INTRODUCTION

1. The Involvement of hGH and its Receptor in Prostate Cancer MaslamaBidoseePharmacology Unit The Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel

2. INTRODUCTION • Although essential, androgens are not sufficient to induce normal growth and functionality of the prostate. Other hormones and growth factors, including pituitary hormones, are known to act on normal prostate and can potentially be involved in abnormal prostate function. • Patients first detected with early stage prostate cancer (PCa) are treated by androgen ablation therapy, until they relapse with a disease that is no longer androgen sensitive. • There is thus a urgent need to improve our knowledge, not only of the central role of androgens, but also of other hormonal and growth factors in initiation of this disease and in progression to its lethal stages.

3. INTRODUCTION (cont.) • Growth hormone (GH) is one of a number of hormones known to play an important role in development and regulation of normal prostate growth and function. GH involvement in prostate function has been studied in experimental animals, particularly rats, but almost not in man. • GH might be involved in regulating prostate function whether directly via its membranal receptors (GHR) or indirectly via GH-induced systemic IGF-I and/or GH-induced prostatic IGF-I receptors. • Nevertheless, there have been only very few studies on its role in development and maintenance of PCa, and this is incompletely understood.

4. INTRODUCTION (cont.) • As part of earlier studies in our laboratory of the GH and GHR involvement in PCa, in human PCa LNCaP cells GHR was characterized at the level of GH binding and of GHR mRNA isoforms expression. • Those studies demonstrated, for the first time LNCaP cell expression of the long (GHR) and the truncated (GHRtr) GHR mRNA isoforms, and GH-BP shedding by these cells. • Moreover, hGH was shown to induce phosphorylation of the tyrosine kinase janus kinase 2 (JAK2), of GHR itself, of the signal transducer and activator of transcription STAT5 and of mitogen-activated protein kinase p42/p44 MAPK. • These findings provided an initial evidence of the functionality of the LNCaP cell GHR.

5. INTRODUCTION (cont.) • In this study, we further studied the role of GH and its receptors in the most common experimental human PCa cell lines: androgen sensitive LNCaP cells, androgen insensitive PC3 and DU145 cells as well as in PCa patient tissue specimens. • In the first part, we studied the binding of 125I-hGH to GHR in the three cell lines. Most subsequent binding studies were performed on the LNCaP cells, since only very low binding was detected in the PC3 and DU145 cell lines. • We then investigated the regulation exerted by potent androgen analog, mibolerone, and by other hormones and growth factors, known to be involved in PCa growth and progression, on the GHR binding to LNCaP cells, and provided additional evidence of the functionality of the LNCaP cell GHR.

6. INTRODUCTION (cont.) • In the second part, we determined the gene expression for the long and for the truncated GHR isoforms in patient tissue samples, androgen-sensitive LNCaP cells and androgen-insensitive PC3 and DU145 cells. • Furthermore, we determined the homologous and heterologous hormonal regulation of gene expression of both GHR isoforms. • In the third part: we investigated the effects of hGH, as well as of androgen, estrogen and thyroid hormone, on the gene expression of IGF-I, IGF-II, their receptors, IGF-IR, IGF-IIR, and estrogen receptors (ERα, ERß) in the three PCa cell lines. • In the fourth part, we studied the proliferative effects of hGH alone or in combination with other hormones/growth factors known to induce PCa cell proliferation.

7. PART I Androgen, Estrogen, Cortisol, IGF-I, IGF-II and EGF-Induced Regulation of Growth Hormone Receptors in LNCaP Human PCa Cells

8. METHODS • Cell Culture:LNCaP, PC3 and DU145 cells were grown with 10% FCS according to recommendations by the ATTC. Hormonal treatments were preceded by 24 h culture in serum-free RPMI-1640 (without phenol red) medium + 0.1% BSA, and performed in the same medium. • The hormones used were: hGH; mibolerone, a potent and long acting androgen analog; estradiol-17ß’cortisol, EGF, IGF-I, IGF-II and triiodothyronine (T3).

