Cytoskeleton, cell growth, apoptosis and disease

1. Cell biology 2014 (12/2 -14) Lecture 11: Cytoskeleton, cell growth, apoptosis and disease A medical perspective • Definitions of disease: • When something is wrong with a bodily function • A state that places individuals at increased • risk of adverse consequences • Etcetera

2. The cause of different diseases Mendelian/Genetic diseases Heart disease Allergy 100% genetics 100% environment Infectious diseases Diabetes Cancer Complex multi-factorial diseases

3. Three cytoskeleton systems: distinct but overlapping functions Intermediate filaments Actin filaments Microtubules - Cell shape and integrity - Motility of the whole cell or cellular appendages - Intracellular organization

4. Intermediate filaments and epithelia blistering 2. 1. Cytosolic intermediate filaments support: 1) Cell-cellcontacts (desmosomes: cadherins) 2) Cell-ECMcontacts (hemidesmosomes: integrins) 1 + 10 5 + 14 Mutations in keratin 5 or 14 cause Epidermolysisbullosa simplex (1/40 000), a disease manifested as blistering of the epidermis. Albert et al Fig. 16-21

5. Consequences of dysfunctional nuclear lamina Lamin A, B & C: intermediate filament proteins stabilize the nuclear envelope Laminopathies are genetic diseases manifested as either: I.Dystrophy of skeletal and/or heart muscles, caused by mutations affecting Lamin A/B or proteins attaching lamins to the nuclear envelope II.Progeria, caused by mutations in the lamin A gene, or in a lamin A processing enzyme. This result in excessive farnesylation.A farnesyltransferase inhibitor, initially developed to target oncogenic Ras, delays progression.

6. The actin cytoskeleton – an overview • Support of the plasma membrane • Cell migration • Contraction Muscle contraction Cytokinesis

7. Arp 2/3 WASP Fanta Fanta Dysfunctional actin regulation in Wiskott-Aldrich syndrome • Wiskott-Aldich syndrome (1/150.000) is caused by mutations • in the Wiskott-Aldrich syndrome protein (WASP) ZZZZ Arp 2/3 WASP • Manifested by: • Thrombocytopenia • Eczema (skin blushing) • Immunodeficiency syndrome Underdeveloped cortical actin results in defective platelets Fanta Fanta Deficient migratory and phagocytotic capacity of immune cells - Can only be cured by a hematopoietic stem cell transplant

8. Pathogenic E. coli: actin dependent colonization Enteropathogenic E. coli induce actin containing pedestals in intestinal epithelia Virulence factors that activate N-WASP Activation of Arp 2/3 Loss of absorptive surface is one cause for the associated diarrhea

9. ActA 1. 2. 3. 4. 5. ActA ActA Listeria: actin dependent motility 1-3) Phagocytosis and escape from phagosome 4) Bacterial multiplication 5) Penetration of a neighboring cell through actin based motility Arp 2/3 video 24.3-listeria_parasites

10. The microtubule system - an overview ER • Intracellular • organization Golgi Organelle positioning Chromosome segregation Chemotactic agent - Cell motility Cell polarisation and transport Movement of cellular appendages

11. Lissencephaly: defective neuron migration Lis1 • Lissencephaly ("smooth brain," 1/30.000) is a disorder characterized by the lack of normal convolutions (folds) in the brain Lissencephalybrain Normal brain mutated in many cases  loss of dynein ( ) dependent centrosomereorientation  defective cell polarization - 1/30 000 births, early death in severe cases

12. Non-functional cilia in Kartagener syndrome • Kartagener syndrome (1/20.000) is caused by mutations • affecting cilia specific dynein • - Manifested by respiratory infections, infertility and situsinversus Upper respiratory epithelia Patient with Kartagener syndrome Normal Goblet cell Bacteria is caught in mucus and cleared by a cilia mediated flow Bacteria is not cleared due to defective cilia

13. Microtubule-poisoning drug: Taxol • Alkaloid ester isolated from the bark of • Taxus brevifolia (Pacific yew) • Stabilization of microtubules Therapeutic uses: Treatment of breast, lung and ovarian cancer Prevention of restenosis of coronary stents (Surface coating of stents  local action) Major side-effects: Bone marrow suppression, gastro-intestinal upset and peripheral neuropathy

14. Microtubule-poisoning drug: Vinca alkaloids • Isolated from Catharanthusroseus • Named: Vinblastine, Vincristine,Vindesine • and Vinorelbine • Sequesters tubulin Therapeutic use: Treatment of leukemia, lymphoma, breast, lung, prostate, skin and testicular cancer Major side-effects: Bone marrowsuppression, gastro-intestinal upset and peripheralneuropathy

15. An oncology perspective on signal transduction, cell growth, checkpoints, apoptosis and the cytoskeletons X X X X X X X X X X X X Progression towards malignancy involves : i) uncontrolled proliferation, ii) resistance to apoptosis, iii) cell migration, iv) tissue invasion • Clonal evolution • Selection of • malignant clones Metastasis animation 20.2 -contact_inhibition video 20.1 -breast_cancer_cells

