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Chemoresistance and Transformation
-ABC family- membrane transport proteinMDR1-P-gp48 different ABC transportersextrude many types of drugs from cancer cells, thereby conferring multidrug resistance
Is there something common?
Chemoresistance Transformation -Oncogenic signal
-Defect in apoptotic pathway
-Many drugs induce apoptotic pathways that defects in this pathway also results in multidrug resistancc
Myc and rasMyc and bcl-2Myc and mutant p53E1A and E1B
Increasing our knowledge of the components involved in the pathways
that mediate cell death and survival…..
Gives us the hope is that targeting specific molecules (Targeted therapy) will impart sensitivity to Chemotherapy —
a combination therapy is possible
Cancer chemotherapeutic drugs that induce apoptosis
Activator Protein 2(AP-2)
AP-2 gene family: AP-2, AP-2, AP-2, AP-2δ and AP-2
5’- GCCNNNGGC -3’
1 437
Activation domainDNA Binding domain
helix-span-helix motif
Dimerization domain
N C
Homozygous deletion for any of the AP-2 genes results in lethality either during embryogenesis or shortly after birth
Implicated in development
Implicated in transformation Progressive loss of expression of these genes has been linked to the progression of human cancers
Breast carcinoma (Gee et al., 1999)
Colon carcinoma (Ropponen et al., 2001)
Melanoma (Several papers from *Bar-Eli’s group)
Prostate cancer (Ruiz et al., 2001Glioma (Bar-Eli’s group)
AP-2 activated p21, inhibited DNA synthesis and stable colony formation (Zeng Y-X., Somasundaram, K., and El-Deiry WS Nature Genet,, 1997)
No genetic alterations in AP-2 have been reported
Epigenetic silencing ?
AP-2 adenovirus (Ad-AP2) makes functional AP-2 protein
-Sequence-specific DNA-binding(5’- GCCNNNGGC -3)
-AP-2 specific reporter activation(3X-AP2-CAT)
-Target gene activation(p21WAF1/CIP1)
Ad-LacZ Ad-AP2
SW48
0H
460
HT
1080
A
F
C D
E
B
0
20
40
60
80
100
120
0 20 40 60
- Ad-LacZ
- Ad-AP2
% V
iab
ilit
y
MOI
AP-2
PARP
Actin
Ad-LacZ Ad-AP20 24 48 72 Hrs0 24 48 72
Hrs
2448
2448
%A %G1 %S %G2 2.04 57.32 26.19 14.45 1.08 58.52 25.48 14.92 1.71 82.45 3.24 12.6042.44 32.51 6.30 16.75
Ad-LacZ
Ad-AP2
Virus
24 hr
48 hr
Ad-LacZ Ad-AP2
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
101
102
103
104
FL1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
101
102
103
104
FL1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
101
102
103
104
FL1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
101
102
103
104
FL1-H
-->
G1A G2
S S
PI-DNA content
Brd
u-D
NA
syn
thes
is
AP-2 inhibits cancer cell growth by inducing cell cycle arrest and apoptosis
ApoptosisTwo broad pathways that lead to apoptosis:
Apoptosis
ExtrinsicIntrinsic
Adapter
Are caspases activated ?If yes, how important they are?How important caspase 3 is?Caspase 8 or 9 or both are important!
