadic colon-cancer cases, we have the same sequence of mutation events. Usually, the first mutated gene is APC Telomerase activation in tumor cells Telomerase activation has been observed in ~90% of all human tumors Telomerase Function: Extends telomeres, the protective caps on chromosome ends. Normal Cells: Low or absent telomerase activity leads to telomere shortening and limits cell division. Cancer Cells: Telomerase is often reactivated, maintaining telomere length and enabling indefinite cell division. Impact on Cancer: Reactivation contributes to cancer cell immortality and tumor growth. Research Focus: Targeting Donate and Blasco, 2011 telomerase could limit cancer cell proliferation. Summary 1. Cancer arises from the uncontrolled proliferation of abnormal cells, which can metastasize to other parts of the body. 2. The development of cancer is a microevolutionary process, requiring multiple genetic mutations and exhibiting genetic instability. 3. Cancers are classified by tissue origin, with carcinomas, sarcomas, lymphomas, and leukemias being the primary types. 4. Hallmarks of cancer include uncontrolled growth, evasion of apoptosis, and the ability to invade tissues and form metastases. 5. Genetic and epigenetic changes, including mutations in oncogenes and tumor suppressor genes, play crucial roles in cancer development.  From Carcinogenesis to delivering Precision Oncology to Patients with Cancer Prof Constantina Constantinou [email protected] Monday October 21, 2024  Learning objectives (LOBs) Define the term 'malignancy', and describe the role of oncogenes and tumour suppressor genes and their contribution to carcinogenesis To define precision oncology and describe the challenges associated with delivering precision oncology to patients with cancer  Overview 1. Introduction 2. Carcinogenesis 3. Oncogenes and Tumour Suppressor Genes 4. Challenges in cancer treatment 5. New technological developments 6. Precision Oncology 7. Challenges with delivering of precision oncology to cancer patients (Mateo et al., 2022 paper) 8. Summary 9. Further Reading 1. Introduction What is cancer? Cancer is an abnormal growth of cells which tend to proliferate in an uncontrolled way and, in some cases, to metastasise (spread) Malignancy is the tumour property to invade nearby tissues and spread (metastasise) to other parts of the body 1. Introduction At least 200 forms of cancer (and many subtypes for each) Cancers with highest incidence: 1. Breast 2. Lung 3. Colorectal https://gco.iarc.fr/today/data/factsheets/cancers/39-All-cancers-fact-sheet.pdf 1. Introduction Almost 10 million deaths in 2020 https://gco.iarc.fr/today/data/factsheets/cancers/39-All-cancers-fact-sheet.pdf 2. Carcinogenesis Internal stimuli External stimuli 2. Carcinogenesis Proto-oncogenes: Ras, Myc S Checkpoint Check for: Cell size Tumour DNA replication Tumour suppressor: suppressor: Rb p53 2. Carcinogenesis Carcinogenesis is a multi-stage process involving multiple hits Colon EFFECTS OF MUTATIONS 1 Loss of tumor- 2 Activation of 4 Loss of suppressor gene ras oncogene tumor-suppressor Colon wall APC (or other) gene p53 3 Loss of 5 Additional tumor-suppressor mutations gene DCC Normal colon Small benign Larger benign Malignant tumor epithelial cells growth (polyp) growth (adenoma) (carcinoma) The Multistep Model of Cancer Development: Colorectal cancer Fearon ER and Vogelstein B. Cell, 1990 2. Carcinogenesis Activation of oncogenes+ inactivation of tumor suppressor Cancer genes Mutations Cell containing Normal excessive cell mutations Uncontrolled cell division / Defective pathway of apoptosis 2. Carcinogenesis Cancer= environment + genetic predisposition Carcinogens: substances and exposures that can lead to cancer Type Example Chemical Benzene Mutations Alkylating agents leading to (chemotherapy) Environment uncontrolled Physical X-rays cell UV light proliferation Viral Hepatitis B Human Papilloma and inhibition of apoptosis = Genetic CANCER Hereditary cancer Li-Fraumeni syndrome predisposition syndromes predisposition 3. Oncogenes and Tumor Suppressor Genes Two kinds of genes are very important in cancer development: Oncogenes and Tumour Suppressor genes 