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From the Lineberger Comprehensive Cancer Center,*
the
Department of Pediatrics,
the Laboratories for
Reproductive Biology,
Department of Surgery,
Division of Urology, and the Department of Pathology and Laboratory
Medicine,
University of North Carolina, Chapel
Hill, North Carolina
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Abstract |
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CWR22 is an androgen-dependent human CaP xenograft propagated subcutaneously in nude mice. CWR22 resembles the majority of human CaPs; CWR22 secretes PSA, undergoes tumor regression after androgen deprivation, and recurs as a palpable, growing and ultimately lethal tumor after several months in the absence of testicular androgen.10-13 We demonstrated that recurrence of CWR22 tumor after androgen deprivation was associated with re-expression of a network of androgen-regulated genes including PSA, human kallikrein-2, Nkx 3.1, AR co-activator ARA-70, cell cycle genes Cdk1 and Cdk2,3 and insulin-like growth factor binding protein-5.14 Recently, Amler and associates15 have reported incomplete reactivation of the androgen response pathway despite androgen absence in recurrent CWR22 using microarray analysis. Similar expression of AR and these androgen-regulated genes in androgen-dependent and recurrent CWR22 tumors suggested a role for AR regulation of gene expression in the development of recurrent CWR22 despite the absence of testicular androgen.
Video image analysis has been used to quantitate AR expression more
precisely than visual scoring.16-19
We developed an
automated method for measuring AR expression in individual cells that
was used to demonstrate the dependence of AR protein levels on serum
androgen levels in the CWR22 model.20
In CWR22
tumor-bearing mice castrated for 6 days, AR mean optical density (MOD)
decreased to 57% of levels in tumors from intact mice. After 72 hours
of exogenous testosterone administration to 6-day castrated mice, AR
MOD in CWR22 returned to the level found in tumors from intact mice.
Cellular proliferation of testosterone-treated tumors reached 
50%
of the original androgen-stimulated CWR22 tumors from intact
mice.14
These data suggested that the majority of CWR22
cells on day 6 after castration had functional AR. In archived radical
prostatectomy specimens, AR protein content was higher in
androgen-dependent, clinically localized CaP and lower in prostate
intraepithelial neoplasia than benign prostatic hyperplasia
(BPH).19,20
AR immunostaining intensity was similar in
androgen-stimulated and recurrent tumors from the CWR22 xenograft and
transurethral resection of the prostate specimens of BPH; all tissues
were small volume and fixed immediately after
procurement.20
Finally, 12 specimens of recurrent CaP and
16 specimens of BPH, all acquired by transurethral resection of the
prostate and fixed immediately, exhibited similar AR immunostaining
(unpublished data). Taken together, these findings suggest that AR is
expressed in androgen-stimulated CaP, diminished but recoverable after
castration, and re-expressed despite androgen absence on CaP
recurrence.
We sought to test the hypothesis that re-expression of AR coincided with the onset of androgen-independent cellular proliferation in CaP. To test this hypothesis, the temporal relationship between AR protein expression and cellular proliferation was determined using the CWR22 xenograft model during tumor regression and recurrence after castration. Quantitative immunohistochemistry and color video image analysis were used to measure precisely the proportion of cells expressing AR and Ki-67 and the intensity of expression of AR associated with response to androgen deprivation and emergence of the recurrent phenotype.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Nude/nude athymic mice were purchased from Harlan Sprague-Dawley,
Inc., Indianapolis, IN. The CWR22 tumor model has been maintained by
continuous passage since December of 1995 from CWR22 cells that were a
gift from Thomas A. Pretlow, MD, PhD, Case Western Reserve
University). CWR22 tumors were transplanted as 1 million
dissociated cells suspended in Matrigel (Collaborative Research Inc.,
Bedford, MA) injected subcutaneously into nude mice 4 to 5 weeks of
age.11,12
A 12.5-mg sustained-release testosterone pellet
(Innovative Research of America, Sarasota, FL) was placed
subcutaneously in each animal 2 days before tumor injection and every 3
months thereafter to maintain consistent serum levels of testosterone
of 4 ng/ml. After tumors reached a volume of 1 cm3
,
animals were anesthetized with methoxyflurane, castrated, and the
testosterone pellets removed. Intact mice bearing tumors and castrated
animals with either regressed or recurrent CWR22 tumors were exposed to
methoxyflurane and sacrificed by cervical dislocation. Tumor height,
width, and depth were measured using calipers and tumor volume was
calculated by multiplying these three measurements and 0.5234. Tumors
were excised and cut into several pieces (125
mm3); half was frozen in liquid nitrogen and half
was fixed in 10% buffered formalin for 24 to 48 hours, washed in
phosphate-buffered saline (PBS) for 24 hours, and paraffin-embedded.