Ad-LacZ Ad-AP2
Caspase 9
p46
p35
p18
p55
p42
Caspase 8
p32
p17
Caspase 3
Actin
AP-2
PARP
p12p10
p20
0 48 72 Hrs0 48 72
Caspase 3, 8 and 9 are activated during AP-2induced apoptosis
Ad-AP2
DM
SO
z-V
AD
-fm
k
AP-2
Actin
p32
p17Caspase 3p20
PARP
PAN Caspase inhibitorz-VAD-fmk
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
060
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
060
0
48hrs 72hrs 48hrs 72hrs
Ad-LacZ Ad-AP2
Con
trol
z-
VA
D-f
mk
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
060
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
PI-DNA content
Essential role of Caspases in AP-2induced apoptosis
MC
F7/
pv
MC
F7/
c3
Caspase 3
Actin
1 2R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL
1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL
1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL
1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL
1-H
-->
MCF-7/pvAd-LacZ Ad-AP2
24h
r48
hr
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL
1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL
1-H
-->
MCF-7/c3Ad-LacZ Ad-AP2
24h
r48
hr
PARP
MCF7/c3Ad-LacZ Ad-AP2 Ad-LacZ Ad-AP2
AP-2
Actin
0 48 72
MCF7/pv
Hrs0 48 72 0 48 72 0 48 72
DAPI BrdU
Ad
-Lac
ZA
d-A
P2
DAPI BrdU
MCF7/pv MCF7/c3
Role of Caspase 3 in AP-2 induced apoptosis
PARP
p32
p17
Caspase 3p20
p55
p42Caspase 8
p12p10
Caspase 9p46
p35
AP-2
Actin
DM
SO
z-IE
TD
-fm
k
Ad-AP2
AP-2
PARP
p55
p42Caspase 8
p12p10
Caspase 9p46
p35
p32
p17
Caspase 3p20
Actin
DM
SO
z-L
EH
D-f
mk
Ad-AP2Channels (FL2-H)
0 30 60 90 120 150
Num
ber
010
020
030
040
050
060
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
060
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
Channels (FL2-H)0 30 60 90 120 150
Num
ber
020
040
060
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
060
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
48hrs 72hrs 48hrs 72hrs
Ad-LacZ Ad-AP2
Con
trol
z-
IET
D-f
mk
z-L
EH
D-f
mk
pCEP4/AP-2pCEP4
pCEP4/AP-2 + z-IETD-fmk
pCEP4/AP-2 +z-LEHD-fmk
pCEP4/AP-2 + z-VAD-fmk
Empty
Extrinsic pathwayCaspase 8 inhibitor
z-IETD-fmk
Intrinsic pathwayCaspase 9 inhibitor
z-LEHD-fmk
Caspase 9, not 8, is needed for AP-2induced apoptosis
Apoptosis
ExtrinsicIntrinsic
Adapter ??
-Ad-LacZ-Ad-LacZ/Mock-Ad-LacZ/Lamin siRNA-Ad-LacZ/FADD siRNA
-Ad-AP-2-Ad-AP-2/Mock-Ad-AP-2/Lamin siRNA-Ad-AP-2/FADD siRNA
0
20
40
60
80
100
120
0 10 20 30 40 50 60
% L
ive
cell
s
MOI
FAD
D si
RNA
Moc
k
Lamin
siR
NA
FADD
GAPDH
1 2 3
Lamin
Actin
FADD
FAD
D si
RNA
Moc
k
Lamin
siR
NA
1 2 3 4
Con
trol
Role of FADD in AP-2 induced apoptosis
hrs
4872
4872
Ad-LacZ Ad-AP2
1.98 1.37
1.54 2.23
33.49
54.68
31.97
48.62
Lamin siRNA
FADD siRNA
siRNA
% Apoptosis
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
020
040
060
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
060
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
040
8012
016
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
040
8012
016
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
040
8012
016
0
Ad-LacZ
Lamin siRNA FADD siRNA
Ad-AP2
Lamin siRNA FADD siRNA
48 h
rs72
hrs
Channels (FL2-H)0 30 60 90 120 150
Num
ber
040
8012
016
0
Apoptosis
ExtrinsicIntrinsic
Adapter?
0
20
40
60
80
100
120
0 10 20 30 40 50 60
-Ad-LacZ-Ad-LacZ/Mock-Ad-LacZ/Lamin siRNA-Ad-LacZ/Apaf1 siRNA
-Ad-AP-2-Ad-AP-2/Mock-Ad-AP-2/Lamin siRNA-Ad-AP-2/Apaf1 siRNA%
Liv
e ce
lls
MOI
Role of Apaf-1 in AP-2 induced Apoptosis
hrs
4872
4872
Ad-LacZ Ad-AP2
1.44 1.23
1.79 1.39
27.96
49.48
5.98
6.23
Lamin siRNA
Apaf1 siRNA
siRNA
% Apoptosis
Ad-LacZ
Lamin Apaf1
Ad-AP2
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
080
160
240
