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JAK-1 Inhibition
Andrew B. Bonner, Hoa Q. Trummell, Eddy S. Yang
Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, Alabama USA
ABSTRACT
Background and Objectives: It is known that the inhibition of
Epidermal Growth Factor Receptor (EGFr) with cetuximab
results in radiosensitization [1]. Also, this inhibition of EGFr
results in partial reduction of activated signal transducer and
activator of transcription-3 (STAT-3). Activated STAT-3 functions
to protect cells from apoptosis. We hypothesized that the
addition of a Janus Kinase (JAK)-STAT-3 inhibitor (JAK1i) may
enhance the radiosensitizing effects of cetuximab by further
inhibiting activated STAT-3 compared to cetuximab alone.
Methods/Results:
The radiosensitization activity of the
combination of JAK-STAT-3 inhibition ([JAK1i]; Calbiochem,
LaJolla, CA) and EGFr inhibition (cetuximab) was assessed.
Four human head and neck cell lines were studied: UM-SCC-1
and UM-SCC-5, and two modified UM-SCC-5 lines; a STAT-3
knockdown line (STAT-3-2.4 created by transfection of short hair
pin RNA against STAT-3) and control (NEG-4.17). Exposure to
either 0.5 g/ml of cetuximab or 1M JAK1i for 8 or 24 hours
resulted in reduced activated STAT-3 (immunoblot), and the
combination treatment showed greater reduction in activated
STAT-3 compared to the individual treatments.
Next, 72h
postradiation exposures to JAK1i (1M) in combination with
cetuximab (0.5 g/ml) resulted in greater radiosensitization
compared to the individual agents with respect to cellular
proliferation, colony formation and apoptosis.
Finally, the
combination treatment of JAK1i (1M) and cetuximab (0.5 g/ml)
following radiation resulted in an increase in unrepaired
radiation (RT) - induced DNA double strand breaks at 6 and 24
compared to the use of JAK1i or cetuximab alone.
Conclusions: These findings suggest that dual inhibition of
EGFr (cetuximab) and JAK-STAT-3 (JAK1i) leads to greater
radiosensitization than with either cetuximab or JAK1i alone.
First Point of
Inhibition
(Cetuximab)
DNA Repair
Figure 4.
The addition of JAK1i (1M) to the
combination of cetuximab (0.5 g/ml) and RT did
not increase the induction of DNA double strand
breaks (dsbs) but resulted in less repair of radiationinduced DNA dsbs at 6 and 24 hours following
radiation, as measured by the neutral comet assay in
all four cell lines. Top. The mean tail moment for 6
hours of treatments alone or proceeded by radiation.
Bottom. The mean tail moment for 24 hours.
Following treatment, exponentially growing cells
were collected and processed for single-cell gel
electrophoresis assay (Trevigens Comet Assay).
Neutral comet assays were performed as previously
described [3].
Conclusions
1.
2.
3.
4.
Cetuximab slows the repair of radiationinduced DNA dsbs and the addition of
JAK1i increases this effect.
5.
Second Point
of Inhibition
(JAK1i)
DMSO
Cetuximab
Figure 1.
Immunoblot analyses revealed that the
phosphophorylation of STAT-3 was significantly reduced
with the dual treatment of cetuximab and JAK1i as
compared to either individual agent alone. UM-SCC-1,
UM-SCC-5, STAT-3 knockdown cells (STAT3-2.4) and
control transfected cells (NEG4.17) were treated with
cetuximab (5g/ml) and/or JAK1i (1M) for either 8 or 24h
with or without 5 minutes of exposure to EGF (60ng/ml)
as indicated in the figure. Protein lysates were subjected
to SDS-PAGE and Western blot analysis for STAT-3,
pSTAT-3(Tyr705) and pSTAT-3(Ser727). GAPDH was used
to control for loading variability.
Representative
immunoblots for the UM-SCC-1 cells with (B) or without
(A) EGF as previously described [2].
JAK1i
Cetuximab + JAK1i
1.
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3.
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hippocampal neurons
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