Intermittent androgen suppression delays progression to androgen-independent regulation of prostate-specific antigen gene in the LNCaP prostate tumour model

ft. Steroid Biochem. Molec. Biol. VoL 58, No. 2, pp. 139-146, 1996

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Intermittent Androgen Suppression Delays Progression to Androgen-independent Regulation of Prostate-specific Antigen Gene in the LNCaP Prostate Tumour Model N a o h i d e S a t o , 1 M a r t i n E. G l e a v e , 1'2 N i c h o l a s B r u c h o v s k y , 1 P a u l S. R e n n i e , 1 S. L a r r y G o l d e n b e r g , 2, P a u l H . L a n g e 3 and Lorne D. Sullivan 2 1Department of Cancer Endocrinology, University of British Columbia, Vancouver, British Columbia, Canada, 2Division of Urology, University of British Columbia, Vancouver, British Columbia, Canada and 3Department of Urology, University of Washington, Seattle, Washington, U.S.A.

In m o s t patients with prostate cancer, continuous androgen suppression (CAS) therapy causes tumour regression and an accompanying decrease in serum prostate specific antigen (PSA). However, with tumour progression, regulation o f both tumour growth and P S A gene expression b e c o m e s a n d r o g e n - i n d e p e n d e n t . Because a n d r o g e n resistance develops, in part, f r o m adaptive cell survival m e c h a n i s m s activated by a n d r o g e n withdrawal, we hypothesize t h a t i n t e r m i t t e n t re-exposure to a n d r o g e n s m a y prolong t i m e to a n d r o g e n - i n d e p e n d e n t progression. The objective o f this s t u d y was to d e t e r m i n e w h e t h e r i n t e r m i t t e n t a n d r o g e n suppression (IAS) could delay the onset o f a n d r o g e n i n d e p e n d e n t PSA gene regulation in L N C a P prostate t u m o u r m o d e l when c o m p a r e d to CAS. Five or six cycles o f IAS were possible before progression developed. IAS prolonged t i m e to a n d r o g e n - i n d e p e n d e n t PSA gene regulation f r o m an average o f 26 days in CAS to 77 days in IAS. S e r u m P S A increased above p r e - c a s t r a t e levels in all mice t r e a t e d with CAS by 28 days p o s t - c a s t r a t i o n , b u t r e m a i n e d below p r e - c a s t r a t e levels in 75% o f I_AS-treated mice by 60 days post-castration. By 15 weeks p o s t - c a s t r a t i o n , s e r u m PSA levels increased 7-fold above p r e - c a s t r a t e levels in C A S - t r e a t e d m i c e c o m p a r e d to 1.9-fold increase in I A S - t r e a t e d mice. PSA m R N A expression levels highly correlated with s e r u m PSA levels in b o t h groups. M a i n t e n a n c e o f a n d r o g e n d e p e n d e n c y t h r o u g h IAS m a y be due to a n d r o g e n - i n d u c e d differentiation a n d / o r d o w n - r e g u l a t i o n o f a n d r o g e n - s u p p r e s s e d gene expression. C o p y r i g h t © 1996 Elsevier Science Ltd.

J. Steroid Biochem. Molec. Biol., Vol. 58, No. 2, pp. 139-146, 1996

INTRODUCTION No therapy exists that surpasses the ability of androgen withdrawal therapy to cause regression of local and distant disease in prostate cancer [1]. Approximately 80% of prostate cancer patients achieve symptomatic and objective responses fi~llowing androgen suppression and prostate-specific antigen (PSA) levels decrease in almost all patients. However, after a variable period of *Correspondence to M. E Cleave, D e p a r t m e n t of Cancer Endocrinology, British Columbia Cancer Agency, 600 West 10th Ave., Vancouver, BC, VSZ 4E6 Canada. Tel.: 604-877-6015 Fax.: 604-877-6011. Received 3 Jul. 1995; accepted[ 18 Dec. 1995.

time averaging 24 months, the tumour inevitably recurs with increasing serum PSA levels and is characterized by androgen-independent growth. Over the past 20 years, most efforts have focused on maximizing the degree of androgen suppression therapy by combining agents that inhibit or block both testicular and adrenal androgens. However, maximal androgen ablation increases treatment-related side-effects and expense while prolonging time to androgen independence by 3-6 months in most patients [2-5]. Autonomous increases in serum PSA after androgen withdrawal therapy are the earliest sign of androgenindependent progression [6] and reflect non-androgen regulation of a previously androgen-regulated gene [7].