9. METHODS • Exposure to androgen was performed in two different serum-free media: 1. Gc complete medium: Chemically-defined culture medium based on RPMI-1640 medium, supplemented with T3, fetuin, insulin, hydrocortisol. 2. RPMI-1640 (without phenol red) + 0.1% BSA. • 125I-hGH (1-2 ng/well) specific bindingdetermined on cultured LNCaP cells ± hGH (1-2 g/well) at 4 C for 24 h. • All values are mean  SEM (n3); p0.05 versus control.

10. :P<0.01 versus Control Dose-Dependent Mibolerone-Induced Inhibition (72h) of 125I-hGH Binding to LNCaP Cells in Gc Medium 125I-hGH Specific Binding (% of control)

11. Time-Dependent Biphasic Effect of Mibolerone (10-8 M) on 125I-hGH Binding to LNCaP Cells in Gc Medium 125I-hGH Specific Binding (% of control) : P<0.01 versus Control

12. Dose-Dependent Mibolerone-Induced (10-8 M; 9h) Stimulation of 125I-hGH Binding to LNCaP Cells in Gc Medium 125I-hGH Specific Binding (% of control) : P<0.01 versus Control

13. Biphasic Dose-Dependent Mibolerone-Induced Stimulation (72h) of 125I-hGH Binding to LNCaP Cells in RPMI + 0.1% BSA Medium 125I-hGH Specific Binding (% of control) : P<0.01 versus Control

14. : P<0.01 versus Control Time-Dependent Mibolerone-Induced (10-11 M) Stimulation of 125I-hGH Binding to LNCaP Cells in RPMI + 0.1% BSA Medium 125I-hGH Specific Binding (% of control)

15. Scatchard Analysis of Specific Binding of125I-hGH to GHR in LNCaP Cells n=3 for all values; * :P<0.01 versus Control

16. Mibolerone-Induced (72h) and Medium-Dependent Regulation of LNCaP Cell GHR

17. : P<0.01 versus Control; : P<0.01 versus Mibolerone T3 (10-10 M; 72h) Modulation of Mibolerone-Induced (10-11 M; 72h) Stimulation of GHR in RPMI+0.1% BSA Medium 125I-hGH Specific Binding (% of control) Control Mibolerone T3 Mibolerone + T3

18. Dose-Dependent T3-Induced Inhibition (72h) of 125I-hGH Binding to LNCaP Cellsin RPMI + 0.1% BSA Medium 125I-hGH Specific Binding (% of control) T3 (log M)

19. 125I-hGH Specific Binding (% of control) Exposure Time (h) Time-Dependent T3-Induced Inhibition (72h) of 125I-hGH Binding to LNCaP Cellsin RPMI + 0.1% BSA Medium

20. : P<0.01 versus Control; : P<0.01 versus Mibolerone Inhibition of Mibolerone-Induced (72h) Stimulation of GHR by Anti-Androgens 125I-hGH Specific Binding (% of control) Control Casodex + Mibolerone Flutamide + Mibolerone Flutamide 10-5 M Casodex 10-5 M Mibolerone 10-11M

21. 125I-hGH Specific Binding (% of control) E2 (log M) Biphasic Dose-Dependent E2-Induced Long Term Stimulation (72 h) of 125I-hGH Binding to LNCaP Cells.

22. Time-Dependent E2-Induced (10-9 M) Stimulation of 125I-hGH Binding to LNCaP Cells 125I-hGH Specific Binding (% of control) Exposure Time (h)

23. Biphasic Dose-Dependent Cortisol-Induced Long Term Stimulation (72 h) of 125I-hGH Binding to LNCaP Cells. 125I-hGH Specific Binding (% of control) Cortisol (log M)

24. Time-Dependent Cortisol-Induced (10-7 M) Stimulation of 125I-hGH Binding to LNCaP Cells 125I-hGH Specific Binding (% of control) Exposure Time (h)

25. Dose-Dependent IGF-I-Induced Long Term Stimulation (72 h) of 125I-hG Binding to LNCaP Cell 125I-hGH Specific Binding (% of control) IGF-I (log M)

