Synergy Team 4

1
Synergy Team 4

• Project F Cholesterol-dependent Prostate Cancer
  Cell Survival Mechanisms Freeman
• Project G Studies on Inhibitors of Mitogenic
  Signal Transduction in Prostate Cancer
• Rosen, Sawyers
• Project H Signal Defects Involved in Recurrent
  Prostate Cancer Hsieh
• Links to TGF-b Signaling in Prostate Cancer
• Guise, Chirgwin

2
Activation of the Phosphoinositide-3-kinase/Akt
Pathway in Prostate Tumor Cells
LNCaP
PC3
MC3T3
Akt
Akt308-P
PTEN
3
Does Cholesterol Play a Role in Prostate Cancer?
Project F Cholesterol-dependent Prostate Cancer
Cell Survival Mechanisms

• Cholesterol accumulates in BPH and PCa
• PCa aggressiveness is linked to a Western diet,
  including high fat diets and consumption of
  animal products
• Caveolin-1 expression is linked to aggressive PCa

4
High Levels of Circulating Cholesterol Promote
Growth Of LNCaP Xenografts
normal
high cholesterol
normal
high cholesterol
2
100
30/32
80
tumor volume (cm 3)
0.5
60
14/32
tumor take (percent)
40
0.25
20
0
normal
high cholesterol
0
normal
high cholesterol
5
EGFR Activation Occurs in Lipid Rafts in LNCaP
Cells




Cholesterol




Filipin




EGF
Tn-S
Tn-S
Tn-S
Tn-S
Raft
Raft
Raft
Raft
p-EGFR
EGFR
Zhuang et al. Cancer Res. 622227-2231, 2002
6
Lipid Rafts and Caveolae
Flat Rafts
GPI
Src
Cytosol
50 - 100 nm
Caveolae
GPI
Src
Cytosol
7
Lipid rafts exist in caveolin-negative prostate
cancer cells
Triton-S
Raft
Triton-S
Raft
Fyn
Gi?3
Cav-1
PC-3
LNCaP
Zhuang et al. Cancer Res. 622227-2231, 2002
8
Targeting Cholesterol In LNCaP Cell Membranes
Inhibits Akt Signaling
Cholesterol






EGF












Filipin
Filipin (µg/ml)
0
0
0.5
2.0
1.0
4.0
EGF (ng/ml)
p-EGFR
p-Akt
EGFR
Akt
p-Akt
Akt
9
1
2
3
4
5
6
8
7
10
11
12
Zhuang et al. Cancer Res. 622227-2231, 2002
9
Targeting Cholesterol In LNCaP Cell Membranes
Induces Apoptosis
LY








Filipin
5.0
4.0
Relative apoptotic index
3.0
2.0
1.0
SFM
LY
LYEGF
Fl
Fl EGF
FlCh EGF LY
1
2
3
4
Zhuang et al. Cancer Res. 622227-2231, 2002
10
Simvastatin (5mM) inhibits Akt activity and
stimulates apoptosis in LNCaP cells
2.5
Control
2
Sim
Akt-P
1.5
Absorbance A405-A490
1
Akt
0.5
0
1
2
3
4
5
Days
11
Addition of cholesterol to cell membranes
inhibits the apoptotic effect of simvastatin
6
5
4
Apoptotic index
3
2
1
0
Control
Sim
Simchol
Treatment
12
Increased Akt activation and reduced apoptosis in
LNCaP tumors in SCID mice fed a high cholesterol
diet
P-Akt
TUNEL
Normal diet
High cholesterol diet
13
The Akt Serine-Threonine Kinase
PH
Catalytic Regulatory
14
Cholesterol depletion from rafts differentially
results in dephosphorylation of Akt at the T308
site
Filipin
FBS
Ctrl
Akt
Akt T308-P
Akt S473-P
15
T308-P form of Akt is enriched in the raft
compartment
Raft
B
Ab
Akt
Akt
IgG H
Akt
T-308P
IgG H
Akt
S-473P
IgG H
Akt 308P
IP
293/T7-Akt WT
16
Akt distribution 24 h serum withdrawal
Triton soluble
Triton soluble
Raft
Raft
Akt
Akt
Akt T308-P
Akt T308-P
Akt S473-P
Akt S473-P
LNCaP
MC3T3
LNCaP
MC3T3
PTEN
Actin
17
Akt Residing in the lipid raft fraction of LNCaP
cells is functionally inhibited
Input
TS
Raft
Akt Kinase Assay
p-S473-Akt
TS
Raft
p-T308-Akt
p-GSK-3 ?/?
0.05
1.0
Akt
?-Tubulin
Akt
0.60
1.0
Gi?-2
IP anti-Akt (1G1)
18
Constitutively activated Akt (Myr-Akt) is
functionally inhibited in the lipid raft
compartment
IP controls
pcDNA3.1
Myr-Akt
Cy
M
Raft
Cy
M
B
Ab
Raft
p-GSK3 a/b
Akt
LNCaP
19
Project H Signal Defects in Recurrent Prostate
Cancer
DOC-2/DAB2 and its interactive protein (DIP1/2)
as tumor suppressors in human prostate cancer
cells
Lymph node
Normal
Primary
Bone
MDAPCa 2b
MDAPCa 2a
PZ-HPV-7
SWNPC-2
TSU-Pr1
LNCaP
SWPC-2
SWPC1
C4-2
PrEC
DOC-2/DAB2
DIP 1/2
Actin
20
The Inhibitory Effect of DOC-2/DAB2 on Human
Prostate Cancer
0.7
C4-2
0.6
neo-4
0.5
p59-18R
0.4
Relative cell number
0.3
0.2
0.1
0
1
2
3
4
5
6
7
8
9
Time (day)
21
Predicted Functional Domains in DOC-2/DAB2
Protein
1
500
600
100
200
300
700
400
768
230
448
25
28
42
180
G493
S494


