Molecular and Cell
Biology of Tumors
Mgr. Lucia Knopfová, PhD.
Prof. RNDr. Jana Šmardová, CSc.
Institute of Experimental Biology
Faculty of Science
MU Brno
Bi9910en
3. Mitogenic signaling I
Proto-oncogenes, oncogenes, signaling pathways,
regulation of cell cycle
Molecular and Cell
Biology of Tumors
(1) Sustaining proliferative signaling
(2)  Evading growth suppressors
(3)  Resisting cell death
(4) Enabling replicative immortality
(5) Inducing angiogenesis
(6) Activating invasion and metastasis
(7) Genome instability and mutation
(8) Tumor-promoting inflammation
(9) Deregulating cellular energetics
(10) Avoiding immune destruction
Hallmarks of cancer
Hanahan D. and Weinberg R.A., Cell 144 (2011) 646-674
Is cancer disease of the cell
cycle?
•  Deregulation of cell cycle is key factor in cell transformation
•  Deregulation of cell cyle is not the only factor in cancerogesis, it is usually
not fully transforming
Cell cycle progression
M - G1 - S - G2
M – DNA copies are separated; condensed
chromosomes
G1 – DNA content: 2N
S – DNA replication
G2 – DNA content: 4N
G0 – senescent or non-dividing cells
Postmitotic phase – without re-entering cell cycle, linked with terminal differentiation
Cell cycle regulation
Term „cell cycle clock“
indicates that in a cell
there is a molecular
„device“ that integrates
all signals coming from
inside and from
extracellular space and
determines whether cell
enters active cell cyle or
quiscent state.
Weinberg RA. The Biology of Cancer. Garland Science 2007
Key molecules regulating cell
cycle
Ø  Cyclin-dependent kinases (Cdk, Cdc; human - 13)
•  Have catalytic and regulatory subunits, kinase activity is dependent on
interaction with respective binding partners – cyclins
•  Cyclins (human - 29)
•  Their expression levels fluctuate during cell cycle
•  They affect the specific phase of the cell cycle by activating respective
CDK kinase, afterwards are quickly degraded
•  Some have other functions not cycle dependent expression
Ø  Inhibitors (CKIs, cyclin-dependent kinase inhibitors)
•  Ink4 family of CKIs (p16, p15, p18, p19) – target mainly Cdk4 and
Cdk6
•  Cip/Kip family (p21, p27, p57) – more promiscuous, often interfere
with binding of cyclin to CDK
Key molecules regulating cell
cycle
Restriction checkpoint
•  mitogenic stimulation induces cell cycle
progression
•  antimitogenic stimulation blocks cell cycle
•  Cell cycle is dependent of exogenous signals
only during the part of G1 phase – until
restriction checkpoint (or G1 checkpoint or
Major checkpoint)
Restriction
checkpoint: key
regulatory point in cell
cycle
pRB – trigger of cell cycle
•  pRB in hypophosphorylated state blocks the transition through
the restriction checkpoint: it interacts with TFs of E2F family (8
genes) and thus prevent transactivation of their target genes
(proteins encoded by these genes are required for S phase)
•  When phosphorylated pRB losses the ability to bind with E2F and
thereby the passage through the restriction checkpoint is possible
⇒ Regulation of the restriction checkpoint = regulation of pRB
phosphorylation
E2F family of transcription factors
•  8 genes + alternatively spliced variants + multiple transcription start
sites
•  regulation: transcriptional, translational, posttranslational, protein
degradation, biding of co-factors, cellular localization
Kent LN a Leone G. Nat Rev Cancer 19: 326-338, 2019
Key molecules regulating cell cycle
Ø  cyclin dependent kinases (Cdk, Cdc; human - 13)
•  Have catalytic and regulatory subunits, kinase activity is dependent
on interaction with respective binding partners – cyclins
•  cyclins (human - 29)
•  Their expression level fluctuates during cell cycle
•  They affect the specific phase of the cell cycle by activating
respective CDK kinase, afterwards are quickly degraded
•  Some have other functions not cycle dependent expression
Ø  inhibitors (CKIs)
•  Ink4 family of CKIs (p16, p15, p18, p19) – target mainly Cdk4 and
Cdk6
•  Cip/Kip family (p21, p27, p57) – more promiscuous, often interfere
with binding of cyclin to CDK
Ø  pRB
Ø  E2Fs
Phosphorylation of pRB
during cell cycle
Extent of pRB
phosphorylation is tightly
controlled during cell cycle:
during M/G1 transition all
phosphate residues are
removed from pRB, after
restriction checkpoint the
extent of pRB phosphorylation
gradually increases with peak
at M phase
(pRB is phosphorylated at
serine and threonine amino
acids)
Weinberg RA. The Biology of Cancer. Garland Science 2007
Cell cycle regulation
Lundberg AS F a Weinberg RA, Eur J Cancer 35: 1886-1894, 1999
Regulation of pRB
phosphorylation
Phosphorylation of pRB
•  Positivelly regulated by:
- complexes of cyclins D (D1, D2 and D3) and CDK4/CDK6
- complexes of cyclin E and CDK2
•  Negativelly regulated by:
- inhibitors of cyclin:CDK complexes: p21WAF1 and p27KIP1
- inhibitors of CDKs: p15INK4B a p16INK4A
Mitogenic signaling leads to increased expression of cyclins D and
donwregulation of protein levels of inhibitors
Antimitogenic signaling leads to overexpression of inhibitor(s) and
decreased expression of cyclins D
What is damaged in
transformed cells?