3. Oncogenes and Tumor Suppressor Genes Proto-oncogenes: Ras, Myc S Checkpoint Tumour Check for: suppressor: pRb Cell size Tumour DNA replication suppressor: p53 Adapted from BioNinja.com 3.1 Oncogenes Proto-oncogene: a normal cellular gene which regulates cell growth and/or division and differentiation. Oncogene: a proto-oncogene that has been activated by mutation or overexpression. David P. Clark, Nanette J. Pazdernik, in Biotechnology (Second Edition), 2016 3.1 Oncogenes Mutations Proto-oncogenes ON Cancer Oncogenes ON/ OFF Controlled cell division  3.1 Oncogenes 1)1) Point Point Mutation: Mutation: variant variant in proto-oncogene in proto-oncogene (KRAS(KRAS in lungincancer) lung cancer) or in or in promoter/regulatory promoter/regulatoryelement element 2) Gene Amplification (c-myc in breast cancer) 2) Gene Amplification: e.g. c-myc in breast cancer 3) Chromosomal Translocation: creation of fusion protein (BCR-ABL in CML) or 3) Chromosomal disruption Translocation: of regulatory elementscreation of fusion protein (BCR-ABL in CML) or disruption of regulatory elements Proto-oncogene Genomic DNA Mutation within Multiple copies Gene moved to new DNA the gene of the gene locus, under new controls Oncogene New promoter Mutant Normal growth- Normal growth- hyperactive stimulating stimulating growth- protein protein stimulating in excess in excess protein in normal amount 3.1 Oncogenes HER2 The ErbB receptor family, also known as the EGF receptor family or type I receptor family, includes the epidermal growth factor (EGF) receptor (EGFR) or ErbB1/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4. HER2/neu/ERBB2 gene encodes for part of the human epidermal growth factor receptor. Pohlmann P et al. Clinical Cancer Research. 2009 HER2 Growth factors bind EGFR (HER1) or HER3 and alter conformation of receptors that become active. Receptor dimerization (heterodimerizaion or homodimerizaion) is required for HER2 function. HER1 Pohlmann P et al. Clinical Cancer Research. 2009 HER2 HER2 protein has intracellular tyrosine kinase activity Mazzucchelli S et al. World J Pharmacol. 2014  HER2 The HER2 gene is amplified in ~20% of the invasive breast cancers and used to be associated with aggressive disease and poor prognosis. Gullo G et al. Investigational New Drugs. 2008  Development of drugs for HER2+ cancers Trastuzumab (Herceptin) and pertuzumab (PERJETA) are monoclonal antibodies that target Her2 (targeted therapy). These drugs are only effective in Her2+ cancers. Boix-Perales H et al. The Oncologist, 2014 3.2 Tumour Suppressor Genes (TSG) TSG encode proteins that maintain the checkpoints and control genome stability. They inhibit replication and proliferation of damaged cells by: ▪ Repairing DNA damage (e.g. BRCA1/2) ▪ Inhibition of cell proliferation (RB1) ▪ Inhibition of cell proliferation, induction of apoptosis (P53) 3.2 Tumour Suppressor Genes (TSG) Mutations Proto-oncogenes ON Cancer Oncogenes ON/ OFF Inactive Tumour suppressor genes Controlled Controlled cell Mutations cell division division Tumour suppressor genes 3.2 Tumour Suppressor Genes (TSG) 2 Protein kinases MUTATION Mutated p53 cannot activate transcription UV 3 Active light form of p53 1 DNA damage in genome DNA Protein that inhibits the cell cycle Fig. 18-21b DNA repair genes: BRCA1/2 3-8% of all women with breast cancer have a mutation in BRCA1 or BRCA2 BRCA1/2 genes produce proteins involved in repair of DNA double strand breaks Defects in DNA repair genes cause genomic instability and accelerate the activation of oncogenes and the loss of tumour suppressors DNA repair genes: BRCA1/2 Toss A and Cortesi L. Journal of Cancer Science & Therapy, 2013 DNA repair genes: BRCA1/2 Talazoparib Olaparib Rucaparib Sonnenblick A et al. Nat Rev Clin Oncol. 