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Since clinical androgen-independent progression is almost invariably associated with a rapidly rising PSA level, PSA is used as a surrogate marker of disease activity and androgen dependence. L N C a P is an androgen-sensitive, PSA-secreting, h u m a n prostate cancer cell line that is able to form tumours in male athymic mice under a variety of conditions [8, 9]. As in h u m a n prostate cancer, serum PSA levels in this model are regulated by androgen and are directly proportional to tumour volume [10]. After castration, serum and tumour-cell PSA levels decrease up to 80% and remain suppressed for 3-4 weeks before increasing again. Increases in serum PSA after castration reflect the spontaneous up-regulation of PSA gene expression in the absence of androgen. Decreases in serum PSA after castration result from apoptosis and decreased androgen-regulated PSA gene expression [10]. T h e ability of a cell to undergo apoptosis and express PSA are acquired as a feature of differentiation of prostatic cells under the influence of androgens. In the absence of androgens, proliferating cells do not differentiate and cannot become preapoptotic again, which results in development of the androgen-independent phenotype and growth [11]. We hypothesize that if tumour cells which survive androgen withdrawal are forced into a normal pathway of differentiation by androgen replacement, then apoptotic potential might be restored and progression to androgen independence may be delayed. It follows that if androgens are replaced soon after regression of tumour, it should be possible to bring about repeated cycles of androgen-stimulated growth, differentiation and androgen-withdrawal regression of tumour. Using the androgen-dependent Shionogi mouse mammary tumour model we have previously demonstrated that 4 or 5 apoptotic cycles are possible with intermittent androgen suppression (IAS) before androgen-independent progression. Furthermore, IAS prolongs time to androgen independence 3-fold when compared to continuous androgen suppression (CAS) [12]. In this study we selected the L N C a P tumour model to investigate whether IAS can delay progression to androgen-independent regulation of PSA gene when compared to CAS. M A T E R I A L S A N D METI-IODS

Assessment of in vivo tumour growth L N C a P cells were kindly provided by D r L. W. K. Chung (The University of Texas M. D. Anderson Cancer Center, Houston, TX) and maintained in R P M I 1640 (Terry Fox Laboratory, Vancouver, BC, Canada) with 5% fetal bovine serum (GIBCO, Burlington, ON, Canada). One x 106 L N C a P cells were inoculated s.c. with 0.1 ml of Matrigel (Becton Dickinson Labware, Bedford, MA) in the flank region of 6- to 8-week-old male athymic nude mice (BALB/c strain, Charles River Laboratory, Montreal, PQ,

Canada) via a 27-gauge needle under methoxyfluorane anesthesia. Turnouts were thereafter measured twice weekly and their volumes were calculated by the formula: L × W x H x 0 . 5 2 3 6 [13].

Intermittent androgen suppression protocol When turnouts became approximately 1 cm in diameter, usually 8 to 12 weeks after injection of L N C a P cells, mice were surgically castrated via abdominal approach under methoxyfluorane anesthesia. Mice were then randomly selected to either IAS or CAS (i.e. castration alone) groups. Protocol-1 For the first IAS group (IAS-1), a testosterone propionate pellet (3.75 mg/mouse/cycle) (Innovative Research of America, Toledo, OH) was buried subcutaneously in the flank region of each mouse 2 weeks after castration. T h e half-life of each pellet was about 2 weeks. T h e testosterone pellet was not removed, and another pellet was inserted at 3-3.5 week intervals. These androgen replacements were continued until serum PSA nadir levels rose above pre-castrate levels, reflecting androgen-independent regulation of PSA gene. In the first CAS group (CAS-1), no androgen replacement was done after castration. Serum PSA levels were measured weekly in both groups. Protocol-2 In the second IAS group (IAS-2), a testosterone propionate pellet was implanted by the same procedure as in protocol-1. In contrast to IAS-1, the testosterone pellet was removed 1 week after insertion, and another pellet was implanted 2 weeks later. Hence, each 3 week cycle consisted of a 1 week " o n " period with normal or elevated testosterone levels and a 2 week " o f f " period with castrated testosterone levels. T h e 3 week cycles continued up to the sixth cycle, and then discontinued because of small numbers of surviving mice. T h e second CAS group (CAS-2) received the same treatment as in protocol-1. Serum PSA levels were obtained once weekly and t u m o u r volume measurements were twice weekly in both groups.

Determination of serum PSA levels Blood samples were obtained with tail vein incisions of nude mice, and after centrifugation the serum was stored at - 2 0 ° C until PSA assay. Serum PSA levels were determined by an enzymatic immunoassay kit with a lower limit of sensitivity of 0.2 ng/ml (Abbott IMX, Montreal, PQ, Canada) according to the manufacturer's protocol. T i m e to androgen-independent PSA regulation was defined as the duration of time required after castration for serum PSA levels to return to or increase above pre-castrate levels.

Northern "blot analysis Total cellular RNA was extracted from tumours by the acid guanidinium thiocyanate-phenol-chloroform method [14]. In both IAS-1 and IAS-2 groups, tumours

