Generalidades de la biología

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Biología del cáncer

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 MIN
 CIN ➔ Aneuploidía
 Pérdida de p53, pRB, BRCA,
HNPCC
Mecanismos oncogénicos
Células
cáncer
Proliferación desregulada
 Pérdida de TSG (pRB, p53)
 Incremento oncogenes (Ras, Myc)
Inhabilidad para diferenciarse
 Paro antes de diferenciación terminal
 Persisten funciones de células madres
Pérdida de la apoptosis
 ↓ p53
 ↑ bcl2
Inestabilidad genómica
Pérdida de la senescencia replicativa
 25-50 divisiones (pRB, p53, p16INK4)
 TELomerasa
Incremento angiogénesis
 ↑ VEGF, FGF, IL-8
 ↓ TSG: endostatina, trombospondina
Invasión
 ↓ gap junctions, cadherens
 ↑ MMP → Epithelial to mesenchymal
Evasión sistema inmune
 ↓ MHC I & II
 T-Cell tolerance / ↓ Dendrítica

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Growth factors
Nutrients & O2
Hormones
Cell-Cell inter.

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Inducción de p53 por daño de
DNA y retenes oncogénicos
mdm2
p53
ATM/ATR
chk1 / chk2
mdm2 mdm2P19ARF
myc, E2F, EIA
Inducción P19ARF
p53 p53
p53 p53
Activación transcripcional de los genes
respondedores a p53

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Extracellular
Domain
Transmembrane
Domain
Intracellular
Domain
EGF Pathway
 EGFR: transmembrane protein
Tyrosine Kinase
Domain
Adapted from:
Ciardiello F, et al. N Engl J Med. 2008;358:1160-1174. www.clinicaloptions.com

10. YOUR LOGO
HER/erbB family
Salomon DS, et al. Crit Rev Oncol Hematol 1995;19:183–232
Woodburn JR. Pharmacol Ther 1999;82:241–50
HER1
EGFR
erbB1
HER2
erbB2
neu
EGF
TGF-α
Amphiregulin
Betacellulin
HB-EGF
Epiregulin Heregulins
NRG2
NRG3
Heregulins
Betacellulin
Cysteine-
rich
domains
Tyrosine-
kinase
domains
HER3
erbB3
HER4
erbB4
Ligands:

11. YOUR LOGO
Y920
Y891Y845
EGF
Stepwise EGFR ligand binding and tyrosine phosphorylation
1
Y1146Phosphotyrosine
EGF
TM
N
C
TM
L1
L2
CR2
CR1
N
C
monomers tethered, inactive
TM
N
C
TM
N
C
EGFR
CR1
L2
CR2
L1
2
predimer extended, symmetric, inactive
EGF
TMTM
EGFR
CR1
L2
CR2
L1
3
dimer extended, asymmetric
EGF
TM
EGFR
CR1
L2
CR2
L1
4
dimer extended, asymmetric, active
EGF
5
dimer extended, asymmetric switched
EGF
CR1
L2
CR2
L1
TMTM
Y845
Y920
Y891
Y992 Y1045
Y1068
Y1086
Y1173
Y1148 Y1148
Y1086
Y1173
Y1068
Y1045Y992
EGF
CR1
L2
CR2
L1
TMTM
Y1148
Y1086
Y1173
Y1068
Y1045Y992
Y845
Y920
Y891
Y1148
Y1086
Y1173
Y1068
Y1045
Y992
Y891
Y920Y845
6
dimer extended, asymmetric active
activated kinase
activating kinase
kinase
inactive
tethered,
inactive
extended,
active
kinase
inactive
receptor kinase
donor kinase activating kinase
activated kinase
receptor kinase
donor kinase
EGF
EGF EGF
EGFR
TM
EGFR