320
Channels (FL2-H)0 30 60 90 120 150
Num
ber
080
160
240
320
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
Channels (FL2-H)0 30 60 90 120 150
Num
ber
070
140
210
280
Lamin Apaf1
48 h
rs72
hrs
siRNA
Apaf1 si
RNA
Mock
Lamin siRNA
Apaf1
GAPDH
1 2 3
Lamin
Apaf-1
Actin
Control
Mock
Lamin siRNA
Apaf-1 si
RNA
1 2 3 4
Apoptosis
ExtrinsicIntrinsic
Adapter
Apoptosis
ExtrinsicIntrinsic
Adapter
Mitochondria
Mitochondrial membrane potential
Mitochondrial Outer Membrane Permeabilization
Pro-apoptotic Bcl-2 member Bax translocates to Mitochondria
Cytochrome C is released into cytoplasm
0102030405060708090
100
Con
trol
-AP
-2
% c
ells p
osit
ive f
or
J-ag
gre
gate
s
+A
P-2
Val
inom
ycin
1 2 3 4
Cyt c
-actin
Cytosolic MitochondrialAd-AP2
Cytosolic Mitochondrial
0 24 36 48
Bax
Oxidative complex 1
0 24 36 48 0 24 36 48 0 24 36 48
Ad-LacZ
Hrs
Measurement of Mitochondrial Membrane Potential using JC-1 Dye
Mitochondria
Cytoplasm
- ---
-
--
--
NucleusHealthy cell
Apoptotic cell
JC-1JC-1
Apoptosis
ExtrinsicIntrinsic
Adapter
HCT116 B
ax -/-
HCT116 W
T
Bax
Actin
1 2
Ad-LacZ Ad-AP2
HCT116 Bax-/-HCT116 WT
PARP
Actin
AP-2
0 48 72 0 48 72 0 48 72 0 48 72
Ad-LacZ Ad-AP2
24 h
r48
hr
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
070
140
210
280
Channels (FL2-H)0 30 60 90 120 150
Num
ber
070
140
210
280
Channels (FL2-H)0 30 60 90 120 150
Num
ber
040
8012
016
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
050
100
150
200
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
080
160
240
320
400
Channels (FL2-H)0 30 60 90 120 150
Num
ber
070
140
210
280
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
070
140
210
280
Channels (FL2-H)0 30 60 90 120 150
Num
ber
070
140
210
280
72 h
r
HCT116 WT HCT116 Bax -/-
Ad-LacZ Ad-AP2 Ad-LacZ Ad-AP2
Role of Bax in AP-2 induced apoptosis
Ad-AP-2 0 24 48 72
Ad-LacZ
Bax
Actin
Con. Bax
AP-2hrs 0 24 48 72
Bcl-2 familyAnti-apoptotic -
Pro-apoptotic Multi-domain - BH3-only -
Bax
Bax Bax
Bcl
-2 Bcl
-2
Bcl
-X-S
Cell death
Cell survival
Cell death
Bax
Bax
Bcl-2
AP-2
Actin
Ad-LacZ Ad-AP2hrs0 24 48 0 24 48
Role of Bcl-2 in AP-2 induced apoptosis
-AP-2-Bcl-2
-1012345678
Nor
mal
ized
Fol
d c
han
ge
-26 12 24hrs after
Tet removal-3
-2
-1
0H
460
SW
480
1 2
Nor
mal
ized
Fol
d c
han
ge
-Bcl-2
Ad-AP2
0
100
200
300
400
500
600
700
Lu
cife
rase
act
ivit
y(C
PM
X 1
000)
Bcl-2 prom-Luc
pSG5
pSG5/AP-2
+
5
-
+
-
5
+
10
-
+
-
10
+
15
-
+
-
15
P1+1 P2 +610AP-2
173 bps (-129 to +44)
-1291
bs
GST-AP-2 Bcl-2 wtSV40 AP2 wtSV40 AP2 m
----1
+---2
+
--3
+
--4
+
--5
+-
-6
+-
-7
+-
-8
+--
9
+--
10
+--
11
5’-CTAATTTTTACTCC CTC TCCC CGC GACTCCTGA-3’-30-62
GCC N(3/4) GGC
5’-CTAATTTTTACTCC tat TCCC aaa GACTCCTGA-3’
Bcl-2 wt
Bcl-2 m
AP-2 consensus binding motif
GST-AP-2 Bcl-2 wtBcl-2 m
+--1
+--2
+
-3
+
-4
+
-5
+-
6
+-
7
+-
8
Lad
der
Bcl
-2 p
rom
Inp
ut
An
ti-L
amin
An
ti-A
P-2
Bcl-2
1 2 3 4 5
AP-2 binds to and represses Bcl-2 promoter
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
050
100
150
200
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
060
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
Channels (FL2-H)0 30 60 90 120 150
Num
ber
080
160
240
320
400
Channels (FL2-H)0 30 60 90 120 150
Num
ber
060
120
180
240
24 h
r48
hr
72 h
r
SW480/NeoAd-LacZ Ad-AP2Ad-LacZ Ad-AP2
SW480/Bcl-2 # 3
Channels (FL2-H)0 30 60 90 120 150
Num
ber
040
8012
016
0
Bcl-2
Actin
SW
480/
Neo
SW
480/
Bcl
-2 #
3
SW
480/
Bcl
-2 #
15
1 3 4
SW480/Neo
0 48 72
Ad-LacZ Ad-AP2
AP-2
Actin
PARP
0 48 72 0 48 720 48 72
1 2 3 4 5 6 7 8 9 10 11 12Ad-LacZ Ad-AP2
SW480/Bcl-2 # 3
Hrs
Vector Bcl-2 AP-2AP-2+Bcl-2
How important Bcl-2 down regulation is?
