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Cancer Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Darlinghurst, Sydney, New South Wales 2010, Australia [S. M. H., D. I. Q., D. R. H., R. L. S.]; Department of Anatomical Pathology, Royal Prince Alfred Hospital, and Department of Pathology, University of Sydney, Camperdown, New South Wales 2050, Australia [C. S. L.]; Departments of Urology [D. G., P. C. B., P. D. S.] and Medical Oncology [J. J. G.], St. Vincent’s Hospital, Darlinghurst, Sydney, New South Wales 2010, Australia; and Douglass Hanly Moir Pathology, North Ryde, New South Wales 2113, Australia [W. D.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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70% positive
nuclei) in the malignant epithelium and loss of AR immunoreactivity in
the adjacent periepithelial stroma (
30%) was associated with higher
clinical stage (P = 0.01), higher
pretreatment prostate-specific antigen level
(P = 0.03), and earlier relapse after
radical prostatectomy (log-rank P = 0.009). These data identify a pattern of AR expression in malignant
epithelium and adjacent stroma that is associated with a poor clinical
outcome in prostate cancer. Equally important, they identify the need
to further investigate the mechanistic basis of loss of AR expression
in the malignant stroma and its potential role in deregulation of
prostate epithelial cell proliferation. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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In the normal adult prostate, circulating androgens act via the
AR3
-positive smooth muscle stromal cells to maintain a differentiated,
quiescent epithelium (3)
. The rapid regression of prostate
epithelium that follows castration is associated with a progressive
dedifferentiation of the smooth muscle cells to relatively
undifferentiated fibroblasts. These fibroblasts respond to castrate
levels of androgen via the AR to promote mitogenesis of the epithelium.
At the same time, the epithelium acts via the AR-positive fibroblasts
to facilitate the reversion of the fibroblastic stroma to a fully
differentiated smooth muscle phenotype. In prostate cancer, it is
hypothesized that altered gene expression in the epithelium results in
aberrant epithelial-stromal cell signaling that ultimately leads to
epigenetic changes in the stroma and augmentation of uncontrolled
proliferation of the epithelium (3)
. For example, loss or
reduced expression of the cellular adhesion molecule, E-cadherin,
because of mutations and deletions in the E-cadherin or
-catenin genes is a common feature of
malignant prostate epithelial cells (3
, 4)
. In addition,
the ability of stromal cells to down-regulate E-cadherin expression has
been implied from experiments in vitro where tumor-derived
human prostatic stromal cells were cocultured with a nontumorigenic
SV40T-immortalized human prostatic epithelial cell line BPH-1,
resulting in markedly decreased E-cadherin expression, loss of contact
inhibition, and phenotypic changes in the epithelial cells
(3)
. Such data emphasize the importance of
stromal-epithelial signaling in the prostate and raise questions on the
potential role of stromal AR expression in phenotypic changes
associated with prostate tumorigenesis.
The emergence of a "tumor stroma" that facilitates prostate carcinogenesis is not a new concept (3) . Thompson et al. (4) demonstrated in an in vivo mouse reconstitution model, the requirement for ras- and myc-induced transformation of both the stromal and epithelial components of the mouse urogenital sinus for carcinoma to develop. In addition, fibroblasts derived from rat urogenital sinus mesenchyme stimulated growth of the prostate cancer cell line, LnCaP, and conversely, LnCaP-conditioned medium stimulated growth of the urogenital sinus mesenchyme (5) . However, the role of AR in the "tumor stroma" and any relationship between alterations in stromal AR expression and prostate cancer progression remain unclear. Thus, we sought to determine whether changes in AR expression in the stromal and epithelial components of prostate cancers were associated with disease progression and a poor clinical outcome.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. Relapse was defined by the following criteria: biochemical disease
progression with a serum PSA concentration at or above 0.4 ng/ml,
rising over a 3-month period; or local recurrence on DRE confirmed by
biopsy or by subsequent rise in PSA.
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Immunohistochemistry.
Immunohistochemical staining for AR was performed on routinely
processed, paraffin-embedded tissue specimens. Four-µm sections of
the tissue and control specimens were cut and mounted on Superfrost
Plus adhesion slides (Lomb Scientific, Sydney, NSW, Australia).