26. Time-Dependent IGF-I-Induced (10-11 M) Stimulation of 125I-hGH Binding to LNCaP Cells 125I-hGH Specific Binding (% of control) Exposure Time (h)

27. Dose-Dependent IGF-II-Induced Long Term Stimulation (72 h) of 125I-hGH Binding to LNCaP Cells 125I-hGH Specific Binding (% of control) IGF-II (log M)

28. Time-Dependent IGF-II-Induced (10-11 M) Stimulation of 125I-hGH Binding to LNCaP Cells 125I-hGH Specific Binding (% of control) Exposure Time (h)

29. SUMMARY - PART I • Mibolerone: • Gc medium: - long term: dose- and time-dependent inhibition of 125I-hGH binding to GHR. - short term: rapid and transientincrease in binding. • RPMI + 0.1% BSAmedium: - Dose- (biphasic) and time-dependent increase in binding: observed between 24-72 h. - The longer term changes in GHR binding reflected changes in number of GHR sites and not in affinity. • This effect of mibolerone is androgen receptor-mediated as it was blocked by the anti-androgens bicalutamide (Casodex) and flutamide.

30. SUMMARY - PART I (cont.) • Triiodothyronine: -T3 reduced basal and mibolerone-induced GHR binding. These effects were dose- and time- (biphasic) dependent. -Thusthe medium-dependent, differential regulation of the LNCaP GHR protein mostly reflects the T3 content of the Gc medium. • Estradiol: -Dose- (biphasic) and time-dependent increase in binding, suggesting that some of the effects of estradiol on androgen-dependent human PCa could involve regulation of GHR protein levels/mRNA levels, presumably acting via the ERβ receptors. • Cortisol, IGF-I, IGF-II: -Dose- (biphasic) and time-dependent increase in binding.

31. CONCLUSIONS - PART I • Our findings bring to light the importance of the coordinated regulation exerted by different hormones and growth factors, on the GHR in human PCa LNCaP cells, and thus provide additional evidence of the functionality of the LNCaP cell GHR.

32. PART II GHR Gene Expression and Regulation in Human PCa Cell Lines and Patient Tissue Samples

33. INTRODUCTION • We have now extended our studies to the determination of gene expression for the long and for the truncated GHR isoforms in: 1. patient benign prostatic hyperplasia (BPH) and PCa tissue samples 2. androgen-sensitive LNCaP cells 3. androgen-insensitive PC3 and DU145 cells • Furthermore, in LNCaP cells, we determined the hormonal regulation of gene expression of both GHR isoforms : 1. homologous regulation, by hGH itself 2. heterologous regulation by androgen, estrogen, and thyroid hormone.

34. METHODS • Semi-quantitative RT-PCR was performed using specific primer sets and non-saturating conditions optimized for full length GHR and for its truncated mRNA isoform (GHRtr). • Quantitation was based on densitometric analysis and normalization versus beta-actin (cell lines) or r18S (patient tissues) house keeping genes. Each experiment was repeated at least three times. • Real Time PCR was performed in order to further confirm the validity of the semi-quantitative estimates of selected experiments of the regulatory effects of hGH on gene expression

35. GHR GHRtr Relative Gene Expression of GHR and GHRtr in Human PCa Cell Lines 1/2.5 1/12.5 1/50 GHR GHRtr β-actin Relative Gene Expression (% of control) # * * * /# LNCaP PC3 DU145 * # :P<0.05 versus GHR :P<0.05 versus LNCaP

36. r18S GHRfl GHRtr BPH CaP BPH CaP Increased Expression of GHR and GHRtrin CaP v’s BPH * Relative Expression (arbitrary units) GHR GHRtr

37. GHR GHRtr * -actin * * * * * GHR GHRtr Dose-Dependent Short-Term (6h) Regulation of GHR and GHRtr mRNA by hGH in LNCaP Cells Relative Gene Expression (% of control) hGH (ng/ml)

