The Influence of Mutations on Ageing and Cancer Progression

1. Ageing 2: Cancer

2. Review: The force of natural selection declines with ageing due to increase in extrinsic mortality (= weakening of natural selection) and reduction in reproduction with age (selection acts at reproduction)

3. Review Selection acts on genetic variation in a population (culling different individuals that have different genotypes) This variation is caused by mutations What are mutations?

4. MutationsThe Raw Material for Evolution Mistakes: Any change in the genetic code, including changes caused by errors in DNA replication or errors in DNA repair

5. MutationsThe Double Edged Sword Cause of many diseases, ageing, cancer Cause of evolutionary novelty, upon which natural selection could act

6. Hierarchical processes that are affected by Mutations • STRUCTURAL • Amino Acid composition (nucleotide substitutions, Amino Acid substitutions) • Gene duplications, deletions; Chromosome duplications, deletions • Secondary, Tertiary, Quaternary structure • REGULATORY • Gene/Protein expression (transcription, RNA processing, translation, etc) • Protein activity (allosteric control, conformational changes, receptors)

7. Amino AcidSubstitutions • Synonymous substitutions: • Mutations that do not cause amino acid change (usually 3rd position) • Nonsynonymous substitutions: • Mutations that cause amino acid change (usually 1st, 2nd position)

8. REGULATORY • Gene/Protein expression • Transcription: Mutations at promoters, enhancers, transcription factors, Histones/acetylation, etc. • RNA Processing: Mutations at splice sites, sites of polyadenylation, sites controlling RNA export • Translation: Mutations in ribosomes, regulatory regions, etc • Protein activity (allosteric control, conformational changes, receptors)

9. Central Dogma (gene expression)

10. Transcription • Could have mutations in promoter or enhancer • Could have mutation in transcription factor • Mutation in repressor • Mutations in factors that alter chromatin structure (histone acetylation)

11. A hallmark of ageing and cancer is the increase of genomic instability with age Selection that acts to preserve genomic stability would diminish with age Somatic mutations (mutations in the body) accumulate with age ≠ mutational accumulation from previous lecture (heritable deleterious mutations that get expressed later in life  diseases expressed later in life)

12. Cancer is very common • Lifetime risk of cancer in human populations is around 1 out of 3 • Each year 10 million cases are diagnosed • We have no global cure for cancer

13. What is Cancer? HeLa cells A problem of multicellular organisms Multicellularity requires social cohesion of cells: All cells must die at some point in multicellular organisms (apoptosis, programmed cell death) Cancer cells revert to unicellular selfishness, and are immortal, and fail to die  tumor

14. Tumor Progression by Clonal Evolution • Cancer cells have a short term evolutionary advantage over wild type cells, because they grow and proliferate at a greater rate • Thus, selection leads to a preponderance of cancer cells over healthy cells • As more mutations accumulate in cancer cells, the greater the competitive edge • In other words, cancer cells evolve towards higher virulence within the body • However, at the whole organismal level there is a cost

15. Cancer Stem Cells? • Unresolved issue: to what extent is cancer (tumor growth) due to proliferation of all cells or a few special cells (cancer stem cells)? • Hypothesis 1: all tumor cells are immortal- every cell in a tumor is the same • Hypothesis 2: only some cells immortal- special lineage of cells (cancer stem cells) in a tumor is really responsible for tumor growth and metastasis • To what extent should treatment focus on targeting cancerstem cells?

