Interactions Between Vitamin D and Androgen Receptor Signaling in Prostate Cancer Cells Nancy L. Weigel, Ph.D. Baylor College of Medicine
Jan 14, 2016
Interactions Between Vitamin D and Androgen Receptor Signaling in Prostate Cancer Cells
Nancy L. Weigel, Ph.D.
Baylor College of Medicine
Androgen Receptor in Prostate
GF
DHTStromal Cell Androgen Receptor is Required for Prostate Development
Androgen Receptor is Active in Adult Stromal and Epithelial cells
AR AR AR AR AR AR AR AR AR AR AR
AR
AR
AR
AR
AR
AR
AR
AR
PSA
DHT
Prostate Cancer
• Primary tumors are androgen dependent and express PSA
• Tumors treated with androgen ablation
• Tumors become ablation resistant—express PSA
• Tumors still androgen receptor dependent?
LNCaP and C4-2 Human Prostate Cancer Cells
• Isolated from lymph node metastasis• Androgen dependent in vivo• Express AR (mutant)• Express wt p53
• Derived from LNCaP cells• Androgen independent in vitro and in vivo
LNCaP
C4-2
Androgen Dependence of LNCaP Cell Growth
Zhao et al. (1997) Endocrinology 138:3290-8 Fig 3A
0
300000
600000
900000
12h 24h 48h
CP
M
C
si
Androgen Receptor is Required for Growth of Androgen Independent C4-2 Cells
AR
AR siRNA Reduces PSA Expression in C4-2 Cells
Actin
PSA
Csi ARsi
0
10
20
30
PC-3
0
100
200
300 Ethanol10nM 1,25D10nM 1,25D (Rec)100nM 1,25D100nM 1,25D (Rec)
Day 9 Day 15Day 12
**** ********
6 9 12
LNCaP
Differential Growth Inhibition of Prostate Cancer Cells
1 α Dihydroxyvitamin D3 (1,25 D) Dependent Growth Inhibition of LNCaP Cells Is Reversed by Casodex
Cel
l Num
ber
(X10
-4)
0
4
8
12
16Control
10nM 1,25 D
1nM DHT
DHT+1,25 D
1uM Cas
Cas+1,25 D
Is Androgen Activity Essential for 1,25 D Growth Inhibition of Prostate Cancer Cells?
• Is this phenomenon unique to LNCaP lineage cells?
• Would 1,25 D effectively inhibit cancers that have failed androgen ablation therapy?
1,25 D Inhibits LN3 and C4-2 Cell Growth In Androgen Depleted Medium
Ce
ll N
um
be
r (X
10-4
)
LN3 C4-2
Control 1,25 D Control 1,25 D0
5
10
15
20
0
6
12
18
Casodex Reverses 1,25 D Mediated Growth Inhibition of LN3 and C4-2 Cells in Medium with Androgens
Cel
l N
um
ber
(%
Co
ntr
ol)
0
20
40
60
80
100
120
LNCaP LN3 C4-2
control
1,25 D
Casodex
Cas + 1,25 D
Non-LNCaP Derived Prostate Cancer Cell Lines Expressing AR
• LAPC4 cells – – Androgen dependent– wt AR– Mutant p53
• PC-3 AR cells –– PC-3 cells stably transfected with wt AR– Lack p53
• 22Rv1 cells – – Derived from androgen dependent CWR22 xenograft– Mutant AR– Functional p53
1,25 D Inhibits PC-3 AR and LAPC4 Cell Growth in Androgen Depleted Medium
0
4
8
12
16
Control 1,25 D Control 1,25 D
Ce
ll N
um
ber
(X
10-4
)
PC-3 AR LAPC4
0
4
8
12
16
20
Casodex Does Not Alter 1,25 D Mediated Growth Inhibition of PC-3 AR, LAPC4 and 22Rv1 Cells in Medium Containing FBS
Cel
l N
um
ber
(%
Co
ntr
ol)
0
20
40
60
80
100
120
PC-3 AR LAPC4 22Rv1
control
1,25 D
Casodex
Cas + 1,25 D
Summary
• Endogenous androgens are not required for 1,25 D mediated growth inhibition
• Reversal of 1,25 D action by anti-androgens is peculiar to LNCaP/ LNCaP-derived cells
Potential Mechanisms for 1,25 D – Androgen Interaction in LNCaP Cells