As a positive tissue control, a benign prostatic hyperplasia specimen
that had high levels of AR expression was included. In addition,
paraffin-embedded cell pellets of the prostate and breast cancer cell
lines LnCaP and MDA-MB-231 were used as positive and negative controls,
respectively. Prior to staining, the sections were dewaxed and
rehydrated and then unmasked in 0.01 M citrate buffer (pH
6.0). The mouse monoclonal anti-AR antibody (Clone 2F12; Novocastra
Laboratories, Newcastle-upon-Tyne, United Kingdom) was applied
to the sections and incubated overnight at 4°C. A streptavidin-biotin
peroxidase detection system was used according to the manufacturer’s
instructions (Vectastain Elite kit; Vector Laboratories,
Burlingham, CA), with 3,3'-diaminobenzidine as substrate.
Counterstaining was performed with Whitlock’s hematoxylin, followed by
Light Green (BDH Laboratory Supplies, Poole, United Kingdom).
The percentage scores were derived from assessing AR positivity in 500
epithelial cells and 200–250 stromal cells in each tumor specimen by
two independent assessors (S. M. H. and D. I. Q.) and by one
pathologist (C. S. L.), all of whom were blinded to patient outcome.
For each slide, the percentage of nuclear AR immunostaining within
areas of stroma and carcinoma was expressed as the ratio of AR-positive
cells to the total number of cells counted. Each specimen was
categorized by AR nuclear immunoreactivity in the cancer and adjacent
periepithelial stroma so that each tumor was categorized on the basis
of < or 70% AR expression in the epithelium
and or >30% AR positivity in the periepithelial
stroma.
Statistical Analysis.
The primary outcome was disease-specific relapse, which was measured
from the date of RP. Data were evaluated for disease relapse using the
method of Kaplan Meier and log-rank test and by univariate and
bivariate analyses in a Cox proportional hazards model for AR status
and other clinical and pathological predictors of outcome. The
multivariate model was produced by assessing AR status with other
baseline covariates of clinical relevance: Gleason grade, pathological
stage, and preoperative PSA, which were modeled as dichotomous or
continuous variables as appropriate. Paired t tests were
used to assess the differences in the mean AR immunoreactivity in the
periepithelial stroma compared with the malignant epithelium and the
nonperiepithelial stroma in the same specimen. The associations between
AR expression and discrete categorical variables were tested using the
2 test. P < 0.05
was required for significance. All reported Ps are
two-sided. All statistical analyses were performed using Statview 4.5
software (Abacus Systems, Berkeley, CA).
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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and 2
. Detectable levels of AR were found in 91 of 96 (95%) cancers, with a
mean level of 72% AR-positivity. In the NPES, a mean of 76%
AR-positive cells was observed in 94 cases (99%) where AR was present.
In contrast, in the PES, which was defined as a 10-cell stromal layer
surrounding the malignant epithelium, a mean of only 25% AR positivity
was observed in 81 cases where AR was detected. This is significantly
lower than that observed in the malignant epithelium
(P < 0.001) or NPES (P < 0.001).
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. Group B represents
53 of 96 (55%) cases with concurrent high AR expression (70%) in
the cancer and low (30%) expression in the PES. Group A represents
all those cases in which alternate patterns are present. The
combinations represented by Group A are 30% AR+ PES and <70% AR+
tumor cells in 27 of 96 cases (28%); >30% AR+ PES 70% AR+ tumor
cells in 11 of 96 cases (12%) and >30 AR+ PES <70 AR+ tumor cells in
5 (5%) cases. Kaplan-Meier analysis of the data categorized by AR
expression demonstrates clearly that the latter three groups
cosegregate into a distinct group of patients with a significantly
better outcome than the patients in Group B (Fig. 3)
.