16. Ageing and Cancer • Cancer Cells • Cancer is considered a disease of ageing because the somatic mutations that accumulate during ageing can sometimes disrupt normal genetic control of cell proliferation and cell death (apoptosis) • Ironically, these mutations make cancer cells immortal, such that they fail to age and die

17. The Irony of Cancer Environmental assaults (oxidative stress, smoking, pesticides, radiation, etc.) cause DNA damage Cancer (immortal) cells that fail to age and die (tend to be expressed in older individuals) This is considered a symptom of “ageing” because as you age the DNA repair and genomic stability mechanisms decline with age Mutations by chance at genes that affect cell growth, proliferation, DNA repair; some of the mutations might have been inherited

19. Types of Mutations that lead to Cancer • Mutations to proto-oncogenes --> leading to oncogenes, or insertions of oncogenes(genes involved in cell growth and development; growth factors, growth factor receptors etc) • Mutations to tumor suppressor genes (e.g. Trp53; Genes whose products block abnormal growth) • Mutations to DNA repair genes (mismatch repair etc) • Telomere shortening leading to chromosome instability and gene deletions

20. Treatment for Cancer can age you further • Once cells have become immortal, there is a tradeoff between killing the cancer cells and accelerating ageing in normal cells • Radiation and chemotherapy kills cancer cells but will age normal cells (and induce mutations) • Cancer is a byproduct of ageing; yet, cancer protection/treatment could accelerate ageing

21. Targets for Cancer Suppression • Reduction in cell proliferation and apoptosis (programmed cell death) • Tyrosine kinase inhibitors: target signal transduction(block signal pathways that cause cells to proliferate; upregulate pathways that cause apoptosis) • Histone deacetylase inhibitors • Angiogenesis inhibitors(choking cells, by cutting off blood supply)

22. Example: p53 Transcription factor -- regulates expression of other genes In humans is encoded by the TP53 gene One of the most commonly mutated genes in human cancers; Regulates the cell cycle; reduce cell proliferation, increases apoptosis; thus, functions as a tumor suppressor that is involved in preventing cancer Activate DNA repair proteins when DNA has sustained damage Regulates genes involved in metabolism (glucose utilization and mitochondrial respiration)-- recall that increased metabolism could lead to faster ageing, as escaped electrons during respiration (e-transport chain) could cause cellular and DNA damage)

23. Sirtuins • In recent years, the increasing interest in Sirtuin protein biology has been astounding • Mechanism of action poorly understood • Important function in maintaining genomic stability, in some cases DNA repair

24. Sirtuins Sirtuins perform NAD+ dependent ADP-ribosylation of histones to interfere with histone acetylation (they are histone deacetylases)

25. Histone Deacetylases/Histone Acetyltransferase Plays important role in regulation of gene expression The ability of a particular transcription factor to bind to its target gene is, in part, dependent on modifications that are made to the histone proteins. Abnormal activity of HDACs has been observed in acute promyelocytic leukemia, acute myelogenous leukemia, non-Hodgkin lymphoma, and some types of colorectal and gastric carcinoma

26. Histone Deacetylases/Histone Acetyltransferase Plays an important role in the regulation of gene expression Histone acetyl transferases (HATs):Acetylate Histones, enhance transcription; acetylation neutralizes positive charges on the histone by changing amines into amides and decreases the ability of the histones to bind to DNA, allowing gene expression Histone Deacetylases (HDACs):Deactylate Histones, repress transcription; remove acetyl groups from an ε-N-acetyl lysine amino acid on a histone Different classes have different effects that either promote or suppress cancer depending on which histones they are affecting (and which genes are expressed or repressed)

27. Defects in acetylation machinery appears to lead to alterations in acetylation and perhaps the development of cancer • An imbalance in histone acetylation may lead to changes in chromatin structure and transcriptional dysregulation of genes involved in the control of cell cycle progression, differentiation, and apoptosis

28. Histone Deacetylases • Certain classes of Histone deacetylases (which affect gene expression) affect insulin regulation (glucose, insulin production, fat metabolism, cell survival) • Such HDACs have the same effects of DR without starving • One class of Histone Deacetylases: Sirtuins are a family of NAD+ dependent histone deacetylases that play important roles in gene silencing, DNA repair, rDNA recombination, and aging