Specimens of BPH prepared identically were used as positive controls.
Blood was obtained on sacrifice of all tumor-bearing mice for
measurement of serum PSA.
Immunohistochemistry
The avidin-biotin-immunoperoxidase technique21 was modified for use in paraffin-embedded tissues that were immunostained using capillary action with a MicroProbe staining station (Fisher Scientific, Pittsburgh, PA).22 Monoclonal antibody (mAb) F39.4.1 (BioGenex, San Ramon, CA) recognizes an epitope in the N-terminal region of human AR.23 mAb MIB-1 (Oncogene, Cambridge, MA) and polyclonal antibody MIB-5 (DAKO Corp., Carpinteria, CA) react with the cell cycle-associated antigen Ki-67 expressed during the proliferative phases (G1, S, G2, and M) but absent in the resting phase (G0) of the cell cycle.24 mAb A67-B/E3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) corresponds to amino acids 1 to 261 representing full length PSA p30 of human origin.25
Paraffin-embedded CWR22 tumor specimens were cut into 6-µm-thick histological sections. After deparaffinization and rehydration, tissue sections were heated to 100°C for 30 minutes in a vegetable steamer in the presence of antigen retrieval solution (CITRA, pH 6.0; BioGenex) and cooled for 10 minutes. Slides were preincubated with 2% normal horse serum for 5 minutes at 37°C and washed with automation buffer (Fisher Scientific).
AR mAb was diluted 1:300 (0.13 µg/ml in PBS containing 0.1% bovine serum albumin, pH 7.4) and sections were stained for 120 minutes at 37°C. Slides were incubated in biotinylated anti-mouse immunoglobulin (IgG) (Vector Laboratories, Inc., Burlingame, CA) for 15 minutes at 37°C (1:200 in PBS, pH 7.4) and in horseradish peroxidase-conjugated avidin-biotin complex (Vector Laboratories, Inc.) for 15 minutes at 37°C (1:100 in PBS, pH 7.4). The immunoperoxidase complexes were visualized using diaminobenzidine tetrahydrochloride (Vector Laboratories, Inc) for 8 minutes at 37°C (0.75 mg/ml in Tris buffer containing 0.03% hydrogen peroxide, pH 7.6). Slides were dehydrated through graded alcohol solutions and cleaned by Hemo-De xylene substitute (Fisher Scientific). Counterstaining was performed with hematoxylin (Gill’s formula, 1:6 dilution; Fisher Scientific) for 12 seconds. Slides were mounted with Permount and coverslips. Two representative slides were selected from each time point and stained with the polyclonal AR antibodies, AR52 and PG-21, following protocols reported previously.5,26 AR52 was provided by Dr. Elizabeth M. Wilson (University of North Carolina at Chapel Hill) and PG-21 was provided by Dr. Gail S. Prins (University of Illinois at Chicago). Slides prepared from a CWR22 tumor on day 6 after castration and human BPH were included as external controls to avoid variation of immunostaining intensity caused during staining procedures. Nonimmune mouse IgG (Vector Laboratories, Inc.) was used instead of AR mAb at the same IgG concentration for negative control slides prepared from the same tissue blocks as specimens; negative control slides were nonreactive.