• Altered transcriptional activation by AR and/or VDR
• Interactions downstream of receptor activity
Androgens AR AR Target Gene Transcription
1,25 D VDR VDR Target Gene Transcription
Co-factors
DHT --- +++
+ Cas + 1,25 D
1,25 D Does Not Alter Transcriptional Activity of AR in LNCaP Cells
RLU
/bga
l (X
10-3)
0
2
4
6
8
10
VDR Activity in LNCaP Cells is Not Altered by Androgens/Anti-androgens
1,25 D +--- ++
+ DHT + Cas
RLU
/bga
l (X
10-3)
0
3
6
9
12
DHT Inhibits Induction of CYP24
Lou and Tuohimaa(2006) J. Steroid Biochem. Mol. Biol. 99:44-49 Fig. 2
VDR Dependent Induction of IGFBP-3 Requires Androgen in LNCaP Cells, but not in PC-3 Cells
- +-+ - +
10% FCS 10% sFCS
R18811,25D
-- - - + +
IGFBP-3
PC3LNCaP C4-2
- - -+ + +
IGFBP-3
Actin
Fold Increase: 21.3 31.9 6.1
Effect of Casodex Co-treatment on Downstream Actions of 1,25 D
• Cell cycle arrest – partially blocked
• Protects against induction of apoptosis
• Protects against bcl-2 down-regulation following 1,25 D treatment
1,25 D / Anti-Androgen Interactions Downstream of Receptor Activation
1,25 D VDR Target Genes
Androgens AR Target Genes
GrowthInhibition
Anti-androgens
A.Common downstream effector for the VDR and AR dependent growth inhibitory pathways.
B. Common (growth inhibitory) target gene induced by 1,25 D and androgens
• Novel gene (Geck et al, Tufts) induced by androgens in LNCaP cells
• Induced only at doses that cause growth inhibition
• Essential for androgen induced growth inhibition of MCF-7/AR cells
• Hypothesis – AS3 may be a common target for androgen and 1,25 D induced growth regulation of LNCaP cells
AS3 (APRIN)– A Gene Involved in Androgen Induced Growth Inhibition of LNCaP Cells
Treatment Relative Expression of
AS3
(w/o CHX)
Relative Expression of
AS3
(+ CHX)
Vehicle 1.00 1.00
R1881 30.15 ± 7.69 23.12 ± 5.56
1,25 D 23.24 ± 6.65 24.49 ± 12.12
AS3 Induction by 1,25 D is Independent of de novo Protein
Synthesis
Hypothetical Model for AS3 Regulation
RXR
Coactivators
VDRE ARE ???
???
ARVDR
VDRE ARE ???
???
RXR VDR
Coactivators
+ Androgens
+ 1,25 D
+ CasodexCoactivators Corepressors
VDRE ARE ???
???
RXR VDR
Coactivators
Treatment Relative Expression of AS3
Vehicle 1.00
R1881 36.24 ± 12.26
1,25 D 28.82 ± 9.74
Casodex 1.29 ± 0.87
1,25 D + Casodex 15.18 ± 3.76
Casodex Blocks 1,25 D Mediated Induction of AS3
*
AS3 is not Induced in 22RV1 Cells Where Casodex Does Not Reverse 1,25D Mediated Growth Inhibition
Casodex
Casodex+ 1,25 D
0
20
40
60
80
100
120
*
**
Ce
ll n
um
be
r (%
Co
ntr
ol)
0
20
40
60
80
100
120
*
*
*
0
0.5
1
1.5
Con(FCS) 1,25D
00.5
11.5
22.5
33.5
44.5
Con(FCS) 1,25D
AS
3/1
8S
PS
A/1
8S
0
0.5
1
1.5
Con(CSS) R1881
F. G.
0
1
2
3
4
5
6
Con(CSS) R1881
AS
3/1
8S
IGF
BP
3/1
8S *
*
Conclusions
• There are functional interactions between androgen receptor and vitamin D receptor signaling.
• Reversal of 1,25 D mediated growth inhibition by anti-androgens is not universal.
• AS3 is a common target for androgens and 1,25 D in LNCaP cells, but not in 22RV1 cells.
Acknowledgments
• Shalini Murthy
• LaMonica Stewart
• Irina Agoulnik
• Tara Polek