MCB Exam 3 Review

1. MCB Exam 3 Review Stewart and Miner lectures

2. In the 1930’s while carrying out classic mutagenesis experiments in flies, Hermann Muller (Nobel, 1938) first noted that chromosome ends had “distinct” properties and named them telomeres (telo meaning ”end” and mere meaning “part”).

3. Telomere Function distinguishes between the chromosome end and a double strand break protects the chromosome from end-to-end fusions

4. Mitosis Progression Prophase Metaphase Anaphase Telophase TRF2DN Normal Anaphase Anaphase Bridge -actin TRF2DN Ctrl Mock Loss of telomere integrity leads to telomere dysfunction characterized by fusions and anaphase bridges

5. Telomere Length 1 2 3 4 5 6 The “telomere” hypothesis (Harley and Greider) Senescence Population Doubling Time

6. The telomere hypothesis ALT Telomere Length Stop Crisis hTERT 1 in ~107 Time

7. TP1 Dyskerin ? ? HSP90 Telomerase adds telomeric repeats to the 3’ termini of the chromosome -many components are required in vivo (this list continues to grow). hTERT p23 hTR CAAUCCCAAUC

8. Proof that telomerase was important in immortality had to wait until the catalytic component was identified… 1997 -- the catalytic component of telomerase (hTERT) is cloned -Weinberg Lab at the Whitehead Institute/MIT -Cech Lab at the University of Colorado-Boulder

9. hTERT “Normal” Immortal Mortal “Normal” Several groups demonstrated that telomerase could immortalize “some” cell types -Bodner, 1998 -Rufer, 1998 -Vaziri, 1998

10. V WT DN V WT DN V WT DN V WT DN + - + - + - + - + - + + - + - + - + - + - + - + - + - + - + - + - + - - + - + - + - + - + - (Dominant Negative) DN-hTERT expression eliminates telomerase activity LoVo HA-1 SW613 36M - + HT IC Hahn and Stewart et al 1999

11. Tumorigenic SV40 T-ag SV40 T-ag H-ras H-ras Not so much Cooperating oncogenes transform mouse cells…what is wrong with human cells Mouse Human

12. T-ag t-ag SV40ER, TERT, and H-ras cooperate to transform normal human cells SV40 ER H-ras TERT Tumors Hahn et al, 1999

13. Functional steps toward cancer Hanahan and Weinberg, Cell 2011 Hanahan and Weinberg, Cell 2000

14. Telomerase activity in human carcinomas Tissue Incidence Head/neck and lung 78-100% GI and pancreas 85-100% Hepatic tissue 86% Breast 75-88% Cervical/endometrial/vaginal/ovarian 91-100% Prostate 90% Kidney/urinary 83-100% Neural* 50-100% Skin 83-95% Hematological tissue 73-100% *retinoblastoma, meningioma, neuroblastoma, Shay 1997

15. Tumors are complex “organs”

16. Is the stroma a participant??? null, Volume 144, Issue 5, 2011, 646–674

17. “Normal” Fibroblast Cancer -associated Fibroblast Pre-neoplastic Cell Tumorigenic Cell “Normal” Fibroblast The role of the stroma in tumorigenesis

18. PTEN inactivation in the stromal compartment accelerates mammary tumorigenesis (Trimboli, Nature 09) MMTV-Her2/Neu (expression restricted to epithelial cells) X FSP-Cre (expression restricted to stroma, mostly) X PTENloxP/loxP + PTEN stroma Epithelium Microarray -increased ECM remodeling -increased immune infiltration -increased angiogenesis - PTEN stroma Stroma Trimboli, 2009

19. Early studies demonstrated that tumors arose in areas prone to wounding…..i.e. - Deelman found tumors arose at the margin of accidental wounds in the skin of mice treated with tar. • Bissell’s group found that RSV infected chickens developed tumors at the site of wounds. • BPV induces tumors at sites of wounds. • Tat and v-jun transgenic mice develop tumors at sites of wounds. • These are but a few examples… For review: Sieweke & Bissell

20. Cancer -associated Fibroblast Increased cancer incidence with age If it takes 5 or more mutations to create a human tumor cell why do we get cancer when we are so young??? DePinho, 2000