29. SIR2 • The deacetylation of histones by SIR2 interferes with recombination in the repeated array of ribosomal DNA genes • Such rDNA recombination could result in a type of mutation (extrachromosomal rDNA circles), which cause ageing in yeast (Sinclair & Guarente, 1997) • Extra copies of or greater expression of SIR2 increases lifespan in yeast, C. elegans, Drosophila, Metazoans (multicellular animals)

30. Mammalian Sirtuins

31. SIRT1 • The most prominent mammalian sirtuin, SIRT1, has received considerable attention because of its link with human metabolism, aging and cancer • SIRT1 has many targets for histone deacetylation, including lysine residues at positions 9 and 26 of histone H1, 14 of H3, and 16 of H4 (next figure)

32. SIRT1 regulates many functions involving apoptosis and cell survival • For example, cooperates with HIC1 to regulate activity of p53 (tumor suppressor gene) and apoptosis

33. Ageing 3: Mechanisms, Mitigation

34. Well, ageing and cancer are difficult issues to resolve because so many potential targets are involved (as mutations are happening all across the genome)

35. Current treatment • Ageing: the tendency is to treat the symptoms, and not the mechanistic root causes • For example with cancer: the treatments are very crude and involve a brute force approach • Let’s just kill off the cells with radiation or chemotherapy: kill both normal and cancer cells; hope that the cancer will die and the patient will survive

36. It might be more effective to take a targeted approach in treating ageing and diseases of ageing, like cancer • This is difficult because there are so many potential targets • Where are the mutations occurring? • What impacts are they having?

37. Related topics that I’ll discuss Next: • Caloric Restriction • Nutrient Signaling • Sirtuins • Resveratrol

38. Caloric Restriction Much research is devoted to understanding why caloric restriction extends lifespan, as such insights would help reveal what genetic mechanisms cause ageing

39. Role of Diet • Dietary restriction (DR) has been found to increase life span in many organisms • Caloric Reduction by 30% greatly increases lifespan • Mechanism is not fully understood

42. Lifespan varies among populations

43. While there are genetic differences among populations, environment (diet) also plays a big role It has been established that certain diets lead to longer lifespan For example, the Japanese live the longest. However, Japanese-Americans on an American diet have shorter (= American) life spans Americans tend to eat large portions (large caloric intake), and have higher animal fat intake, less vegetables

44. Evolutionary Hypothesis: tradeoff between reproduction and maintenance (longer lifespan) Why should caloric restriction slow aging and increase lifespan? During years of famine, it may be evolutionarily favorable for an organism to halt reproduction and to upregulate protective and repair enzyme mechanisms to try to extend lifespan to allow for reproduction in future years.

45. Mechanism? Physiological mechanisms are poorly understood Hypotheses (some examples): Energy: Decreased oxidative damage: less food consumption, less metabolic damage Nutrient Signaling: Altered glucose utilization, Reduction of IGF/insulin signaling activity Enhanced stress responsiveness Changes in gene expression; Increased levels of sirtuins Bishop, N.A. & L. Guarente. 2007. Genetic links between diet and lifespan: shared mechanisms from yeast to humans. Nature Reviews Genetics 8:835-844.

47. Mechanisms of life span extension might differ between moderate vs severe caloric restriction

48. Severe Caloric Restriction(famine) Upregulate AMPK and downregulate TOR genes

49. Severe Caloric Restriction Under severe caloric restriction, downregulating TOR1 and SCH9 (genes encoding two protein kinases involved in nutrient sensing), as well as other genes of these nutrient-sensing pathways, extend the life span of yeast (not know yet if important in mammals) Sirtuin independent extension of lifespan

50. mTOR(downregulated with severe DR) is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription • AMPK(upregulated with severe DR) suppresses mTOR; acts as a metabolic master switch regulating several intracellular systems, including cellular uptake of glucose, β-oxidation of fatty acids and the biogenesis of glucose transporter 4 (GLUT4) and mitochondria.