2015 Mechanism of action of PARP inibitors Jacob et al. Urology Times Urologists in Cancer Care, UCC December 2020, Volume 09, Issue 04. https://www.urologytimes.com/view/parp-inhibitors-treating-mcrpc-from-a-genetic-basis Development of drugs (PARP inhibitors) for germline BRCA1/2 cancers 1. Talazoparib, germline BRCA1/2, HER2- breast cancer 2. Olaparib, germline BRCA1/2, pancreatic cancer and prostate cancer 3. Rucaparib, germline or somatic BRCA1/2 prostate cancer TP53 and RB1 CDK inhibitor (p21) p53 Rb TP53 2 Protein kinases MUTATION Mutated p53 cannot activate transcription UV 3 Active light form of p53 1 DNA damage in genome DNA Protein that inhibits the cell cycle Fig. 18-21b TP53 “The Guardian of the Genome” The TP53 gene encodes for p53 protein The p53 protein: o Detects DNA damage o Induces G1-G2 cell cycle arrest o Induces apoptosis Over 50% of cancers contain mutations in the TP53 gene Most commonly affected tumour suppressor gene in human cancer Levine and Oren. Nat Rev Cancer, 2009 Rb1 Encodes Rb protein which prevents Retinoblastoma: cell growth by inhibiting cell cycle until 1 in 20,000 children cell is ready to divide 90% present before 5 years of age Malignant tumour of the eye(s) that Treatment: surgery & radiotherapy originates from the retina (light 98% of cases are cured sensitive lining of the eye) Mitogens Rb Rb1 Why does the cancer develop specifically in the retina? Cell Type Specificity: The retina has rapidly dividing cells during early development. Developmental Timing: Active development in early years increases susceptibility to RB1 mutations. Tissue-Specific Factors: Unique genetic or environmental factors in retinal cells heighten vulnerability. Individuals with hereditary RB1 mutations may develop other cancers later in life (e.g. osteosarcomas, melanoma, lung cancer) 4. Challenges in cancer treatment Cancer is caused Cancer by the is caused accumulation of by accumulation of multiple multiple mutations mutations. Heterogeneity of tumours Diverse physiological pathways and tissue specificity Understanding of Cancer Landscape and Identification of Common Targets 4. Challenges in cancer treatment Bert Vogelstein et al. Science 2013;339:1546-1558 5. New technological developments - Next Generation Sequencing (NGS) - Liquid biopsies  6. Precision Oncology Precision Oncology: The integration of molecular profiles into clinical decision making in cancer treatment (Yates et al., 2020) The science of using a patient’s genetics to create a treatment plan targeted to the molecular characteristics of their cancer Precision oncology is a rapidly developing area of research that is making its way, more and more, into mainstream oncology practice https://na.geneseeq.com/precision-oncology/#what-is-precision-oncology 6. Precision Oncology The aim of Precision Oncology is to improve patient outcomes Chemotherapy can be toxic and kill healthy cells in addition to cancer cells Precision oncology is targeting a specific biomarker and therefore prevents cancer cells with that genetic biomarker from dividing and growing while preventing damage in healthy cells (less side effects) https://na.geneseeq.com/precision-oncology/#what-is-precision-oncology 7. Challenges with delivering precision oncology to patients with cancer Mateo J, et al. Delivering precision oncology to patients with cancer. Nat Med. 2022 Apr;28(4):658-665. doi: 10.1038/s41591-022-01717-2. Epub 2022 Apr 19. PMID: 35440717. The increasing development of MGTOs New era of precision medicine led to an increasing number of molecularly guided treatment options (MGTOs) receiving approval for use in patients The increasing development of MGTOs is the result of our increased understanding of cancer biology but also the use of high throughput technologies such as Next Generation Sequencing (NGS) 1. Talazoparib 2. Olaparib 3. Rucaparib Gap between anti-cancer drug development and delivery of drugs to patients Despite rapid development of MGTOs, several challenges are faced by healthcare systems 23% of patients with newly diagnosed advanced non small cell lung cancer (NSCC) did not receive genomic testing for any of the four guideline recommended therapeutic targets (ALK, BRAF, EGFR and ROS alterations) before first line treatment (Gondos et al., 2020) Gondos et al. Genomic testing among patients (pts) with newly diagnosed advanced non small cell lung cancer (aNSCLC) in the United States: a contemporary clinical practice patterns study. J. Clin Oncol Abstract 9592 (2020). Gap between anti-cancer drug development and delivery of drugs to patients Drug development