Intracrine Metabolism of Testicular
Androgens by Castration-Recurrent
Prostate Cancer – Are There
Opportunities for Novel Treatments?
James L. Mohler, MD
Associate Director and Senior Vice President for Translational Research
Chair, Department of Urology
Professor of Oncology
Roswell Park Cancer Institute
Professor, Department of Urology
University at Buffalo, State University of New York
Buffalo, New York
2010 NCCN Guidelines Update
Castration-Recurrent CaP
• Chemotherapy Simplified
• Docetaxel 1st line regimen
• No best second line regimen
• Sipuleucel-T (Provenge®)
• Asymptomatic or minimally
symptomatic
• ECOG 0-1
New Theories for Prostate Cancer
(CaP) Recurrence
• Androgen Receptor (AR) responds to
castration with molecular and
biochemical alterations that cause
hypersensitivity to low levels of ligand
• CaP responds to castration by
synthesizing DHT from weaker androgens
and/or cholesterol
AR Hypersensitized
• AR 10,000 times more sensitive in
androgen-independent than androgensensitive CaP cell lines
• AR coactivators change from SRC-1 to
TIF-2 in cell lines, xenografts, and
clinical specimens
• AR phosphorylated by SRC or Ack1
tyrosine kinases
Gregory CW, et al. Cancer Res. 2001;61(7):2892-2898.
Agoulnik IU, et al. Cancer Res. 2006;66(21):10594-10602.
Guo Z, et al. Cancer Cell. 2006;10(4):309-319.
Mahajan NP, et al. Proc Natl Acad Sci U S A. 2007;104(20):8438-8443.
Activated Ack1 Promotes Androgen-Independent
Growth of LNCaP Xenografts
LNCaP cells (2 X 106 cells/injection) stably expressing caAck or vector control were
injected subcutaneously into the flanks of castrated nude mice.
Mahajan NP, et al. Proc Natl Acad Sci U S A. 2007;104(20):8438-8443.
New Theories for CaP
Recurrence
• AR responds to castration with molecular
and biochemical alterations that cause
hypersensitivity to low levels of ligand
• CaP responds to castration by
synthesizing DHT from weaker
androgens and/or cholesterol
in Benign Prostate (n = 32; gray)
vs
Castration-Recurrent CaP (n = 23;
white)
Mohler JL, et al. Clin Cancer Res. 2004;10(2):440-448.
LC-MS/MS of DHT and T in
Benign Prostate Tissue
DHT
MW 291
T
MW 289
Titus MA, et al. Clin Cancer Res. 2005 ;11(13):4653-4657.
Benign Prostate (n = 18)
T (nM)
1.8
2.5
2.9
13.0
1.2
2.9
1.4
1.6
2.7
2.8
2.8
3.2
3.3
3.9
Mass Spec
RIA
DHT (nM)
ADT
Castration-Recurrent CaP (n = 18)
T (nM)
3.4
23.6
LHRH+flu
0
14.5
orch
1.2
16.8
orch+flu
11.3
LHRH
1.2
12
LHRH+flu
1.7
0.0
20.5
orch
3.8
17.1
LHRH 5.4
3.9
13.2
orch
8.6
9.8
1° hypogonad
14.3
flu 11.4
1.2
11.2
orch
1.1
2.0
6.5
orch
10.7
LHRHDES
7.2
13.7
Lupron
0.0
13.7
orch
1.6
0.7
20.3
orch
6.7
38.3
DESorch
9.1
12.4
fluDES
1.1
2.8
3.2
13.7
8.1
1.6
3.7
13.6
4.6
DHT (nM)
0.0
0.0
4.9
7.8
6.7
9.8
2.8
0.0
2.5
0.4
1.3
0.0
5.2
1.5
0.0
3.8
2.8
1.3
1.5
Titus MA, et al. Clin Cancer Res. 2005 ;11(13):4653-4657.
Testicular Androgen Levels in
Castration-Recurrent CaP
Mass Spec
Titus 2005
T
AS-BP (n=18)
2.75
CR-CaP (n=18)
3.75
Montgomery 2008
T
AS-BP (n=6)
0.04
AS-CaP (n=4)
0.23
CR-Met CaP (n=8)
0.74
Titus MA, et al. Clin Cancer Res. 2005;11(13):4653-4657.
Montgomery RB, et al. Cancer Res. 2008;68(11):4447-4454.
Mohler JL, et al. Clin Cancer Res. 2004;10(2):440-448.
Geller J, et al. Prog Clin Biol Res. 1979;33:103-111.
Labrie F, et al. Br J Urol. 1989;63(6):634-638.
