1. Figure 8. Tumor growth curves of NCI-H1299
cell line mouse xenograft after in vivo
treatment. Each curve represent different
treatment combination: control, GSI only,
docetaxel only, and GSI/docetaxel combination.
Statistically significant growth inhibition was
seen in GSI/docetaxel as compared to control.
Abstract
Background
The Epithelial-Mesenchymal Transition (EMT) is a key
developmental program that is often activated during
cancer invasion and metastasis. EMT cells possess the
characteristics of cell motility, invasiveness and
chemotherapy resistance. EMT expression profile
correlates with poor outcome in multiple tumors and
there is evidence suggesting an integral role of Notch
pathway in mediating EMT. In this study, we examined
Notch activity and EMT status in correlation with
chemoresistance in lung adenocarcinoma.
Results
Transduction of multiple NSCLC cell lines, NCI-H1299,
NCI-H460, NCI-H358, NCI-H441 with a Notch GFP-
reporter construct identified a subset of cells with high
Notch activity (GFP-pos). We compared the tumor
invasiveness capacity of GFP-pos and GFP-neg cells
utilizing a transwell invasion assay. Cells with high
Notch activity, as evidenced by GFP expression, had
multiple fold higher invasion capability compared to
GFP-neg cells (p=0.0001). To evaluate whether Notch
activity correlates with resistance to chemotherapy, we
treated GFP-pos and GFP-neg cells with docetaxel or
cisplatin and assessed Annexin V expression using
flow cytometry. GFP-pos cells had five fold less
apoptotic cells as compared to GFP-neg cells
indicating resistance of GFP-pos cells to
chemotherapy. When GFP-pos cells were exposed to a
Notch signaling inhibitor, gamma secretase inhibitor
(GSI), these cells were rendered sensitive to cisplatin
and docetaxel. RNA microarray analysis and RT-PCR
analysis revealed an increased expression of EMT
related genes (Vimentin, N-Cadherin, Snail-1,….) in
GFP-pos cells as compared to GFP-neg cells. Upon
treatment with GSI, GFP-pos cells had decreased
invasion capability, decreased expression of
mesenchymal-related genes and increased expression
of epithelial genes. Furthermore, NOD/SCID mice with
subcutaneous lung tumor xenografts treated with GSI
showed decreased expression of EMT genes at level of
RNA and protein. The combination of GSI and
docetaxel reduced tumor growth in mouse xenografts
compared to GSI and docetaxel as single agents.
Conclusion
These studies suggest that the Notch pathway is an
important regulator of EMT status and that its inhibition
may alter EMT chemo-resistance properties. The
addition of a Notch inhibitor may enhance the efficacy
of chemotherapy in the treatment of NSCLC.
Conclusions
 Notch activity is associated with
mesenchymal morphology.
 Notch activity correlates with increased
invasiveness and resistance to
chemotherapy.
 Notch activity correlates with
overexpression of mesenchymal and
decreased expression of epithelial genes.
 Blocking Notch activity by GSI decreases
the expression of mesenchymal genes and
increased the expression of epithelial
genes.
 Notch inhibition by GSI decreases the
invasion capabilities of mesenchymal cells.
 Notch inhibition renders resistant cells
sensitive to chemotherapy.
 Sequential treatment of GSI and traditional
chemotherapy might lead to better
response.
 Notch activity is essential for an EMT status
and inhibiting Notch pathway makes these
cells more epithelial like.
Blocking the Notch pathway inhibits the epithelial-mesenchymal transition (EMT)
status in lung cancer and alters chemoresistance.
Khaled Hassan, Luo Wang, Paige O’Dowd, Gwangil Kim, Hasan Korkaya, April Davis, Suling Liu, , Gregory P. Kalemkerian, Max S. Wicha.
Division of Hematology Oncology, University of Michigan, Ann Arbor, Michigan
Figure 7. Upper panel. RT-PCR relative mRNA expression of mesenchymal and
epithelial genes in NCI-H1299 xenograft tumor treated with Docetaxel or GSI.
Lower panel. IHC staining for Vimentin a) control, B) docetaxel, c) GSI treated.
a) b)
Figure 3 a) MTT assay after treatment of various doses of GSI followed
by single dose of docetaxel. B) AnnexinV apoptosis assay with varying
doses of sequential GSI and docetaxel treatment.
Figure 4. a) Relative mRNA expression of mesenchymal and epithelial
genes in NCI-H1299 Notch positive and negative cells by RT-PCR. b)
Relative mRNA expression of mesenchymal genes in NCI-H441 Notch
positive and negative cells by RT-PCR. c) Relative mRNA expression of
epithelial genes in NCI-H441 Notch positive and negative cells by RT-
PCR.
a)
Figure 5. Relative mRNA expression RT-PCR of mesenchymal a) and
epithelial b) genes in NCI-H441 Notch-positive cells treated with different GSI
concentrations.
Figure 6. NCI H441 Notch positive invasion Assay. a, b) Control untreated
cells. c, d) cells treated with 0.1uM GSI. e, f) cells treated with 1uM GSI.
Figure1. Upper panel; Notch positive H-441 trans-well invasion after
48hrs. Cells appear to have more spindle-like morphology. Lower panel;
Notch negative H-441 trans-well invasion after 48hrs. Less invasive cells
were detected in Notch negative H-441 that appeared to have more round
morphology as compared to Notch positive cells.
Figure 2. . AnnexinV apoptosis assay. A) Untreated GFP-pos and GFP-neg.
B) 1.5% AnnexinV in GFP-neg. C) 0.5% AnnexinV in GFP-pos. D) Docetaxel
treated cells. E) 66% AnnexinV in GFP-neg. F) 11% AnnexinV in GFP-pos. G)
Cisplatin treated GFP-pos and GFP–neg cells. H) 37% AnnexinV in GFP-neg.
I) 7.6% AnnexinV in GFP-pos. Annexin V apoptosis assay after exposure to
Docetaxel and Cisplatin in different cell lines (average of 3 experiments); J)
NCI-H1299, K) NCI-H358, L) NCI-H460, M) A549, N) NCI-H441.
Results
AA
B C
0.5%1.5%
D
E F IH
G
11%66% 7.6%37%
a) b) c)
0
20
40
60
80
100
120
0uM 1uM 2uM 5uM
%Viability
GSI Dose
GFP-pos
GFP-pos
a)
b)
b)NCI-H1299 GFP-pos NCI-H1299 GFP-pos NCI-H441 NCI-H441
b)
a) b)
c) d)
e) f)
a) b)
b)
a)
c)
NCI-H1299 GFP-pos/GFP-neg
NCI-H441 NCI-H441
Relativeexpression
Relativeexpression
Relativeexpression
GSI
Docetaxel
# 3309