Transcript Cancer Genes

Cancer Genes and Targets for
Therapy
Helen C Hurst
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Molecular Oncology Unit
Charterhouse Square
Cancer Treatment
Primary Tumour
Metastasising cells
Surgery
Chemotherapy
Radiotherapy
Targeted
Therapy
“Cancer Genes”
Metastatic
Tumours
Cells in multicellular organisms are continually receiving signals
from each other and their environment
This leads to proliferation, differentiation or even cell death (apoptosis)
as appropriate to the needs of the organism as a whole
In cancer, this normal balance goes awry  Cancer Genes
Cancer progression in ductal carcinoma
of the pancreas
….progressive mutation/activation of “cancer genes”
What is a “Cancer Gene”?
• Proliferation: Oncogenes and Tumour suppressor genes
• Cell survival: Apoptosis vs DNA repair
• Epithelial-stromal interactions: Angiogenesis, Invasion and
Metastasis
• Cell surface markers: Immune Evasion
• Membrane pumps: Drug resistance and response to therapy
• Metabolism: allow more rapid growth (e.g. ribogenesis)
 virtually any gene product may be a target for therapy as long as:
• It’s expression level/structure/activity is sufficiently different
between normal and tumour cells
• It is required for continued growth/survival of the tumour cells
• Many are involved in cellular signalling pathways:
Signalling Pathways
Growth/survival
GFs
2nd messenger
cascade
Gene expression/
Small
Molecules
Arrest/apoptosis
Examples of Targeted Therapies in
Clinical Use
• Anti-endocrine therapies:
– Tamoxifen (anti-ER therapy) - breast cancer
– Anti-androgen therapy - prostate cancer
• Anti-ErbB therapies:
– Herceptin - immunotherapy against HER2/ ERBB2
in breast cancer
– Iressa - small molecule tyrosine kinase inhibitor
against EGFR for solid tumours
• Glivec - small molecule tyrosine kinase
inhibitor against Bcr-abl for CML
Oestrogen Receptor in Breast Cancer
Normal - only a few
cells express ER
ER +ve tumour
~65% of breast tumours are ER +ve  show proliferative
response to oestrogens (ovaries)  benefit from
anti-oestrogen therapy
The ER is a ligand dependent
transcription factor
oestrogen
++++
ERE
(oestrogen response element)
Proteins that 
Growth/survival
Use of anti-oestrogens in treating breast
cancer
• Anti-oestrogens block the binding of
oestrogen to the ER  proliferative gene
expression and signalling are blocked
• Giving early stage, ER+ve patients Tamoxifen
for 5 years immediately after surgery has
 mortality by 28%
• Tamoxifen use in early stage disease  UK
annual breast cancer mortality rate fell from
~16,000 to 12,800 in 12 years (1988-2000)
But…...
…. there are problems:
• Tamoxifen is associated with a ~2-fold  risk
of blood clot formation (thromboembolism)
• Tamoxifen is linked to a ~2.5-fold  risk of
endometrial cancer
• Significant numbers of ER +ve patients never
respond to Tamoxifen (de novo resistance)
• Those that do respond initially, can relapse
with resistant disease (acquired resistance)
because oestrogen has a bad and a good side….
Pluses and minuses
• Anti-oestrogens like Tamoxifen
and Raloxifene are “partial
agonists”  block oestrogen
action in breast; allow some
signalling in other organs
• This has consequences that
are both positive and negative:
– Tamoxifen and Raloxifene are
both agonists in bone 
protect against osteoporosis
– In the endometrium Tamoxifen
(but not Raloxifene) is an
agonist, hence  endometrial
cancer
Alternative strategies
• Use total oestrogen agonists like Faslodex that block
all oestrogenic activity and result in down-regulation
of the ER
• Remove oestrogens altogether using aromatase
inhibitors which prevent synthesis of oestrogens
Clinical trials have shown aromatse inhibitors to be effective
and well-tolerated and resistance is slower to develop….
…however, resistance to these agents is an issue and develops
for largely similar reasons as Tamoxifen resistance:
Signal pathway cross-talk  oestrogen-independence
 target 1 or more of these pathways in addition
(combination therapy)
AR in Prostate Cancer
•All PC initially respond
to anti-androgen therapy
•After 2-5 years tumours
become resistant
•Various mechanisms e.g.
mutation of AR and/or
gene amplification
•Increased signalling via
other pathways (as in
breast cancer) also
important
What are ErbB proteins?