21. “Normal” Fibroblast Cancer -associated Fibroblast Pre-neoplastic Cell Tumorigenic Cell “Old/Senescent” Fibroblast “Normal” Fibroblast “Old” stroma is phenotypically similar to CAFs

22. Other characteristics of senescent cells • Absence of proliferation markers • Flattened cell morphology • Increased cell volume • Senescence-associated β-galactosidase • p16INK4a tumor suppressor expression • Senescence-associated heterochromatin foci • Altered secretory profile (SASP)

23. RS SIS 763 genes 2362 genes 2094 genes Candidate Genes Validation by RT-PCR Test in growth assays Microarray Analysis • Harvested RNA from 72h-starved fibroblasts • BJ young • BJ old (RS) • BJ Bleo (SIS) • Possible candidates were selected based on: • Secretion pattern • Involvement in growth promotion • Implication in tumorigenesis

24. SIPS RS Young Mitogens & Regulation of Proliferation Immune & Inflammation SIPS RS Young SIPS SIPS RS Young SIPS Extracellular Matrix & Secreted Factors SIPS RS Young SIPS Pazolli et al. 2009 Growth factors, ECM, and inflammatory genes are highly upregulated in senescent fibroblasts

25. Whether cells undergo telomere-based senescence or stress-induced senescence, p53 and Rb are the downstream effectors AND activation results in a permanent growth arrest (a.k.a potent tumor suppressor mechanism. Campisi 2005

26. Elimination of senescent cells increases life long tumor latency (i.e. reduced tumors) D J Baker et al. Nature 1–8 (2016) doi:10.1038/nature16932

27. If the elimination of senescent cells reduces life long tumor rates how does that explain earlier data suggesting senescence is a tumor suppressor??? restrains tumor growth Chen et al., 2005

28. Senescent-derived IL6 is required for suppressive granulocyte-mediated tumor growth Antibody Depletion shRNA Knockdown Ruhland, in revision

29. Current wisdom argues that primary tumor cells release “factors” that prep the premetastatic niche for the arrival of tumor cells and their outgrowth. Yang Liu, Xuetao Cao Cell, 2016

30. The Extracellular MatrixNovember 29, 2016 Jeff Miner, Ph.D. Renal Division 7717 Wohl Clinic 362-8235 minerj@wustl.edu

31. Why do all multicellular animals have ECM? • Acts as structural support to maintain cell organization and integrity (endothelial tubes of the cardiovascular system; mucosal lining of gut; skeletal muscle fiber integrity) • Compartmentalizes tissues (pancreas: islets vs. exocrine component; skin: epidermis vs. dermis) • Provides hardness to bone and teeth (collagen fibrils become mineralized/calcified) • Presents information to adjacent cells: • Inherent signals (e.g., RGD motif in fibronectin) • Bound signals (BMP7, TGFβ, FGF, SHH, etc.) • Serves as a highway for cell migration during development (neural crest migration), in normal tissue maintenance (intestinal mucosa), and in injury or disease (wound healing and cancer)

32. Types of ECMs • Basement membrane (basal lamina) • Epithelia, endothelia, muscle, fat, nerves • Elastic fibers • Skin, lung, large blood vessels • Stromal or interstitial matrix • Bone, tooth, and cartilage • Tendon and ligament

33. Cells Need Receptors to Recognize and Respond to ECM • Integrins • Dystroglycan • Syndecans • Muscle-Specific kinase (MuSK) • Discoidin domain receptors (DDRs) 1 and 2 • Others

34. Types of ECM Components • Collagens • Proteoglycans • Perlecan, aggrecan, agrin, collagen XVIII • Hyaluronan (no protein core) • Large Modular Glycoproteins • Laminins, nidogens, fibronectin, vitronectin • Fibrillins, elastin, LTBPs, MAGPs, fibulins • “Matricellular” Proteins • SPARC, Thrombospondins, Osteopontin, tenascins

35. Basement Membranes • Specialized layers of extracellular matrix surrounding or adjacent to all epithelia, endothelia, peripheral nerves, muscle cells, and fat cells • In general, basement membranes appear very similar to each other by EM. • But all are not alike! • There is a wealth of molecular and functional heterogeneity due primarily to isoform variations of BM components.