Caspase 3 is essential
Intrinsic pathway Caspase 8 and FADD adapter not neededCaspase 9 and Apaf1 are essentialMitochondrial membrane potential is lostBax translocates to mitochondriaCytochrome c is released from mitochondriaBax is essential
AP-2 binds to Bcl-2 promoter and represses its transcription
Apoptosis
Intrinsic
AP-2Bcl-2
Bax
BaxBax
How does AP-2induce apoptosis?
Does AP-2 has any role in cancer cell Chemosensitivity ?
p53
50% of primary tumor have mutated p53
Major chemosensitivity determinant
Gets activated upon DNA damage and induces apoptosis
irradiation
Chemotherapy
Need for identification of other determinants
- Chemodrug- Ad-LacZ + Chemodrug - Ad-AP2 + Chemodrug
0
20
40
60
80
100
120
Adriamycin
Etoposide
Cisplatin
Taxol
Carboplatin
Per
cen
t IC
50
Cells
O/N
Mock/Ad-LacZ/Ad-AP-2
6 hrs
Add chemo
48 hrs
% Live cells
Chemosensitivity of cancer cells over expressing AP-2
Tet-Off system
Transcription is turned off by tet
- Tet
AP-2under Tet-responsive element
P min CMVTRE AP-2
tTA
active
tTA expressing adenovirus
pCMV
tetR VP16
tTA
Tet bound tTA
Tet
+ Tet
inactive
tTA – tetracyclin controlled transactivator
AP-2
tTA + + + + + +
SW480-1 SW480-2 SW480-3
1 2 3 4 5 6
Tet + - + - + -
-Chemodrug-Chemodrug + tTA + Tet-Chemodrug + tTA + 0.1 g Tet
0
20
40
60
80
100
120
Per
cen
t IC
50
Adriamycin
Etoposide
Cisplatin
Taxol
Carboplatin
Ad-tTA + Tet Ad-tTA
% A % S % S% AHrs
0122448
40.2844.8538.7537.20
40.2813.36 5.71 2.35
2.641.011.400.25
2.64 0.54 3.3436.02
0.00
1
tTA
1 0.1
0.01
-
Tet (g/ml)
+ + + + +
AP-2
Actin
1 2 3 43X-AP2CAT pSG5pSG5/AP2tTATet
++ - - -
+ -+ - -
+ - - ++
+ - - + -
% C
AT
Con
vers
ion
0
5
10
15
20
25
AP-2 binding sites
CAT
3X AP2-CAT
Controlled expression of AP-2increases the cancer cell chemosensitivity
Adria tTA Tet
IC25
IC50
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
B
+--
++1 g
++0.1 g
AP-2 expression sensitizes cells to undergo apoptosis upon chemotherapy
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
Control/ 0 hr
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
10
11
02
10
31
04
FL1
-H -
->
tTA - +Tet - 0.1 g
MOCK AP-2
A
G1 G2A
S S
AdriatTATet
---
-+
0.1 g
0
5
10
15
20
25
30
35
40
45
1 2 3 4 5 6 7 8IC25
--
IC50
--
IC25
+1.0 g
IC50
+1.0 g
IC25
+0.1 g
IC50
+0.1 g
AP-2Adria Adria Adria+ AP-2
- % <G1
- % S
% A
pop
tosi
s/%
DN
A s
ynth
esis
0
10
20
30
40
50
60
70
ChemotTATet
---
-+
0.1 g
IC25
--
IC50
--
IC25
+1.0 g
IC50
+1.0 g
IC25
+0.1 g
IC50
+0.1 g
AP-2Chemo Chemo Chemo+ AP-2
- Cisplatin
- Taxol
- Etoposide
1 2 3 4 5 6 7 8
% A
pop
tosi
s
AP-2 expression sensitizes cells to undergo apoptosis upon chemotherapy
Taxol
Cisplatin
Adriamycin
0 24 48 72 hrs
AP2
ActinEtoposide
AP2
Actin
AP2
Actin
AP2
Actin
What is the role of endogenous AP-2 in chemosensitivity ?
