Cell cycle and tumors
Oncogenes
 Oncogene is a gene that
encodes a protein which
hyperactivity is one step to
cancer
 A proto-oncogene is a
normal gene that can become
an oncogene due to
mutations changing structurefunction abilities of its product
or increasing its expression
 Oncogene is a dominant
allele of a proto-oncogene
= loose of a (normal)
function is dominant
Mutations that result in protooncogene – oncogene transformation
 Structural that cause
 an increase in protein (enzyme )
activity
 a loss of regulation (constant
activity)
 Regulatory due to
 an increase of protein expression
 an increase of protein (mRNA)
 Chromosomal translocation
Proto-oncogene activation
TRKs as proto-oncogenes
 EGFR signaling
causes increased
proliferation,
decreased
apoptosis, and
enhanced tumor
cell motility and
neo-angiogenesis.
 The EGFR is
expressed or
highly expressed
in a variety of
human tumors of
epithelial origin.
Carlos L. Arteaga The Oncologist
2002;7:31-39
 Increased expression.
 Ligand autocrine loop.
 Heterodimerization
 Mutations in the inhibition system
 Mutations in EGFR
(phosphatase)
Heterologic
control
Carlos L. Arteaga The Oncologist 2002;7:31-39
 GPCRs can exert positive effects on EGFR signaling in several ways, such as
- activation of matrix metalloproteinases, which cleave membrane-tethered EGFR
ligands
- GPCRs indirectly activate Src, which can phosphorylate the EGFR at tyrosines.
 Steroid hormones can also influence EGFR signaling by activating the transcription of
genes encoding EGFR ligands
 Estrogen can transactivate EGFR via the GPCR GPR30, potentially explaining the
EGF-like effect of estrogen
EGFR Mutations
 The EGFR mutation, EGFRvIII, involves deletion of exons 2
to 7 and loss of residues 6 to 276 in the receptor’s
ectodomain.
 This mutant yields a constitutively active receptor that is not
downregulated by endocytosis and is potently transforming
Ras-protooncogene
 Mutations of the Ras proto-
oncogene occurs in 20% of
human cancers
 Most of mutations results in loss
of the response of oncogenic
Ras to GTPase-activating
proteins (GAPs).
 The constitutively activated,
GTP-bound Ras activates
downstream effectors including
the MAP kinase.
 In addition, Ras activity in
tumor cells can be increased by
other mechanisms, including
mutation of GAP proteins and in
downstream proteins.
 Raf serine/threonine
Proto-oncogene Raf




kinase=MAPKKK
Activation of the Raf serine/threonine
kinase is induced by binding to Ras
and leads to activation of the MAP
kinase pathway
66% of melanomas and 10% of colon
cancers harbor activating mutations in
the BRAF proto-oncogene
The mutations lead to constitutive
activation of the downstream MEK (
MAPKK) and extracellular signalregulated kinases (ERK1/2=MAPK).
This results in the phosphorylation of
MAPK's cytoplasmic and nuclear
targets and alters the pattern of
normal cellular gene expression.
Protooncogenes of the
PI3K/Akt signaling
pathway
 The PI3K/Akt pathway
is activated in 30–40% of
human cancers and is
thought to play a critical
role in tumor cell
survival, proliferation,
growth, and glucose
utilization.
 Amplification or activating point mutation of the gene encoding the
catalytic subunit of PI3K (p110) is observed in 20–30% of cancers, and
amplification of the Akt2 (p85) gene occurs in others
 Akt promotes cell survival by activation of the transcription factor NFkB; preventing inactivation of Myc, -catenin, cyclin D1, and cyclin E,
and blocking upregulation of p27Kip1 and Bim.
 Furthermore, the growth of cancer cells requires the activation of
mTOR
Oncogene silencing by anticancer drugs
 Inhibitors of TRKs –



Harrison’s Internal Medicine

monoclonal antibodies; Imatinib,
Erlotinib – block ATP binding to
tyrosine kinase active site.
Ras requires posttranslational
modifications, including
addition of a farnesyl lipid
moiety. - Inhibition of RAS
farnesylation.
