# Presentation: 523866

## 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**

**DHT**

- Stromal 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

## Androgen Receptor is Required for Growth of Androgen Independent C4-2 Cells

**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

## Differential Growth Inhibition of Prostate Cancer Cells

**PC-3**

-    **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

- Cell Number (X10-4)

- 0

- 4

- 8

- 12

- 16

- Control

- 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

- Cell Number (X10-4)

- LN3

- C4-2

- Control

- 1,25 D

- 0

- 6

- 12

- 18

## Casodex Reverses 1,25 D Mediated Growth Inhibition of LN3 and C4-2 Cells in Medium with Androgens

**Cell Number (% Control)**

**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

**Cell Number (X10****-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

**Cell Number (% Control)**

**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

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

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

- RLU/bgal (X10-3)

- 0

- 2

- 4

- 6

- 8

- 10

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

- RLU/bgal (X10-3)

## 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

## 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

**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

- 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**

## AS3 Induction by 1,25 D is Independent of de novo Protein Synthesis

| 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 |

## Hypothetical Model for AS3 Regulation

- + Androgens

- + 1,25 D

- + Casodex

- Coactivators

- Coactivators

## Casodex Blocks 1,25 D Mediated Induction of AS3

| 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 |

- *

## 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

**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