Intermittent Androgen Suppression in LNCaP were harvested at either' peak or nadir phases of cycle as was indicated in Fig:. 2. In CAS groups, tumours were extirpated beginning 4 weeks after castration and RNA was extracted. R~ange of typical yields of total cellular RNA was 2 0 0 - 3 0 0 p g / 1 0 0 m g tissue as quantified by spectrophotometry. Five pg of total cellular RNA was denatured in 50% formamide/2.2% formaldehyde at 60°C and fractionated by electrophoresis on a 1% agarose gel containing 6.7% formaldehyde. Samples were transferred onto Nitran membrane (S and S Inc., Keene, N H ) using the capillary m e t h o d in 20 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium citrate) and the membrane was baked at 80°C for 2 h. Hybridiization was carried out at 42°C overnight in 6 x SSC, 50% formamide, 5 m M E D T A , 0.1% sodium dodecyl sulfate (SDS), 5 x Denhardt's solution, 1 0 m M sodium phosphate (pH 6.5), 0.2 mg/ml sonicated salmon sperm D N A with a c D N A probe labeled with [~32p]-dCTP using oligolabelling kit (Pharmacia Biotech, PQ, Canada). T h e D N A probe for PSA was a 1.4 kb EcoK[ fragment o f P S A c D N A [15]. Finally, the membrane was washed in 0.1 x SSC and 0.1% SDS at 65°C for 60 rain. Autoradiograms were prepared by exposing Kodak X-Omat AR film at - 8 0 ° C in the cassette 'with intensifying screens. T h e films were scanned by GS-300 densitometer (Hoefer Scientific Instruments, San Francisco, CA) at least three times per lane. Density of bands for PSA was normalized by that of 18 s rRNA and shown as a mean value of a relative PSA expression ratio from two N o r t h e m analyses which were carried out separately.

RESULTS Changes in serum P S A of L N C a P tumour-bearing mice Protocol-1 In both IAS-1 and CAS-1, serum PSA levels decreased an average of 3-fold by 2 weeks after castration [Fig. 1 (A)]. However, within 4 weeks after castration, serum PSA in all mice in CAS-1 increased above their pre-castrate PSA level, thereby demonstrating androgen-independent PSA regulation. In contrast, L N C a P tumours treated with IAS-1 maintained androgen-regulated PSA production up to the fifth cycle of IAS-1. Serum PSA levels in IAS-1 decreased by 6 0 - 7 0 % by 2 weeks post-castration and increased 8-10-fold above nadir levels 1 week after the first androgen replacement. Serum PSA levels reached their peak 1 week after androgen replacement and fell more gradually to a nadir 2-2.5 weeks later as the testosterone pellet was resorbed. By the end of the fifth cycle, L N C a P tumour,; in IAS-1 progressed to an androgen-independent autonomous state and their PSA nadir levels no longer decreased below pre-castrate levels even in the androgen-deprived condition. In summary, average time to androgen-independent regulation PSA regulation, defined as the duration of time required after castration for serum PSA levels to

141

return to or increase above pre-castrate levels, was 77.0 (range: 33-122) and 26.3 days (range: 21-28) in IAS-1 and CAS-1 group, respectively (Table 1). Serum PSA levels exceeded pre-castrate PSA by 28 days postcastration in all mice of CAS-1. In contrast, the nadir serum PSA in 75% of mice treated with IAS remained lower than pre-castrate PSA by 60 days post-castration. By 15 weeks post-castration, average serum PSA in IAS-1 increased only 1.9-fold (range: 0.6-2.5) above pre-castrate levels, while 7.0-fold (range: 4.2-7.7) in CAS-1. Similarly, the rate of increase in serum PSA (PSA velocity) was 9-fold faster in CAS-1 (20.4 ng/ml/week, range: 11-53) compared to IAS-1 (2.4 ng/ml/week, range: 1.9-7.9). Protocol-2 In IAS-2, the testosterone pellet was removed 1 week after implantation to achieve faster and lower nadir levels of serum testosterone; a new pellet was inserted 2 weeks after removal of pellet [Fig. 1 (B)]. Each 3 week cycle therefore consisted of 2 weeks of castrate levels of testosterone and 1 week of normal or elevated levels. Serum PSA decreased by 90-95% by 2 weeks post-castration and increased 30-fold above nadir levels after the first androgen replacement. With each successive cycle, PSA decreased by 80-90% during the second to sixth cycle. This intermittent treatment and complete removal of each testosterone pellet enabled L N C a P turnouts to respond more sharply to androgens than in IAS-1. L N C a P tumours in IAS-2 maintained androgen-regulated PSA production up to the sixth cycle of intermittent treatment, while tumours in CAS-2 were characterized by autonomously PSA production similar to CAS-1. Serum PSA levels exceeded pre-castrate PSA by 54 days post-castration in all mice of CAS-2. In contrast, nadir serum PSA in 80% of mice in IAS-2 remained lower than pre-castrate PSA by 119 days post-castration (Table 1). By 16 weeks post-castration, average nadir serum PSA in IAS-2 remained below pre-castrate levels, while average serum PSA in CAS-2 group increased 5-fold. Similarly, PSA velocity was more than 10-fold faster in CAS-2 (16 ng/ml/week) compared to IAS-2 ( - 0.14 ng/ml/ week). P S A m R N A expression correlates with serum P S A in L N C a P tumours

L N C a P turnouts in intact mice express abundant PSA m R N A [Fig. 2(A)]. In the IAS-treated group PSA m R N A levels decrease by 75% at first nadir (N1), 2 weeks post-castration, and then dramatically increase 4-5-fold at first peak (P1), 1 week after the first androgen replacement [Fig. 2(B)]. With each successive cycle of IAS, PSA m R N A expression returns to basal levels at nadir stages (N3, N5) of serum PSA, and increases again with re-exposure to androgens at each following peak stage (P3, P5). Importantly, PSA m R N A levels remained well below pre-castrate expression levels up to the fifth nadir (N5) in IAS-treated groups, demonstrating continued androgen-regulated

