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

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100

120

*

**

Ce

ll n

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be

r (%

Co

ntr

ol)

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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

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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