Inhibitors of Raf kinases (e.g.,
sorafinib)
Inhibitors of MEK (MAPKK)
Inhibitors of TOR - repamycin
Tumor suppressor genes
 Tumor suppressor gene is a gene
encoding a protein which
function is required to prevent
cancer
 Loose of function by both tumor
suppressor gene alleles is one
step to cancer
 Unlike oncogenes, cancerassociated mutations in
tumor suppressor genes are
recessive = loose of a
(normal) function is
recessive
 Tumor-suppressor
geneseither have a repressive
effect on the regulation of the
cell cycle or promote
apoptosis, and sometimes do
both including
 Genes that are active at
checkpoints
 Genes that products are
involved in DNA reparartion
 Some genes that product are
involved in cell adhesion
 p53 is a key tumor
p53
p53 signaling pathways




Gasco et al. Breast Cancer Res 2002 4:70-76
suppressor
p53 is a transcription factor
p53 activity is regulated by the
inhibitor MDM2 and by
phosphorylation
Under the non-stressed
condition, the p53 protein is
maintained at a low level in
cells by the proteasome
degradation pathway due to
activity of MDM2, an E3
ubiquitin ligase.
Once activated, p53 binds to a
specific DNA sequence, termed
the p53-responsive element, in
its target genes to regulate their
expression.
5
 The ataxia–telangiectasia-
mutated protein (ATM) abd
Rad3-related protein kinase
(ATR) are kinases that get
active in response to DSB
and phosphorylate histone
H2AX, p53 and some other
targets
 ATM is active at G1/G0
phase
 ATR is active at G2 phase
Lucy C. Riches et al. Mutagenesis 2008;23:331-339
 Activation of the G2/M
checkpoint after DNA damage.
 In response to DNA damage, the
ATM/ATR phosphorylate and
activate Chk1 and Chk2
 Chk1 and Chk2 phosphorylate
Cdc25.
 Phosphorylated Cdc25 is
sequestered in the cytoplasm by 143-3 proteins, which prevents
activation of cyclinB/Cdk1 by
Cdc25 and results in G2 arrest.
 ATM/ATR also activates p53dependent signaling. This
contributes to the maintenance of
G2 arrest by upregulating 14-3-3.
 In addition, p53 induces p21, a Cdk
inhibitor that binds to and inhibits
cyclinB/Cdk1 complexes. P:
phosphorylation.
Wang et al. Molecular Cancer 2009
8:8 doi:10.1186/1476-4598-8-8
pRB –retinoblastoma protein
 Genetic lesions that render the
retinoblastoma pathway
nonfunctional are thought to
occur in all human cancers.
 Loss of function of pRB as
guardian of the G1 restriction
point enables cancer cells to
enter a mitotic cycle without
the normal input from
external signals.
The Hallmarks of Cancer
Figure 1 Acquired
Capabilities of Cancer.
 We suggest that most if not
all cancers have acquired
the same set of functional
capabilities during their
development, albeit
through various
mechanistic strategies.
 Douglas Hanahan ,
Robert A Weinberg. The
Hallmarks of Cancer.
Cell, Volume 100, Issue 1,
2000, 57 - 70

Hallmark 1. Self-sufficiency in growth signals
Mechanisms developed by
tumor cells
Examples
Selfproduction of
corresponding mitogens
PDGF (platelet-derived growth factor) and TGFα (tumor growth
factor α) by glioblastomas and sarcomas, respectively
Overexpression or structural
alternations in mitogen
receptors
EGFR is upregulated or mutated in stomach, brain, and breast
tumors
Changes in the mitogenactivated cascades
Up to 25% human tumors activate MAPK w/o upstream signaling
via mutations in Ras, Raf, MEK
Mutations in inhibitory systems
Mutations in EGDR controlling phosphatase
Cooperation with normal
neighbors
Hallmark 2. Insensitivity to Antigrowth Signals
Mechanism
Example
Disruption of the
retinoblastoma protein
(PRb) pathway
Loose of functional PRb
mutations in CDK4
PRb sequestration by viral oncoproteins
Insensibility to contact
inhibition
Fibroblasts
 Antigrowth signals act in two
different ways, either to force
cells to enter a quiescent phase
or to differentiate
 .
Transforming growth factor-β (TGF-β)
 TGF-β regulates
differentiation and
proliferation of
different cell types.