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Fig. 1. C h a n g e s i n s e r u m P S A o f L N C a P t u m o u r - b e a r i n g m i c e t r e a t e d w i t h I A S o r C A S . A, P r o t o c o l - l : L N C a P tumour-bearing mice were castrated when tumours became approximately 1 cm in diameter. In IAS-1, a t e s t o s t e r o n e p e l l e t w a s i m p l a n t e d s.c. 2 w e e k s a f t e r c a s t r a t i o n , a n d a d d i t i o n a l p e l l e t s w e r e i m p l a n t e d a t 3-3.5 w e e k i n t e r v a l s ( A ) . N o a n d r o g e n r e p l a c e m e n t w a s d o n e a f t e r c a s t r a t i o n in C A S - 1 . S e r u m P S A levels w e r e m e a s u r e d weekly in both groups. Serum PSA in IAS-1 increases and decreases with each cycle of testosterone replacement. B y t h e e n d o f t h e fifth cycle, P S A n a d i r n o l o n g e r falls b e l o w p r e - c a s t r a t e level, i n d i c a t i n g p r o g r e s s i o n to a n d r o g e n - i n d e p e n d e n t P S A r e g u l a t i o n (ALP). S e r u m P S A i n C A S - 1 d e c r e a s e d to a n a d i r 2 w e e k s a f t e r c a s t r a t i o n but then steadily increased at a rate 9 times faster than IAS-1 despite continued androgen-deprivation. Data show a v e r a g e s e r u m P S A levels f r o m 5 m i c e i n b o t h I A S - 1 a n d C A S - 1 . B , P r o t o c o l - 2 : I n I A S - 2 , i n c o n t r a s t to I A S - 1 , t h e t e s t o s t e r o n e p e l l e t w a s r e m o v e d 1 w e e k a f t e r i m p l a n t a t i o n (A), a n d a n o t h e r p e l l e t w a s i m p l a n t e d 2 w e e k s l a t e r ( A ) . C A S - 2 r e c e i v e d t h e s a m e t r e a t m e n t a s p r o t o c o l - 1 . S e r u m P S A levels w e r e o b t a i n e d o n c e w e e k l y in b o t h g r o u p s . N o t e t h a t L N C a P t u r n o u t s i n I A S - 2 m a i n t a i n e d t h e i r a n d r o g e n - s e n s i t i v i t y u p to t h e s i x t h c y c l e o f i n t e r m i t t e n t t r e a t m e n t , w h i l e t u m o u r s i n C A S - 2 s h o w e d a n a u t o n o m o u s l y i n c r e a s i n g p a t t e r n s i m i l a r to C A S - 1 . S e r u m P S A levels e x c e e d e d p r e - c a s t r a t e P S A b y 7-8 w e e k s p o s t - c a s t r a t i o n i n all m i c e o f C A S - 2 . I n c o n t r a s t , s e r u m P S A in 80% o f m i c e i n I A S - 2 r e m a i n e d l o w e r t h a n p r e - c a s t r a t e P S A b y 17 w e e k s p o s t - c a s t r a t i o n a n d a n d r o g e n - i n d e p e n d e n t P S A r e g u l a t i o n w a s n o t o b s e r v e d u n t i l 23 w e e k s p o s t - c a s t r a t i o n i n I A S - 2 . D a t a s h o w a v e r a g e s e r u m P S A levels f r o m 5 m i c e in b o t h I A S - 2 a n d C A S - 2 . N 1 - N 6 , f i r s t - s i x t h n a d i r o f s e r u m P S A ; P 1 - P 6 , first--sixth p e a k o f s e r u m P S A ; ALP, s t a g e o f a n d r o g e n - i n d e p e n d e n t P S A r e g u l a t i o n .

Table 1. Effect of I A S on time to androgen-independent P S A regulation in the L N C a P tumour model Protocol- 1

T i m e to androgen-independent PSA regulation t (Days) Post-castrate PSA > pre-castrate PSA* Fold increase in s e r u m PSA ~ PSA velocity" (ng/ml/week)

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41.8 All t u m o u r s by day 54 5.0 16.0

*Duration of time required for s e r u m PSA levels to return to or increase above pre-castrate levels. *Percentage of t u m o u r s a n d time w h e n post-castrate s e r u m PSA levels exceeded pre-castrate levels. Ratio of s e r u m PSA at 15 weeks (protocol-I) or 16 weeks (protocol-2) post-castration to that of pre-castration. Rate of increase in s e r u m PSA from pre-castration to 15 weeks (protocol-i) or 16 weeks (protocol-2) post-castration. *, P < 0 . 0 5 ; **, P < 0 . 0 0 5 ; ***, P < 0 . 0 0 1 w h e n compared to each CAS group (t-test, one-tail).