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p27
E2F 1-3
KSR
Growth Factor signaling modules
CR1GF
L1
L2
CR2
CR1
Y845
Kinase
Y1173
Y1086
Y891
Y992
Y1148
Y1045
Y920
Y1068
L1
L2
CR2
Y845
Kinase
Y1173
Y1086
Y891
Y992
Y1148
Y1045
Y920
Y1068
GFCR1
PI3K
PDK
aPKC
AP-1AP-1
STAT 3
P
STAT 3
P
PP
Grb2
SOS
Ras
SHC
Src STAT 3
P
STAT 3
P
STAT 3
P
p70S6K
P
P
SRFElk Ets
P
TCFCRE NFkBCRE
PP
NFkB
P P
MEK1/2
ERK1/2S217 S221
T202
Raf1
S338
Y341
14-3-3
GSK-3
-Catenin
S9
Glycogen
syntahse
CRMP-2
WNK-1
P
P
P
P
APC
P
MAP1B
P
PKB
T308 S473
Bad
P
Cas 9
P
XIAP
P
P
PFK-2
ATP-citrate
lyase
PKC
P
PKC
P
PKC
P
PLC1
p90Rsk
MEKK2
JNK1/2
MKK7
MKK4
PP
Grb2
SOS
Rac/Rho
PP
DAG
IP3
PKC
RKIP
S153 I-1
P
PP1
MARCKS
Ca
Ca
Ca Ca
Ca
Ca
Ca
Ca
Ca
CaM
CamKIICaM MLCKCaM
P
DAPKCaM
P
P
Fascin
P
P
S129
Bcl-2
G1
S
G2
M
mTOR
P
Raptor
GL FKBP12
4EBP1
P
S6
p70S6K
P
P
AAAAA
60S
40S
PTEN
P
P
Cot
P
FOXO1
Foxa2
P
P
P
C-Myc
E2F 1-3
ATM
Cyclin D1
CDK4/6
pRb
HDM2
P
p53
P
GRK5CaM
FOXO1
P
P
P
P

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ProliferationApoptosis Resistance Transcription
TGFα Interleukin-8
bFGF VEGF
MetastasisAngiogenesis
Shc
PI3K
RafMEKK-1
MEKMKK-7
JNK ERK
Ras
mTOR
Grb2
AKT
Sos-1
EGF Pathway

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Angiogenesis is the process of new blood
vessel formation from existing vasculature
Sturk, Dumont. In: Basic Science of Oncology 2005

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Angiogenesis is essential to
tumor development
 An independent blood supply is required for a tumor to grow beyond 2mm
in diameter1,2
 Larger tumors rely on their vasculature for survival and further growth1,2
1. Ferrara, Henzel. Biochem Biophys Res Commun 1989; 2. Folkman. NEJM 1971
Small avascular tumor
Tumor
Blood vessels
Large, highly
vascularized tumor
Growth
factors

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Angiogenesis is involved throughout tumor
formation, growth and metastasis
Stages at which angiogenesis plays a role in tumor progression
Premalignant
stage
Malignant
tumor
Tumor
growth
Vascular
invasion
Dormant
micrometastasis
Overt
metastasis
(Avascular
tumor)
(Angiogenic
switch)
(Vascularized
tumor)
(Tumor cell
intravasation)
(Seeding in
distant organs)
(Secondary
angiogenesis)
Adapted from Poon, et al. JCO 2001
Tumour growth depends on angiogenesis

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 Also known as vascular permeability factor (VPF)
 aka: VEGF-A; related molecules are VEGF-B, C, and D
 Central mediator of angiogenesis
 Mitogen for endothelial cells
 45KDa heparin binding homodimeric glycoprotein
 Regulates angiogenesis
 Promotes survival of immature vasculature
 Binds to membrane receptor tyrosine kinases
 Four molecular species arising from the same gene
- VEGF121, VEGF165*, VEGF189, VEGF206
*Predominant molecular species
VEGF is at the center of the
angiogenic pathway
1. Ferrara, et al. Biochem Biophys Res Comm 1989
2. Leung, et al. Science 1989; 3. Keck, et al. Science 1989

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The VEGF family of isotypes and
receptors
Angiogenesis Lymphangiogenesis
VEGF-A, -B, PlGF
VEGFR-1 VEGFR-2
VEGF-A, -C, -D
VEGFR-3
VEGF-C, D
Disulfide bonds
Adapted from Hicklin, Ellis. JCO 2005

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Tumor vasculature is abnormal
Konerding et al. Blood Perfusion and Microenvironment of Human Tumors 2002
Normal colon Nearby colorectal cancer
Tumor vasculature is dilated, highly
chaotic, and tortuous, with a lack of
hierarchical vessel arrangement
VEGF INDEPENDENT.
VEGF DEPENDENT.