48 hrs
GPDH
AP-2
72 hrs
Unt
reat
ed
Adr
iam
ycin
Cis
plat
in
Eto
posi
de
Tax
ol
Mar
ker
Unt
reat
ed
Adr
iam
ycin
Cis
plat
in
Eto
posi
de
Tax
ol
- Control
- Mock
- Lamin siRNA
- AP-2 siRNA
0
10
20
30
40
50
60
70
80
90
EtoposideAdria Cisplatin Taxol%
Via
bili
ty
What is the role of Chemotherapy induced AP-2in Cancer cell chemosensitvity?
GAPDH
AP-2
Con
trol
Moc
k
siRN
A L
amin
A/C
siRN
A A
P-2
1 2 3 4
Lamin A/C
- Moc
ksiR
NA Lam
in A
/C
siRNA A
P-2
Contro
l
AP-2
Actin
- + + + + Adria
1 2 3 4 5
AP-2
Actin
- + + + + Cisplatin
AP-2
Actin
- + + + + Taxol Chemotherapy induced AP-2 contributes to chemosensitivity
Chemotherapy induced AP-2 contributes to chemosensitivity
Plate cells
TransfectsiRNA
Add Adria
Stain the colonies
O/N
2 days
2weeks
Control
Mock
Lamin/siRNA
AP-2siRNA
0 0.1 0.2 0.4 0.8 1
Adriamycin g/ml
AP-2expression
+
+
-
In cell culture, silenced AP-2 is re-expressed by methylation inhibitor (5-aza-2 deoxycytidine)
Normal breast epithelium
Ductal carcinoma in situ (DCIS)
Invasive breast tumors
AP-2 expression and Breast Cancer progression
Effect of re-expression of silenced AP-2 on chemosensitivity ?
Hypermethylation of AP-2promoter
-
+ 75% (12/16)
(Douglas et al., 2004)
+ 16% (3/19)
5aza2dC induced re-expression of epigenetically silenced AP-2 in MDA-MB-231 cells increases the chemosensitivity and is AP-2 dependent
- + - +SW480MDA-MB-231
5aza2dC
AP-2
Actin
1 2 3 4
MDA-MB-231
- MockLamin siR
NA
AP-2 siR
NA
Control
- + + + + 5aza2dC
1 2 3 4 5AP-2
Actin
0
10
20
30
40
50
60
70
1 2 3 4 5Chemotherapy5aza2dCMockLamin siRNAAP-2 siRNA
% V
iab
ilit
y
- Adriamycin
- Cisplatin
5aza2dC fails to induce apoptosis in AP-2 siRNA transfected MDA-MB-231 cells upon chemotherapy
ControlMockLamin siRNAAP-2 siRNA
2.01.41.20.8
5.47.35.46.3
0.70.70.20.8
64.756.457.213.9
% Apoptosis
Adria5aza2dC
--
+-
-+
++
Con
trol
Moc
k
Lam
in s
iRN
A
AP
-2s
iRN
A
AP-2
PARP
Actin
Con
trol
Moc
k
Lam
in s
iRN
A
AP
-2s
iRN
A
Con
trol
Moc
k
Lam
in s
iRN
A
AP
-2s
iRN
A
Con
trol
Moc
k
Lam
in s
iRN
A
AP
-2s
iRN
A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
- adria + adria - adria + adria
+ 5aza2dC- 5aza2dC
Channels (FL2-H)0 50 100 150 200
Num
ber
01
00
20
03
00
40
05
00
Channels (FL2-H)50 100 150 200 250
Num
ber
070
14
021
0280
Channels (FL2-H)0 50 100 150 200
Num
ber
01
00
20
03
00
40
0
Channels (FL2-H)0 50 100 150 200 250
Num
ber
070
14
021
028
0
Channels (FL2-H)0 50 100 150 200
Num
ber
01
00
20
03
00
40
05
00
Channels (FL2-H)0 50 100 150 200 250
Num
ber
070
14
021
028
0
Channels (FL2-H)0 50 100 150 200
Num
ber
01
00
20
03
00
40
05
00
Channels (FL2-H)0 30 60 90 120 150
Num
ber
080
160
240
320
-Ad
ria
+A
dri
a
+ 5
aza2
dC
AP-2siRNA
Control MockLamin siRNA
+A
dri
a
- 5a
za2d
C
Channels (FL2-H)0 50 100 150 200
Num
ber
08
01
60
24
03
20
Channels (FL2-H)0 50 100 150 200
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 50 100 150 200
Num
ber
08
01
60
24
03
20
Channels (FL2-H)0 50 100 150 200
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 50 100 150 200
Num
ber
08
01
60
24
03
20
-Ad
ria