Gap Delivery of new drugs to patients Challenges to implementing precision medicine and critical steps to address these 1. Facilitation of equal access to genomic testing through a patient centred approach Transition of molecular testing from centers of academic excellence to populations receiving care in the community European Medicines Agency (EMA) is responsible for approval of all medicines in Europe but access to medication is heterogenous across European countries Access to advanced diagnostics should be in a patient centred and not an institution centered manner Deliver testing where the patients are but refer patients to centres where they can access advanced diagnostics Challenges to implementing precision medicine and critical steps to address these 2. Ensuring that clinical studies provide robust evidence for new drugs and technologies Clinical trials on the prevalence of a specific biomarker focus on selected populations which may not be representative of the wider population Ethnic or socio economic groups or people with comorbidities may be under-represented Investigate the genomics of real world populations enriching for groups under represented in clinical trials to determine the prevalence of precision oncology targets and to inform allocation of resources Challenges to implementing precision medicine and critical steps to address these 3. Enabling physicians to interpret genomics data The complexity and data generated through comprehensive genomics profiling increases Studies have shown that many clinicians are concerned with the interpretation of results Education of healthcare professionals, development of decision making tools and access to multi-disciplinary teams are important for improving clinical management Challenges to implementing precision medicine and critical steps to address these 4. Empowering patients towards shared decision making Healthcare professionals face challenges in interpreting genomic test results Healthcare professionals should communicate accurate information so that the patients are empowered to consider their options and make their decisions 8. Summary Cancer is a genetic disease and occurs in a multistep manner Activation of oncogenes and inhibition of TSG lead to cancer development Precision oncology is the integration of molecular profiles into clinical decision making in cancer treatment (Yates et al., 2020) Gap between anti-cancer drug development and delivery of drugs to patients Challenges remain in delivering precision oncology to patients with cancer 9. Further Reading 1. Biology: a global approach. Campbell et al, (2020) 12th Edition. 2. Molecular Biology of the Cell, Alberts, B. et al. (2015) 6th Ed. 3. The Biology of Cancer, Weinberg, RA (2014) 2nd Ed. 4. Hanahan D and Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell. 2011. doi.org/10.1016/j.cell.2011.02.013 5. National Research Council (US) Safe Drinking Water Committee, “Mechanisms of Carcinogenesis” Thomas RD, editor. Washington (DC): National Academies Press (US); 1986. https://www.ncbi.nlm.nih.gov/books/NBK219109/ 6. Lee E and Muller WJ. Oncogenes and Tumor Suppressor Genes. Cold Spring Harb Perspect Biol. 2010. www.ncbi.nlm.nih.gov/pmc/articles/PMC2944361/ 7. Ke X and Shen L. Molecular targeted therapy of cancer: The progress and future prospect. In Frontiers in Laboratory Medicine. Volume 1, Issue 2, June 2017, Pages 69-75, doi.org/10.1016/j.flm.2017.06.001 8. Mateo J, et al. Delivering precision oncology to patients with cancer. Nat Med. 2022 Apr;28(4):658-665. doi: 10.1038/s41591-022-01717-2. Epub 2022 Apr 19. PMID: 35440717. Thank you! DNA sequencing and precision medicine Konstantinos Voskarides Learning objectives 1. Define personalised medicine 2. Describe the basis of sequencing techniques such as Sanger and Next Generation Sequencing and discuss clinical applications 3. Interpret a basic electropherogram 4. Explain the limitations of genomic risk profiling and the pitfalls of direct-to-consumer genetic testing 5. Discuss the implications of variable access and utilisation of genetic testing DNA sequencing technologies  Sanger sequencing The dideoxy method of sequencing DNA relies on