Transcript Thomas Luo - Science - Madison Metropolitan School District

Identification of Therapeutic Targets in Triple Negative Breast
Cancer Cells: β-Catenin, a Critical Chemoresistant Mediator
Thomas Luo1, 2 with Bhawna Sharma2 PhD and Dr. Vincent Cryns2 MD
Madison West High School, Madison, Wisconsin1;
Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin2
Results
Introduction
Breast cancer affects over 200,000 new patients in the United
States every year. One particularly challenging subtype is triplenegative breast cancer (TNBC), occurring in 15-25% of breast
cancer and garnering its name from a lack of estrogen receptor,
progesterone receptor, and human epidermal growth factor
receptor 2 (HER2). TNBC is clinically characterized to be more
aggressive and largely unresponsive to clinically available
targeted therapies resulting in poor prognosis making
understanding the pathways and characteristics of TNBC to be
vital importance for urgent need of developing future therapies.
Increasing evidence suggests that multiple signaling pathways are
abnormally regulated and involved in pathogenesis of TNBC—two
of which are Wnt/-catenin pathway and Notch pathway. Abnormal
activation of Wnt/-catenin signaling has been associated with
TNBC: several important oncogenes (FZD7, LRP6, TCF) in Wnt/catenin were up-regulated in TNBC cells. Normally, -catenin
plays a critical role in intercellular adherent junction. However,
after losing its association with cellular junction, -catenin released
from membrane will gain transcriptional function potentially
promoting tumor cell invasion. Also strongly related to TNBC cells,
the B-crystallin gene is linked to neoplastic-like changes and loss
of cell growth control in basal-like breast cancer/TNBC. In light of
the poor outcome of TNBC case, there is an urgent clinical need of
its therapeutic approaches; therefore we designed experiments to
attempt to identify potential therapeutic targets in those complex
pathways.
Wnt Pathway off
Results
Effects of β-catenin Inhibitor
Reduction of Cell Density by ICG001
A.
B.
Figure 2. Inhibitory effects of
ICG001(ICG),
Doxorunicin
(DOX), and Paclitaxel (PAC) on
cell viability of MDA-MB-231
cells. A. Cells transfected with
control vector (132-V) or vector
expressing αB-crystallin (αB)
(231-αB). Cells were treated
with
drugs
and
their
combinations for 48 hours. B.
Cells were treated for 72 hours.
C. Cells without transfection
were used. Cells were treated for
72 hours. DMSO was used as
control treatment. Cell viability
was detected with MTS assay.
Results showed that ICG + DOX
markedly reduced cell viability
in the two types of transfected
cells compared with treatment
with ICG or DOX alone.
Furthermore, ICG + DOX also
reduced cell viability in nontransfected cells.
Figure 3. Reduction of cell
densities by ICG, DOX, and PAC
in MDA-MB-231 cells. DMSO was
used as control treatment. Cell
number was detected with
crystal violet assay. A. Cells
were not transfected with αBcrystallin (αB). B. Cells were
transfected with αB. Cells were
treated with drugs and their
combinations for 48 hours. C.
Cells densities of each treatment
groups were measured with a
NIH image J software, and the
cell density was expressed as
mean intensity detected.
C.
Results showed that ICG + DOX
or PAC markedly reduced cell
confluence in the two types of
transfected cells compared with
treatment with ICG, DOX or PAC
alone.
C.
Inhibition of Cell Migration/Proliferation by ICG001
Wnt Pathway on
Wnt
Frizzled
LRP5/6
P
Dvl
Actin
P
α-catenin
CK1 APC
β-catenin
GSK3
Actin
Axin
GSK3
Frizzled
LRP5/6
Axin
CK1 APC
α-catenin
β-catenin
E-cadherin
Figure 3. Inhibition of cell cell
migration /proliferation by ICG
in MDA-MB-231 cells. A. the
wound at 0 hour after scratch. B.
wound at 24 hours after scratch,
and cells were cultured in
control medium. C. Cells were
treated 5 μM ICG for 24 hours
after scratch. D. Cells were
treated 10 μM ICG for 24 hours
after scratch. E. the wound of
each treatment groups were
measured with a NIH image J
software, and the effects of
treatment was expressed as
percentage of wound closure
(%).
Dvl
Effects of Notch Pathway Inhibitor
β-catenin
β-catenin
β-catenin
β-catenin
β-catenin
Proteasome
HDAC
Groucho/TLE
CBP/p300
Off
TCF
A.