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2 analysis of the
relationship between the patterns of AR expression and other
clinicopathological variables showed that concurrent overexpression of
the AR (70%) in the malignant epithelium and loss of AR in the
adjacent PES (30%; Group B) was associated with higher clinical
stage (P = 0.01), higher pretreatment PSA
level (P = 0.03), and early tumor recurrence
(P = 0.009). Of the 36 men (35%) who
experienced PSA relapse, 26 (76%) demonstrated this altered pattern of
stromal-epithelial AR expression (Table 1)
. In agreement with other
published series (7)
, significant predictors of relapse on
univariate analyses in this cohort were pretreatment serum PSA levels,
pathological stage, Gleason grade, and clinical stage (Table 2)
. In addition, elevated expression of AR (70%) in the malignant
epithelium and loss of AR in the adjacent PES (30%; Group B) was a
significant predictor of relapse after radical prostatectomy on
univariate analysis (P = 0.016). Concordant
with this observation, Kaplan-Meier product limit analysis of
disease-free survival in patients stratified on the pattern of
stromal-epithelial AR levels demonstrated a significant difference in
relapse between the two strata (log-rank P = 0.012; Fig. 3
), with patients exhibiting concurrent overexpression of
AR (70%) in the malignant epithelium and loss of AR immunoreactivity
in the PES (30%) showing an earlier time to relapse after radical
prostatectomy. In addition, reanalysis of the data using different
cutoffs in AR positivity in the PES at 20 or 25% showed that the
relationship of altered AR expression and poor prognosis was maintained
(both log-rank P = 0.01).When stepwise
multivariate analyses were constructed with backward elimination, the
factors most predictive of relapse were, in descending order: Gleason
grade, pathological stage, with pretreatment PSA concentration and AR
strata equally predictive in this group.
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70%) in the
cancer and low (30%) expression in the adjacent PES
(P = 0.059; Table 1
). In addition, 4 of these
patients had developed hormone-resistant disease, and of these, three
cases (75%) showed this altered pattern of AR expression. |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Two common molecular mechanisms potentially responsible for prostate
epithelial cell proliferation in the absence of high levels of
circulating androgens have been proposed previously. These involve
alterations of the signals that activate the AR pathway and
deregulation of AR itself (8)
. Activation of the AR
signaling pathway by heregulin and other ligands for receptor tyrosine
kinases, e.g., insulin-like growth factor-1, transforming
growth factor , and keratinocyte growth factor, have been implicated
recently in disease progression (9, 10, 11)
. During androgen
deprivation, growth factors expressed by stromal cells can stimulate
epithelial cell proliferation by activating the AR in a
ligand-independent manner. For example, signaling via HER-2/neu
activates the AR pathway in the absence of androgens and acts
synergistically with low concentrations of androgen to activate PSA
transcription (11)
. HER-2/neu activation of PSA
transcription is not inhibited by antiandrogen, consistent with
an androgen-independent mechanism (11)
. These findings
have important clinical ramifications because they implicate tyrosine
kinase receptors as potential targets for therapeutic intervention in
the management of advanced prostate cancer.
The AR itself is also a target for deregulation. Mutations in the AR gene can alter steroid binding specificity and transactivational properties of the AR protein (8) . An association between reduced numbers of a polymorphic CAG repeat in the AR gene has been correlated with increased transcriptional activity of the AR and poor clinical outcome (12) . In addition, AR protein overexpression as a result of AR gene amplification may contribute to loss of growth control by enabling tumor cells to become hypersensitive to castrate levels of androgen in the prostate (13) . The immunocytochemical findings to date have been equivocal with regard to the clinical usefulness of AR levels in predicting prostate cancer outcome. Numerous studies investigating the relationship between AR immunoreactivity and other known markers of clinical significance have reported varying conclusions. In the studies where outcome data are available, AR status was (14 , 15) or was not (16, 17, 18) prognostic. However, there are no published studies to date that have addressed the relationship between stromal AR levels and clinical progression in this disease.
The data presented in this study support altered AR expression in the tumor-associated stroma as another potential mechanism of achieving androgen-independent prostate epithelial cell proliferation. In agreement with other published series, AR was present in the majority of prostate cancers examined and was not associated with tumor stage or grade (17) . However, the loss of AR in the malignant PES implies that the regulation of AR expression in the tumor stroma is altered in prostate cancer. In addition, the recent finding that the stroma surrounding high-grade prostatic intraepithelial neoplasia also lacks AR expression suggests that this alteration in stromal AR expression is an early event in prostate cancer progression (19) . Although the mechanisms responsible for this loss of AR expression remain undefined, there is evidence from studies in breast cancer that the estrogen receptor and the epidermal growth factor receptor are reciprocally regulated (20) . Thus, loss of AR in the stroma may be associated with up-regulation of receptor tyrosine kinase expression if similar mechanisms are operative in prostate cancer. In addition, growth factors may function synergistically with low concentrations of androgens and low AR expression to activate the AR pathway (11) . Hence, identification of factors that can regulate stromal AR expression and activate the AR pathway is a priority. There is increasing evidence that the multistep progression to malignancy involves both genetic alterations to the epithelium and epigenetic effects from the tumor stroma (3) . Thus, altered AR expression in the stroma may contribute to uncontrolled epithelial cell proliferation resulting from genetic alterations in the AR itself and in key cell cycle regulatory genes, e.g., p53 (6) and p16INK4A 4 leading to a highly aggressive tumor phenotype.