chain-terminating dideoxyribonucleoside triphosphates (ddNTPs). These ddNTPs are derivatives of the normal deoxyribonucleoside triphosphates (dNTPs) that lack the 3′-hydroxyl group. When incorporated into a growing DNA strand, they block further elongation of that strand Automated dideoxy sequencing relies on a set of four ddNTPs, each bearing a uniquely colored fluorescent tag Electropherogram Automated dideoxy sequencing relies on a set of four ddNTPs, each bearing a uniquely colored fluorescent tag. A tiny part of the data from such an automated sequencing run. Each colored peak represents a nucleotide in the DNA sequence. Nucleotide substitution Hom Hom Het Nucleotide deletion Hom Het Hom Next Generation Sequencing Principles of Illumina sequencing An image of the surface of the Illumina flow cell, showing individual DNA clusters after a round of DNA synthesis with colored NTPs. Precision medicine, sometimes known as "personalized medicine" is an innovative approach to tailoring disease prevention and treatment that takes into account differences in people's genes, environments, and lifestyles. The goal of precision medicine is to target the right treatments to the right patients at the right time. (FDA, 09/27/2018) Inherited diseases - Direct Sanger sequencing for a candidate gene to find the responsible mutation - Screening of a panel of candidate genes through a Next Generation Sequencing approach - Whole Genome Sequencing (WGS) or Whole Exome Sequencing (WES) Who needs genetic testing for an inherited disease? n A newborn, because his/her parents are affected or carriers n Presympomatic genetic testing n A patient, for diagnostic purposes https://www.athenadiagnostics.com/view-full-catalog/h/hearing- loss-advanced-sequencing-and-cnv-evaluatio Pharmacogenetics or Pharmacogenomics Pharmacogenomics can play an important role in identifying responders and non-responders to medications, avoiding adverse events, and optimizing drug dose. FDA list a big list of drugs for which pharmacogenomic markers may give useful information https://www.fda.gov/drugs/science-and-research-drugs/table- pharmacogenomic-biomarkers-drug-labeling  Abacavir, a drug used for treating HIV infection, can cause a severe and occasionally a life threatening hypersensitivity reaction Martin et al, 2012 Carr and Cooper, 2000  CYP genes: Very important for pharmacogenetics Warfarin is a frequently used oral anticoagulant, that may cause bleeding as a side-effect CYP2C9 and VKORC1 gene polymorphisms affect warfarin metabolism and dosage Restrepo et al, 2018 Multifactorial diseases and other traits Predicting individuals’ probabilities for developing multufactorial diseases (diabetes, cancer, obesity etc) through sequencing data is still very risky There are exceptions, like genetic variants related with thrombophilia (genes: MTHFR, F5, prothrombin gene) and Alzheimer’s disease (APOE gene) Prof. George Churge  Note: Human Genome Project (HGP) took more than 13 years to accomplish (1990-2003) and costed more than $3 billion Population carriers’ screening  Tay-Sacs and sickle cell disease The impact of carrier screening in lowering the incidence of a genetic disease can be dramatic. Carrier screening for Tay-Sachs disease in the Ashkenazi Jewish population has been carried out since 1969. Screening followed by prenatal diagnosis, when indicated, has already lowered the incidence of Tay-Sachs disease by 65% to 85% in this ethnic group. In contrast, attempts to screen for carriers of sickle-cell disease in the U.S. African American community have been less effective and have had little impact on the incidence of the disease so far. The success of carrier screening programs for Tay-Sachs disease, as well as the relative failure for sickle cell anemia, underscores the importance of community consultation, community engagement, and the availability of genetic counseling and prenatal diagnosis as critical requirements for an effective program. What are many clinical features of the above diseases? Why Tay-Sachs and sickle-cell disease are so frequent in the above populations? Beta-thalassemia prevention in Cyprus by carrier screening Dr Michael Angastiniotis Newborn