143

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Fig. 2. Changes in PSA mRNA levels of LNCaP tumours treated with IAS or CAS. A, LNCaP tumours w e r e harvested at the following time: pre-castration (Pre-Cx, intact group); first, third and fifth nadir (N1, N3 and N5) and each following peak 0P1, P3 and P5) of serum PSA as well as stage of androgen-independent PSA regulation (PAP, cf. Fig. 1A) in IAS-1 and -2 (IAS group); 4 weeks post-castration in CAS-1 (CAS group). Five /~g of total RNA was fractionated on 1% agarose gel and hybridized with cDNA probe of PSA (upper panel) and 18s rRNA (lower panel). PSA mRNA expression strongly correlates with changes of serum PSA levels in both IAS and CAS groups. PSA mRNA in the CAS group increased back to pre-castrate expression levels by 4 weeks post-castration, wlfile PSA mRNA expression in the IAS group remained well below pre-castrate expression levels up to N5, demonstrating continued androgen dependence ofPSA gene regulation. Arrows; position of 28 s and 18 s rRNA. B, Quantitation of relative PSA mRNA expression. Each density of PSA mRNA expression was normalized by that of 18 s rRNA. Note that PSA mRNA expression returns to p r e - c a s t r a t e levels b y 4 weeks post-castration in all tumours in the CAS group, but is only 10% of pre-castrate levels at N5 in IAS group by 14 weeks post-castration, suggesting that androgen-dependence of PSA gene regulation still maintained.

P S A gene expression. ]in contrast, P S A m R N A levels increase b a c k to pre--castrate expression levels i n C A S - t r e a t e d g r o u p s by 4 weeks p o s t - c a s t r a t i o n , illustrating m o r e r a p i d d e v e l o p m e n t o f n o n - a n d r o g e n r e g u l a t e d P S A gene expression. T h e s e data clearly d e m o n s t r a t e that P S A m R N A expression highly correlates with changes of s e r u m P S A levels in b o t h IAS a n d C A S groups.

Effect of androgen withdrawal on size and histology of L N C a P tumour I n the C A S group, a m i n o r i t y of t u m o u r s decreased slightly i n size b y 2 weeks after castration, b u t s u b s e q u e n t g r o w t h rates were n o t affected b y c a s t r a t i o n a n d c o n t i n u e d to increase gradually i n m o s t t u m o u r s (data n o t s h o w n ) . M o r e o v e r , n o evidence of necrosis or c a s t r a t i o n - i n d u c e d apoptosis was o b s e r v e d histo-

morphologically, a n d t e s t o s t e r o n e - r e p r e s s e d prostatespecific message ( T R P M - 2 ) expression was n o t observed o n N o r t h e r n analysis [10]. As i n the C A S group, L N C a P t u m o u r s i n the IAS g r o u p did n o t regress significantly after c a s t r a t i o n a n d c o n t i n u e d to grow g r a d u a l l y d u r i n g i n t e r m i t t e n t a n d r o g e n t r e a t m e n t . N o o b v i o u s difference i n histology was a p p a r e n t i n L N C a P t u m o u r s treated with IAS or CAS. T h e s e data, a l o n g with the r a p i d f l u c t u a t i o n s i n s e r u m a n d t u m o u r m R N A P S A levels, suggest that t u m o u r cell d e a t h i n d u c e d b y a n d r o g e n w i t h d r a w a l is n o t r e s p o n s i b l e for the decrease in s e r u m P S A of L N C a P t u m o u r - b e a r i n g mice treated with C A S or IAS. C h a n g e s i n the r e g u l a t i o n of P S A m R N A expression a p p e a r to be p r i m a r i l y r e s p o n s i b l e for changes i n s e r u m P S A levels after c a s t r a t i o n a n d t e s t o s t e r o n e r e p l a c e m e n t , a n d with p r o g r e s s i o n to a n d r o g e n i n d e p e n d e n c e i n this t u m o u r model.

144

N. Sato et aL DISCUSSION

In the normal prostate, the cycle of androgeninduced cell growth and castration-induced apoptosis and regression can be continued through multiple cycles of androgen replacement and withdrawal. Data from animal studies and observations from the long term follow-up of Chinese eunuchs [16] demonstrate that normal prostatic epithelial cells undergo apoptotic regression and do not develop the ability to regenerate and grow in an androgen-depleted environment. In contrast, progression to androgen independence nearly always occurs following androgen ablation therapy for patients with prostatic carcinoma. The inability of androgen withdrawal therapy to be curative may therefore result from pre-existing clones of androgen-independent cells in which the apoptotic process does not begin (clonal selection) [17], or from the up-regulation of androgen-repressed adaptive mechanisms capable of aborting the apoptotic process in subpopulations of cells (adaptation) [18]. Genetic instability along with clonal selection, rather than adaptation, appears to be the mechanism that mediates androgen-independent progression in the Dunning R-3327 rat prostatic adenocarcinoma model [19]. Genetic instability increases tumour heterogeneity by increasing the number of distinctly different clones of cells. The Dunning R-3327 model is androgen-sensitive and its doubling time is prolonged after castration but there is minimal or no tumour regression [20]. In this model, IAS may be harmful because it would result in stimulation of residual androgen sensitive ceils when testosterone levels increase. Maximal androgen ablation with the addition of cytotoxic agents that kill remaining androgenindependent cells appears to be the most effective therapy in this model [21]. Investigations from our laboratory using the androgen-dependent Shionogi mouse tumour model stress the importance of adaptive mechanisms in androgen-independent progression [11, 12, 22]. Following castration of mice bearing Shionogi tumours, time-dependent progression from an androgendependent to -independent state occurs as reflected by coordinated changes in the expression of genes and proteins that regulate tumour cell proliferation and differentiation. Androgen withdrawal precipitates apoptosis and tumour regression in a highly reproducible manner; however, despite complete remissions, castration fails to result in cure and androgen-independent progression invariably occurs within 60 days following castration. Using an in vivo limiting dilution assay to determine the proportions of androgen-dependent and -independent tumourigenic stem cells in parent and recurrent tumours, Bruchovsky et aL observed that most androgen-dependent daughter and tumourigenic stem ceils are eliminated after castration, and that surprisingly, no enrichment of androgen-independent