MIB-1 mAb staining was performed at an IgG concentration of 0.5 µg/ml (1:50). All other steps were as described for AR immunostaining. Serial sections adjacent to the sections stained for AR were obtained from tumors on day 120 after castration and stained with MIB-1 mAb. Colon cancer tissue served as positive controls and 0.5 µg/ml of nonimmune mouse IgG was used instead of MIB-1 mAb at the same IgG concentration for negative control slides prepared from the same tissue blocks as specimens; negative control slides were nonreactive.
PSA mAb (1:50, 4 µg/ml) was biotinylated and blocked in vitro using the Iso-IHC kit (InnoGenex, San Ramon, CA) to avoid background staining caused by infiltrated murine cells in CWR22 tumors harvested from castrated animals. Sections were digested in Proteinase-K (20 µg/ml, DAKO Corp.) for 6 minutes at room temperature. Sections were incubated in the blocking solution and labeled with PSA mAb for 1 hour at 37°C and in streptavidin-peroxidase (InnoGenex) for 5 minutes at 37°C. Immunoreaction was visualized by diaminobenzidine tetrahydrochloride for 8 minutes at 37°C. Double immunohistochemistry was performed on additional CWR22 slides to co-localize PSA expression among Ki-67-positive tumor cells. Sections were eluted by glycine buffer (pH 2.3) for 5 minutes three times at room temperature and antigen-retrieved as described previously. A mixture of normal goat serum (2%) and avidin (1:50 in PBS, Vector Laboratories, Inc.) was used for blocking for 5 minutes at 37°C. Sections were reacted with MIB-5 (1:50, 20 µg/ml) mixed with biotin (1:50 in PBS, Vector Laboratories, Inc.) for 2 hours at 37°C. The same avidin-biotin-peroxidase complex technique used for MIB-1 was performed. Immunoreaction was visualized by 3-amino-9-ethylcarbazole (AEC) (Vector Laboratories, Inc.) for 10 minutes at 37°C. BPH and CaP specimens were used as positive controls. For the negative control slide, nonimmune rabbit IgG (Vector Laboratories, Inc.) was used instead of PSA mAb at the same IgG concentration; appropriate biotinylated IgGs were replaced with PBS in PSA and MIB-5 steps to check against cross-reactions. Negative control slides showed neither nonspecific reaction nor cross-reactions.
Automated Digital Image Analysis
Automated digital image analysis was performed as described
previously.20
Briefly, imaging hardware consisted of a
Zeiss Axioskop microscope, a 3-chip charge-coupled device camera
(C5810; Hamamatsu Photonics Inc., Hamamatsu, Japan), a camera control
unit (Hamamatsu Photonics Inc.), and a Pentium-based personal computer.
Each field of view for AR-stained slides was digitized at total
magnification x1200 using a x40 objective (numerical aperture, 1.3).
For MIB-1- and PSA-stained slides, a x20 objective (numerical
aperture, 0.6) was used for total magnification at x600. Twenty images
that contained 200 to 250 nuclei at x1200 and 400 to 500 nuclei at
x600 provided an adequate sample size for each tumor because the
deviation of average intensity values of randomly chosen immunopositive
areas became stable (within ±5%).
Immunopositivity and immunonegativity were determined using a linear discriminant analysis based on hue, saturation, and intensity of 100 immunostained cells of an intact CWR22 specimen and 100 cells of a negative control slide, respectively. The positivity for AR, Ki-67, and PSA was defined as the total number of pixels from immunopositive areas divided by the total number of pixels from all nuclear areas detected in a given specimen.
Differences in MOD and percentage of AR-, Ki-67-, and PSA-positive cells from all images from all tumors at various time points were evaluated using Wilcoxon rank sum tests. Correlations between features were examined using the Pearson’s product moment correlation test. F-tests were performed to compare the variances among samples. Statistical significance was achieved if P < 0.05.