DHT
13.7
1.25
DHT
1.92
2.75
0.25
RIA
Mohler 2004
T
AS-BP (n=30)
3.26
CR-CaP (n=15)
2.78
Geller 1979
T
AS-BP (n=17)
CaP orch ± DES (n=9)
CaP DES 1 mg (n=6)
Labrie 1989
T
human CaP (n=?)
orch (n=5, 2-12m)
orch+fl (n=4, 2m)
-
DHT
8.13
1.45
DHT
17.6
4.47
12.4
DHT
18.6
9.29
ND
Intracrine Metabolism of
Testicular Androgens
• DHT from weak adrenal
androgens
• DHT from cholesterol
Increased Levels of Enzymes
that Make Testosterone
Stanbrough M, et al. Cancer Res. 2006;66(5):2815-2825.
Intracrine Metabolism of
Testicular Androgens
• DHT from weak adrenal
androgens
• DHT from cholesterol
Testicular Androgen Production
from Cholesterol
• LuCap xenografts and clinical specimens show upregulation of key enzymes required for metabolism of
progesterone to adrenal androgens and then
testosterone (Montgomery, Cancer Res, 2008)
• LNCaP cells up-regulate enzymes required for cholesterol
influx, synthesis, and metabolism to produce DHT by
“back door” metabolism (Leon, Prostate, 2010)
Montgomery RB, et al. Cancer Res. 2008;68(11):4447-4454.
Leon CG, et al. Prostate. 2010;70(4):390-400.
Testicular Androgen Production
from Cholesterol
•
14C-cholesterol
appears as 14C-DHT in LNCaP cells thru
up-regulation of StAR, the rate-limiting enzyme in steroid
synthesis (Locke, Prostate, 2010)
• DHT synthesis persists in spite of CYP17A1
(ketoconazole) and 5α-reductase-2 (finasteride)
inhibition in A-I LNCaP cells and C-R LNCaP xenografts
(Locke, J Steroid Biochem Mol Biol, 2009)
Locke JA, et al. Prostate. 2010;70(3):239-251.
Locke JA, et al. J Steroid Biochem Mol Biol. 2009;115(3-5):126-136.
New Treatment Paradigm for
Castration-Recurrent CaP
•
•
•
•
•
Prevent synthesis of tissue androgens
Enhance degradation of tissue androgens
Inactivate or destroy AR
Destroy prostate vasculature
Prevent ligand-independent AR activation
Origin of Tissue DHT in
Castration-Recurrent CaP
Barriers to Understanding
Androgen Metabolism
Mohler JL, Wilson EW, unpublished.
Pathways to
DHT Synthesis
Intact pathway
Adrenal androgen
pathway
Cholesterol
pathway
Backdoor
pathway
Modified from Locke JA, et al. Cancer Res. 2008; 68(15):6407-6415.
What exciting new drugs are in clinical trials that:
1) block the metabolism of adrenal androgens
into testicular androgens, or
2) block the affect of testicular androgens (better
anti-androgen)?
A.
B.
C.
D.
E.
Abiraterone
TAK-700
VN124-1
MDV3100
All of the above
What exciting new drugs are in clinical trials that:
1) block the metabolism of adrenal androgens
into testicular androgens, or
2) block the affect of testicular androgens (better
anti-androgen)?
A.
B.
C.
D.
E.
Abiraterone
TAK-700
VN124-1
MDV3100
All of the above
CYP21 Inhibition
• Abiraterone
– Attard, J Clin Oncol, 2008
– Cougar/Johnson & Johnson
• TAK-700
– Millenium/Takeda
• VN124-1
– Handratta, J Steroid Biochem Mol Biol, 2004
– Vasaitis, Mol Cancer Ther, 2008
– Tokai Pharmaceuticals
Attard G, et al. J Clin Oncol. 2008;26(28):4563-4571.
Handratta VD, et al. J Steroid Biochem Mol Biol. 2004;92(3):155-165.
Vasaitis T, et al. Mol Cancer Ther. 2008;7(8):2348-2357.
Phase II Study of Dutasteride in
Prostate Cancer Recurrent During
Androgen Deprivation Therapy
• 25 evaluable men with asymptomatic castration-recurrent
CaP (mean age 70, PSA 62, GS 8, and 15 M1b)
• Safety
– Grade 3 or higher adverse events using NCI criteria in 8 men
– All judged unrelated to treatment
• Responses
– 14 progressed
– 9 stable (3, 3, 3, 4, 4, 5, 5, 9, 9 mo)
– 2 partial response [PSA decline > 50%] (5, 11 mo)
Shah SK, et al. J Urol. 2009;181(2):621-626.
5α-Reductase Type 3
Gene
NCBI Blast search revealed a gene located at
4q12 spanning
3.1 kb with possible AP-1 sites
Protein
Widely distributed protein (318 aa) with
homology to Type 1 (20%) and Type 2 (25%)
isozymes
5α-Reductase Type 3
5α-reductase activity shifts from Type 2 in AS-BP →
Type 1 in AS-CaP → Type 3 in CR-CaP
Type 1
Type 2
Type 3
AS-CaP
0.71 ± 0.55
0.047 ±
0.032
0.69 ± 0.36
CR-CaP
0.26 ± 0.11
0.003 ±
AS-CaP 0.006
0.56 ± 0.15
qRT-PCR
AS-BP
IHC
Godoy A, Mohler JL, unpublished.