tumourigenic cells was evident in regressed parent turnouts; rather, the proportion of tumourigenic cells decreased [11]. However, androgen-independent recurrences are characterized by more aggressive and poorly differentiating turnouts with a massive increase in the proportion of androgen-independent tumourigenic stem cells [11]. These results suggest that progression to androgen-independence following castration may result from the ability of a small number of initially androgen-dependent tumourigenic stem cells to adapt to an androgen-depleted environment. The LNCaP turnout model system is an androgensensitive, PSA-producing, human-derived prostate turnout that closely mimics the clinical course of prostate cancer after androgen withdrawal therapy. Serum PSA levels are directly proportional to tumour volume in intact mice and both androgens and turnout volume are important co-determinants of circulating PSA levels[10,23]. Immediately after castration, serum PSA levels decrease rapidly by 80% and increase up to 20-fold following androgen supplementation without detectable castration-induced turnout cell death or concomitant changes in tumour volume [10]. These changes in PSA production in vivo reflect changes in androgen-regulated PSA gene expression. Beginning 4 weeks after castration, PSA production increases above pre-castrate levels in the absence of testicular androgens, reflecting the onset of androgenindependent regulation of the PSA gene [10, 24-26]. Although changes in serum PSA in this model mimics the clinical course of human prostate cancer, changes in the hormonal milieu created in this model (castration with androgen supplementation) differs from that created clinically (reversible androgen suppression), the former resulting in supranormal serum testosterone levels during times of testosterone replacement. In the LNCaP model, changes in serum PSA following castration, during IAS, and with progression to androgen independence, are due to changes in PSA gene regulation at the transcriptional level, rather than to changes in tumour volume with clonal selection and expansion. Following castration, consistent tumour regression does not occur, castration-induced apoptosis is not seen histologically, and TRPM-2 mRNA expression is not measurable [10]. Similarly, in men with prostate cancer treated with androgen withdrawal therapy, serum PSA levels decrease into the normal range (< 4 ng/ml) or become undetectable (< 0.2 ng/ml), despite having significant residual tumour volume [6]. PSA production in the regressed tumour is decreased, but increases again over time as tumour cells become androgen independent. Indeed, an increasing serum PSA following androgen ablation therapy in men with prostate cancer is the first sign of androgenindependent prostate cancer progression [6]. In the LNCaP tumour model, rising PSA levels 4 weeks after castration reflect escape from androgen-regulated PSA production partly through up-regulation of previously

Intermittent Androgen Suppression in LNCaP androgen-repressed alternative pathways of signal transduction [7, 10, 24-26]. T h e spontaneous recovery of PSA synthesis in an androgen-independent L N C a P t u m o u r appears to be clue to production of a soluble non-androgenic factor that stimulates PSA production in an autocrine fashion [7]. Kokontis et al. [27] reported that after long-term androgen deprivation in L N C a P cells g r o w n in vitro, adaptational changes resulted in increased AI~',expression and transcriptional activity. This m a y represent another m e c h a n i s m for a u t o n o m o u s PSA production following progression to androgen independence. Observations from the Shionogi and L N C a P t u m o u r models suggest that androgen-independent t u m o u r recurrences are associated with adaptational changes in gene expression in turnout cells that are precipitated by androgen withdrawal. T h e adaptive process is scattered and constitutive, rather than diffuse and readily reversible. Hence, clonal expansion of initially androgen-dependent stem cells m a y represent a normal physiological response in abnormal (malignant) cells that is genetically p r o g r a m m e d and precipitated or directed by epigenetic factors. This process occurs in benign tissues in the organs and has been termed clonal constitutive adaptation by Farber in reference to toxin-induced hepatocyte regeneration [28]. Androgen resistance may, therefore, be a primary but quiescent property of some prostate cancer ceils which is activated in response to androgen withdrawal. If androgen-independent progression does result from adaptive cell survival mechanisms in response to androgen withdrawal with up-regulation of alternative growth regulatory pathways, then intermittent re-exposure to androgens m a y prevent or down-regulate these mechanisms. This concept was proposed by Noble 20 years ago using the bib rat model, who stated " a reduction in hormone, levels sufficient to prevent advancing t u m o u r growth, but adequate to reduce the extent of regression, could possibly reduce the frequency or prevent the development of a u t o n o m o u s change" [29]. T h e results of this study using the L N C a P t u m o u r model add to the growing b o d y of evidence that suggest that IAS m a y help delay progression to androgen independence. T h e effects of IAS on progression was investigated using the Shionogi t u m o u r model, where time to progression was prolonged 3-fold from 50 to 150 days [12]. Unlike the Shionogi model, which uses loss of castration-induced apoptosis as the measurable endpoint of progression, the L N C a P model uses loss of maintenance of androgen-regulated PSA gene expression as its endpoint of progression. Clinicians managing patients wiLth prostate cancer similarly use PSA as an intermediate endpoint of androgenindependent progression [30]. In this study, I.AS helped maintain androgen-regulated PSA gene expression and prolonged the time to androgen-independent PSA regulation in L N C a P tumours. IAS prolonged time