Western Immunoblot Analysis of AR
Lysates were prepared from frozen CWR22 tumors. Tumor tissue (100 mg) was pulverized in liquid nitrogen, thawed on ice, and mixed with 1.0 ml of RIPA buffer with protease inhibitors (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.5 mmol/L phenylmethylsulfonyl fluoride, 10 µmol/L pepstatin, 4 µmol/L aprotinin, 80 µmol/L leupeptin, and 5 mmol/L benzamidine). Tissue was homogenized on ice for 30 seconds using a Biohomogenizer (Biospec Products, Inc., Bartlesville, OK). Two µl of 0.2 mol/L phenylmethylsulfonyl fluoride were added and homogenates incubated 30 minutes on ice. Homogenates were centrifuged at 10,000 x g for 20 minutes; supernatants were collected and centrifuged to prepare final lysates. Supernatant protein (100 µg) from each sample was electrophoresed in 12% sodium dodecyl sulfate-polyacrylamide gels and electroblotting to Immobilon-P membrane (Millipore Corp., Bedford, MA). Immunodetection used AR mAb F39.4.1 at 1:10,000 dilution. Secondary antibody (goat anti-mouse IgG conjugated to horseradish peroxidase; Amersham Corp., Arlington Heights, IL) was used for detection by enhanced chemiluminescence (DuPont-NEN Research Products, Boston, MA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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. The
majority of nuclei in CWR22 tumors from intact nude mice (98.5 ±
0.2%) showed intense staining of AR (MOD was 0.26 ± 0.01)
(Figure 1)
. On day 1 after castration, AR
MOD decreased and remained low on day 4 after castration whereas AR
percent positivity declined to a minimum on day 2 (2%) and remained
low on day 4 (10%). On day 6 after castration, AR-positive nuclei
increased sixfold to 63% and were distributed evenly throughout all
tumor sections. AR positivity increased further to 71% on day 12. AR
MOD decreased to a low of 0.11 on day 4 and remained low at 0.15 to
0.17 on days 12 through 120 after castration. Recurrent CWR22 tumors
obtained 150 days after castration exhibited lower percent AR
positivity (72.1 ± 7.6%) than CWR22 tumors from intact,
androgen-stimulated mice (P = 0.03). However,
among malignant nuclei expressing AR, MOD was similar
(P = 0.99) in the original CWR22 under androgen
stimulation and recurrent CWR22 in the absence of testicular androgens.
Immunostaining of CWR22 tumors before and after castration yielded
similar results when the polyclonal antibodies AR-52 and PG-21 were
used instead of AR mAb F39.4.1 (data not shown). Western blotting
(Figure 2)
of CWR22 tumor lysates
revealed similar AR levels in androgen-dependent and recurrent CWR22
tumors and reduced AR levels after castration until tumor recurrence.
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. Ki-67 positivity was high in
CWR22 tumors from intact nude mice (Figure 1)
, decreased gradually on
days 1 to 4 after castration, and remained at low or undetectable
levels on day 6 through day 90 after castration. Although most CWR22
cells were in growth arrest, proliferating cells occurred randomly at a
frequency <1% beyond day 6 until day 120 when multiple foci of 5 to
20 proliferating cells were detected throughout the tumors (Figure 1)
.
Recurrent CWR22 tumors, compared to the original CWR22 tumors,
exhibited lower cellular proliferation (49.1 ± 7.4%,
P = 0.006). Mean serum PSA levels (261.5 ± 123.0
ng/ml) of mice bearing recurrent tumors (n = 6)
increased to levels similar to those of tumor bearing intact mice
(246.7 ± 55.3 ng/ml) (Table 1)
.