screening Depending on the frequency of genetic diseases per population, the test list could inlcude among others: Hearing test Phenylcetonuria Hypothyroidism Cystic fibrosis Congenital adrenal hyperplasia (CAH) RNA sequencing Global Analysis of mRNAs by RNA-sequencing Provides a Snapshot of Gene Expression  Global Analysis of mRNAs by RNA-seq Provides a Snapshot of Gene Expression Human fibroblasts were deprived of serum for 48 hours; serum was then added back to the cultures at time 0, and the cells were harvested for mRNA measurements at different time points. red indicates an increase in expression; green is a decrease in expression. Useful videos n Direct-to-consumer genetic testing (5 mins) https://www.youtube.com/watch?v=crsxdOnJg1I n Interpreting results (5 mins) https://www.youtube.com/watch?v=mDRuI8hxS0c Introduction to Epidemiology and Public Health Epidemiology and Global Public Health Stream GEMD-101 October 31st, 2024 Christiana Demetriou Associated Professor, Epidemiology and Public Health Learning objectives Define the terms 1 `Epidemiology’ and `Public Health’ Outline the potential value of studying health and disease in populations 2 Distinguish between 3 descriptive and analytic epidemiology Explain the concepts of DALYs, QALYs, Life Expectancy (LE), Health Adjusted LE (HALE), incidence, prevalence, incidence rate and mortality as metrics for the study 4 of disease in populations Define the different levels 5 of prevention and appreciate their value in Public Health practice  Why am You are studying to be a medical I here? doctor. This involves learning how to assess, diagnose, and treat your patients. Figure source: Zdenek Sasek Why am I here? Video link: https://www.youtube.com/watch?v=xYeAmafTGCA  Why am You are studying to be a medical I here? doctor. This involves learning how to assess, diagnose, and treat your patients. Epidemiology and Public Health are the cornerstone disciplines that allow you to do that effectively, safely and efficiently. Figure source: Zdenek Sasek So what is Epidemiology? epi-demi-ology (επί + δήμος + λόγος) study of (disease) upon the public “…the study of the distribution and determinants of health-related states or events in specified populations, and the application of this study to the control of health problems” ~ John M. Last 1983  3 plus 1 Frequency of Disease Take Effective Action Distribution for the Control of of Disease Disease Determinants of Disease The definition of epidemiology includes three (3) components and one (1) main goal  Frequency of Frequency of disease, disability and death = Burden of Disease Disease What is the burden of a particular illness on population health? mortality (deaths) morbidity (ill-health) Figure source: Institute for Health Metrics and Evaluation; Global burden of Disease Study  Frequency of Measures of Disease Burden (1) Disease Cumulative Incidence or risk Occurrence of new cases arising in a given period in a specified population 𝐼𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑛𝑒𝑤 𝑐𝑎𝑠𝑒𝑠 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒 ℎ𝑒𝑎𝑙𝑡ℎ 𝑒𝑣𝑒𝑛𝑡 𝑑𝑢𝑟𝑖𝑛𝑔 𝑎 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑒𝑑 𝑡𝑖𝑚𝑒 𝑝𝑒𝑟𝑖𝑜𝑑 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑜𝑝𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑟𝑖𝑠𝑘 𝑎𝑡 𝑡ℎ𝑒 𝑏𝑒𝑔𝑖𝑛𝑛𝑖𝑛𝑔 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑒𝑑 𝑡𝑖𝑚𝑒 𝑝𝑒𝑟𝑖𝑜𝑑 Prevalence or prevalence rate Frequency of existing cases in a defined population at a given point in time 𝑃𝑟𝑒𝑣𝑎𝑙𝑒𝑛𝑐𝑒 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑒𝑥𝑖𝑠𝑡𝑖𝑛𝑔 𝑐𝑎𝑠𝑒𝑠 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒 ℎ𝑒𝑎𝑙𝑡ℎ 𝑒𝑣𝑒𝑛𝑡 𝑑𝑢𝑟𝑖𝑛𝑔 𝑎 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑒𝑑 𝑡𝑖𝑚𝑒 𝑝𝑒𝑟𝑖𝑜𝑑 𝑜𝑟 𝑎𝑡 𝑎 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑒𝑑 𝑡𝑖𝑚𝑒 − 𝑝𝑜𝑖𝑛𝑡 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑜𝑝𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑟𝑖𝑠𝑘 𝑑𝑢𝑟𝑖𝑛𝑔 𝑡ℎ𝑒 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑒𝑑 𝑡𝑖𝑚𝑒 𝑝𝑒𝑟𝑖𝑜𝑑 𝑜𝑟 𝑎𝑡 𝑎 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑒𝑑 𝑡𝑖𝑚𝑒 − 𝑝𝑜𝑖𝑛𝑡 Frequency of Measures of Disease Burden (2) Disease Incidence rate A measure of how fast new cases of disease are