Harnessing

Cell signalling in cancer

cell death mechanisms and
cancer therapy
to improve therapy
Gianmaria Liccardi
 Lecture Plan:

• Lecture 1:
– EGFR Oncogenic signaling
• Lecture 2:
– Cell death in Cancer therapy
 Cell Death Survival

Inflammation Inflammation

Tissue Homeostasis
Tissue Repair
 Survival Cell Death

Inflammation Inflammation

Cancer Hallmark Chronic Inflammatory Diseases

-Inflammatory Bowel Diseases

-Psoriasis

-Rheumatoid Arthritis

SARS-Cov2
 Cancer is a mutltifactorial disease
• Cancer cells proliferate uncontrollably
• Cancer cells loose their ability to die
• Cancer therapy aims to:
– inhibition of cancer cell proliferation and or
promote cancer cells death
 Conventional anti-cancer therapy is to
damage DNA

1. Chemotherapy- cisplatin
2. Radiotherapy- Ionising radiation

1,2: Induced DNA damage –

Unrepaired DNA damage may induce cell death
 Monoadduct Ionizing radiation

Interstrand
crosslink

cisplatin

Intrastrand
crosslink
DNA-protein
crosslink

Very toxic

Single and double strand breaks

Despite the success in survival and organ conservation
conventional anticancer approaches alone are limited:
Dose and toxicity and low specificity of the approach

MOLECULAR TARGETING
RADIO/CHEMOTHERAPY
TARGETING
SPECIFIC MOLECULES

CHEMOTHERAPY RADIOTHERAPY

• MOLECULAR RADIO/CHEMOTHERAPY IS THE

COMBINATION OF RADIO/CHEMOTHERAPY PLUS
TARGETING SPECIFIC MOLECULES
 Activation of Survival
Pathways in cancer
cells

DRIVER ACTIVATION OF
CANCER
OF CANCER SURVIVAL
CELL
PATHWAYS IN THE
DOES NOT
CANCER CELLS
DIE

LOSS OF CELL DEATH

TARGETING MOLECULAR THERAPY
COMPONENTS
 MOLECULAR RADIO/CHEMOTHERAPY

STEP1 EGFR
Identify Biological Marker: this is often an oncogenic driver that leads to
1) Tumorigenesis 2) first line therapy resistance and/or 3) secondary
acquired resistance

STEP2 EGFR INHIBITORS

UTILIZE Targeted therapy aimed at inhibit the driver
often in combination with conventional therapy or Ionising radiation

DIRECT ACTIVATION
OF CELL DEATH
STEP3
MACHINERY
CELL DEATH Cancer cell death
(cytotoxic effect) or
inhibition of Cancer cells
proliferation (cytostatic
effect)
 EGF receptor Family -ERBBs

ERBBs include:
EGFR, Her2, Her3, Her4
 ErbB receptors and cancer
• ErbB receptors are aberrantly activated in a wide range of human
tumours
• As EGFR is involved in cell proliferation motility and survival an
imbalanced EGFR system can easily lead to CANCER
– Nearly 50% of all human tumours overexpress EGFR (or
mutated versions)
– The proliferative and anti-apoptotic pathways activated by
EGFR signalling can contribute to tumorigenesis
– Play a key role in cellular radio- and chemo-resistance
– Offer a therapeutic target for the treatment of cancer
 EGFR Expression in
Selected Human Tumors
Colon 25-77%
Head and Neck 95-100%
Pancreatic 30-89%
NSCLC 40-80%
Renal cell carcinoma 50-90%
Breast 14-91% (45%)
Ovarian 35-70%
Glioma 40-63%
Bladder 31-48%
 Oncogenic EGFR: How and Why?
• Modulation of EGFR ligand system
1. Autocrine production of Ligands
2. Overexpression: gene amplification or defective
downregulation
• By-passing inherent controls
1. Acquisition of somatic mutations giving rise to constitutive
active EGFR variants
• EGFR nuclear expression
As EGFR is involved in cell proliferation motility and survival an
imbalanced EGFR system can easily lead to a neoplastic
transformation
Modulation of EGFR ligand system
-Autocrine production of ligands
• EGF and TGFα are usually co expressed with EGFR in a variety
of human Cancers
• Autocrine production of these ligands in EGFR overexpressed
tumours has been associated with high proliferation and poor
clinical outcome
• TGFα induced EGFR activation evades EGFR degradation
maintaining recycling and signalling activation
Modulation of EGFR ligand system
-Overexpression
• Spontaneous receptor dimerisation due to
abundance induce constitutive activation of
downstream signalling, ligand independent
increased motility – aggressive phenotype
• Increased activity of the EGFR promoter via a
P53 mediated mechanism
• Gene copy amplification (NSCLC, GBM)
• EGFR has intrinsic enhancer ability
Modulation of EGFR ligand system
-Defective downregulation

