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

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

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

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

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

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

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

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

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

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

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

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)
PTENloxP/loxP
+ PTEN stroma
Epithelium
Microarray
-increased ECM remodeling
-increased immune infiltration
-increased angiogenesis
- PTEN stroma
Stroma
Trimboli, 2009

19. BPV induces tumors at sites of wounds.
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. 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???
Cancer -associated
Fibroblast
DePinho, 2000

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

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)
Remember – senescent cells are alive and metabolically active

23. 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
SIS RS
2094 genes
2362 genes
763 genes
Candidate Genes
Validation by RT-PCR
Test in growth assays

24. Growth factors, ECM, and inflammatory genes are highly upregulated in senescent fibroblasts
SIPS RS
Young
Pazolli et al. 2009
Mitogens & Regulation of Proliferation
Immune & Inflammation
Extracellular Matrix & Secreted Factors
Not surprisingly, analysis of the microarray data revealed that numerous factors were up and down regulated following induction of senescence. Many of the regulated factors included various mitogens, factors known to mediate the inflammatory response which I am sure we’ll hear more about throughout this meeting as well as factors that impact the extracellular matrix including Lox which Valerie Weaver spoke about this morning.
After interrogating the microarray list we generated a priority list based on factors that were likely to function in a paracrine manner. Follwing validation of these factors we choose to focus our attention on Osteopontin.

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. D J Baker et al. Nature 1–8 (2016) doi:10.1038/nature16932
Elimination of senescent cells increases life long tumor latency (i.e. reduced tumors)
ore, ATTAC lacks Ink4a promoter elements required for transgene induction in replicating pre-neoplastic cells that robustly express endogenous Ink4a owing to Rb inactivation and AP fails to kill thesore, ATTAC lacks Ink4a promoter elements required for transgene induction in replicating pre-neoplastic cells that robustly express endogenous Ink4a owing to Rb inactivation and AP fails to kill these cells (Extended Data Fig. 2a–g). e cells (Extended Data Fig. 2a–g).
D J Baker et al. Nature 1–8 (2016) doi: /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
In human prostate cancer and in a mouse model of prostate cancer, PTEN tumor suppressor loss is thought to drive cancer development.
In the mouse model, loss of PTEN triggers a robust senescence response as shown by p53 activation and SAbeta-gal staining. Deletion of p53 in the mouse completely abrogates this senescence response.
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 Matrix November 29, 2016
Jeff Miner, Ph.D.
Renal Division
7717 Wohl Clinic

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) Elastic fibers
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
Type I
fibrils
Skin, tendon, bone, ligaments, dentin, interstitium
Type II
Fibrils
Cartilage, vitreous humor
Type III
Skin, muscle, bv
Type IV
2D sheets
All basement membranes
Type V
Fibrils with globular end
Cornea, teeth, bone, placenta, skin, smooth muscle
Type VI
Fibril-assoc. (I)
Most interstitial tissues
Type VII
Long anchoring fibril
Skin--connects epidermal basement membrane/hemidesmosome to dermis
Type IX
Fibril-assoc. (II)
Type XIII
Transmembrane
Hemidesmosomes in skin
Type XV
HSPG
Widespread; near basement membranes in muscle
Type XVII
Hemidesmosomes in skin (aka BPAG2 or BP180)

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 hydroxylase mutations; 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.

51. Scurvy
Liver spots on skin, spongy gums, bleeding from mucous membranes, immobility, depression
Caused by Vitamin C deficiency
Ascorbate is required for prolyl hydroxylase and lysyl hydroxylase activities
Acquired disease of fibrillar collagen
Illustration from Man-of-War by Stephen Biesty (Dorling-Kindersley, NY, 1993)

52. Osteogenesis Imperfecta (brittle bone disease)
Clinical:
Ranges in severity from mild to perinatal lethal
bone fragility, short stature, bone deformities, teeth abnormalities, gray-blue sclerae, hearing loss
Biochemical:
Reduced and/or abnormal Type I collagen
Molecular Genetics:
Mutations in either Type I collagen gene, COL1A1 or COL1A2, resulting in haploinsufficiency or disruption of the triple helical domain (dominant negative: glycine substitutions most common)