Channels (FL2-H)0 30 60 90 120 150
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 50 100 150 200
Num
ber
010
020
030
040
0
Channels (FL2-H)0 30 60 90 120 150
Num
ber
080
160
240
320
0
1.0
2.0
3.0
4.0
Tu
mor
vol
um
e (1
000
X m
m3 )
Days
5.0
6.0
0 5 10 15 20
8.0
7.0
25 30
5aza2dC treatment inhibits the tumorigenicity of MDA-MB-231 cells upon chemotherapy in an AP-2 dependent manner
- Control
- 5aza2dC - Adria- Mock + Adria + 5aza2dC- Lamin siRNA + Adria + 5aza2dC- AP-2 siRNA + Adria + 5aza2dC
MDA-MB-231
Treatment No of tumors/ Mean volume (%)No of mice mm3 ± SE
Control 3/3 6426 ± 1118 1005aza2dC 3/3 5100 ± 291 79Adria 3/3 2348 ± 1172 37Mock + 5aza2dC +Adria 0/4 0 ± 0 0Lamin siRNA + 5aza2dC+ Adria 0/4 0 ± 0 0AP-2 siRNA + 5aza2dC + Adria 5/5 1832 ± 300 29
5aza2dC treatment inhibits the tumorigenicity of MDA-MB-231 cells upon chemotherapy in an AP-2 dependent manner
AP-2 overexpression increases the chemosensitivity of cancer cells
Conclusions
5aza2dC induced re-expression of AP-2 in breast cancer cells increases chemosensitivity and inhibits tumorigenicity upon chemotherapy
Chemotherapy induces endogenous AP-2, which contributes to chemosensitivity
AP-2 sensitizes cancer cells undergo apoptosis upon chemotherapy
0
20
40
60
80
100
120
0 20 40 60
- Ad-LacZ
- Ad-AP2
% V
iab
ilit
y
MOI
Hrs
2448
2448
%A %G1 %S %G2 2.04 57.32 26.19 14.45 1.08 58.52 25.48 14.92 1.71 82.45 3.24 12.6042.44 32.51 6.30 16.75
Ad-LacZ
Ad-AP2
Virus
24 hr
48 hr
Ad-LacZ Ad-AP2
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
101
102
103
104
FL1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
101
102
103
104
FL1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
101
102
103
104
FL1-H
-->
R2
2 3
4 5
10 30 50 70 90 110 130 150 170 190 210 230 250
FL2-H -->
101
102
103
104
FL1-H
-->
G1A G2
S S
PI-DNA content
Brd
u-D
NA
syn
thes
is
AP-2 inhibits cancer cell growth by inducing cell cycle arrest and apoptosis
Wajapeyee and Somasundaram, 2003 JBC
Apoptosis
Intrinsic Extrinsic
Adapter
Apoptosis induction-two pathways
AP-2Bcl-2
Bax
BaxBax
Wajapeyee and Somasundaram, 2006 JBC
How does AP-2 induce apoptosis?
How does AP-2 inhibit cell cycle progression?
Normal cells: HEL299 cells – human normal lung fibroblasts
0
24
48
2448
>G1 G1 SG2/Mhr
4.30
4.58
2.17
3.781.59
53.10
54.46
54.26
70.9075.86
18.56
20.36
19.61
9.08 6.34
23.76
19.73
25.02
16.6016.20
Mock
Ad-LacZ
Ad-AP2
Virus
Cell cycle profile
Ad-LacZ Ad-AP2
DA
PI
AP
-2
Brd
U
a b
c d
e f
Brdu incorporation
Serum Starvation
-48hrs
Serum Stimulation (Release)
0 hr
Virus infection
-16
FACS &Western
+24 hrs
FACS &Western
+18 hrs
Moc
k
Release 0 hrs Release 18 hrs Release 24 hrs
G1: 81.70S: 9.21G2: 9.09
G1: 73.68S: 9.74G2: 16.58
G1: 42.20S: 26.42G2: 31.38
Ad
-Lac
ZA
d-A
P2
G1: 87.54S: 6.11G2: 6.35
G1: 75.41S: 8.22G2: 16.36
G1: 42.06S: 26.71G2: 31.23
G1: 89.95S: 4.54G2: 5.62
G1: 90.61S: 3.61G2: 5.71
G1: 85.57S: 5.25G2: 9.18
Channels (FL2-H)0 50 100 150 200
Num
ber
020
040
060
0
Channels (FL2-H)0 50 100 150 200
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 40 80 120 160 200