•EGFR overexpression may saturate

the endocytic machinery
•Active EGFR is a poor substrates for
lysosomal degradation
•Her2-EGFR heterodimer escapes
unbiquitylation
•Cbl zinc finger domain mutation impairs
EGFR ubiquitylation
•RTK-Chaperone association is often
found in tumour cells rendering the
protein a poor substrate for lysosomal
degradation
•EGFR- Src shields EGFR from
degradation by redirecting Cbl ubiquitin
ligase activity against Cbl itself
 By-passing inherent controls
-Somatic mutations
• The somatically acquired mutations in the EGFR gene
are not randomly distributed but rather clustered in
specific areas
• There are 1380 mutations of the EGFR gene
sequenced from tumour samples. 30% (413 of 1380)
are found in adenocarcinoma, 2% in NSCLC (16 of
1380) EGFR mutation target in particular
adenocarcinoma.
• The number of sequenced EGFR mutations is
incredibly high and will be categorized according to
their location within the EGFR domains
 Somatic mutations
-Extracellular mutations

•Expressed in Glioblastoma
•EGFR vI (deletion of most of the
extracellular domain)
•EGFRvII ( in frame deletion of 83
amino acids)
•EGFRvIII (deletion of exon 2-7 )
•Impaired ligand binding and
constitutive activation
•EGFRvIII is internalised but fails
to be ubiquitylated.
 Somatic mutations -
Intracellular
mutation 1/2
•This group targets the residues
around the ATP binding pocket
within the kinase domain (lung
cancer)
• 3 classes:
•Class I 50% ( in frame deletion in
exon 19 : DL747–E 749)
•Class II (single point missense
mutation):
•L858R- 40%
is located next to the
conserved DFG sequence
that has the function to
stabilise the activation loop
•G719S/A/C - 5%
•Class III 5% ( in frame
duplications or insertions in exon
20)
 Somatic mutations -
Intracellular mutation 2/2

This group targets the C-terminal

domain and are often found in
GBM

•EGFRvIV: D exon 25-27

•Oncogenic potential via depriving
the inhibitory regulation of the
carboxyl tail on the kinase domain
•EGFRvV: D958 and exon 25-28
•EGFRTDM 18-25: duplication of
exon 18-25
•EGFRTDM 18-26 : duplication of
exon 18-26
 Conclusion 1
• EGFR is per se a potent regulator of cellular
fate
• EGFR aberrant regulation induces cellular
transformation- cancer (ONCONGENIC DRIVER)
• EGFR oncogenic signalling is achieved by:
1. Overexpression- gene aplification, defective
downregulation
2. Hyperactivation- autocrine production of ligands
cross talk and somatic mutations
Constitutive activation of downstream signalling
pathways
CYTOSOLIC EGFR role in cancer

Baselga. Eur J Cancer 2002:37 (Suppl. 4):S16–S22.

 EGFR nuclear translocation
• Following activation
the conformational
change of the
intracellular domain
result in the EGFR
NLS sequence to be
exposed and
recognised by the
importin machinery
into the nucleus

EGFR constitutive activation and defective downregulation in cancer

cells promote EGFR nuclear translocation/expression
NLS sequence= NUCLEAR LOCALISATION SIGNAL SEQUENCE
EGFR nuclear translocation steps
 Monoadduct Ionizing radiation

Interstrand
crosslink

cisplatin

Intrastrand
crosslink
DNA-protein
crosslink

Very toxic

Single and double strand breaks

 Nuclear EGFR modulation of DNA
repair
Nuclear EGFR is required for the repair of
• DNA strand breaks following IR treatment
• Repair of DNA interstrand crosslinks following
cisplatin

EGFR BINDS TO DNAPKcs (catalytic subunit of

the DNA protein kinase complex- major
component of the Non homologous end
joining pathway)
Prediction:
MUTANT EGFR that cannot undergo nuclear translocation should affect the repair
Of strand breaks following chemotherapy

