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ABCC10 loss enhances docetaxel retention, increases apoptosis and diminishes tumor growth
ABCC10 loss Sensitizes cells to docetaxel, paclitaxel, gemcitabine and vinorelbine.
ABCC10 is associated with diverse signaling proteins including EGFR and AKT
Decreases EGFR, SRC, AKT and ERK activation
Acknowledgements
Role of ABCC10 in Docetaxel Resistance in NSCLC
Dicle Özel, Janet Wangari-Talbot, Jhoneil Cooper, James Matthew Denshaw, Bruce Zhang, and Elizabeth Hopper-Borge
Developmental Therapeutics Program, Fox Chase Cancer Center, Philadelphia PA.
Materials and Methods
Abstract Results
Conclusions
ABCC10 is a multidrug resistance protein of the ATP binding cassette (ABC) transporter family. It has been documented that it increases resistance to anti-
cancer agents in vitro including taxanes, vinka alkaloids, and nucleoside analogues. It has been observed that, in vivo, loss of ABCC10 in a mouse model
increases tissue sensitivity to the drug paclitaxel. Contributions of ABCC10 to multidrug resistance and tumorigenic signaling in non-small cell lung carcinoma
(NSCLC) were investigated in this study. shRNA knock down of ABCC10 in A549 and H1299 NSCLC cells demonstrated that ABCC10 loss increased
sensitivity to the anti-cancer agents: docetaxel, paclitaxel, gemcitabine and vinorelbine. To demonstrate increased sensitivity, we performed MTT assays after
ABCC10 loss and treated the cells with docetaxel, paclitaxel, gemcitabine and vinorelbine. The ABCC10 shRNA cells showed reduced viability compared to
the controls. We also examined PARP cleavage by western blotting to demonstrated enhanced apoptosis. We observed more PARP cleavage in the shRNA
cells compared to the controls. In vivo, we found that xenograft tumors of ABCC10-shRNA cells showed more potent suppression of tumor growth by
docetaxel than controls. Additionally, we investigated possible effects caused by the loss of ABCC10 on the expression and activation of mitogenic and
apoptotic signaling cascades. We performed Reverse Phase Protein Array Analysis to identify changes in protein expression and phosphorylation. Decreased
activation of EGFR, AKT and SRC was correlated with the loss of ABCC10. We confirmed the RPPA analysis by showing the decreased phosphorylation of
EGFR, SRC and AKT by western blots. PI3K-AKT signaling was identified as the major signaling pathway inhibited due to the loss of ABCC10. This report
identifies ABCC10 as a mediator of multidrug resistance protein and oncogenic signaling in lung cancer. These new findings of ABCC10’s connection to
activation of signaling cascades that are necessary for tumor growth may lead to new discoveries in lung cancer development. Further research is needed to
explore how ABCC10 interacts with other mechanisms and PI3K-AKT signaling proteins and to utilize this protein for drug development.
Cell culture:
A549, H1299, NCI-1975 , HCC827, EKVX, H358 lung cancer cells were obtained from ATCC. HEK C18 was previously described.
1 x 106 cells were seeded in T25 flasks and incubated overnight, next day medium was changed to serum free medium
Knockdown of ABCC10 in A549 and H1299, was performed with three different sh-RNA cell lines. Several clones were generated for each parental cell
line
Western Immunoblots:
Crude cell lysates were collected with RIPA buffer with protease inhibitors. Routinely, 30µg separated on 7% tris acetate gels, transferred to PVDF
membrane and probed with indicated antibodies.
PARP cleavage Apoptosis Assay:
1 x 106 cells were seeded in T25 flasks and incubated overnight, next day medium was changed to serum free medium
After 12-16 hours, cells were treated with complete medium containing drugs for 72 hrs. Crude lysates were collected and analyzed by western
immunoblotting.
MTT Cytotoxicity Assay:
2x105 cells were seeded in 96 well plates and incubated overnight; the next day, drugs docetaxel, paclitaxel, vinorelbine, and gemcitabine were added to
wells to get the following well concentrations: 0, 1, 3, 10, 100, 300, 1000, 3000, 10000 nm
The cells were left in the incubator for three days, then MTT solutions 1 and 2 were added to the wells
The cells were left in the incubator overnight and read with a spectrophotometer
Reverse Phase Protein Assay:
Protein lysates were collected as described previously and analyzed at MD Anderson Cancer Center for expression of 162 phosphorylated and non-
phosphorylated proteins. The average change in expression is presented as a heat map.
Grant support: JWT: 2T32-CA009035-36, EHB: NIH core grant CA06927(FCCC) and FCCC Lung Cancer Research Fund. MD Anderson Cancer Center RPPA Core Facility.
Investigate how ABCC10 affects major signaling pathways
Mechanisms for modulation of cell signaling by ABCC10.