 The TGF-β family
play a key role in
control of
maintenance of
metabolic
homeostasis in the
bone tissue and in
the fracture healing
process.
Poniatowski et al., 2015
Antigrowth signaling
 TGFBRs (I and II types)
are Ser/Thr kinase
 TGFBRs phosphorylate
Smads on C-end
 Phosphorylated SMADs
interact with the common
signaling transducer
Smad4, and translocate to
the nucleus.
 SMAD complex activate
genes required for
chondrocyte
differentiation
Osteoarthritis and Cartilage 2009 17, 1539-1545DOI: (10.1016/j.joca.2009.06.008)
TGFRB control
Zhao and Chen, 2014
 TGF-β ligand-
bound receptor
complexes are also
subject to
constitutive control
related to their
internalization on
the clathrindependent or lipidraft-dependent
endocytic pathway,
and this ensures
their correct
physiological
response, activity,
and distribution on a
cell surface
Contact inhibition of locomotion (CIL)
 In 1954 Abercrombie & Heasyman found that when moving




fibroblasts met, they stopped moving in the direction that led to
contact, and little overlap between opposing populations was
observed.
Besides fibroblasts, neural crest cells and haemocytes exhibit CIL
When a collision occurs between the leading edges of two
fibroblasts, both cells stop moving and retract their leading edges
from the point of contact
When the leading edge of one fibroblast contacts the rear side of
another, the movement of the second fibroblast is not affected
As for cancer cells, while many, but not all, cancer cells fail to
undergo CIL towards normal cells, the same cancer cells do display
CIL during collisions with one another
Journal of Microscopy
Volume 251, Issue 3, pages 232-241, 15 MAR 2013 DOI: 10.1111/jmi.12024
http://onlinelibrary.wiley.com/doi/10.1111/jmi.12024/full#jmi12024-fig-0001
 Quantification of CIL. CIL is measured by comparing the contact
acceleration indices (Cx) for free moving (F) and contacting (C) cells.
Cells were tracked for 50′ before collision (A) and 50′ after collision (B).
Free moving cells were tracked for the same time periods. The component
Cx of vector B–A represents the difference between how far the cell has
progressed in the direction of A′ and how far it would have gone had there
been no collision. CIL is indicated by a negative Cx value because cells
change direction and move backwards following the collision. A more
negative Cx value indicates a more significant CIL response.
Molecular mechanisms  Upon contact, cells stop migrating,
retract their actin-driven
of CIL
protrusions, repolarize and form a
new protrusion to reinitiate
migration in a new direction.
 CIL is mediated by Eph-ephrin
signaling
 Eph receptor–ephrin ligand
interactions depend upon direct cell–
cell interactions and are unique in
that they trigger bidirectional
signalling within the receptor and
ligand-expressing cell.
 Activation of EphA receptors leads to
repulsive migratory behaviour via the
GTPase RhoA whereas EphBs can
trigger Cdc42 activation and this can
facilitate attractive migration
Many cancer cells hyperproduced EphB receptors
Contact-unimpeded migration could facilitate tumour invasion through the stroma
Hallmark 3. Evading Apoptosis
Mechanism
Example
Inactivation of p53
More than50% of all cancers
Titration of the FAS ligand via
hyperexpression of the nonsignaling
FAS receptor homolog (oncogene)
Lung and colon carcinoma
Upregulation of Bcl-2 (oncogene)
B cell lymphomas
p53 and cancer
Fifty percent
of cancer
harbours p53
mutations,
while in the
remaining
50%, the wild
type p53 is
deemed to lose
its function via
various
mechanisms
that affect the
expression and
activity of p53.
Xu-Monette et al., 201
p53 and cancer
Bortezomib inhibits the proteasome
Nutlin competitively displaces
p53 from the binding on
MDM2 and effectively causes
the stabilization of p53.
RITA binds to the amino
terminal on p53 domain
instead of on MDM2
protein.
 The main inhibition
mechanism of p53 is
amplification or
overexpression of its
negative regulator MDM2.
 MDM2 is an E3 ubiquitin
ligase that induces
polyubiquitination of p53
protein and subsequently
promotes its proteolytic
degradation in the 26S
proteasome complex
 Overexpression of MDM2
will lead to a high
production of
polyubiquitinated p53 ready
proteasomal degradation.