CR-CaP
5α-Reductase Type 3
NOT inhibited by finasteride or dutasteride
1000
CWR22
CWR22R
750
Metabolism of
T to DHT
(pmol/mg/min)
500
250
0
Control
Titus MA, Mohler JL, unpublished.
5 nM Dutasteride
5-reductase-3 immunostaining in androgen-stimulated benign
prostate (AS-BP), androgen-stimulated high grade intraepithelial
neoplasia (AS-HGPIN), androgen-stimulated CaP (AS-CaP), and
castration-recurrent CaP (CR-CaP) tissue sections
Godoy A, Mohler JL, unpublished.
New Treatment Paradigm
for Castration-Recurrent
CaP
•
•
•
•
•
Prevent synthesis of tissue androgens
Enhance degradation of tissue androgens
Inactivate or destroy AR
Destroy prostate vasculature
Prevent ligand-independent AR activation
Inactivate AR Using Antiandrogens
• Old and relatively ineffective
– Flutamide
– Bicalutamide
– Nilutamide
• New and perhaps more effective
– Small molecule AR antagonist (MDV3100)
• Tran, Science, 2009
• Medivation, Inc.
– AR-specific histone deacetylase inhibitors
• Vorinostat, panobinostat, romidepsin
• ie, Welsbie, Cancer Res, 2009
Tran C, et al. Science. 2009;324(5928):787-790.
Welsbie DS, et al. Cancer Res. 2009;69(3):958-966.
“Destroy” AR using AR Dominant
Negatives or si/shRNA
ÆU3 LTR
ÆTR
ÆU3 LTR
pTK511
ÆU3 LTR
LacZ
ÆU3 LTR
pTK478
Lentiviral vectors containing Delta TR (AR dominant negative) or LacZ (control)
Kafri, Wilson, UNC, Titus, Mohler, RPCI
Median Tumor Volume
(to 1st Death)
Kafri, Wilson, UNC, Titus, Mohler, RPCI
Survival
100
(P = 0.033)
Days
80
60
40
20
0
Delta TR + T
Kafri, Wilson, UNC, Titus, Mohler, RPCI
LacZ + T
New Treatment Paradigm
for Castration-Recurrent
CaP
•
•
•
•
•
Prevent synthesis of tissue androgens
Enhance degradation of tissue androgens
Inactivate or destroy AR
Destroy prostate vasculature
Prevent ligand-independent AR activation
Prostate Endothelial Cells Express
Androgen
Receptor
AS-Benign
AS-CaP
Kidney
Organ-Specific in
Clinical Specimens
DHT Translocates AR to Nucleus
in Primary Cultures of Human
Prostate
Endothelial Cells
- DHT
+ DHT
AR / vWF
Godoy A, et al. Endocrinology. 2008;149(6):2959-2969.
Human Prostate Endothelial Cells
are Androgen Responsive
Endothelial Apoptosis Peaks on Day 2 after Castration
in Human Prostate Xenografts
Castration (d2)
CD34
Control
Godoy A, et al. Endocrinology. 2008;149(6):2959-2969.
MVD
Endothelial Cell AR is Functional and
Targetable in vivo
Fluorescent
Platelets
Adhere to
Damaged
Endothelium
Castration (d2)
CD34
Control
Ad-ARE-MMTV
Adenoviral Vectors
Administered i.v. Confirm
Endothelial Cell Uptake
and AR Function
Godoy A, et al. Endocrinology. 2008;149(6):2959-2969.
ImmunoAnalysis
Mutations
O. Harris Ford, III, MS
Swaroop Singh, PhD
Diana Mehedint, MD
Antony Jeyaraj, PhD
Desok Kim, PhD
Andrew B. Smitherman, MD
Sheila Greene
Olga Kozyreva, PhD
Yousef Sharief, PhD
Androgen Receptor
Frank S. French, PhD
Elizabeth Wilson, PhD
Peter Petrusz, PhD
Susan J. Maygarden, MD
Michael J. Schell, PhD
Christopher W. Gregory, PhD
Androgen Metabolism
Mark Titus, PhD
Elzbieta Kawinski, PhD
Carol Wrzosek
Yun Li
Gene Therapy
Tal Kafri, MD, PhD
Brian Ziethamel
Roswell Park
NIEHS
Support
Gary J. Smith, PhD
Alejandro Godoy, PhD
Viviana Montecinos, PhD
Michael Moser, PhD
Gregory Wilding, PhD
Shaozeng Zhang, MD, PhD
Kenneth Tomer, PhD
Fred Lih
Pat Stockton
Julie F. Foley
Gordon Flake, MD
NCI
NIA
DOD
GSK