Foci of recurrent cellular proliferation, as indicated by Ki-67
staining, appeared on day 120 day after castration. When these
proliferating foci were immunostained for PSA expression using
double-staining immunohistochemistry, positive cytoplasmic staining for
PSA was observed in the foci that contained Ki-67-positive cells
(Figure 3)
. Moreover, the appearance of
proliferating cells on day 120 after castration was associated with
increased (P = 0.01) serum PSA levels (24
± 3 ng/ml) compared to day 90 after castration (11 ± 2 ng/ml).
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; A, B, and
C) than CWR22 tumors from intact mice before castration
(F-tests; P < 0.001, 0.05, and 0.05,
respectively). Intertumor heterogeneity of cellular proliferation in
recurrent CWR22 was reflected in serum PSA levels that ranged from 25
to 740 ng/ml when measured in recurrent tumor-bearing mice. Throughout
the study period, AR MOD showed a parallel trend with Ki-67 positivity
(r = 0.64, P = 0.03) and PSA
levels (r = 0.77, P = 0.02)
(Table 1)
. In addition, Ki-67 positivity was highly correlated with PSA
levels (r = 0.90, P = 0.002).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In recurrent tumors 150 days after castration, AR expression and cellular proliferation were more heterogeneous than in tumors from intact mice; recurrent tumors exhibited lower percent-positive nuclei and greater variation between tumors than CWR22 tumors from intact mice. However, among nuclei immunostained for AR, there was no difference in AR MOD between intact and recurrent tumors. Previous studies have established that AR activation subsequent to binding of androgen results in increased nuclear levels of AR,29 homodimerization of AR,30 and binding of AR to DNA sequences that function as enhancers for AR-induced transcriptional activation.1,2,31 The similar levels of AR expression in intact and recurrent tumors suggests that nuclear AR may be stabilized despite the absence of testicular androgen in recurrent CWR22 by a ligand-independent or synergistic mechanism. AR activation is linked closely to stabilization of AR protein; binding of androgen stabilizes AR causing it to have a slower rate of degradation.32 AR MOD, by its definition, represents a mean nuclear staining intensity relative to cytoplasm.20 Therefore, increased AR MOD may reflect AR activation. Another possible mechanism of increased AR expression in recurrent CWR22 is AR gene amplification. AR gene amplification was detected in 7 of 23 cases of recurrent human CaP whereas none was detected before androgen-deprivation therapy.33 However, AR gene amplification in recurrent CWR22 tumors was not detected using Southern blot analysis and competitive reverse transcriptase-polymerase chain reaction.34
Because AR was expressed in a lower percentage of cells and at a lower
MOD in those cells expressing AR on days 1 to 4 after castration and AR
re-expression occurred on gross tumor recurrence, the temporal
relationship between recovery of AR expression and the onset of
cellular proliferation should provide insight into the role of AR in
CaP recurrence after castration. On day 120 after castration, when
recurrent tumor growth was indicated by increased serum PSA but tumor
sites did not yet demonstrate growth grossly, small foci of tumor cells
were recognized in harvested samples that immunostained for Ki-67 and
expressed PSA. These findings suggested these foci were precursors of
the recurrent CWR22 tumor that appears grossly 150 days after
castration. Two groups of investigators have reported on the
relationship between serum PSA and tumor recurrence after castration in
the CWR22 model. Serum PSA increase before gross tumor recurrence in
the CWR22 model was reported first by Nagabhushan and
associates.12
The time course of these events cannot be
compared to our results because they performed castration at higher
tumor volumes. Agus and associates28
castrated CWR22
tumor-bearing mice at tumor volumes similar to our studies. They
reported increased serum PSA 115 days after castration that preceded
tumor recurrence recognized grossly 20 days later. We reported
previously that PSA is one of several known androgen-regulated genes
whose mRNA was expressed at increased levels in recurrent CWR22 despite
the absence of testicular androgen.3
In the current study,
these RNA findings were confirmed by PSA protein quantitation at the
tissue level. PSA is a well known androgen-regulated
gene,35,36
however, other factors such as vitamin
D37
and transforming growth factor-ß138
have been shown to be involved in transcriptional regulation of the PSA
gene. One or more of these same factors may also be an initiator of
recurrent growth. Nonetheless, coincidental increased PSA serum levels
and tissue expression and the recurrence of tumor cellular
proliferation might be caused by the same mechanisms, one of which is
reactivation of AR.