Future Directions
References
Hopper-Borge, E, et al., 2004). Cancer Res 64, 4927-4930.
Hopper-Borge, E, et al., (2009). Cancer Res 69, 178-184
Hopper-Borge, E, et al., (2009). Cancer Res 71, 3649-3657
Chen, Z.S., et al., Mol Pharmacol, 2003. 63(2): p. 351-8
Figure 1. ABC transporter expression in NSCLC cell line and shRNA knockdown. A. Whole cell lysates collected from NSCLC cells and analyzed by western immunoblotting against ABCC10. HEKMRP7-C18 was used as a positive control for ABCC10. B.
Knockdown of ABCC10 by shRNA. Figure (2A) A549 and (2B) H1299 cells targeted with a non-silencing control (NSC1) or ABCC10 specific shRNA (shRNA1-3). Expression of ABCC10 was detected by western immunoblotting. The total protein was normalized
to β-Actin.
A549
H1299
HC
C827
H358
NC
I1975
EK
VX
HE
K-M
RP
7 C
18
β-actin
ABCC10
1A.
ABCC10
β-actin
1.0 1.0 0.35 0.23 1.0 1.0 0.5 0.3
1B.
Drug A549 NSC1
A549 sh501B
A549 sh901D
H1299 NSC1
H1299 sh331D
H1299 sh902C
A549 fold Sensitivity
H1299 fold Sensitivity
Docetaxel 16.48+/-0.98
3.677+/- 0.79
4.80+/- 0.100
73.04+/- 0.4
11.68+/-1.288
16.14+/- 3.389
3.5 to
4.5
4.5 to 6.3
Paclitaxel 75.42+/- 1.313
10.95+/- 0.315
19.56+/- 0.369
57.33+/-5.207
16.29+/- 0.8832
25.11+/- 2.54
3.9 to 6.8
2.3 to
3.5
Drug A549 NSC1
A549 sh501B
A549 sh901D
H1299 NSC1
H1299 sh331D H1299 sh902C
A549 fold resistance
H1299 fold resistance
Vinorelbine 412+/-1.17
34.96+/-1.684
37.43+/-0.7535
102.4+/-2.457
20.4+/- 0.8701 29.95+/- 1.3
11.1 to
11.8
3.4 to 3.6
Gemcitabine 559+/-0.555
109.9+/-1.451 155.8+/-3.591
1182+/- 7.190-
339.5+/- 7.997
319.6+/- 4.483
3.6 to
5.1
3.5 to 3.7
2.
Figure 3. Effects of ABCC10 loss on in vitro apoptosis. Western immunoblots assessing the cleavage of poly
(ADP-Ribose) polymerase, a hallmark of apoptosis after docetaxel treatment in A549 (3A) and H1299 (3B) non-
silencing control cells and ABCC10- shRNA cells. The total protein was normalized to β-Actin.
3A.
A549 NSC1 sh501B sh901D
0 3 10 0 3 10 0 3 10
nM Docetaxel
A549 NSC1 sh501B sh901D
0 5 20 0 5 20 0 5 20
nM Gemcitabine
A549 NSC1 sh501B sh901D
0 5 20 0 5 20 0 5 20
nM Vinorelbine
0 3 10 0 3 10 0 3 10
nM Docetaxel
H1299 NSC1 sh331D sh902C
0 5 20 0 5 20 0 5 20
nM Gemcitabine
H1299 NSC1 sh331D sh902C
0 5 20 0 5 20 0 5 20
nM vinorelbine
H1299 NSC1 sh331D sh902C
A5
49
NSC
-1
shA
BC
C5
0-1
B
shA
BC
C9
0-1
D
H1
29
9
NSC
-1
shA
BC
C3
3-1
D
shA
BC
C9
0-2
C
3B.
Figure 4. Signaling changes associated with ABCC10 loss. Western immunoblots showing a decrease in
various signaling proteins in A549 or H1299 parental cells, or control shRNA and ABCC10 shRNA
cells. β-actin was used as a loading control.
Fibronectin
pAKT S473
AKT
pmTor S2448
mTOR
SRC
SRCY416
pSRC Y416
A549
A549 N
SC
1
A549 s
h501B
A549 s
h901D
H1299
H1299 N
SC
1
H1299 s
h331D
H1299 s
h902C
β-actin
A549
A549 N
SC
1
A549 s
h501B
A549 s
h901D
H1299
H1299 N
SC
1
H1299 s
h331D
H1299 s
h902C
pEGFR Y1068
EGFR
β-actin
pERK
ERK
4
Figure 2. Docetaxel, paclitaxel, vinorelbine, gemcitabine cytotoxicity assays. 2A. Effects of ABCC10 loss on
sensitivity to docetaxel and paclitaxel. 2B. Effects of ABCC10 loss on sensitivity to vinoreline and gemcitabine.
IC50 values and fold sensitivity is indicated in the tables below each set of curves.