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required for post-castrate PSA m R N A and serum levels to increase back to pre-castrate levels by 3-fold or m o r e in two separate experiments. Furthermore, PSA levels rose 9 - 1 0 times faster after castration with CAS c o m p a r e d to IAS. T h e slower and delayed rise in nadir PSA levels after castration using IAS suggests that transcriptional factors that regulate PSA gene expression in androgen-independent tumours can be repressed by periodic re-exposure to androgens. Intermittent exposure to androgen m a y maintain androgen-regulated PSA gene expression and delay the onset of a u t o n o m o u s PSA production by suppressing the up-regulation of alternative non-androgen transcriptional factors like the PSA autocrine factor [7]. Extrapolation of animal model data to the h u m a n disease m u s t always be done with caution, but data from the L N C a P and Shionogi t u m o u r models suggests that progression to androgen independence can be delayed with IAS. Whether IAS will enhance progression-free survival or overall survival in patients with prostate cancer is unknown, although the possibility of an improved quality of life and outcome has been considered by others [31]. However, an earlier clinical trial at Memorial Sloan-Kettering Cancer Center was conducted prior the availability of safer reversible agents for androgen suppression and also prior to PSA, both of which now permit a more tailored approach to hormonal therapy to those patients who are most likely to respond to it. We have applied IAS to 47 cases of patients on the basis of our animal model studies, and observed that the median time to progression and survival of 14 m e n with stage D2 prostate cancer were similar to the expected results with continuous androgen ablation [2, 32]. Quality of life is improved with reduced toxicity from medication, recovery of sexual function and normal sense of well-being during " o f f " treatment cycles. M o r e information on whether IAS enhances progression-free survival or overall survival will b e c o m e available from phase III clinical trials which are in the planning stages now. Acknowledgements--The authors thank Mary Bowden and Virginia

Yago for their excellent technical assistance. This work was supported in part by grant 6-73535 from the National Cancer Institute of Canada and Lotte and John Hecht Memorial Foundation.

REFERENCES

1. Huggins C. and Hodges C. V.: Studies on prostate cancer. I. The effect of castration, of estrogen, and androgen injection on serum phosphatases in metastatic carcinoma of the prostate. CancerRes. 1 (1941) 293-297. 2. Crawford E. D., Eisenberger M. A., McLeod D. G., Spaulding J. T., Benson R., Dorr F. A., Blumenstein B. A., Davis M. A. and Goodman P. J.: A controlled trial of leuprolide with and without flutamide in prostatic carcinoma. New Engl. J. Med. 313 (1989) 419-424. 3. B~land G.: Combination of Anandron with orchiectomy in treatment of metastatic prostate cancer. Results of a double-blind study. Urology 37, Suppl 2 (1991) 25-29. 4. Denis L. J., Carnelro de Moura J. L., Bono A., Sylvester R., Whelan P., Newling D. and Depauw M.: Goserelin acetate and

146

5.

6.

7.

8.

9.

10,

11.

12.

13.

14.

15. 16.

17.

N. Sato et al. flutamide versus bilateral orchiectomy: a phase III EORTC trial Urology 42 (1993) 119-129. Janknegt R. A., Abbou C. C., Bartoletti R., Bernstein-Hahn L., Bracken B., Brisse J. M., Da Silva F. C., Chisholm G., Crawford E. D. and Debruyne F. M.: Orchiectomy and Anandron (Nilutamide) or placebo as treatment of metastatic prostatic cancer in a multinational double-blind randomized trial. J. Urol. 149 (1993) 77-83. Miller J. I., Ahmann F. R., Drach G. W., Emerson S. S. and Bottaccini M. R.: The clinical usefulness of serum prostate specific antigen after hormonal therapy of metastatic prostate cancer. J. Urol. 147 (1992) 956-961. Hsieh J.-T., Wu H.-C., Gleave M. E., von Eschenbach A. C. and Chung L. W. K.: Autocrine regulation of prostate-specific antigen gene expression in a human prostatic cancer (LNCaP) subline. Cancer Res. 53 (1993) 2852-2857. Gleave M., Hsieh J.-T., Gao C., von Eschenbach A. C. and Chung L. W. K.: Acceleration of human prostate cancer in vivo by factors produced by prostate and bone fibroblasts. Cancer Res. 51 (1991) 3753-3761. Gleave M. E., Hsieh J.-T., von Eschenbach A. C. and Chung L. W. K.: Prostate and bone fibroblasts induce human prostate cancer growth in vivo; implications for bidirectional tumor-stroreal cell interaction in prostate carcinoma growth and metastasis. J. Urol. 147 (1992) 1151-1159. Gleave M., Hsieh J.-T., Wu H.-C., yon Eschenbach A. C. and Chung L. W. K.: Serum prostate specific antigen levels in mice bearing human prostate LNCaP tumors are determined by tumor volume and endocrine and growth factors. Cancer Res. 52 (1992) 1598-1605. Bruchovsky N., Rennie P. S., Coldman A. J., Goldenberg S. L., To M. and Lawson D.: Effects of androgen withdrawal on the stem cell composition of the Shionogi carcinoma. Cancer Res. 50 (1990) 2275-2282. Akakura K., Bruchovsky N., Goldenberg S. L., Rennie P. S., Bucldey A. R. and Sullivan L. D.: Effects of intermittent androgen suppression on androgen-dependent tumors; apoptosis and serum prostate-specific antigen. Cancer 71 (1993) 2782-2790. Janek P., Briand P. and Hartman N. R.: The effect of estrone-progesterone treatment on cell proliferation kinetics of hormone-dependent GR mouse mammary tumors. Cancer Res. 35 (1975) 3698-3704. Chomczynski P. and Sacchi N.: Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Analyt. Biochem. 162 (1987) 156-159. Lundwall A. and Lilja H.: Molecular cloning of human prostate specific antigen cDNA. F E B S Lett. 214 (1987) 317-322. Wu, C.-P. and Gu, F.-L.: The prostate in eunuchs. In EORTC Genitourinary Group Monograph 10 - - Urologic Oncology; Reconstructive Surgery, Organ Preservation, and Restoration of Function. Wiley-Liss Inc., New York (1992) pp. 249-255. Isaacs J. T. and Coffey D. S.: Adaptation versus selection as the mechanism responsible for the relapse of prostatic cancer to androgen ablation therapy as studied in the Dunning R-3327-H adenocarcinoma. Cancer Res. 41 (1981) 5070-5075.