16. Two distinct types of ”cancer genes” Oncogenes Tumor suppressors On On Gene X Gene Y Off Gene X Gene Y Off On On Off X On Gain-of-function Loss-of-function A single genetic change Two independent events Dominant phenotype

17. CKI Ras Bcl-2 Rb Definitions: oncogenes and tumor suppressors An oncogene is a gene that when mutated, or overexpressed, contributes to converting a normal cell into a tumor cell (constitutive activity dominant phenotype) Onc point mutation overexpression A tumor suppressor-gene is a gene whose loss, or inactivation, contributes to converting a normal cell into a tumor cell (recessive phenotype) TS p53 Inactivating point mutations or loss of the entire gene (germ line mutation in one allele and/or acquired somatic mutations)

18. Cell type specific proliferative signals Cells from different tissues express distinct sets of growth factor receptors and signaling proteins Cell type B Cell type C Cell type A Major mitogen signaling pathway: RTK Wnt Hedgehog Alterations in tumors:RTK signals Wntsignals Hedgehog signals

19. Wnt RTK Frizzled Ras APC Aberrant proliferative signals in tumors XGF Hedgehog Onc Onc TS Patched Smoothened Onc Dishevelled TS TS Raf Axin GSK-3b Fused Onc SuFu Erk TS b-catenin Onc myc Onc myc G1 Gli Gli Onc Onc Onc Onc myc Onc Onc G1 G1

20. P P Cdk Cdk G1 S TGF-b DNA replisome Insensitivity of tumors to anti-growth signals Mitogen signaling p15 p21 p16 TS TS viral The retinoblastoma pathway HPV E7 Onc Rb E2F Cdc6 ORC = germ line mutations identified

21. p53 BH3 only TS TS Onc Bax Bcl-2 Cyt. C Evading cell death (apoptosis) Ligand TS Survival factor signaling Onc Death receptor TS Adaptor Caspase 8 Caspase 9 Caspase 3 Apoptosis

22. Randomly acquired oncogenic mutations drives tumor progression Mutation 1. Self-sufficiency in proliferative signals 1 2. Insensitivity to anti-growth signals 2 3. Evading cell death (apoptosis) 3 4. Limitless replicative potential 4 5. Sustained angiogenesis 5 6. Metastasis capability 6

23. Same “diagnosis” but different set of mutations Patient A with diagnosis X 1 2 3 4 5 6 X X X X Patient B with diagnosis X 1 2 3 4 5 6 X X X

24. How many somatic mutations during a life time? Cell death and replacement  risk for mutations & chromosomal instability Year 1-15 Controlled and co- ordinated divisions Uncontrolled divisions  Tumors 1013 – Human diploid genome: ~6 x109bp – Only some few errors per replication cycle – Average t½ of cells is 7 years (range: 24h to >100 years)

25. Normal cells have a very low rate of mutations Cancer related genes Other genes Random mutation Time Due to the low normal mutation frequency, progression to a fully malignant tumor is statistically improbable How come that malignant tumors have either a lot of mutations (~10 %) or chromosomal aberrations (90 %)? (~400 genes are frequently altered in tumors, 6 to 80 genes per “patient”)

26. Genomic instability: Two distinct levels 1. Defective DNA repair (MIN) Mutation in a gene encoding some enzyme required for DNA-repair X X No repair  many mutations  accelerated tumor progression 2. Chromosome segregation errors (CIN) Mis-segregation due to a defective a gene that encodes some protein essential for high fidelity chromosome segregation

27. 1 2 3 4 5 6 MIN reflects an escalated mutation rate Cancer related genes Other genes DNA repair(TS) Genetic alteration Time ?? Often uncertain which ones of all the mutations that contribute to tumor progression MIN: mini-satellite DNA instability (due to defective DNA repair)

28. CIN through excessive centrosomes Two centromes More than two centrosomes Kinetochore attachments satisfy the spindle checkpoint miss-segregation  aneuploidy CIN: Chromosomal instability

29. APC CIN through loss of APC The tumor suppressor gene product APC functions as a MT plus-end stabilizing protein ( ) that facilitates stable MT-kinetochore connections Satisfied spindle checkpoint Centromere Centrosome AC Kinetochore CIN: Chromosomal instability

30. CIN through a defective spindle checkpoint 1) A normal cell Delayed anaphase until all kinetochores are attached 2) A tumor cell with a (partially) defective spindle checkpoint

31. Cancer x x Genomic instability and tumor progression A stable genome Too much genetic instability ”Optimal” genetic instability ”Selection barriers”

32. Principles of cancer treatment Surgery- Impossible to remove all cancer cells Radiation- Targets both cancer- and normal cells Chemotherapy- Side-effects • General chemotherapy: drugs that interferes with: • DNA-replication • DNA structure • The function of the microtubule-system • Chemotherapy may also include cell type specific drugs. E.g. inhibition of hormone dependent tumor growth

33. x x Selective killing of tumor cells by chemotherapy Mutations that inactivate various checkpoints are common in malignant tumors  no cell cycle arrest in patients treated by chemotherapy!