•  Tumor suppressor pRB (retinoblastoma, ..)
•  Tumor suppressor p16INK4A (melanoma, ..)
•  protooncogenes cyclins D
–  amplification or overexpression of cyclin D1 in more than 50% of
breast cancer, HNSCC
–  Chromosomal translocations including cyclin D1 locus in mantle
cell lymphomas: t(11;14)
•  „periferal players“ – components of mitogenic and antimitogenic
pathways
Amplification of cyclin D1 locus
in head and neck cancer
•  Example of detection of
CCND1 amplification (11q13)
in head and neck squamous
cell carcinoma (HNSCC) by
FISH. There are 3 to 5 copies
per cell.
•  Approx 1/3 of HNSCC have
amplification of CCND1 and
thereby enhanced expression
level of cyclin D1. As result
regulation of pRB
phosphorylation is disrupted
Weinberg RA. The Biology of Cancer. Garland Science 2007
Cyclin D1 and mantle cell
lymphoma (MCL)
MCL is characterised by t(11;14)(q13;q32) translocation
which juxtaposes Ig heavy chain gene (IGH) sequences (enhancer)
with the CCND1 locus, leading to up-regulation of cyclin D1
expression.
Note: CCND1=BCL1=PRAD1
What is damaged in
transformed cells?
•  Tumor suppressor pRB (retinoblastoma, ..)
•  Tumor suppressor p16INK4A (melanoma, ..)
•  protooncogenes cyclins D
–  amplification or overexpression of cyclin D1 in more than 50% of
breast cancer, HNSCC
–  Chromosomal translocations including cyclin D1 locus in mantle
cell lymphomas: t(11;14)
•  „periferal players“ – components of mitogenic and antimitogenic
pathways
Mitogenic signaling
Classification of protooncogenes
I
I. Growth factors
II. Receptors for growth factors
III. Proteins Ras
IV. Non-receptor tyrosine kinases
V. Transcription factors
This classification reflects steps of signaling pathways:
1. Binding of ligand (growth factor) with receptor
2. Activation of receptor protein kinase
3. Signal transduction to nucleus via cascade of protein kinases
4. Activation of transcription factor
Schematic outline of signaling
pathway
Schematic outline of signaling
pathway
Signaling pathways: examples
Growth factors and receptor
tyrosine kinases (RTK)
Signaling via tyrosine phosphorylation is used mainly in
mitogenic signaling. Other signaling pathways use serine and
threonine residues.
Growth factors and receptor
tyrosine kinases (RTK)
Growth factors: polypeptides that are produced by cells and triggers
signaling for either start or stop of proliferation, differentiation and
survival by binding to their receptors at the cell surface
•  Evidence for involvement of extracellular signaling molecules in cell
transformation: factors secreted by cancer cells are able to induce
neoplastic transformation in vitro
Stimulation:
•  autocrine – growth factor stimulates producing cell
•  paracrine – growth factor stimulates neighbourging cells
•  endocrine – stimulation of distant cells
Signal transduction by RTK
(EGFR)
After ligation with
signal molecues
receptors form
dimers (all but IGF
receptor that is
convalently bound
homodimer) and
autophosphorylation
is induced.
Phosphorylation induces conformation change – thus
catalytic domain is opened and substrates may bind to
the binding domain. Binding of the substrates is
mediated mostly by SH2 and SH3 (Src-homology)
domains.
Weinberg RA. The Biology of Cancer. Garland Science 2007
Type of mutations affecting
growth factors and RTK
•  Overproduction of growth factors (→ autocrine stimulation)
•  Amplification of receptor: (a) concentration of receptors at the cell
surface is so high that they cluster randomly and dimerize without
interaction with ligand; (b) extremely efficient capturing of the
extracellular signal: cells with high levels of receptor are
stimulated for cell division even though the concentration of
signal molecule is low and normally insufficient to activate
mitogenic pathway
•  Structural changes of receptor: activation is not dependent on
binding of ligand (point mutations, short deletions, shortened
forms as results of gene fusions, etc.)
Alterations of RTK
v-ErbB has unlike EGFR
shortened extracellular domain ⇒
it transduces mitogenic signal
constitutively (without the
interaction with ligand)
(1984 – discovery of the structural
similarity of EGFR and v-ErbB)
Weinberg RA. The Biology of Cancer. Garland Science 2007
Alterations of RTK
v-ErbB has unlike EGFR
shortened extracellular domain ⇒
it transduces mitogenic signal
constitutively (without the
interaction with ligand)
(1984 – discovery of the structural
similarity of EGFR and v-ErbB)
Weinberg RA. The Biology of Cancer. Garland Science 2007
Alterations of RTK
Fusion of genes for RTK with
other genes
Constitutive dimerization
of receptor as a result of
gene fusion with a gene
that normally form
dimers or oligomers.