53. Cell-Matrix Interactions December 1, 2016
Jeff Miner, Ph.D.
Division of Nephrology
7717 Wohl Clinic
Twitter: @JeffMinerPhD

54. Fibronectin
A glycoprotein associated with many extracellular matrices and present in plasma/serum
Alternative splicing generates many isoforms that heterodimerize covalently via S-S bonding
Fibroblasts make it, assemble it, stick to it, and respond to it
FN harbors the “RGD (Arg-Gly-Asp)” motif (in domain III-10) that serves as a ligand for various integrins, especially α5β1
Fn-/- mouse embryos die at E8.5 due to defects in the vasculature and in heart development
Mao and Schwarzbauer, Matrix Biol. 2005

55. Fibronectin and Branching Morphogenesis
Sakai et al., Nature 2003

56. Fibronectin is Required for Somitogenesis: Mesenchyme to Epithelium Transition (MET)
Dr. Christoph Winkler, Wurzburg

57. Integrins Direct FN Fibril Formation
Secreted compact soluble FN binds integrin
FN binding induces reorganization of actin and signaling
Cell contractility leads to changes in FN conformation, exposing FN interaction domains and allowing fibril formation
Mao and Schwarzbauer, Matrix Biol. 2005

58. Integrins
Large family of transmembrane receptors for extracellular matrix and cell surface proteins.
Consist of an α and a β subunit, both with a single-pass transmembrane domain.
16 different α chains and 8 different β chains associate to form 22 distinct heterodimers.
Cytoplasmic tails of both α and β chains mediate cell signaling events in response to ligand binding.

59. Integrins
Some integrins bind to a specific site on matrix proteins, such as Arg-Gly-Asp (RGD), which is found in fibronectin, vitronectin, tenascin, et al.
Ligand binding absolutely requires divalent cation**
As mechanotransducers, integrins link the extracellular matrix to the force generating actin-myosin cytoskeleton. This both allows the cell to influence the nature of the extracellular matrix, and allows the ECM to influence cellular architecture and behavior.

60. Integrin Family Members and Their Ligands
Hynes (2002) Cell 110:673

61. Integrins Need to be Activated
Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling.
Ligand binding transduces signals from the cellular environment to the interior of the cell through outside-in signaling.
Protein structure analyses have provided insights into the mechanisms whereby integrins become activated to bind ligand and how ligand binding translates to changes in intracellular signaling.
Adair and Yeager, Meth. Enzymol. 2007

62. Anoikis
Apoptosis induced by inadequate or inappropriate cell/matrix interactions.
Resistance to anoikis can lead to metastasis of epithelium-derived cancer cells.

63. Focal Adhesions are Organized by Integrins and Form Along Actin Stress Fibers
Anchors
Feet
Sensors
Do they exist in tissues in vivo?

64. Receptors for the Basement Membrane
Cells are thought to recognize the basement membrane through receptors that interact with specific basement membrane components, primarily with laminin.
Integrins
Dystroglycan
Binding of receptors to the basement membrane can result in signal transduction and alterations in cell behavior.

65. Laminin-Binding Integrins
α3β1, α6β1, α7β1, and α6β4
They are found on the surface of many epithelial (α3 and α6), endothelial (α3, α6), and muscle (α7) cells.
They bind primarily to laminin α chains and demonstrate some specificity.
Their activities are modulated by members of the tetraspanin family of 4-pass transmembrane proteins
CD9, CD81, CD151
Tetraspanin

66. Integrins Regulate Basement Membrane Architecture
Control Integrin α3 Knockout
(Itga3 expressed by the upper cell)
Kreidberg et al., Development 1996

67. The Dermal-Epidermal Junction

68. The Dermal-Epidermal Junction

69. Collagen-Binding Integrins
Integrin α1β1 and α2β1 bind to type IV, as well as to other collagens.
Itga1 and Itga2 knockouts have mild phenotypes, but they show that α1β1 downregulates collagen IV synthesis, whereas α2β1 upregulates it, at least in the setting of injury and fibrosis.

70. ECM and Cell/Matrix Interactions
Fibronectin
Laminin