38. GHR GHRtr -actin * * * * * * * * * * * * GHR GHRtr Time-Dependent Regulation of GHR and GHRtr mRNA by hGH (100 ng/ml) in LNCaP Cells Relative Gene Expression (% of control) Exposure to hGH (h)

39. GHR GHRtr β-actin * * * * * * * GHR GHRtr Dose-Dependent Regulation of GHR and GHRtr mRNA by hGH (24 h) in LNCaP Cells -actin Relative Gene Expression (% of control) hGH (ng/ml)

40. GHR GHRtr Dose-Dependent Regulation of GHR and GHRtr mRNA by hGH (24 h) in PC3 Cells GHR β-actin GHRtr β-actin * PC3 Relative Gene Expression (% of control) hGH (ng/ml)

41. Dose-Dependent Regulation of GHR and GHRtr mRNA by hGH (24 h) in DU145 Cells GHR GHR β-actin GHRtr β-actin GHRtr * * * * * * DU145 Relative Gene Expression (% of control) hGH (ng/ml)

42. GHR GHRtr β-actin * * * * * GHR GHRtr Time-Dependent Stimulation of LNCaP Cell GHR and GHRtr mRNA by Androgen Analog, Mibolerone (10-11 M), in LNCaP Cells Relative Gene Expression (% of control) Exposure to Mibolerone (h)

43. GHR GHRtr β-actin * * # * * * # GHR GHRtr Control E2 E2+hGH T3 T3+hGH Short-Term (6h) Regulation of GHR and GHRtr mRNA by E2 (10-9 M) and T3 (10-10 M), ± hGH (100 ng/ml), in LNCaP Cells Relative Gene Expression (% of control)

44. SUMMARY - PART II • Relative Gene Expression of GHR and GHRtr: - In human PCa cell lines: GHR and GHRtrboth expressed: - LNCaPPC3>DU145 cells - GHR 4-7-fold>GHRtr - In patient tissue samples: - GHR and GHRtrboth expressed in BPH and PCa. - GHR and GHRtrboth increased in PCa compared to BPH. -  inGHR > in GHRtr

45. SUMMARY - PART II (cont.) • Regulation of GHR and GHRtr Gene Expression : - Homologous regulation: LNCaP: rapid dose-dependent  in GHR and GHRtr. PC3: GHR – no effect ; GHRtr  DU145:  GHRtr >GHR - Heterologous regulation: Mibolerone (10-11M): in GHRtr > in GHR 17 estradiol (10-9 M): GHR – no effect GHRtr rapid  Triiodothyronine (T3 10-10 M): rapid  GHR= GHRtr

46. CONCLUSIONS – PART II • Our findings of differential expression of the two GHR isoforms in the androgen-sensitive and androgen-insensitive PCa cell lines, as well as of the differential regulation exerted by hGH and by steroid hormones in the PCa cells, provide additional evidence of the functionality of the PCa cells GHR.

47. PART III Hormonal Regulation of IGF-I, IGF-II, IGF-IR, IGF-IIR and ERßGene Expressions in Human Prostate Cancer Cell Lines

48. INTRODUCTION • The aim of next stage of our study was to investigate the effects of hGH, as well as of androgen, estrogen and thyroid hormone, on gene expression in LNCaP cells, as well as in androgen-insensitive PC3 and DU145 cells. • The gene expression of IGF-I, IGF-II, their receptors, IGF-IR, IGF-IIR, and estrogen receptors (ERα, ERß)was studied using specific primer sets for each of these genes.

49. * * * * * Time-Dependent Regulation of IGF-I Gene Expression by hGH (100 ng/ml) in LNCaP Cells IGF-I -actin Relative Gene Expression (% of control) Exposure to hGH (h)

50. PC3 PC3 β-actin DU145 β-actin * * DU145 * * * Dose-Dependent Regulation of IGF-II mRNA by hGH (24 h) in PC3 and DU145 Cells IGF-II Relative Gene Expression (% of control) hGH (ng/ml)