Num
ber
050
100
150
200
Channels (FL2-H)0 50 100 150 200
Num
ber
030
060
090
012
00
Channels (FL2-H)0 50 100 150 200
Num
ber
010
020
030
040
050
0
Channels (FL2-H)0 50 100 150 200
Num
ber
050
100
150
200
Channels (FL2-H)0 50 100 150 200
Num
ber
020
040
060
080
010
00
Channels (FL2-H)0 50 100 150 200
Num
ber
020
040
060
080
0
Channels (FL2-H)0 50 100 150 200
Num
ber
020
040
060
0
Regulation of Cell cycle
E2F
pRB
SenescenceSerum starvationCell-to-cell contactGrowth inhibition
E2F
P P P
pRB
DNA damagep53
CDC2
CDC2
CDC2
CDK4
Ad-LacZ Ad-AP2
CDK6
CDK2
1 2 3 4 5 6 7 8 9 10
Cyclin D2
Cyclin B
Cyclin D1
Cyclin E1
Cyclin E2
p21
p27
p57
AP-2
Actin
pRB
AP-2 represses Cyclin D2
Hrs after serum release
Mock Ad-LacZ Ad-AP2
AP-2
Cyclin D2
Actin
0 18 24 0 18 24 0 18 24
Con
trol
Con
trol
Con
trol
1 2 3 4 5 6 7 8 9 10 11 12
Cyclin E1
Cyclin B
Cyclin D1
AP-2 represses Cyclin D2
Hrs after virus infection
-10
-5
0
5
10
15
Fol
d c
han
ge
0 6 12 24 36
-AP-2
-Cyclin D2
Cell cycle
E2F
pRB
SenescenceSerum starvationCell-to-cell contactGrowth inhibition
E2F
P P P
pRB
DNA damagep53
CDC2
CDC2
100 96 24 12
CDK4
Cyclin D2
32P-GSTpRB
Ad
-Lac
Z
Ad
-AP
2
Con
trol
Ad
-p21
IP: CDK4
1 2 3 4
0
20
4060
80
100
120
Inte
nsit
y
100 86 54 21
Ad
-Lac
Z
Ad
-AP
2
Con
trol
Ad
-p21
IP: CDK6
1 2 3 4
CDK6
Cyclin D2
32P-GSTpRB
0
20
4060
80
100
120
Inte
nsit
y
32P-Histone H1
Cdk2
Cyclin E1
Ad
-Lac
Z
Ad
-AP
2
Con
trol
Ad
-p21
IP: CDK2
1 2 3 4
100 100 90 400
20
4060
80
100
120
Inte
nsit
y
100 96 84 32
Ad
-Lac
Z
Ad
-AP
2
Con
trol
Ad
-p53
IP: CDC2
1 2 3 4
0
20
4060
80
100
120
Inte
nsit
y
32P-Histone H1
Cdc2
Cyclin B1
AP-2 inhibits CDK4 and CDK6
Plate cells
Infect withviruses
IP: CDK
O/N
24 hrs
Kinaseassay32P-ATP
Substrate
AP-2failed to inhibit transcription from Cyclin D2 promoter
How does AP-2 repress cyclin D2 ?
E-box Cyclin D2Promoter of
Cyclin D2
C-myc
AP-2 site
AP-2
x
E-box Cyclin D2Promoter of
Cyclin D2
C-myc
J
C-myc mediated activation of cyclin D2 is inhibited by AP-2
0
20
40
60
80
100
1 2 3 4 5
Lu
cife
rase
act
ivit
y(C
PM
X 1
000)
Cyc D2 Promoterc-MYCAP-2
+--
++-
++
++
++
120
MYC
AP-2
Actin
Ad-MYC
0 24 36 48
Ad-AP2+ Ad-MYC
0 24 36 48 Hrs
Cyclin D2
Ad-LacZ
0 24 36 48
Cyclin D1
0 2 3 4 5 6 7 8 9 10 11 12
LUCCyc D2
promoter
AP-2binds to a overlapping AP-2/c-myc binding sequence
-1886 AP-2
E-box-1596 to -1591
AP-2-1604 to -1595
197 bps-1645 to -1448
+1
5'-GCCcgctGCACGTG-3
E-box
GST-AP-2 Cyc D2 wtSV40 AP2 wtSV40 AP2 m
----1
+---2
+
--3
+
--4
+
--5
+-
-6
+-
-7
+-
-8
+--
9
+--
10
+--
11
Cyc D2 wt 5'-GCCAT GCC CGCT GCA CGTGCC AGCTTGGC-3'
GCC N(4) GGC AP-2 consensus binding motif
E-box
AP-2 site is a bonafide element
Mar
ker
Gen
omic
DN
A
Inp
ut
Lam
in
AP
-2
Myc
Ad-Myc
Ad-Myc+Ad-AP2
1 2 3 4 5 6
AP-2 binding interferes with c-myc
CycD2
Actin
SW
480/
Neo
SW
480/
Cyc
D2
# 15
SW
480/
Cyc
D2
# 5
SW
480/
Cyc
D2
# 11
SW
480/
Cyc
D2
# 26
1 2 3 4 5
SW480/Neo SW480/CycD2 # 15
% B
rdU
in
corp
orat
ion
0
10
20
30
40
50
60
70
80
90
-Ad-AP2
-Ad-LacZ
1 2 3 4
How important cyclin D2 repression is?
