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 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: Fig 3A

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

PC Ethanol 10nM 1,25D 10nM 1,25D (Rec) 100nM 1,25D 100nM 1,25D (Rec) Day 9Day 15Day 12 ************ LNCaP Differential Growth Inhibition of Prostate Cancer Cells

1 α Dihydroxyvitamin D 3 (1,25 D) Dependent Growth Inhibition of LNCaP Cells Is Reversed by Casodex Cell Number (X10 -4 ) 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 Control1,25 D Control1,25 D

Casodex Reverses 1,25 D Mediated Growth Inhibition of LN3 and C4-2 Cells in Medium with Androgens Cell Number (% Control) LNCaPLN3C4-2 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 Control1,25 DControl1,25 D Cell Number (X10 -4 ) PC-3 ARLAPC

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) PC-3 ARLAPC422Rv1 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/bgal (X10 -3 )

VDR Activity in LNCaP Cells is Not Altered by Androgens/Anti-androgens 1,25 D DHT+ Cas 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 % FCS10% sFCS R1881 1,25D IGFBP-3 PC3LNCaP C IGFBP-3  Actin Fold Increase:

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 ARTarget Genes Growth Inhibition 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

TreatmentRelative Expression of AS3 (w/o CHX) Relative Expression of AS3 (+ CHX) Vehicle1.00 R ± ± ,25 D23.24 ± ± AS3 Induction by 1,25 D is Independent of de novo Protein Synthesis

Hypothetical Model for AS3 Regulation RXR Coactivators VDRE ARE ?? ? AR VDR VDRE ARE ?? ? RXR VDR Coactivators + Androgens + 1,25 D + Casodex Coactivators Corepressors VDRE ARE ?? ? RXR VDR Coactivators

TreatmentRelative Expression of AS3 Vehicle1.00 R ± ,25 D28.82 ± 9.74 Casodex1.29 ± ,25 D + Casodex15.18 ± 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 + 1,25 D * * * Cell number (% Control) * * *   AS3/18S PSA/18S F. G. AS3/18S IGFBP3/18S * *

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