Weinberg RA. The Biology of Cancer. Garland Science 2007
Structure and main classes of
RTK
Weinberg RA. The Biology of Cancer. Garland Science 2007
EGF family
Family of EGFR receptors:
erbB-1 (EGFR); erbB-2 (HER-2/
neu); erbB-3 (HER-3); erbB-4
(HER-4)
Growth factors, that binds to
EGFR receptors: EGF, TGF-α,
amphiregulin (AR), HB-EGF
(heparin-binding EGF-like growth
factor), cripto, betacellulin (BTC),
epiregulin
EGF family
EGF (epidermal growth factor)
- 53 amino acids
- First identified and purified growth factor (Stanley Cohen – Nobel
prize 1986)
- Produced as a precursor (1217 AA)
- Stimulates proliferation and differentiation of various cell types
TGF-α (transforming growth factor α)
- 50 AA, 40% homology with EGF, also mitogen
- First identified autocrine growth factor
- Produced as a membrane bound precursor, released by proteolytic
cleavage in two steps
- Expressed by many cancer cell lines and in many tumors that express
EGFR
EGFR family
EGFR (EGF receptor)
•  Is encoded by c-erbB (ERBB) gene – homologous with v-erbB
(carried by retrovirus avian erythroblastosis virus: viral counterpart
has deleted extracellur domain)
•  Sometimes denoted as erb-B receptors or HER (human EGF
receptor)
•  Function as homodimers and heterodimers
- Overexpression of EGFRs detected in breast, esophagus, stomach,
ovarian, endometrial, cervical and other cancer; associated with poor
prognosis in breast, esophagus and ovarian cancer
-  Point mutations of EGFR1 (ERBB1) in lung cancer : EGFR - target of
anti-cancer therapy by small inhibitors of kinase domain (TKI), point
mutations predict good response to gefitinib
EGFR family
HER2/Neu encoded by gene ERBB2
Ø  amplification and/or overexpression of c-erbB2 (ERBB2) found in
breast adenocarcinomas, stomach, salivary glands, ovarian and
colorectal cancer
Ø  In breast and ovarian cancer is amplification/overexpression of
HER2/Neu associated with worse prognosis
Ø  Amplification of HER2/Neu is prognostic and predictive marker in
breast cancer: patients with HER2/Neu amplifications undergo more
radical treatment and may receive Herceptin – monoclonal
antibody specific for protein HER2/Neu
Amplification of HER2/neu in
breast cancer
•  Amplification of HER2/neu first described in breast cancer in 1987
•  Amplification resulting in minumum 5 copies of HER2/Neu gene per
cell is associated with poor prognosis
Weinberg RA. The Biology of Cancer. Garland Science 2007
EGFR-targeted anti-cancer therapy
A.  Monoclonal antibodies
Herceptin (Trastuzumab) – anti ErbB2 (HER2): therapy of
metastatic breast cancer with overexpression (amplification)
of HER2/neu
Cetuximab (Erbitux) – anti EGFR: therapy of advanced CRC
positive for EGFR
(Cetuximab binds EGFR with approx 50times higher affinity
compared to EGF – mechanism of action is direct competition with
ligand)
K-ras status needs to be determined! (Why?)
EGFR-targeted anti-cancer therapy
B.  Small molecules – inhibitors of TK domain
Gefitinib (Iressa), Erlotinib (Tarceva) – block ATP binding
site of EGFR: therapy of advanced non-small lung
carcinomas; gefitinib is efficient only in a subgroup of patients
with specific mutations of TK domain of EGFR
Lapatinib (Tyverb®)
– 4-anulinochinazolin
- dual inhibitor of HER1 and HER2
- inhibits intracellular tyrosinekinase domains of HER1 and HER2
- Used as a therapy of advanced metastatic breast cancer with
overexpression of HER2
PDGF family
PDGF (platelet-derived growth factor) – efficient mitogen – stimulates
mesenchymal cells (fibroblasts, adipocytes, endothelial cells,
smooth muscle cells)
-  homodimer (AA, BB) or heterodimer (AB) of two related chains A and
B: B is encoded by c-sis proto-oncogene
-  In 1983 the homology between B chain and v-sis oncogene
transduced by Simian sarcoma virus: altered B chain is constitutively
expressed that leads to the sustained stimulation of cells with PDGF
receptor
-  Elevated expression of PDGF A or B or both frequently detected in
sarcomas, gliomas, lung, breast, esophagus, stomach and colorectal
carcinomas
VEGF family
VEGF (vascular endothelial growth factor) / VPG (vascular
permeability factor)
•  Mitogenic effect on endothelial cells, important for
angiogenesis
•  VEGF/VPG has 18% sequence homology with PDGF A and B
FLT (FLT1 =Fms-Related Tyrosine Kinase 1, VEGFR1) encodes
receptor for VEGF/VPG, binds VEGF with high affinity and
mediates its biological function
other receptors for VEGF described: KDR (VEGFR2) and FLT4
(VEGFR3, modulates KDR signaling by forming heterodimers)
receptor encoded by FLT3 binds FLT3L not VEGF, expressed by
haematopoetic stem cells, mutations in AML)
FGF family
•  Large family –many polypeptides: FGF1 to FGF18, rather original
names are used (not all of them stimulate fibroblasts for
proliferation!!)
