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Drug screening and cancer resistance: EGFR
From the gene card activity, let’s investigate a
specific gene: egfr
The egfr gene has mutations in 18-20% of patients
who develop lung adenocarcinoma (see image
below). Patients with lung adenocarcinomas tend
to have unfavorable prognoses and high stage
tumors often cannot be surgically removed.
Instead, patients are treated with chemotherapy
drugs and typically have a less than 2-year
survival rate.
EGFR (epidermal growth factor receptor) is
a receptor tyrosine kinase. Upon binding
its specific ligand (for example, EGF) the
EGFR dimerizes and its receptor tyrosine
kinase activity is activated. Kinases add
phosphate groups to proteins. In the case
of EGFR, this kinase autophosphorylates,
adding phosphates to its own tyrosine
amino acids. Once phosphorylated, a
number of signaling cascades are activated
(including the MAPK, AKT and JNK
pathways). In general, these pathways
promote cell proliferation, angiogenesis,
migration, adhesion and invasion while
inhibiting apoptosis (see adjacent figure).
1. What is the normal function of the protein
encoded by the egfr gene?
Encodes the EGFR membrane protein that binds
extracellular ligands (ex. EGF) and activates (via
phosphorylation) pathways that promote cell
proliferation, angiogenesis, migration, adhesion and
invasion.
2. How might you prove a particular mutation in egfr caused cell proliferation?
Mutate the egfr gene in a cell line and observe the cells for hallmarks of cancer.
Certain EGFR mutations cause its kinase domain to be constitutively active, meaning
it can no longer be turned “off”. This causes unregulated cell proliferation, angiogenesis,
migration, adhesion and invasion, all of which contribute to cancer.
Receptor tyrosine kinases are common targets for chemotherapy drugs, due to their
pivotal role in cancer progression. One such drug is gefitinib, which specifically binds
EGFR and interrupts its signaling cascades. In a process called oncogene addiction,
cancer cells tend to depend upon a single oncogenic pathway for proliferation and
survival (in this case, EGFR and its downstream cascades). When this oncogenic
pathway is disrupted, it leads to cancer cell death. For this reason, gefitinib is only
effective on cancers with overactive/mutated EGFR. These mutations therefore render
EGFR susceptible to gefitinib treatment. Gefitinib specifically inhibits the ATP binding
site of the EGFP kinase domain, preventing autophosphorylation and the activation of
downstream pathways.
Let’s observe a lung adenocarcinoma cell line, in which various egfr mutations have
been made… (image analysis activity here: 26 available alleles for EGFR).
Now we have a cell culture system
in which we can test chemotherapy
drugs. Chemotherapy drugs with
the least side effects tend to be
targeted therapies, which inhibit a
particular cancer-promoting pathway
(like receptor tyrosine kinases).
3. View the figures below (PDB:
4WRG, 4WKQ) of EGFR wild-type
and bound to gefitinib (show with
black sticks). To what part of the
protein does gefitinib bind?
It binds in the ATP binding site
Unfortunately, there are also EGFR
mutations that prevent gefitinib from
binding and inhibiting the kinase’s
ATP binding site (see PDB 4I1Z,
4I22, 3UG2). These EGFR mutants
render their cancer cells resistant
to the gefitinib chemotherapy.
Sometimes, when patients are
treated with gefitinib, their EGFR
already contains these resistant
mutations. These people are called
primary non-responders. In other
cases, gefitinib is very effective
when treatment begins, but
becomes ineffective when a
patient’s EGFR gains a resistant
mutation. These patients have
acquired resistance.
4. Where would you predict resistance mutations typically arise on the EGFR protein?
How would this affect the natural function of the protein? (Interpret the figure below)
Resistance mutations would affect the ATP binding site, decreasing the affinity for ATP.
5. Do the structures below confirm your hypothesis from question 4? The images below
show EGFR co-crystallized with gefitinib (black sticks), the first is the “wild-type”
(unmutated) structure, two structures with a susceptibility mutation (L858R and G719S,
dark green sticks) and a resistance mutation (T790M, red sticks). The fourth image is a
zoom in on gefitinib (black sticks) and the wild-type (light green) residues compared to
the susceptibility (dark green) and resistance (red) mutations.
Yes or no. Resistance mutations are found in the ATP binding domain.
EFRG wild type (PDB:4wrg)
EFRG + gefitinib (PDB:4wkq)
EFRG (L858R/T790M)+ gefitinib (PDB:4i22)
Thr
790
Ser
Met
719
Gly
Arg
858
EFRG (G719S/T790M)+ gefitinib (PDB:3ug2)
Leu
6. When a patient seeks treatment for lung adenocarcinoma, when do you think an
oncologist should prescribe gefitinib? When should gefitinib not be prescribed (use the
table below)? How would the oncologist acquire this information?
Sensitive/responders
Exon 19 deletions
Exon 21 L858R single mutation
Exon 18 G719C single mutation
Exon 21 L861Q single mutation
Resistance/Nonresponders
Exon 20 insertion
Exon 20, S768I single mutation
Exon 20, T790M single mutation
Should be prescribed to the sensitive/responders above, should not be prescribed to the
resistant/nonresponders above. This information would be acquired by sequencing parts of the patient’s
genome (specifically, exons 18-21 of egfr).
7. Use the figure below to determine how common it is for a patient to be a primary nonresponder with the mutation S768I in exon 20. 0.55% of non-responders have this mutation.
8. What might be done to combat resistance mutations?
Combine multiple drug treatments; don’t use any one for an extended amount of time.
9. Write a paragraph that explains the relationship between the following terms: egfr
gene, EGFR protein, gefitinib, receptor tyrosine kinase, cancer and oncogene addiction.
The egfr gene encodes the protein EGFR, which is a receptor tyrosine kinase. In certain cancers, EGFR
has a mutation that causes it to be constitutively active, causing unregulated cell proliferation,
angiogenesis, migration, adhesion and invasion and therefore cancer. The cancer cells depend upon this
pathway in a process called oncogene addiction, therefore, disrupting this pathway by targeting EGFR
with gefitinib kills only cancer cells.
References:
Cortot, A. B., and P. Janne A. "Molecular Mechanisms of Resistance in Epidermal Growth Factor
Receptor-mutant Lung Adenocarcinomas." European Respiratory Review 23.133 (2014): 356-66.
PyMol Molecular Viewer. Palo Alto, CA: DeLano Scientific, n.d. Computer software.
RCSB Protein Databank "RCSB Protein Data Bank - RCSB PDB." RCSB Protein Data Bank - RCSB
PDB. http://www.rcsb.org/pdb/home/home.do. Web. 20 Sept. 2016.
Siegelin, Markus D., and Alain Borczuk C. "Epidermal Growth Factor Receptor Mutations in Lung
Adenocarcinoma." Lab Invest Laboratory Investigation 94.2 (2013): 129-37.
Yang, James Chih-Hsin, Yi-Long Wu, Valorie Chan, Johan Kurnianda, Kazuhiko Nakagawa, Nagahiro
Saijo, Masahiro Fukuoka, Gael Mcwalter, Rose Mccormack, and Tony Mok S.k. "Epidermal Growth
Factor Receptor Mutation Analysis in Previously Unanalyzed Histology Samples and Cytology Samples
from the Phase III Iressa Pan-ASia Study (IPASS)." Lung Cancer 83.2 (2014): 174-81.