18. van Weerden, W. M.: Animal models in the study of progression of prostate and breast cancers to endocrine independency. In Mechanisms of Progression to Hormone-lndependent Growth of Breast and Prostate Cancer (Edited by P. M. J. J. Berns, J. C. Romijin and F. H. Schr6der). Parthenon Publishers, New Jersey (1991) pp. 55-70. 19. Isaacs J. T., Wake N., Coffey D. S. and Sandberg A. A.: Genetic instability coupled to clonal selection as a mechanism for progression in prostatic cancer. Cancer Res. 42 (1982) 2353-2361. 20. Russo P., Liguori G., Heston W. D. W., Huryk R., Yang C.-R,, Fair W. R., Whitemore W. F. and Herr H. W.: Effects of intermittent diethylstilbestrol diphosphate administration on the R3327 rat prostatic carcinoma. Cancer Res. 47 (1987) 5967-5970. 21. Issacs J. T.: The timing of androgen ablation therapy and/or chemotherapy in the treatment of prostatic cancer. Prostate 5 (1984) 1-17. 22. Rennie P. S., Bruchovsky N., Buttyan R., Benson M. and Cheng H.: Gene expression during the early phases of regression of the androgen-dependent Shionogi mouse mammary carcinoma. Cancer Res. 48 (1988) 6309-6312. 23. Gleave M. E., Hsieh J.-T., Wu H.-C., von Eschenbach A. C. and Chung L. W. K.: Evaluation of the pharmacokinetics of PSA using the LNCaP tumor model. J. Urol. 147 (1992) 386A. 24. Wu H.-C., Hsieh J. T., Cleave M. E., Brown N. M., Pathak S. and Chung L. W. K.: Derivation of androgen-independent human LNCaP prostate cancer sublines: role of bone stromal cells. Int. J. Cancer 57 (1994) 406-412. 25. Hsieh J.-T., Gleave M. E., Wu H.-C., Wang X.-H. and Chung L. W. K.: Deranged control of androgen-regulated gene in the relapse stage of human prostatic cancer after castration; Implication for the derivation of androgen-independent cells by adaptive mechanisms. J. Urol. 147 (1992) 414A. 26. Gleave M. E., Bowden M., Goldenberg S. L., Bruchovsky N. and Sullivan L. D.: Predictors of androgen independent PSA progression in the LNCaP model. J. Urol. 151 (1994) 248A. 27. Kokontis J., Takakura K., Hay N. and Liao S.: Increased androgen receptor activity and altered c-myc expression in prostate cancer cells after long-term androgen deprivation. Cancer Res. 54 (1994) 1566-1573. 28. Farber E.: Clonal adaptation during carcinogenesis. Biochem. Pharmacol. 39 (1990) 1837-1846. 29. Noble R. L.: The development of prostatic adenocarcinoma in the Nb rat following prolonged sex hormone administration. Cancer Res. 37 (1977) 1929-1933. 30. Oesterling J. E.: Prostate specific antigen: a critical assessment of the most useful tumor marker for adenocarcinoma of the prostate. J. Urol. 145 (1991) 907-923. 31. Klotz L. H., Kerr H. W., Morse M. J. and Whitmore W. F., Jr.: Intermittent endocrine therapy for advanced prostate cancer. Cancer 58 (1986) 2546-2550. 32. Goldenberg S. L., Bruchovsky N., Gleave M. E., Sullivan L. D. and Akakura K.: Intermittent androgen suppression in the treatment of prostate cancer: a preliminary report. Urology 45 (1995) 839-845.