G1 S G2/MHr
0
121824
121824
60.77
58.79 63.13 57.63
74.97 78.91 86.55
36.44
38.63 30.90 36.79
22.51 17.85 9.74
2.79
2.58 5.97 5.59
2.52 3.21 4.71
Ad-AP2
Virus
Ad-LacZ
Control
SW480/Neo
0
121824
121824
56.88
61.40 60.50 65.17
58.38 57.25 58.86
39.19
34.91 33.30 31.02
35.94 35.60 34.90
3.93
3.69 4.07 3.80
5.67 7.15 6.14
G1 S G2/MHr
Ad-AP2
Virus
Ad-LacZ
Control
SW480/Cyc D2 # 15
D-type cyclin-CDK4 complexes can suppress the skeletal muscle differentiation
What is the significance?
Irreversible cell cycle arrest is a key component of myogenic differentiation
Cyclin D1, cyclin D2 and to some extent cyclin D3 overexpression can block differentiation of myoblasts to myotubes
Myoblast Myotube
DM
C2C12 MUSCLE DIFFERENTATION MODEL SYSTEM
DMDM+
NS siRNADM+
AP-2 siRNAGM10
X40
X10
X10
0X
AP-2 is is needed for differentiation
Days in DM
AP-2
MYC
Cyclin D2
MHC
GAPDH
C2C12 NS siRNA
C2C12 AP-2 siRNA
0 3 5 7 0 3 5 7
1 2 3 4 5 6 7 8
C2C12 NS siRNA
C2C12 AP-2 siRNA
1 2 3 4 5 6 7 8
Days in DM
AP-2
Cyclin D2
MHC
GAPDH
0 3 5 7 0 3 5 7
Cyclin D2
C-myc
AP-2
A possible simple hypothesis !!!
Myoblast Myotube
Myoblast
Myotube
GM
GM + AP-2
DM
DM + c-myc
DM + cyc D2
DM + AP-2 + c-myc
DM + AP-2 + cyc D2
DM + c-myc + cyc D2 siRNA
√
ΧΧ
Χ
√
√
√
Χ
GM
GM
+ A
P-2
10X 100X
a
b
c
d
Forced AP-2 expression induces myogenic differentiation
DM
DM + Cyclin D2
DM + cMYC
10X 100Xa g
b h
c i
DM + MYC + AP-2
DM + Cyclin D2 + AP-2
DM + MYC + Cyclin D2 siRNA
d j
e k
f l
10X 100X
AP-2downregulates c-Myc mediated induction of cyclin D2
Cancer of striated muscle tissue
There are three major forms:
alveolar rhabdosarcoma - most often afflicts adolescents, typically develops in the extremities, body or eye cavities.
embryonal rhabdosarcoma – occurs in infants and young children, develops in the head, neck, extremities or lower genitourinary tract.
pleiomorphic rhabdosarcoma – occurs in adults and typically develops in the extremities
Rhabdosarcoma
AP-2 is induced during muscle differentiation
De-differentiation results in cancer
Loss of AP-2 leads to rhabdosarcoma !!!
-1000 +800
1 47N
P1
P2
P3
P4
Bisulfite sequencing
N P1 P2 P3 P4
AP
-2
fol
d do
wnr
egul
atio
n
0
5
10
15
20
25
30
AP-2
AP-2 is silenced by promoter methylation
AP-2 is induced during muscle differentiation and is needed for differentiation
Loss of AP-2 expression leads to rhabdosarcoma development
Can one reverse the AP-2 expression-methylation/ase inhibitors?
Will it reverse the tumorigenic phenotype of rhabdosarcoma?
Therapeutic value!!!