•  bFGF (FGF2) a aFGF (FGF1) (basic / acidic fibroblast growth
factor)
•  approx 17 kDa, they have 55% homology, similar function, name
derived from different isoelectric points
•  Mitogens of mesenchymal, neuroectodermal and epiderimal
origin, involved in angiogenesis
•  Autocrine growth factor in gliomas and meningiomas
•  Overexpression found in advanced astrocytomas
FGF family
FGF receptors - 4 genes: FGFR1 (flg nebo fms-like gen), FGFR2,
FGFR3 and FGFR4 – in addition genes can be alternativelly
spliced, resulting in complex group of receptors (almost 100
combinations) with different affinity to FGF factors
K-sam –FGFR2
-  Overexpression correlates with invasiveness and poor
prognosis of stomach cancer, amplifications in breast and lung
cancer: tumors with over-active FGFR signaling treated with
targeted therapeutics: ATP competitive TKI inhibitors dovitinib,
ponatinib (also inhibits mutant FGFR2)
HGF family
HGF (hepatocyte growth factor; hepatopoietin A)
•  Stimulates proliferation of hepatocytes (not exclusively), involved in
liver regeneration
•  Is one of the scatter factors (SF): participates in loosening of the
cell-cell interactions in epithelial tissues and blood vessels.
c-met (MET) encodes receptor for HGF
•  Amplification of c-met (stomach carcinomas, melanomas)
•  mutant oncogenic form of c-met, constitutively active (without
binding of HGF): point mutations, shortened extracellular domain;
gene fusions
•  HGF produced by stromal cells (overexpression) and c-met is
expressed on stromal and cancer cells: cooperation in growth and
morphogenesis of some tumors
Oncogenic alteration of MET
Comoglio et al. Nat Rev Cancer 18: 341-357, 2018
Hereditary papillary renal
cancer (HPRC)
•  autosomal dominant syndrome; highly penetrant
•  Caused by inherited mutations of c-met; (locus 7q31)
•  Papillary renal carcinoma represents cca 15 % of kidney cancer
•  Predispose to multifocal, bilateral papillary renal tumors, not early
metastatizing
•  Not accompanied by congenital developmental defects
•  Most germinal (as well as somatic) mutations are point missense
mutations in TK domain of MET
•  Met activation blocks terminal differentiation of renal tubular cells
•  protooncogen c-met is mutated in more than 10 % of sporadic
papillary renal cancers
RET
•  orphan receptor
•  potential ligands: GDNF (glial cell line derived neutrotrophic factor),
neurturin, persephin, artemin
•  Highest similarity with FGFR
•  Expressed mainly in neural, neuroendocrine and nephrogenic
tissues
Ø  2 types of mutations described:
•  loss-of-function mutations (inactivation of RET receptor) –
associated with congenital disorder Hirschprung‘s disease
(absence of some or all ganglion cells in myenteric and submucosal
plexus of colon, causing chronic constipation)
•  gain-of-function = oncogenic mutation – activation of RET
Hirschprung‘s disease
•  1886 – Harald Hirschprung described
the disorder causing death of two infants
•  Absence of inervation leads to
insufficient peristaltics and chronic
constipation
•  appears in 18.6 per 100,000 of live
births
•  Combination of mutations in two genes
RET (CH 10) and EDNRB (CH 13)
accounts for most cases
•  RET receptor assists cells of the neural
crest in their movement through the
digestive tract during the development of
the embryo
•  EDNRB connects these nerve cells to
the digestive tract
Mutations in these two
genes could directly lead
to the absence of certain
nerve fibers in the colon
•  nearly 20% of affected children have associated
developmental disorders and syndromes: often associated
with Down syndrom (7%), deafness, medullary thyroid
carcinoma, MEN2, neurofibromatosis, neuroblastoma
•  Most cases are sporadic, familial burder increases risk 200x
•  Multigenic disease with more than 10 involved genes, RET
proto-oncogene is the most studied
•  RET mutations: half of hereditary and 7-35% of sporadic
cases
Hirschprung‘s disease
Ježová M et al. Cesk Patol 55 (1): 53-59, 2019
RET
•  gain-of-function = oncogenic mutations
gain-of-function is caused by several types of mutations:
§  germline missense mutations
•  Cause constitutive activation of RET
•  Linked with hereditary cancer syndromes with increased
malignancy risk (autosomal-dominant inheritance):
MEN type 2A and 2B (multiple endocrine neoplasia)
and
FMTC (familial medullary thyroid carcinoma)
Multiple endocrine neoplasia
type 2 (MEN2)
•  Autosomal dominant inheritance associated with mutations of
RET
•  gen locus: 10q11.2
•  type A: high risk of medullary thyroid carcinomas, often
combined with feochromocytomas or benign adenomas of the
parathyroid glands
•  type B: Additional features include mucosal neuromas of the lips
and tongue.