Reactivation of p53 mutants
 PRIMA-1 (p53 Reactivation and Induction of Massive
Apoptosis) is a small molecule drug that reactivates mutant
p53 by restoring its wild type conformation and
transcriptional functions, by addition thiols in mutant p53
 MIRA-1 structurally distinct from PRIMA-1, is a maleimide
compound that shifts the equilibrium between the native and
unfolded conformation of p53 towards the native
conformation
Bcl-2 family and intrinsic
pathway in cancer
 Mitochondrial outer membrane
permeabilization (MOMP)
 Cancer cells can modulate
apoptotic pathways
transcriptionally, translationally,
and post-translationally.
Hallmark 4. Limitless Replicative Potential
Mechanism
Telomere maintenance
Example
All kind of cancers
 The T-loop is stabilized by telomere-
specific proteins TRF1 and TRF2
 Mammalian
telomeres end has
single-stranded
(TTAGGG)-rich
3'-overhangs that
are tucked back
into the preceding
double stranded
region to form a Tloop.
Telomere shortening
 The
hexanucleotide
repeat TTAGGG
tandomly repeated
from less than one
hundred to several
thousand times and
telomere-binding
proteins. T
 Every division, the
cell looses 50-200
repeats
Telomere shortening
 T-loop
Telomerase
Cell 2004 116, 273-279DOI: (10.1016/S0092-8674(04)00038-8)
 Telomerase is not expressed in most somatic cells
 Telomerase is expressed in most (>90 %) cancer cells
 The catalytic TERT subunit includes RNA and possesses reverse
transcriptase activity
Hallmark 5. Induced angiogenesis
 Virtually all cells in a tissue have to reside within 100 μm of a capillary blood
vessel
 In order to progress to a larger size, malignant tumors develop angiogenic
ability
 Angiogenesis is regulated by positive and negative signaling circuits
 Angiogenesis-regulating growth factors
Positive - vascular endothelial growth factor (VEGF) and acidic and basic
fibroblast growth factors (FGF1/2).
Negative - thrombospondin-1, β-interferon
 Angiogenesis-regulating receptors - Integrins
Mechanism
Example
Increased expression of VEGF and/or Many tumors
FGFs
Decreased expression of
thrombospondin-1, β-interferon
 (A) Endothelial sprouting is
Angiogenesis
SpannuthWA et al. (2008) Angiogenesis as a strategic target for ovarian cancer therapy
Nat Clin Pract Oncol doi:10.1038/ncponc1051
the dominant process of
vessel growth. Luminal
endothelial cells migrate
through the vessel basement
membrane into the
underlying extracellular
matrix, developing an
elongated 'sprouting'
morphology.
 (B) Vasculogenic mimicry is
the development of
microvascular channels by
aggressive tumor cells.
 (C) Vessel co-option involves
the use of the pre-existing
vasculature in the host tissue.
 (D) The process of tumor
neovascularization, involving
the release of proangiogenic
factors (e.g. VEGF) by tumor
cells to cause endothelial
activation, blood vessel
growth, and subsequent
tumor expansion.
Regulation of neovascularization
 Vascular endothelial growth factor
(VEGF) activates the eNOS, SRC,
RAS-ERK and PI3K-AKT signaling
cascades through VEGFR2 receptor on
endothelial cells, which induce vascular
permeability, endothelial migration,
proliferation and survival, respectively
 JAG1-Notch signaling promotes
angiogenic sprouting.
 TGF-β signaling through
TGFBR1/ALK5 receptor to the
Smad2/3 cascade inhibits endothelial
cell activation, maintaining endothelial
quiescence, whereas TGF-β signaling
through the ACVRL1/ALK1 receptor
to the Smad1/5 cascade promotes the
migration and proliferation of
endothelial cells
 To inhibit VEGF/VEGFR
human/humanized
monoclonal antibodies are
used: Bevacizumab (Avastin)
- anti-VEGF ; Sunitinib
(Sutent) and sorafenib
(Nexavar) are multi-kinase
inhibitors, targeting
VEGFR2
TGFβ paradox
Hallmark 6. Tissue Invasion and
Metastasis
 Most types of human cancer are able to release malignant cells
that move out, invade adjacent tissues, and spread to remote
body parts to found new colonies (metastases).
