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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. 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

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  1. Interactions Between Vitamin D and Androgen Receptor Signaling in Prostate Cancer Cells Nancy L. Weigel, Ph.D. Baylor College of Medicine

  2. 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

  3. 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?

  4. 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

  5. Androgen Dependence of LNCaP Cell Growth Zhao et al. (1997) Endocrinology 138:3290-8 Fig 3A

  6. Androgen Receptor is Required for Growth of Androgen Independent C4-2 Cells AR

  7. AR siRNA Reduces PSA Expression in C4-2 Cells Actin PSA Csi ARsi

  8. 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

  9. 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

  10. 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?

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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)

  20. DHT Inhibits Induction of CYP24 Lou and Tuohimaa(2006) J. Steroid Biochem. Mol. Biol. 99:44-49 Fig. 2

  21. 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

  22. 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

  23. 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

  24. 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

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

  26. 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

  27. Casodex Blocks 1,25 D Mediated Induction of AS3 *

  28. 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) * * * *

  29. 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.

  30. Acknowledgments • Shalini Murthy • LaMonica Stewart • Irina Agoulnik • Tara Polek

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