•  type A is more frequent compared to type B
•  Medullary thyroid carcinomas occur at different age - in 70
years there is a 70% penetrance
Multiple endocrine neoplasia
type 2 (MEN2)
Pheochromocytomas
•  tumor of the adrenal medulla (80%), approx 20% outside of
adrenal gland (i.e. paraganglioma)
•  90% benign, approx 10% malignant-metastatic
•  These neuroendocrine tumors are capable of producing and
releasing massive amounts of catecholamines, metanephrines,
without treatment it is a life-threatening disease („malignant“?)
•  Mostly sporadic
•  around 20% hereditary, in this case multifocal; occur as a part of
cancer syndroms: MEN II (A, B), FMTC, VHL, NF1
Familial
medullary thyroid carcinoma FMTC
•  Less frequent disease compared to MEN, manifestation later in life
and less aggressive
•  Associated with specific RET mutations
RET mutations in MEN2 vs.
FMTC
1.  Mutations occur mostly in extracellular part of receptor in cystein
rich domain: C609, 611, 618, 620, 634 (in MEN 2A and FMTC)
causing permanent dimerization (by disulfidic bonds between
cystein residues) thus activation of receptor without ligand binding
2.  In some FMTC families mutations found in catalytic - kinase domain
(codons 768, 804).
3.  MEN 2B syndrom is associated with mutations in second part of
kinase domain (codons 883, 918)
RET mutations in MEN2 vs.
FMTC
RET
•  gain-of-function
§  missense mutations
§  Translocations resulting in deregulation of chimeric protein; RET
discovered in 1985 as a proto-oncogene that underwent
rearrangement during the transfection of DNA extracted from human
T-cell lymphoma into NIH-3T3 cells (RET – REarranged during
Transfection)
Ø  Translocation of 3´- terminus of RET with 5´- part of other gene
occurs in thyroid papillary carcinomas: chimeric protein contains
tyrosine kinase domain of RET and polypeptide that allows
permanent dimerization
Ø  radiation-induced human papillary thyroid cancer (PTC) is
associated with chromosomal inversions that involve the genetic loci
H4 and RET on chromosome 10
RET
RTK: Kit
•  Originally identified as an
oncoprotein endoced by feline
sarcoma retrovirus
•  Ligand for this receptor is SCF
(stem cell factor); stimulates
some blood cells but also nonhaematopoietic
cells (e.g.
melanocytes)
•  Mutant forms of Kit transduce
the signal constitutively, without
ligation, typical mutations occur
in juxtamembrane
cytoplasmatic domain
Weinberg RA. The Biology of Cancer. Garland Science 2007
Activation of Kit in cancer cells
•  juxtamembrane (JM) domain
negatively regulates Kit receptor
•  Binding of ligand leads to
transphosphorylation of tyrosine
residues of JM domain. That opens
JM domain to interact with Nterminal
part of catalytical domain
resulting in phosphorylation
(activation) of kinase domain
•  Mutations in JM domain result
dimerization of Kit receptor, but
receptor activity may be inhibited
by TKI (unlike receptors with
mutated catalytic domain)
Sheikh E et al. Bosn J Basic Med Sci, 22(5):683-698, 2022
Mutation of c-kit
•  GISTs (gastrointestinal stromal tumors) originate in
mesenchymal cells of lower gastointestinal tract: normally
function to ensure peristaltic contractions of smooth muscle cells
(experimental mutations of Kit in these cells prevent this function
in mice)
•  activating mutation of c-kit found in almost all GISTs, frequently
found in leukemias as well; often point mutations of JM domain,
sometimes deletions of JM domain (worse prognosis) and
mutations in catalytic domain.
•  (hereditary inactivating Kit mutations – piebaldisms – absence of
melanocytes in certain areas of skin and hair)
Familial gastrointestinal
stromal tumors (GISTs)
•  hereditary GISTs associated with activating c-kit mutations in
germinal lineage
•  Set of germinal mutations is similar to somatic mutations occuring
in sporadic GISTs
•  Gen occupies locus 4q11-12
•  Example of a family with
germinal Kit mutation:
•  Deletion of 1 AA in JM domain
•  Manifestation of the mutation
late in life, thereby present in
4 generations
Signaling pathways activated
by RTK
Signaling pathways activated
by RTK
Ras proteins
ras/Ras – first identified
oncogene
Originally thought to function as
mediator of Raf-1 binding to
plasmatic membrane
Current knowledge show that:
•  Raf-1 activation is a complex multistep process. Direct
interaction between Ras and Raf-1 is mediated by several
binding sites in Ras and at least 2 binding sites of Raf-1
•  Raf-1 is not the only substrate (downstream effector) of Ras
proteins.
•  Ras superfamily consists of 35/65 different members (RAW/
2000). All have some sequence homology, but differ in signaling
capacity.
Proteiny Ras
Signal transmission from RTK to
Ras
•  Even transmission process is more complex than originally
estimated
•  activated RTKs recruit the adaptor protein GRB2 that binds
phosphotyrosine consensus sequences via its SH2 domain. Via its
SH3 domains, GRB2 then recruits the guanine nucleotide
exchange factor SOS to the plasma membrane, where it activates
the membrane-localized small G protein RAS by exchanging GTP
for GDP
Weinberg RA. The Biology of Cancer. Garland Science 2007
Ras proteins
Ras superfamily has at
least 65 members:
Families: Ras, Rho, Rab,
Ran, Rad, Arf
Ras family has
subfamilies: Rap, Ral, RRas,
Rheb, H-Ras, Ras
Ø Together called small
G proteins
Proteins H-Ras, N-Ras, K-Ras4A and K-Ras4B have 188/189 AA.