Understanding the mechanisms driving recurrent growth is one of the most important issues in CaP research.39-41 Because AR is a growth-stimulating transcription factor in CaP, reactivation of AR in the absence of testicular androgen could be one of the molecular events that initiates cellular proliferation and leads to tumor recurrence. Several mechanisms have been proposed for activation of AR in the absence of testicular androgen. AR mutations that alter ligand specificity may influence tumor progression subsequent to androgen deprivation by making AR more responsive to adrenal androgens. CWR22 cells express a mutant AR (His 874 to Tyr) that has normal transcriptional activity in response to testosterone and dihydrotestosterone but has altered ligand specificity making it more sensitive to activation by adrenal androgens including dehydroepiandrosterone.13 Alternatively, a ligand-independent mechanism might cause transcriptional activation of AR. Protein kinase A and C modulators might activate AR in the absence of ligand by altering phosphorylation of AR42-45 or AR co-activators.46,47 Stimulation of PKA activity resulted in activation of the N-terminal domain of AR in LNCaP cells.48 Transfected AR was reported to be activated by insulin-like growth factor I, epidermal growth factor, and keratinocyte growth factor in DU-145 cells and PSA was increased by insulin-like growth factor I in LNCaP cells.49 Overexpression of HER-2/neu receptor tyrosine kinase was reported to increase expression of PSA and enhance growth in the androgen-dependent human CaP LAPC-4 xenograft.50 These effects required AR expression and seemed to occur through cross-talk between the AR and HER-2/neu pathways. We reported recently that high-level expression, increased stability, and nuclear localization of AR in recurrent tumor cells were associated with increased sensitivity to the growth-promoting effects of dihydrotestosterone at concentrations as low as the femtomolar range.34 Additionally, we have shown that high expression of transcriptional intermediary factor 2 and steroid receptor coactivator 1 in recurrent CaP increases AR transactivation in response to physiological concentrations of adrenal androgens or other steroids with affinity for AR.51 A single event or combination of events that affect AR function may lead to recurrent tumor growth in the absence of testicular androgen.
The re-expression of AR and known androgen-regulated genes in the 150-day recurrent CWR223,14,15 suggests that AR reactivation has a role in stimulating recurrent tumor growth. AR MOD decreased after castration and failed to increase until sometime between 120 days after castration and gross tumor recurrence. At the 120-day time point, if proliferating cells exhibited increased AR MOD, their rarity would preclude detection because <5% of malignant cells were proliferating. Quantitation of antigens using double-staining immunohistochemistry is technically difficult; our attempts to measure AR expression in Ki-67-immunopositive versus immunonegative cells have been unsuccessful thus far. However, if the small foci of tumor cells on day 120 after castration are precursors of recurrent tumors, direct comparison of AR, androgen-regulated gene products, and other molecules in these foci of cellular proliferation versus other regions of the tumor may be useful to evaluate specific mechanisms driving recurrent growth. Further study of this transition from androgen-dependent to androgen-independent growth in CWR22 may provide valuable insights into the mechanism of androgen-deprivation treatment failure in patients.
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Acknowledgements |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Footnotes |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Supported by the National Institutes of Health [grants RO1-AG-11343 (to F. S. F., J. L. M.), RO1-CA-64865 (to G. J. S., J. L. M.), P01-CA-77739 (to J. L. M., F. S. F., G. J. S.) and P30-HD-18968 (DNA and Tissue Culture Cores)], the United States Army Medical Research and Materiel Command (grant 98-1-8538 to J. L. M.), and the American Foundation for Urologic Disease and Merck U.S. Human Health (to C. W. G.).
D. K. and C. W. G. contributed equally to this work.
Accepted for publication October 8, 2001.
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