An alternative interpretation of our data is that the epithelial cells
in the poor prognosis cohort are overexpressing AR because of AR
amplification, so that it only appears that the PES has low expression.
However, whereas AR amplification occurs in up to 30% of
hormone-refractory local recurrences and metastases of
hormone-refractory prostate cancer, it appears to be a rare event in
primary prostate cancer, with AR amplification detected in only 1% of
205 primary prostate cancers (21)
. Furthermore, the data
presented here demonstrated similar levels of AR staining intensity in
the NPES and in the malignant epithelium (Fig. 1)
. Thus, it appears
unlikely that AR overexpression in the epithelium could explain
relative changes in epithelial to AR expression in PES described here.
Although it is clear that metastatic disease ultimately kills the patients, there is increasing evidence that the genetic changes predisposing tumors to an aggressive course may occur early in the carcinogenic process (22) . Although our data show that altered AR expression is associated with early biochemical relapse and clinical recurrence, there is insufficient follow-up to determine whether such expression is associated with early development of hormone-refractory disease. Because it is not possible to study relative levels of epithelial-stromal AR expression in metastases, this issue will only be resolved by further follow-up of cohorts with surgically treated localized prostate cancer. However, despite these limitations, of the 4 patients in this cohort that developed hormone-resistant disease coupled with local recurrence or distant metastases, three cases (75%) show this altered pattern of AR expression in the radical prostatectomy specimen. Thus, because an androgen-independent phenotype is an almost inevitable consequence of advanced prostate cancer, the possibility, suggested by this and another study (19) , that AR expression in the tumor stroma has a pivotal role early in prostate cancer progression and the subsequent development of androgen independence, may have major clinical implications. The targeting of key paracrine growth factor signaling pathways in locally advanced prostate cancer may be one way of conferring androgen sensitivity to hormone-refractory tumors. Thus, a more detailed understanding of the role of AR signaling mediated by the tumor stroma may provide new insight into the development of androgen-independent disease that will lead ultimately to better clinical management of hormone-refractory prostate cancer and potential new targets for therapeutic intervention.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was supported by grants from the
National Health and Medical Research Council of Australia (NHMRC), New
South Wales Cancer Council, Freedman Foundation, RT Hall Trust, Ronald
Geoffrey Arnott Foundation, St. Vincent’s Hospital, and St. Vincent’s
Clinic Foundation. D. I. Q. is the holder of a NHMRC Medical
Postgraduate Research Scholarship and recipient of the Vincent Fairfax
Family Foundation Fellowship from the Royal Australasian College of
Physicians. 
2 To whom requests for reprints should be
addressed, at Cancer Research Program, Garvan Institute of Medical
Research, 384 Victoria Street, Darlinghurst, New South Wales 2010,
Australia. Phone: 612-9295-8322; Fax: 612-9295-8321; E-mail: r.sutherland{at}garvan.unsw.edu.au
3 The abbreviations used are: AR, androgen
receptor; DRE, digital rectal examination; PSA, prostate-specific
antigen; RP, radical prostatectomy; PES, periepithelial stroma; NPES,
non-PES; TNM, Tumor-Node-Metastasis.
4 S. M. Henshall, D. I. Quinn, C. S. Lee, D. R.
Head, D. Golovsky, P. C. Brenner, W. Delprado, P. D. Stricker, J. J.
Grygiel, and R. L. Sutherland. Overexpression of the cell cycle
inhibitor p16INK4A in high grade prostatic intraepithelial
neoplasia predicts early relapse in prostate cancer patients. Clin.
Cancer Res., in press, 2001.
Received 3/27/00. Accepted 11/28/00.
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REFERENCES |
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in the progression of prostatic neoplasms. Clin. Cancer Res., 1: 545-550, 1995.[Abstract]
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