They reside at the cytosolic side of plasma membrane and
function there as molecular switches during signal transduction
to cytoplasm.
Ras proteins are GTPases: are inactive in GDP-bound form and
active if GDP is exchanged for GTP. Capacity of Ras proteins to
exchange of GTP/GDP and hydrolyse GTP is small, thereby
other proteins participate:
•  Guanine nucleotide exchange factors (GEFs), facilitate
formation of active GTP-bound Ras protein, help to GDP to
dissociate from small GTPase (e.g. SOS1/2, RasGRF/
mCDC25,...)
•  GAPs: GTPase-activating proteins, act antagonistically to
inactivate GTPases by increasing their intrinsic rate of GTP
hydrolysis (p120 GAP, NF1/neurofibromin,...)
Ras proteins
GTPase cycle of Ras proteins
GEF
GAP
GEFs help to dissociate
GDP from Ras – active Ras
GAPs facilitate GTP
hydrolysis – formation of
inactive Ras
Posttranslational modifications:
Interaction with inner part of cytoplasmatic membrane (required
for Ras activation) is mediated by posttranslational modifications
of CAAX motif at C-terminus of Ras
Cys186 of motif CAAX is farnesylated
Ras proteins
Intermezzo: Prenylation
•  Prenylation – the covalent attachment of a lipid
consisting of either three (farnesyl) or four
(geranylgeranyl) isoprene units (intermediates of
cholesterol biosynthesis) to a free thiol of a cysteine
side chain at or near the C-terminus of a protein.
Prenyl group
Intermezzo: Prenylation
farnesylation – attachment of farnesyl group (15 C)
geranylgeranylation – attachment of geranylgeranyl group (20 C)
•  Catalysed by 3 prenyltransferases: farnesyltransferase (FT),
geranylgeranyltransferases I and II (GGTI and GGTII).
•  There is more than 300 proteins in humans with Cystein at C-terminus,
but it is unknown how many are prenylated
•  Farnesylation confirmed for: H-, K- and N-Ras, nuclear lamina,
CENPE (kinetochors); geranylgeranylation: Rho A and C, Rac1,
cdc-42;
•  RhoB – farnesylated and geranylgeranylated;
•  K-Ras – geranylgeranylovated, if FT is inhibited
Ras proteins
Posttranslational modifications:
Interaction with inner part of cytoplasmatic membrane (required
for Ras activation) is mediated by posttranslational modifications
of CAAX motif at C-terminus of Ras:
•  Cys186 of CAAX motif is farnesylated
•  proteolytic cleavage of AAX sequence
•  terminal Cys186 is carboxymethylated
- protein with these modification is more hydrophobic and has
higher affinity to cell membrane
For oncogenic activation of Ras is crucial
farnesylation!
Stucture of Ras proteins
•  Heterogenous domain: Ras proteins are highly homologous in first
164AA, the C-terminus (25AA) is variable
•  Effector domain (AA 32-40) mediates interaction with effector
molecules
•  Switch I (30-38) major binding site for GAPs
•  Switch II (60-76) binding of GEFs
Oncogenic activation of Ras
Siqi et al. Nat Rev Cancer, 2018 Dec;18(12):767-777.
Oncogenic activation of Ras
Oncogenic mutation of Ras detected in almost 35% of human cancer.
•  Mutation of H-ras mostly in skin cancer and head and neck
squamous carcinoma
•  Mutation of K-ras mainly in adenocarcinomas, colorectal carcinomas
and pancreatic cancer
•  Mutation of N-ras frequent in haematopoetic cancers, mostly in AML
and MDS
Oncogenic mutations modify Ras activity:
•  In codons: 12, 13, 59, 61, 63 – altered proteins have impaired
GTPase activity and are resistant to GAPs
•  In codons: 16, 19, 116, 117, 119, 144, 146 – resulting proteins have
facilitated exchange of GDP for GTP
•  Some tumors overexpress Ras
Oncogenic activation of Ras
1982: Ras mutation in codon 12 identified (at the same time in 3
laboratories!). It is the first point mutation causally associated with
cancer development.
Weinberg RA. The Biology of Cancer. Garland Science 2007
Glycine 12 and glutamine
61 are critical for binding of
GFP to Ras
That is why these codons
are mostly mutated in
cancer
Oncogenic activation of Ras
Weinberg RA. The Biology of Cancer. Garland Science 2007
Inhibition of Ras proteins
•  for oncogenic features of Ras is crucial farnesylation!