Experiment:
Expression of EGFR Nuclear Localisation Signal mutant (NLS123 –CANNOT GO TO
THE NUCLEUS) and study DNA repair
EGFR MODULATION OF DNA REPAIR FOLLOWING
CISPLATIN TREATMENT
Repair over time
AMOUNT OF DNA DAMAGE

Fully repaired

Liccardi G. et al 2011 Cancer Res. 2011 Feb 1;71(3):1103-14

EGFR and DNAPKcs expression following cisplatin
SCENARIO: EGFR radio-protector role

H. P. Rodemann, 2006
EGFR dampens chemotherapy and radiotherapy effects by hypercativating DNAPKcs
In the nucleus

Hence:

1) Inhibition of EGFR should prevent the “DNAPKcs effect”

2) DNAPKcs inhibition or loss should have the same result as EGFR inhibition
EXPERIMENT:

COMPARISON OF IONISING RADIATION INDUCED DAMAGE IN CELL THAT

ARE PROFICIENT OR DEFICIENT IN DNAPKcs expression
MO59K:DNAPKcs positive (They express DNAPKcs)
MO59J:DNAPKcs negative (They do not express DNAPKcs)
MO59K treated with Gefitinib should behave like M059J
Do they?
MO59K treated with Gefitinib should behave like M059J
Do they?

Yes they do!

MO59J treated with Gefitinib should behave like M059J
Do they?
MO59K treated with Gefitinib should behave like M059J
Do they?

NO they don’t!
MO59K treated with Gefitinib should behave like M059J
Do they?

NO they don’t!

This difference
Shows that EGFR
Can modulated something
Else in absence of DNAPKcs
EGFR MORE EFFICIENT
DOUBLE STRAND BREAK REPAIR

ERCC1

Open AccessMolecules 2018, 23(12), 3205; doi:10.3390/molecules23123205

 EGFR targeted therapy:
how and why?

• TKIs (Tyrosine kinase inhbitor) compete for ATP binding

necessary for kinase activation and downstream signaling
• Monoclonal antibodies prevent ligand binding inhibiting
receptor activation however inducing internalisation and
degradation as conventional ligands
• Aberrantly activated TKs may be a driving force for
tumour growth and survival
• Their constitutive activation may result in an ONCOGENE
ADDICTION. Depletion of Oncogene activity may result in
cellular death.
 Molecular intervention

cetuximab
Dimerisation
Conformational change
X activation

TKI X Phosphorylation

Eg:
Gefitinib
 ErbB Receptor tyrosine kinase signalling
Pharmacological intervention - TKIs
• Majority are ATP
mimetics (quinazoline
based)
ErbB/ErbB2 • EGFR specific:
Receptor Dimer
P P Gefitinib (Iressa)
Erlotinib (Tarceva)
ATP • Dual inihibitors:
(EGFR/ErbB2)
ErbB receptor tyrosine Lapatinib
kinase inhibitors
 EGFR targeted therapy:
• Tumours harbouring
oncogenic EGFR acquire
dependency on the
survival signals such that
acute disruption leads to
rapid death of tumour
cells
• Majority of the most
aggressive forms of
cancer show nuclear
EGFR and EGFR
somatically acquired
mutations (more active
EGFR) Sharma et al. Nature Reviews Cancer 7, 169–181 (March 2007) | doi:10.1038/nrc2088
EXAMPLE OF MOLECULAR TARGETING WITH
I.R.
TARGETING
SPECIFIC MOLECULES
cetuximab
RADIOTHERAPY
 Objective response rate in L858R EGFR mutation
positive and negative patients
Overall
response 71.2%
rate (%) Gefitinib TKI
80 Chemotherapy Cisplatin

60 47.3%

40

20 23.5%
0
1.1%
(n=132) (n=129)
Mutation positive patients (n=91) (n=85)
Mutation negative patients

Odds ratio >1 implies greater chance of response on gefitinib

Mok et al ESMO LBA 2, 2008
 Conclusion 2
• Nuclear EGFR is required for the repair of cisplatin
interstrand crosslinks and IR induced strand
breaks
• EGFR-DNAPKcs binding is involved in repair of
induced damage via the upregulation of DNAPK
kinase activity
– Survival
• EGFR-ERCC1 interaction also enhaces repair of
double strand breaks
 Conclusions 2
• Oncogenic EGFR will therefore enhance the
response to conventional therapy by
modulating DNA response and upregulating
survival mechanisms
• EGFR Oncogenic ‘addiction’ can be exploited
by cancer therapy
– EGFR targeting via TKI and monoclonal
antibodies has been shown to improve
conventional monotherapy