36. The Major Basement Membrane Proteins LM-511 α1α1α2 Perlecan

37. Fredrik Skarstedt and Carrie Phillips

38. Basement Membranes are Involved in a Multitude of Biological Processes • Cell proliferation, differentiation, and migration • Cell polarization and organization, as well as maintenance of tissue structure • Separation of epithelia from the underlying stroma/mesenchyme/interstitium, which contains a non-basement membrane matrix • Kidney glomerular filtration (barrier between the bloodstream and the urinary space)

39. Primary Components of All Basement Membranes • Collagen IV 6 chains form α chain heterotrimers • Laminin 12 chains form several α-β-γ heterotrimers • Entactin/Nidogen 2 isoforms • Sulfated proteoglycans Perlecan and Agrin are the major ones; Collagen XVIII is another History: The Engelbreth-Holm-Swarm (EHS) tumor: A blessing with a caveat.

40. Laminin Trimers Polymerize • Laminin chains assemble into trimers in the ER and are secreted as trimers into the extracellular space. • Full-sized laminin trimers can self-polymerize into a macromolecular network through short arm-short arm interactions. • The α chain LG domain on the long arm is left free for interactions with cellular receptors.

41. Laminin Mutations in Mice (M) and Humans (H) Have Consequences Lama1, Lamb1, Lamc1: Peri-implantation lethality (M) Lama2: Congenital muscular dystrophy (M, H) Lama3, Lamb3, Lamc2: Junctional epidermolysis bullosa (skin blistering) (M, H) Lama4: Mild bleeding disorder, moto-nerve terminal defects (M); cardiac and endothelial defects (H) Lama5: Neural tube closure, placenta, digit septation, lung, kidney, tooth, salivary gland defects (M) Lamb2: Neuromuscular junction and kidney filtration defects (M); Iris muscle, neuromuscular, kidney filtration defects (H; Pierson syndrome) Lamc3: Brain malformations, autism spectrum disorder? (H)

42. Perlecan • Found widely in basement membranes and in cartilage. • Contains domains similar to LDL receptor, laminin, and N-CAM • Binds to Collagen IV and to Entactin/Nidogen

43. The Collagens • The most ubiquitous structural protein. A triple helical protein containing peptide chains with repeating Gly-Xaa-Yaa (usually Pro) triplets. • The triple helix forms through the association of three related polypeptides (α-chains) forming a coiled coil, with the side chain of every third residue directed towards the center of the superhelix. Steric constraints dictate that the center of the helix be occupied only by Glycine residues. • Many Proline and Lysine residues are enzymatically converted to hydroxyproline and hydroxylysine. • ~28 distinct collagen types; each is assigned a Roman numeral that generally delineates the chronological order in which the collagens were isolated/characterized.

44. Diversity of Collagens

45. Collagen IV Network Trimers (aka protomers) associate with each other, four at the N-terminus and two at the C-terminus (hexamer), to form a chicken wire-like network that provides strength and flexibility to the basement membrane.

46. Type IV Collagen Mutations and Human Disease • COL4A1 mutations • Small vessel disease/retinal vascular tortuosity • Hemorrhagic stroke • Porencephaly • HANAC syndrome • COL4A3/A4/A5 mutations • Alport syndrome/hereditary glomerulonephritis Kidney Glomerular BM

47. Fibrillar Collagens (I, II, III, V)

48. Fibrillar Collagens (I, II, III, V) • Connective tissue proteins that provide tensile strength • Triple helix, composed of three α chains • Glycine at every third position (Gly-X-Y) • High proline content • Hydroxylation required for proper folding and secretion • Found in bone, skin, tendons, cartilage, arteries

49. Biosynthesis of Fibril-forming Collagens Prolyl hydroxylases Lysyl hydroxylase Glycosyltransferases Procollagen N- and C- proteinases Lysyl oxidase Adapted from: Keilty, Hopkinson, Grant. In: Connective Tissue and Its Inheritable Disorders, Wiley-Liss, 1993.

50. Collagen Crosslinking • If crosslinking is inhibited (Lysyl hydroxylasemutations; vitamin C deficiency), collagenous tissues become fragile, and structures such as skin, tendons, and blood vessels tend to tear. There are also many bone manifestations of under-crosslinked collagen. • Hydroxylation of specific lysines governs the nature of the cross-link formed, which affects the biomechanical properties of the tissue. Collagen is especially highly crosslinked in the Achilles tendon, where tensile strength is crucial.