β-catenin
P
X
β-catenin
On
TCF
Figure 1. Wnt/β-catenin signaling pathway and intercellular adheren junction
B.
Figure 3. Effects of DAPT, DOX,
and PAC on cell viability of MDAMB-231
cells.
A.
Cells
transfected with (231-αB) or
without αB-crystallin (αB) (231αB). Cells were treated with
drugs and their combinations
for 48 hours. B. Cells were
treated for 72 hours. C. Cells
without transfection were used.
Cells were treated for 72 hours.
DMSO was used as control
treatment. Cell viability was
detected with MTS assay.
Results showed that DAPT +
DOX did not reduce cell viability
of the three types of cells.
Hypothesis
E.
Results showed that ICG +
or
PAC
reduced
migration/proliferation of
compared with treatment
control.
DOX
cell
cells
with
Discussion: Proposed Mechanism
aB-Crystallin has long been associated with response to DNA damage
and cell stress; however, recent data shows that aB-Crystallin actually
works in complex with -catenin. The down-expression of -catenin in
this study may actively disrupt this complex in TNBC.
Abnormal activity of Wnt/-catenin pathway and/or Notch pathway
leads to pathogenic alteration in TNBC cells rendering them more
aggressive and chemoresistant.
C.
Conclusions
Objective
 We demonstrated that combination treatments with ICG and
DOX or PAC (high dose) a possible reduced the cell viability and
cell number of MDA-MB-231 TNBC cells and inhibited
migration/proliferation of those cells.
To identify potential target molecules for TNBC, then testing the
therapeutic value using a cell culture model and pathway
inhibitors with cytotoxic chemotherapy drugs.
 These finding showed that inhibition of β-catenin activities might
sensitize TNBC to the treatment with conventional chemotherapy
drugs.
Methods
Cell Culture Procedures: MDA-MB-231 TNBC cell line and its
modified line that were transferred with vectors cloned with and
without B-crystallin were cultured in MDEM Medium supplemented
with 10 % FBS in an incubator at 37 oC with 5% CO2.
Cell Viability Assay (MTS assay): Cell viability after drug treatments
was determined using MTS assay. For the assay, cells were treated
with individual drugs or drug combinations for 48 hours. Pathway
inhibitors and cytotoxic drugs used were Paclitaxel (200 nM),
Doxorubicin (1 µM), and Temozolomide (50 µM), β-catenin inhibitor
ICG001 (5 µM), and Notch pathway inhibitor DAPT (50 µM).
Cell Number-Crystal Violet Assay: The effects of drug treatments on
cell number were further measured with Crystal Violet assay. In this
assay, cells were treated with drug and their combinations for 48
hours. The doses of the inhibitors and cytotoxic drugs were the same
as those used in MTS assay experiments stated above.
Wound Healing/Scratch Assay: In assessing cell migration, growth,
and proliferation after drug treatment, a wound healing/scratch assay
was used. Confluent cells were scratched and then treated with and
without ICG (5 or 10 µM). Cell migration was monitored at 0 and 24
hours post-scratch.
The facts that TEM and DAPT did not affect those cells further
showed that the effects of ICG, DOX, and PAC were specific
actions.
Effects of Temozolomide
A.
B.
Figure
4.
Effects
of
temozolomide (TEM), ICG, and
DAPT on cell viability of MDAMB-231
cells.
A.
Cells
transfected with (231-αB) or
without αB-crystallin (αB) (231αB). Cells were treated with
drugs and their combinations
for 72 hours. B. Cells without
transfection were used. Cells
were treated for 72 hours. DMSO
was used as control treatment.
Cell viability was detected with
MTS assay.
Results showed that TEM + ICG
or DAPT did not reduce cell
viability of the three types of
cells.
 Our data indicate that β-catenin is an important signaling
molecule that mediates drug-resistance and tumorigenic effects in
TNBC. Thus, Targeting of β-catenin and Wnt signaling pathway
could be a promising strategy for finding novel therapy approches
for treating TNBC.
Acknowledgements
• Extended thanks to Rachel Egan, Carmen Lombard, Lisa Watchel and the Madison
Metropolitan School District for providing this incredible opportunity
• I would like to thank Vincent Cryns and Bhawna Sharma for their patience, insight, and
assistance.
• A thanks to Chris Schoenholz and Vincent Cryns for writing recommendations to the
SRI program