Transcript Document

Rad51 over-expression contributes to
chemoresistance in human STS; A role
for p53/AP2 transcriptional regulation
J. Hannay, J. Liu, S. Bolshakov, D. Yu,
A. Lazar, R. Pollock, D. Lev
Sarcoma Research Center
University of Texas, MD Anderson Cancer Center
Overall 5YS rate – 46.5%
Have we made much progress?
Overall 5YS – 50%
STS are chemoresistant: Modest
Response Rates; Significant Toxicity
DTIC 15%, IFF 20%, DOX 20-30%
Combination therapies 20-50% - (short lived responses,
only limited improvement in survival):
•DOX/IFF combinations
•Sequential hi dose IFF/hi dose DOX
+ DTIC + /- CY
•MAID + growth factor
•Gem + taxotere
Tumor cell properties/ tumor microenvironment
The complexity of fighting chemoresistance:
• Tumors are heterogeneous
• Multiple chemoresistance mechanisms may be operative
in any specific tumor cell
• Tumor cells are genetically unstable
• New molecular mechanisms develop during the
progression of the tumor
p53 and STS chemoresistance
• p53 gene mutation or dysfunction (e.g., MDM2)
are the most frequent known alteration in STS
• Found more frequently: metastases vs. primary
tumor, high-grade vs. low-grade.
•Significant negative impact on both overall and
sarcoma-specific survival
• Studies from other tumor systems suggest a
connection between mutp53 and resistance to
chemotherapy
Re-expression of wtp53 in mutp53 STS cell lines
results in significant sensitization to Dox – in vitro
And in vivo…
PKC-α
P-gp
Midkine
Mut.
p53
Rad-51
SKLMS1
SW872
A204
TKR
Anti-Rad51
Hs27
SK
EGFR
PDGFR
HT
RD
A204 SW684 SW 872 HUVEC
STS chemoresistance: Rad51
• Homologous
recombination (HR) is crucial for
the repair of complex forms of DNA damage such
as double-strand breaks (DSBs)
• Over-expression of Rad51, the key factor of HR
has been observed in a number of tumors such as
breast and pancreatic cancer to correlate with
chemoresistance
Rad-51 is overexpressed in STS Cell lines, Xenografts
and Tumors
Normal rat
testis
SKLMS-1
xenograft
Rad51
β-actin
MFH - p
MFH - p
ASPS - r
FS - m
RMS - p
RMS - m
LPS - p
LPS - r
Prognostic marker?
•Rad-51 expression possibly correlates with survival:
High and moderate expressers had a 31% and 47% 5YS
rate, whereas 66% of low expressers survived five years
(Fisher’s exact; p < 0.05).
• In most of the IHC positive tumors Rad-51 could be
identified in the cytoplasm
Hs27
SKLMS1
SW872
A204
Rad-51 impacts on STS chemosensitivity:
SKsiRad51
SHAR51 Lipofect’ 14-3-3ζ scram
-c1
alone +ve cont -ve cont 100nM 200nM 400nM
A
β-actin
HARad51
Rad51
B
SKLMS1 chemosensetivity to 48h Low Dose Doxorubicin
Following 20nM siRNA Rad51 pretreatment.
β-actin
HARad51
Rad51
140
120
% viability
100
80
60
No therapy
20nM SCR
20nM siRad51
40
20
0
0
0.001
0.01
Doxorubicin (uM)
0.1
SKSHAR51 scram
-c1
-ve cont
siRad51
100nM 50nM
20nM
10nM
1nM
Rad51 translocates to the nucleus within 1 hour
of Dox exposure in SKLMS-1
DAPI
Rad51
No DOX
1µM DOX – 1h
No Dox, t=0
24h Dox
48h Dox
24h
0
Rad51
β-actin
0
Dox
48h
0
Dox
p53/Rad51
• p53 mutant cells exhibit elevated rates of
spontaneous and induced HR and increased
Rad51 expression
• Rad51 regulation by wt p53 is thought to be
transcription-independent; binding of wt p53
to Rad51 inhibits its function and results in
increased degradation of the protein complex
Induction of wt p53 leads to suppression of Rad51
protein levels
AdEV
MOI
FLAGp53
0
1
AdFLAGp53
10 50 100 500
0
1
10
50 100 500
MOI
FLAGp53
Rad51
Rad51
p21
p21
ß -actin
ß -actin
AdFLAGp53 - RD
AdFLAGp53 - A204
0
0
1
10 50 100 500
1
10 50 100 500
Rad51 suppression by wt p53 does not appear to be
strictly due to enhanced proteosomal degradation
AdLacZ
DMSO
MG132
AdFLAGp53
DMSO
MG132
Hours mk 0 2 4 6 0 2 4 6 mk 0 2 4 6 0 2 4 6
FLAGp53
Rad51
ß-actin
Induction of wt p53 leads to suppression of
Rad51 mRNA transcript generation
SK
neo
AdLacZ
Ala10 Ala14 Ala21
°C 32 38 32 38 32 38 32 38 32 38
VP:cell mock 20
Rad51
Rad51
p21
p21
GAPDH
GAPDH
AdFLAGp53 wt
200 2000 20
• Next we showed the wt p53 did not shorten Rad51
mRNA half life
•Does wt p53 transcriptionally represses Rad51
promoter?
200 2000
-403
Genebank sequence of the Rad51
promoter was screened
 (DNASys software): no classical p53
binding sites were found
-295
-185
-50 +1 +63
Rad51/Luc

Potential AP-2 binding site
Potential SP-1 binding site
Potential core enhancer
Potential Ets-1 binding site
Potential E2A binding site
Reported p53 responsive element
wt p53 leads to suppression of rad51 promoter activity
40
SKLMS1/38C
SKLMS1/32C
Ala14/38C
Ala14/32C
Relative luciferase activity
35
30
25
20
15
10
5
0
pRad-403Luc
Using truncation mutants of the rad51 promoter we were further able
to show that the p53 responsive element is in the –295 to –185 region
Relative luciferase activity
0
-403
+1 +63
Luc
pRad-403Luc
Luc
pRad-295Luc
Luc
pRad-185Luc
Luc
pRad-50Luc
Luc
Basic control
-295
-185
putative AP2 site
-50
200
300
400
500
600
700
800
900
mutant p53
putative p53 site
putative SP1 site
100
wild-type p53
This region contains an
AP-2 binding site which
when mutated, the wtp53
effect is eliminated,
suggesting that the wtp53
repressive effect is
mediated via AP-2
Relative luciferase activity
10
Mock
9
AdLacZ
8
AdFLAGp53
7
6
5
4
3
αAP2/
AP2/
probe
2
1
0
AP2wt
AP2del
Basic
AP2/
probe
pRad-403LUC based promoter constructs
Reintroduction of wtp53 increases
AP-2 binding to the rad51 promoter
Free
probe
Future investigations:
• How do p53 and AP-2
cooperate to repress the
Rad-51 promoter?
• Does AP-2 play a role
in STS chemoresistance?
Many thanks to:
Theresa Nguyen
Jeffery Liu
Parimal Das
Jonathan Hannay
Svetlana Bolshakov
Dhana Kotillingam
Borys Korchin
Wen-Hong Ren
Raph Pollock
Alex Lazar
Zeming Jin