663
667
619
624
714
719
Y50
Y38
Y136
Y170
Y342
Y366
Y385
504
507
512
515
Actin binding motif
SH3-binding site (proline-rich)
ERK phophorylation site
Dab homolog domain
Splicing variant
22
The inhibitory action of DOC-2/DAB2 on c-Src
kinase activation
DOC-2/DAB2
DOC-2/DAB2
Proline-rich domain
SH3
SH3
Kinase
Kinase
Inactive c-Src
Y416
Y416
SH2
SH2
Y527
Y527
EGF treatment
Kinase
Y416
Active c-Src
Substrate
527
SH2
Y
Y non-phosphorylated Y phosphorylated
23
Competition between AR DOC-2 to the binding of
Src
DOC-2 - - -
- (0.2 µg)
AR (µg) - 1.6 - 0.4 0.8 1.6 - 1.6
- 0.4 0.8 1.6
R1881 (10nM) - -
- -
GST-Src (SH3) IB DOC-2/DAB2
WB AR
WB DOC-2/DAB2
24
The structure of hDAB2IP (DIP1/2) protein
1
21
110
135
438
727
736
842
861
967
C2
RasGAP
PR
LZ
21-110 C2 (protein kinase C conserved region 2 )
135-438 GAP (Ras-GTP--gtRas-GADP)
727-736 proline rich domain (protein/protein
interaction)
842-861 Leucine zipper (dimerization)
25

Inhibition of Ras-mediated Raf-1 activation by
DIP1/2 in human prostate cancer cells
- -
EGF
Ras
- -

DIP 1/2
IP aRaf-1 IB aRAS
Control IB

aRAS
aDIP1/2
aRaf-1
26
Structure of hDAB2IP gene (9q33.1-33.3)
419H3
298 A17
Ib
II III IV V VI
VII VIII IX X XI
XII XIII XIV XV
Exon
Ia
12
185
48
49
79
67
60
296
51
65
29
24
2
Intron
1a 1b 2
3 4 5 6 7
8 9 10 11
12 13 14

• 15 exons and 14 introns (9.6kb)
• 967 amino acids
• 94.2 homology with rDIP1/2 protein

27
Down regulation of hDAB2IP in prostate cancer
cells
Bsu36I
NotI
EcoRI
SmaI
KpnI
Sfi
NheI
XhoI
EcoRI
Kpn2I
-
229 636
2848
598 -359 -141 44 129
981
-4621
Exon Ia
(1-88)
Exon Ib
(1228- 1732)
Full-length
-598
981
pGL3-1.6S
Luc
P2
-598
44
pGL3-0.6S
Luc
P1
229
981
pGL3-0.8S
Luc
pGL3-1.6S(full-length)
pGL3-0.6S(P2)
pGL3-0.8S(P1)
cell lines
17.89

2.41
45.00

4.41
33.25

6.21
PZ-HPV-7
2.39

0.90
2.90

0.57
4.45

0.35
PC 3
3.99

0.60
5.18

0.34
0.94

0.11
LNCaP
28
2003 Publications from Synergy Team 4 of
Relevance to the Consortium

• Yang et al., MicroPET imaging of prostate cancer
  in LNCAP-SR39TK-GFP mouse xenografts. Prostate
  5539-47, 2003
• Bianco et al. Loss of PTEN/MMAC1/TEP in EGF
  receptor-expressing tumor cells counteracts
  antitumor action of EGFR tyrosine kinase
  inhibitors. Oncogene 222812-2822, 2003
• Solit et al. Inhibition of heat shock protein 90
  function downregulates Akt kinase and sensitizes
  tumors to Taxol. Cancer Res. 632139-2144, 2003.
• Chiosis et al. 17AAG Low target binding affinity
  and potent cell activity-finding an explanation.
  Mol Cancer Ther. 2123-129, 2003
• Lin and Freeman, Transactivation of ErbB1 and
  ErbB2 receptors by angiotensin II in normal human
  prostate stromal cells. Prostate 541-7, 2003
• Zhou et al. Characterization of a novel negative
  regulator (DOC-2/DAB2) of c-Src in normal
  prostatic epithelium and cancer. J. Biol. Chem.
  2786936-6941, 2003
• Chen et al. Epigenetic regulation of a novel
  tumor suppressor gene (hDAB21P) in prostate
  cancer cell lines. J. Biol. Chem. 2783121-3130,
  2003
• Kang et al. A multigene program mediating breast
  cancer metastasis to bone. Cancer Cell 3537-549,
  2003

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