Ø  Inhibitors of farnesyl transferases (FTIs)
(e.g. CAAX peptides – cell-permeant, specific and efficient, low
cytotoxicity)
•  By inhibiting farnesylation they prevent post-translational
modifications of Ras and thereby its activity
•  Potential anti-cancer therapeutics, but:
–  Ras proteins have many functions in cells and FTIs blocks all of
them (side effects)
–  Ras proteins may be also geranylgeranylated (and FTIs do not
inhibit geranylgeranyltransferases)
–  Ras proteins are not the only targets of FTIs (Rho)
FT inhibitors (FTIs)
At least 6 inhibitors tested in clinical trials:
1.  BMS-214662 (Bristol-Myers Squibb, Princeton, NJ)
2.  L778123 (Merck and Co., Inc., Whitehouse Station, NJ)
3.  lonafarnib (experimental name SCH66336; Sarasar™;
Schering-Plough Corporation, Kenilworth, NJ)
4.  FTI-277 (Calbiochem, EMD Bio-Sciences, San Diego)
5.  L744832 (Biomol International L.P., Plymouth Meeting, PA)
6.  tipifarnib (experimental name R115777; Zarnestra®; Ortho
Biotech Products, L.P., Bridgewater, NJ); most advanced testing
Lonafarnib: FT inhbitors
•  synthetic tricyclic halogenated carboxamide with antineoplastic
properties
•  used primarily for cancer treatment
•  A coctail of lonafarnib and two other drugs proved beneficial for
patients with progeria.
Tipifarnib: FT inhibitors
•  Clinical trials: administration of Tipifarnib to older patients with
AML (aged 65 and more) – not approved by FDA, not efficient
anti-cancer activity (2005)
•  Further tested for efficiency in breast cancer, neurofibromatosis,
and other malignacies
FT inhibitors (FTIs)
•  Not satisfactory outcome in clinical trials so far
•  Side effects
•  Not clear whether they function primarily via Ras proteins
Ras proteins have many
effectors
Ras triggers 3 key effector
pathways
1.  Raf pathway (activation of genes stimulating cell growth/
division, anchorage-independent growth, loss of contact
inhibiton)
2.  PI3K pathway (anti-apoptotic effect)
3.  Ral pathway (facilitates invasion and metastatic dissemination
of tumors)
1. Ras ⇒ Raf
Raf proteins
•  Raf: cytosolic proteins, recruited to the cell membrane by activated
Ras – and activated itself
•  active Raf stimulates MEK (MAP-kinase extracellular signalregulated
kinase) by serine phosphorylation
•  active MEK is dual kinase: phosphorylates both tyrosine and serine/
threonine of target molecules: including MAP kinases p44 ERK1 a
p42 ERK2 (extracellular signal-regulated kinases)
•  ERKs have many substrates in cytosol (Rsk, SOS, MEK,...) and
(upon nuclear translocation) also nuclear (Ets, Elk-1, SAP-1,...)
•  Jun and Fos belong among the target TFs of this pathway
•  almost 60-70% melanomas have mutated B-Raf „instead
of“ Ras
BRAF
•  BRAF gene (7q34) has 18 exons, mRNA is 2478 bp long
•  Mutations of BRAF are associated with many different cancers: :
colorectal carcinoma, malignant melanoma, Non-hodgkins lymfoma,
thyroid papillary carcinoma, non-small cell lung carcinoma, lung
adenocarcinoma
•  80% of mutations is caused by T1799A
transversion that leads to amino acid exchange
V600E
•  This mutation is transforming, because it
simulates T599 phosphorylation and/or S602
phosphorylation in activating domain of BRAF;
mutated BRAF is thus in permanently active
mode, independent on Ras signaling
Krajsová I. Dabrafenib v léčbě metastazujícího melanomu. Remedia 24 (3): 215-219, 2013
RASopathy
•  RAS/MAPK Syndromes
•  Group of neuro-cardio-facio-cutaneous syndromes (NCFC)
•  Specific group of developmental disorders caused by germinal
mutations of genes encoding proteins of RAS/MAPK signaling
cascade
•  phenotypically similar group of syndromes: symptoms- e.g.
characteristic facial features, cardiac defects, cutaneous
abnormalities, neurocognitive delay and a predisposition to
malignancies
•  Noonan syndrome, neurofibromatosis type 1, Legius syndrome,
Costello syndrome a další
•  Each syndrome exhibits unique phenotypic features, however,
there are numerous overlapping phenotypic features (early
diagnosis is difficult)
•  Overall RASopathies are one of the largest group of malformation
syndromes affecting 1 in 1000 subjects
RASopathy
•  somatic mutations in RAS/MAPK pathway lead frequently to
malignant diseases. That is why much higher rate of cancer
development was expected in RASopathies: risk is only 3,5x higher
than in normal population; the most prevalent are haematologic
malignancies
•  Some activating somatic mutations are the same as germinal
mutations, but RASopathy-associated mutations are less activating:
e.g. The most frequent somatic oncogenic mutation of BRAF
(V600E) does not occur in cranio-facio-cutaneous syndrome
(typically caused by BRAF mutations)
Ø  It is assumed that strongly activating oncogenic mutations are
lethal in germ cells
Svobodová E. Molekulární diagnostika syndromu Noonanové a přidružených RASopatií pomocí NGS. DP PřF, 2018
Niemeyer CM, Haematologica. 2014 Nov;99(11):1653-62
RASopathies: overview of syndromes
Germinal mutations of proto-
oncogenes?!
Why tolerated??