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25. Telomeres
Ends of linear chromosomes
Centromere
TelomereTelomere
Repetitive DNA sequence
(TTAGGG in vertebrates)
Specialized proteins
Form a 'capped' end structure

26. Telomeres 'cap' chromosome ends

27. TELOMERE STRUCTURE
5’ 3’
5'
3'
Telomeric
t loop
Telomeric
proteins:
TRF1
TRF2
TIN2
RAP1
TANKS 1,2
POT1
etc
NUCLEAR
MATRIX

28. Why are telomeres important?
Telomeres allow cells to distinguish chromosomes
ends from broken DNA
Stop cell cycle!
Repair or die!! Homologous recombination
(error free, but need nearby homologue)
Non-homologous end joining
(any time, but error-prone)

29. Why are telomeres important?
Prevent chromosome fusions by NHEJ (non-homologous end joining)
NHEJ
Mitosis
FUSION
BRIDGE
BREAKAGE
Fusion-bridge-breakage cycles
Genomic instability
Cell death OR neoplastic transformation

30. Telomere also provide a means for
"counting" cell division
Proliferativecapacity
Number of cell divisions
Finite
Replicative
Life Span
"Mortal"
Infinite
Replicative
Life Span
"Immortal"
How do cells "know" how many
divisions they have completed??

31. The End Replication Problem:
Telomeres shorten with each S phase
OriDNA replication is bidirectional
Polymerases move 5' to 3'
Requires a labile primer
3'
5'
3'
5'
5'
5' 3'
3' 5'
Each round of DNA
replication leaves
50-200 bp DNA unreplicated
at the 3' end

32. TelomereLength(humans)
Number of Doublings
20
10
Cellular (Replicative) Senescence
Normal
Somatic
Cells
(Telomerase
Negative)
Telomere also provide a means for "counting"
cell division: telomeres shorten with each cycle
Telomeres shorten from 10-15 kb
(germ line) to 3-5 kb after 50-60 doublings
(average lengths of TRFs)
Cellular senescence is triggered when
cells acquire one or a few
critically short telomeres.

33. How do replicatively immortal cells
avoid complete loss of telomeres
(how do they solve the end-replication problem)?

34. TELOMERASE:
Key to replicative immortality
Enzyme (reverse transcriptase) with
RNA and protein components
Adds telomeric repeat DNA directly to
3' overhang (uses its own RNA as a template)
Vertebrate repeat DNA on 3' end:
TTAGGG
Telomerase RNA template:
AAUCCC

35. TELOMERASE:
Key to replicative immortality
+ TELOMERASE
Overcomes telomere shortening and the end-
replication problem
Expressed by germ cells, early embryonic cells
Not expressed by most somatic cells (human)
May be expressed by some stem cells, but highly controlled
Expressed by 80-90% of cancer cells
Remaining still need to overcome the end replication problem;
do so by recombinational mechanisms --
ALT (alternative lengthening of telomeres) mechanisms

36. TelomereLength(humans)
Number of Doublings
20
10
Cellular (Replicative) Senescence
Normal
Somatic
Cells
(Telomerase
Negative)
Germ Cells (Telomerase Positive)
+ Telomerase
Telomere Length and Cell Division Potential

37. HOWEVER,
CELLS THAT EXPRESS TELOMERASE
STILL UNDERGO SENESCENCE
(E.G., IN RESPONSE TO DNA
DAMAGE, ONCOGENES, ETC.)
Inducers of cellular senescence
Cell proliferation
(short telomeres)
DNA damage
Oncogenes
Strong mitogens/
stress
Potential Cancer Causing Events

38. Telomerase:
Biomedical uses
Expand cells for replacement therapies
(burns, joint replacements, etc)
Telomerase inhibitors to selectively kill cancer cells

39. The telomere hypothesis of aging
Telomeres shorten with each cell division
and therefore with age
TRUE
Short telomeres cause cell senescence and
senescent cells may contribute to aging
TRUE
HYPOTHESIS:
Telomere shortening causes aging and
telomerase will prevent aging
TRUE OR FALSE?

40. The telomere hypothesis of aging
Telomere length is not related to life span
(mice vs human; M musculus vs M spretus)
Telomeres contribute to aging ONLY if
senescent cells contribute to aging
Telomerase protects against replicative
senescence but not senescence induce by
other causes

41. SUMMARY
Telomeres are essential for chromosome stability
Telomere shortening occurs owing to the biochemistry of
DNA replication
Short telomeres cause replicative senescence
(other senescence causes are telomere-independent)
Telomerase prevents telomere shortening and
replicative senescence
The telomere hypothesis of aging depends on the
cellular senescence hypothesis of aging