occurring 𝐼𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 𝑟𝑎𝑡𝑒 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑛𝑒𝑤 𝑐𝑎𝑠𝑒𝑠 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒 ℎ𝑒𝑎𝑙𝑡ℎ 𝑒𝑣𝑒𝑛𝑡 𝑑𝑢𝑟𝑖𝑛𝑔 𝑎 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑒𝑑 𝑡𝑖𝑚𝑒 𝑝𝑒𝑟𝑖𝑜𝑑 = 𝑇𝑖𝑚𝑒 𝑒𝑎𝑐ℎ 𝑝𝑒𝑟𝑠𝑜𝑛 𝑎𝑡 𝑟𝑖𝑠𝑘 𝑤𝑎𝑠 𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 𝑡𝑜𝑡𝑎𝑙𝑙𝑒𝑑 𝑓𝑜𝑟 𝑎𝑙𝑙 𝑝𝑒𝑟𝑠𝑜𝑛𝑠 (𝑝𝑒𝑟𝑠𝑜𝑛 − 𝑡𝑖𝑚𝑒) Mortality rate Number of new deaths in a defined population at a given point in time 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑛𝑒𝑤 𝑑𝑒𝑎𝑡ℎ𝑠 𝑑𝑢𝑟𝑖𝑛𝑔 𝑎 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑒𝑑 𝑡𝑖𝑚𝑒 𝑝𝑒𝑟𝑖𝑜𝑑 𝑀𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 𝑟𝑎𝑡𝑒 = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑜𝑝𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑎𝑡 𝑟𝑖𝑠𝑘 𝑑𝑢𝑟𝑖𝑛𝑔 𝑡ℎ𝑒 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑒𝑑 𝑡𝑖𝑚𝑒 𝑝𝑒𝑟𝑖𝑜𝑑  Frequency of Measures of Disease Burden - recap Disease Figure source: High-yield Biostatistics, Epidemiology, and Public Health (High-yield Series) 4th Revised edition by Anthony N. Glaser (ISBN: 9781451130171)  Frequency of Measures of Disease Burden (3) Disease Disability Adjusted Life Years (DALYs)  A measure of the overall burden of disease = years of life lost due to early death PLUS years spent living with disability = (years spent living with disability x the disability factor) + (years of life lost x 1) Figure source: Public Health England  Frequency of Measures of Disease Burden (4) Disease Quality Adjusted Life Years (QALYs)  Measure health outcomes by weighting years of life by a factor (Q)  Q represents health-related quality of life  Anchored between 1 (perfect health) and 0 (a health state considered to be as bad as death)  Estimated for all health states between these extremes, but also for some states that are considered worse than death = no. years a patient spends in each health state, multiplied by the quality of life weight, Q, for that state Figure source: http://www.med.uottawa.ca Frequency of Measures of Disease Burden (5) Disease Life expectancy Defined as the mean number of years of life remaining at a given age, assuming age-specific mortality rates remain at their most recently measured levels A population-based average, combining everyone who may die either in early, middle or later life Life expectancy at birth Average number of years that a newborn is expected to live if current mortality rates continue to apply Health adjusted life expectancy (HALE) Obtained by subtracting years of ill health from standard life expectancy Reflects also the burden of diseases that might not affect life expectancy, but affect the quality of life Distribution of Disease Distribution of Disease Does the disease affect particular populations and individuals? Describing Patterns: Descriptive Epidemiology variation between PLACES Answers who, where, when, in relation to what (outcome) variation over TIME Making comparisons and asking questions: variation between PERSONS generate hypotheses Distribution of Disease - Place Distribution of Disease Figure source: Institute for Health Metrics and Evaluation; Global burden of Disease Study Distribution of Disease - Time Distribution of Disease Figure 2 – Age-standardised incidence, prevalence, mortality, and DALY rates (per 100 000 people per year) in seven GBD super regions, 1990–2019, for both sexes and all ages Figure source: The Lancet Neurology; GBD 2019 Stroke Collaborators et al. VOLUME 20, ISSUE 10, P795-820, OCTOBER 01, 2021  Distribution of Disease - Persons Distribution of Disease Figure 1 – Diabetes prevalence by age and sex in 2019 Figure source: Diabetes Research and Clinical Practice 2019 157DOI: (10.1016/j.diabres.2019.107843)  Determinants of Disease Determinants of Disease Understanding the causes or risk factors for a health outcome Analytic Epidemiology Answers why and how Testing hypotheses generated through descriptive epidemiology Appreciating the Wider Determinants of Health as risk factors for disease  Study designs used in descriptive and analytic epidemiology Cross sectional and Ecologic studies can also be analytic !! Figure source: DC Field Epidemiology Training