 Metastases arise as admixture of cancer cells and normal
supporting cells mobilized from the host tissue.
 Metastases are the cause of 90% of human cancer deaths
Epithelial–mesenchymal transition
(EMT)
Singh and Settleman, 2010
 Tumor cells achieve the potential to invade neighboring tissue and to
disseminate throughout the body by activating an epithelial–
mesenchymal transition (EMT) program
 The tissue remodeling processes occurring during EMT are shared by
embryonic development, wound healing, and metastasis formation.
 Molecular hallmarks of EMT are the loss of cell–cell adhesion, the gain
of cell migration capabilities to evade from the primary tumor
 Among many growth factors, TGF-β is one of the most potent
inducers of EMT.
EMT features
Jonathan M. Lee et al. J Cell Biol
2006;172:973-981
 The loss of cell polarity, the loss of epithelial markers, such as E-cadherin
and ZO-1, the gain of the expression of mesenchymal markers, such as Ncadherin, vimentin, and fibronectin, a dramatic cytoskeletal reorganization
accompanied by the change from an epithelial, differentiated morphology
to a fibroblast-like, motile, and invasive cell behavior.
Signaling events during EMT
Jonathan M. Lee et al. J Cell Biol 2006;172:973-981
 Cleavage of E-cadherin
 activation of Snail1
 Snail1 localization to the





nucleus
Repression of E-cadherin by
Snail1, Twist, or other
repressors
expression of vimentin and
other mesenchymal gene
products, partly because of
β-catenin/Tcf–Lef1
activation.
Translocation of β-catenin
to the nucleus
TGF-β activate Tcf–Lef1
transcription complex
The c-Met receptor tyrosine
kinase, through the Crk
adaptor, also stimulates
EMT.
Hallmark 7. Genomic instability
 Genomic instability is defined as the tendency of the genome to
acquire mutations and epimutations as well as alterations in gene
or chromosome dosage
 Age-related telomere shortening increases genome instability:
with short telomeres dividing cells undergo end-to end
telomeric fusions which can lead to chromosomal breakage at
mitosis
 More generally - Age-related alterations in nuclear architecture
and chromatin structure input in acquisition of genomic
instability
 Defects in the integrity of mitochondria profoundly impact on
the stability of our genome, by generating ROS that damage
DNA and oxidize proteins.
Genomic instability associated with
lamins
 Mutations or alterations
in the processing of
lamins affect ability to
maintain the integrity of
the genome.
 Lamins associate with
telomeres, laminsdeficient cells exhibit
accumulation of
telomeres towards the
nuclear periphery during
interphase.
Gonzalo, 2014
Cancer stem cells
 Cancer stem cells are pluri-potent progenitor cells that can self-renew
and divide by asymmetric cell division to give rise to differentiated or
committed progenitors.
 CSCs can be isolated or identified by distinct marker expression
profiles
 CSCs arise from existing stem/progenitor cells or from acquisition of
oncogenic lesions in terminally differentiated cells resulting in
dedifferentiation to a primitive stem cell-like state
Strategies to combat a cancer
miRNAs and cancer
Georgia Sotiropoulou et al. RNA
2009;15:1443-1461
 MicroRNAs regulate
gene expression by
forming duplexes
with the 3’ UTRs of
target mRNAs,
facilitating their
degradation by the
RISC complex of
proteins.
 miRNAs are
transcribed as
precursors, which
are subsequently
processed by the
DROSHA complex
miRNAs regulates oncogene activity
 miRNAs might play oncogenic or





tumor suppressor roles
Each miRNA recognizes hundreds of
different transcript targets.
Most miRNAs in animals function to
inhibit effective mRNA translation
through imperfect base pairing at
their 3′-untranslated region
miRNAs are up- or down-regulated
in various forms of cancer, including
prostate, breast , ovarian ,colorectal,
kidney, and bladder cancer
let-7 microRNA negatively regulates
Ras expression
Smad proteins can directly regulate
microRNA processing by binding to
the DROSHA complex
Sotiropoulou et al., 2009
The Ames test
 The Ames test is a biological assay to assess the mutagenic
potential of chemical compounds
 Technically, it uses bacteria (auxotrotrophs on histidine) to test
whether a given chemical can cause mutations in the DNA.