• ErbB = family of trans-membrane glycoproteins with
an extracellular ligand binding domain and an
intracellular tyrosine kinase domain
• Referred to as “receptor tyrosine kinases”
• Ligand binding  receptor dimerisation, kinase
activation, auto-phosphorylation (on Y)  signalling
cascade initiation
• Normal function  mediate cell-cell interactions in
organogenesis and during adulthood
Ligand
Docking sites for signalling proteins
P
P
The ErbB Network
IHC
•Slow ligand dissociation
•Relaxed ligand specificty
•Slow endocytosis
•Rapid cycling
•Prolonged “firing”
• proliferation
• migration
•  resistance
to apotptosis
FISH
ERBB2 overexpressed in many
solid tumours e.g. 25% breast
carcinomas  correlates with ER
negativity and poor prognosis
The development of Herceptin®
(Trastuzumab)
• Researchers at Genentech raised mouse monoclonal
antibodies against the extra-cellular domain of ErbB2
• One of these, 4D5, potently inhibited growth of ErbB2
overexpressing cultured human breast tumour cells
• Murine antibodies are limited clinically due to being
immunogenic
•  Recombinant, humanised antibody created:
• Herceptin has a higher affinity for ErbB2 than 4D5
and has a cytostatic growth inhibitory effect against
ErbB2+ve breast cancer lines
“Humanising” an antibody
Mouse hybridoma
specificity determining
(variable) regions
Consensus human IgG1
framework
Genetic engineering
V gene cloning
CDR grafting
Eucaryotic expression
Herceptin in the clinic
• Shown to: be well-tolerated; have anti-tumour activity
• In randomised trials - improved survival in patients
with amplification of the ERBB2 gene
• Approved for use in metastatic ErbB2+ve breast
tumours (1998)
• Largely used in combination with chemotherapy
drugs (taxol, cisplatin; cardiac side-effects with dox)
• Mode of action: ErbB2 downregulation; prevents
cleavage of extracellular domain (causes activation);
activates patient’s own immune response
Future improvements
• Herceptin has no activity on tumours that express
moderate levels of ErbB2  limits its use
• 2C4 binds a different epitope  blocks ErbB2
dimerisation with other ErbB receptors  prevents
signalling in low- and high-expressing lines
• Anti-tumour effects in xenografts of breast and
prostatic tumours
• Shown to be safe (Phase I); now in Phase II (efficacy)
trials
• May be useful in a wide range of ErbB2 +ve solid
tumours.
No signalling
Proliferation/Survival
Iressa®
(Gefitinib ; ZD1839)
• Selective and reversible small molecule inhibitor of
EGFR tyrosine kinase activity (from AstraZeneca)
• Also inhibits signalling via EGFR dimerisation with
other ErbB family members
• Preclinical studies - inhibited growth of various
tumour lines and xenografts
• Synergised with cytotoxic chemotherapy agents (e.g.
paclitaxel) and radiation therapy in sensitive lines
• Paradox: senisitive lines could not be predicted from
their level of EGFR expression
Mode of Action of Iressa
Iressa in the clinic
• Good oral bio-availability and well-tolerated  can be
taken once daily (Phase I)
• Good anti-tumour responses in mono- and
combination therapy in a variety of solid tumours:
NSCLC, colorectal, breast, head & neck (Phase II/III)
• Approved for use in patients with advanced, chemoresistant NSCLC
• Assays to determine which patients (NSCLC and
other) will benefit most being developed
• Combination therapies being optimised
Chronic Myeloid Leukaemia (CML)
• Characterised by a massive clonal proliferation of
myeloid cells
• Accounts for 15-20% of all leukaemia cases
• Has 3 phases: chronic (or stable); accelerated; blast
• Chronic phase: excess numbers of myeloid cells that
still differentiate (i.e. cease dividing) as normal
• In 4-6 years disease progresses to blast crisis 
accumulated mutations  ability to differentiate is lost
• Transplantation can cure (but problematic)  less
than 20% of cases can be cured
What mutations cause this?
Chromosome 1
Gene A
Gene B
Chromosome 2
Fusion Gene
Primary transcript
Fusion mRNA
Unique Properties
Chimaeric
protein
Altered Pattern of gene expression
Acts as an oncogene
65% of leukaemias are characterised
by particular somatically acquired
chromosome translocations
Differentiation Blocked
Continued self-renewal
Bcr-abl = constitutively
active tyrosine kinase
(The protein product from this fusion
gene only found in ~70% of patients)
Chronic myeloid leukaemia (CML) is characterised by
the t(9;22)(q34;q11) reciprocal translocation
**
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Bcr-abl inhibitor, Glivec®
(Gleevec; Imatinib; ST1571)
• Rationally designed small molecule that binds to an
inactive form of Bcr-abl and prevents ATP recruitment
 tyrosine kinase activation is blocked
• Pre-clinical studies  growth inhibition and induction of
apoptosis specifically in Bcr-abl expressing cells
• Shown to be orally active and well tolerated
• Effective therapy for all stages of CML inducing
remission in 80% of patients
• Approved in May 2001 < 3yrs after first Phase I study
• >95% patients with chronic phase (stable) disease
 durable response
…but
The downside
• ~all patients with advanced disease will relapse 
develop resistance to Glivec
• Main mechanism: reactivation of Bcr-abl kinase via
point mutations that  drug sensitivity 3- to >100-fold
The solution
• Combination therapy using Glivec with cytotoxic
agents and/or interferon
• Use rational drug design to make similar drug that
binds more avidly: AMN107 with >20-fold higher
affinity for wt and mutant Bcr-abl, published Feb 2005
Summary
• Targeted therapies can be more selective and show
improved efficacy with minimal toxicity
• Almost invariably, initial response and latency
followed by disease resistance
•  inherent weakness of monotherapy
• Combination therapy with cytotoxic drugs is being
assessed but the mutagenic nature of these may
accelerate the development of resistance
• Simultaneous use of multiple targeted agents may 
faster responses and more durable remissions
• Need yet more detailed knowledge of the molecular
changes during cancer progression  TARGETS
Suggested Reading
• “Tamoxifen: a most unlikely pioneering medicine”
Jordan VC (2003) Nat. Rev. Cancer 2, 205-13
• “Aromatase Inhibitors for breast cancer: lessons from
the laboratory” Johnston SRD & Dowsett M (2003)
Nat. Rev. Cancer 3, 821-31
• “Untangling the ErbB signalling network” Yarden Y &
Sliwkowski MX (2001) Nat. Rev. Cancer 2, 127-37
• “STI571 (Gleevec) as a paradigm for cancer therapy”
BJ Drucker (2002) Trends Mol. Medicine 8, S14-18