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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. Androgen Receptor is Active in Adult Stromal and Epithelial cells. DHT. PSA. GF. AR. AR. AR. AR. AR. AR. AR. AR.

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interactions between vitamin d and androgen receptor signaling in prostate cancer cells

Interactions Between Vitamin D and Androgen Receptor Signaling in Prostate Cancer Cells

Nancy L. Weigel, Ph.D.

Baylor College of Medicine

slide2

Androgen Receptor in Prostate

Androgen Receptor is Active in Adult Stromal and Epithelial cells

DHT

PSA

GF

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

AR

Stromal Cell Androgen Receptor is Required for Prostate Development

DHT

prostate cancer
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 and C4-2 Human Prostate Cancer Cells

LNCaP

C4-2

androgen dependence of lncap cell growth
Androgen Dependence of LNCaP Cell Growth

Zhao et al. (1997) Endocrinology 138:3290-8 Fig 3A

differential growth inhibition of prostate cancer cells

300

Ethanol

10nM 1,25D

10nM 1,25D (Rec)

100nM 1,25D

200

100nM 1,25D (Rec)

100

0

*

*

*

*

*

*

*

*

*

*

*

*

Day 9

Day 12

Day 15

Differential Growth Inhibition of Prostate Cancer Cells

LNCaP

PC-3

6 9 12

1 dihydroxyvitamin d 3 1 25 d dependent growth inhibition of lncap cells is reversed by casodex
1 α Dihydroxyvitamin D3 (1,25 D) Dependent Growth Inhibition of LNCaP Cells Is Reversed by Casodex

Control

16

10nM 1,25 D

1nM DHT

12

DHT+1,25 D

Cell Number (X10-4)

1uM Cas

8

Cas+1,25 D

4

0

is androgen activity essential for 1 25 d growth inhibition of prostate cancer cells
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?
slide11

20

15

10

5

0

Control

1,25 D

1,25 D Inhibits LN3 and C4-2 Cell Growth In Androgen Depleted Medium

C4-2

LN3

18

12

Cell Number (X10-4)

6

0

Control

1,25 D

casodex reverses 1 25 d mediated growth inhibition of ln3 and c4 2 cells in medium with androgens
Casodex Reverses 1,25 D Mediated Growth Inhibition of LN3 and C4-2 Cells in Medium with Androgens

control

120

1,25 D

100

Casodex

Cas + 1,25 D

80

Cell Number (% Control)

60

40

20

0

LNCaP

LN3

C4-2

non lncap derived prostate cancer cell lines expressing ar
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

16

12

8

4

0

Control

1,25 D

Control

1,25 D

1,25 D Inhibits PC-3 AR and LAPC4 Cell Growth in Androgen Depleted Medium

20

16

12

Cell Number (X10-4)

8

4

0

PC-3 AR

LAPC4

slide15
Casodex Does Not Alter 1,25 D Mediated Growth Inhibition of PC-3 AR, LAPC4 and 22Rv1 Cells in Medium Containing FBS

control

120

1,25 D

100

Casodex

Cas + 1,25 D

80

Cell Number (% Control)

60

40

20

0

PC-3 AR

LAPC4

22Rv1

summary
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 Potential Mechanisms for 1,25 D – Androgen Interaction in LNCaP Cells

Androgens

AR Target Gene Transcription

AR

Co-factors

1,25 D

VDR Target Gene Transcription

VDR

  • Interactions downstream of receptor activity
1 25 d does not alter transcriptional activity of ar in lncap cells

DHT

-

+

-

+

-

+

+ Cas

+ 1,25 D

1,25 D Does Not Alter Transcriptional Activity of AR in LNCaP Cells

10

8

6

RLU/bgal (X10-3)

4

2

0

vdr activity in lncap cells is not altered by androgens anti androgens

12

9

6

3

0

1,25 D

+

+

+

-

-

-

+ DHT

+ Cas

VDR Activity in LNCaP Cells is Not Altered by Androgens/Anti-androgens

RLU/bgal (X10-3)

dht inhibits induction of cyp24
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

C4-2

LNCaP

PC3

-

+

-

+

-

+

10% FCS

10% sFCS

IGFBP-3

-

-

-

-

+

+

R1881

-

+

-

+

-

+

b Actin

1,25D

Fold Increase:

21.3

31.9

6.1

IGFBP-3

VDR Dependent Induction of IGFBP-3 Requires Androgen in LNCaP Cells, but not in PC-3 Cells
effect of casodex co treatment on downstream actions of 1 25 d
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

Target Genes

VDR

Growth

Inhibition

AR

Target Genes

Androgens

Anti-androgens

1,25 D / Anti-Androgen Interactions Downstream of Receptor Activation
  • 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

as3 aprin a gene involved in androgen induced growth inhibition of lncap cells
AS3 (APRIN)– A Gene Involved in Androgen Induced Growth Inhibition of LNCaP Cells
  • 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
hypothetical model for as3 regulation

Coactivators

AR

VDR

RXR

VDRE

???

ARE

???

Coactivators

VDR

RXR

ARE

???

VDRE

???

Corepressors

VDR

RXR

VDRE

ARE

???

???

Hypothetical Model for AS3 Regulation

+ Androgens

+ 1,25 D

Coactivators

Coactivators

+ Casodex

as3 is not induced in 22rv1 cells where casodex does not reverse 1 25d mediated growth inhibition

G.

F.

*

PSA/18S

AS3/18S

*

AS3/18S

IGFBP3/18S

AS3 is not Induced in 22RV1 Cells Where Casodex Does Not Reverse 1,25D Mediated Growth Inhibition

Casodex

Casodex

+ 1,25 D

*

*

Cell number

(% Control)

*

*

*

*

conclusions
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
Acknowledgments
  • Shalini Murthy
  • LaMonica Stewart
  • Irina Agoulnik
  • Tara Polek