Program Descriptive vs. analytic epidemiology - recap Descriptive Epidemiology Analytic Epidemiology  Take Effective Action Public Health for the Control of Disease “The art and science of preventing disease, prolonging life and promoting health through the organized efforts of society” ~ WHO (1998) Health = “Health is a state of complete physical, mental and social wellbeing and not merely the absence of disease or infirmity” ~ WHO (1948) Deals with preventing disease and enhancing health and wellbeing in human populations Differs from clinical/medical practice in the following: The emphasis is in prevention rather than cure The primary focus is on populations/communities not individuals Public Health is about Prevention – Levels of Prevention Primordial Prevention Prevent the Onset of Clinical development of disease diagnosis risk factors. No disease Asymptomatic disease Clinical course Primary Prevention Secondary Prevention Tertiary Prevention Manage the risk factors. Early diagnosis and Reduce complications, Prevent the onset of prompt treatment. disability and further disease. impairment. The three fundamental domains of Public Health Practice (1) Health Improvement Develop primary prevention programmes e.g. 5-a-day Health Improvement Behaviour change SIDS Substance misuse Behaviour improvement plans i.e. smoking bans, physical activity promotion initiatives i.e. bike lanes etc. Healthcare Health inequalities Health Disability Public Ethnic/Geographic life expectancy Protection Health Wider determinants of health Housing Education The three fundamental domains of Public Health Practice (2) Health Protection Infectious disease Health Childhood vaccination Improvement Immunisation Environmental hazards Flooding Radiation Healthcare Health Public Emergency response Protection To infectious disease outbreaks Health To natural or technological disasters The three fundamental domains of Public Health Practice (3) Healthcare Public Health Health Set up secondary and tertiary prevention programmes eg. Improvement Mammography or Bowel cancer screening Healthcare quality Implement evidence based practice Cost-effectiveness of medical interventions Healthcare Health Health policy Public Implement evidence based guidance Protection Health Commissioning and priority setting Public Health top 10 Largest contribution to improvement in health in 20th Century (US CDC) Figure source: https://hpa.princeton.edu/sites/g/files/toruqf2006/files/public_health.pdf Benefits of Studying Health/Disease ​in the Population​ For any particular illness, studying at population level helps to understand: how much ill-health does an illness cause? does the illness affect particular populations and individuals? what is/are the cause(s) of the illness? how can the illness be prevented? how can the illness be effectively treated? is everyone who could benefit receiving treatment? how should treatment services be organized? Questions? Additional optional resources Hennekens CH, Buring JE, “Epidemiology in Medicine”, Little, Brown & Co, 1987, chapters 1 & 2. Available in the Medical School Library Rothman KJ, Greenland S, Lash TL, “Modern Epidemiology” (3rd edn.), Lippincott, Williams & Wilkins, 2008, chapters 1 & 3 Available in the Medical School Library BMJ – “What is Epidemiology?” http://www.bmj.com/about-bmj/resources-readers/publications/epidemiology-uninitiated/1-what-epidemiology Centers for Disease Control and Prevention – “Introduction to Epidemiology” https://www.youtube.com/watch?v=4oaQUAnA6nY&t=49s Centers for Disease Control and Prevention – “Introduction to Public Health” https://www.youtube.com/watch?v=-dmJSLNgjxo&t=35s Boston University School of Public Health, “Measures of Disease Frequency” https://sphweb.bumc.bu.edu/otlt/mph-modules/ep/ep713_diseasefrequency/ep713_diseasefrequency_print.html The Institute for Health Metrics and Evaluation (IHME) – Global Burden of Disease (GBD) https://www.healthdata.org/gbd/2019 Murray CJL. – “Global burden of disease and injury series: the global burden of disease”, Chapter I “The GBD’s approach to measuring health status” https://apps.who.int/iris/bitstream/handle/10665/41864/0965546608_eng.pdf