Ø  Expression limited to a certain tissue (cell type) (RET, c-kit)
Ø  Expression of the proto-oncogene occur later during organogenesis
thus enabling normal embryogenesis (c-met)
Ø  „mildly“ activating mutation (rasopathy, c-kit)
Ø  specific types of mutations (cdk 4)
2. Ras ⇒ PI3K
PI3K
Phosphatidyl inositol 3 – kinase - lipid kinase
Ø  Composed of 2 subunits: regulatory p85 and catalytical p110
Ø  catalyze the phosphorylation of the D3 position of the inositol ring of
phosphoinositides to produce phosphatidylinositol 3-phosphate
(PtdIns3P = PIP3) –secondary messenger triggering downstream
signaling
PI3K a PIP3
•  PIP3 level is tightly regulated. PIP3 is produced by PI3K and converted
back to PIP2 by phosphatases: PTEN, SHIP1, SHIP2
•  PTEN = PIP3 lipid phoshatase; tumor suppressor (breast cancer,
glioblastoma, Cowden syndrome)
•  PI3K may be activated e.g. by PDGF, NGF, IGF-1, interleukin-3,
•  PI3K produces PIP3 and
PIP3 interacts with Akt
•  Thereby Akt is recruited to
the cell membrane,
phosphorylated and thus
activated
Ø  Two mechanisms leading to
the Akt hyperactivation :
1.  Aktivation of PI3K
2.  Inactivation of PTEN
AKT/PKB
•  One of the most common activated protein kinases in cancer
•  Hyperactivation of Akt is linked with (1) resistence to apoptosis, (2) enhanced
proliferation and (3) motility
•  serine/threonine kinases of PKB family (mammalian cells express 3 highly
homologous (80%) isoforms of Akt encoded by 3 different genes - AKT1, AKT2
and AKT3 (alias PKBα, PKBβ and PKBγ)
Activated AKT ...
•  May phoshorylate more than 9000 proteins
•  induces antiapoptotic signals, prevents release of cytochromu C from
mitochondria, inactivates TF (Forkhead that transactivates FasL),
inactivates pro-apoptotic factor Bad and procaspase-9
•  Activates cell cycle: regulates cyclin D stability, blocks transport of
p21WAF1 and p27KIP1 into nucleus
•  stimulates activity of telomerase via TERT
•  induces translocation of MDM2 into nucleus (thereby p53
degradation)
•  inactivates GSK3 which leads to increase of β-catenin levels
•  inactivates TSC2 (tuberous sclerosis complex 2) that functions as
mTOR inhibitor; mTOR is associated with proliferation rate
•  AKT often changes subcellular localization = regulates
(compartmentalisation) of its substrates
Activated AKT ...
•  May phoshorylate more than 9000 proteins
•  induces antiapoptotic signals, prevents release of cytochromu C from
mitochondria, inactivates TF (Forkhead that transactivates FasL),
inactivates pro-apoptotic factor Bad and procaspase-9
•  Activates cell cycle: regulates cyclin D stability, blocks transport of
p21WAF1 and p27KIP1 into nucleus
•  stimulates activity of telomerase via TERT
•  induces translocation of MDM2 into nucleus (thereby p53
degradation)
•  inactivates GSK3 which leads to increase of β-catenin levels
•  inactivates TSC2 (tuberous sclerosis complex 2) that functions as
mTOR inhibitor; mTOR is associated with proliferation rate
•  AKT often changes subcellular localization = regulates
(compartmentalisation) of its substrates
Activated AKT ...
AKT regulates compartmentalisation
of its substrates
•  amplification and overexpression of AKT2 in 10-20% ovarian and
pancreatic cancer, increased AKT2 activity in 40% of ovarian cancer
•  Amplification of AKT1 found in stomach cancers
•  Increased activity of AKT1 in prostate and breast cancer –
associated with worse prognosis (increased activity in breast
carcinomas may be caused by HER2/neu amplification)
•  inactivating mutations or deletions of PTEN (prostate and
endometrial cancer, glioblastomas, melanomas)
•  Amplifications or overexpression of catalytical subunit p110 of PI3K
•  activating mutations of ras (around 1/3 of all epithelial tumors)
•  Enhanced activity of RTKs, mainly ErbB2/ErbB3 (ErbB3 has
docking site for PI3K)
AKT in cancerogenesis
PI3K pathway alterations in human
cancers
3. Ras ⇒ Ral
•  Third of the most important Ras effectors: Ral A and Ral B; belong
to Ras family, have 58% sequence homology with Ras
•  Signaling from Ras to Ral goes through Ral-GEF: activated Ras directly
binds to Ral-GEF followed by:
a) recruitment of Ral-GEF to the cytosolic side of cell membrane
b) conformation change of Ral-GEF → stimulation of GEF activity →
activation of Ral-A and Ral-B
•  Activated Ral-A and Ral-B activate their target molecules (Rho – actin
dynamics)
•  Outcome of this activation is increased invasion and metastatic
activity of cancer cells
3. Ras ⇒ Ral
Ras effector pathways
Besides the three described
pathways there is more than 8
others Ras effectors.
Overall the (Ras) signaling is more
complex – rather signaling nets
than pathways
Thank you for attention!