(Received for publication, September 26, 1994; and in revised form, November 17, 1994)
From the
Prostate-specific antigen (PSA) is widely used as a tumor marker of prostatic adenocarcinoma. We recently found that 30% of breast tumors produce PSA and that PSA is a favorable prognostic marker in female breast cancer. We measured immunoreactive PSA in cytosolic extracts of normal breast tissue from eight women receiving no medication and one woman who was receiving the progestin-containing oral contraceptive Brevicon. None of the eight cytosolic extracts of normal breast tissue contained appreciable amounts of immunoreactive PSA. However, left and right breast tissues from the woman receiving Brevicon contained high levels of PSA. This immunoreactive species was shown to have a molecular weight identical to that of seminal PSA. Furthermore, reverse transcription of RNA and polymerase chain reaction amplification produced a 571-base pair cDNA that hybridized to a labeled cDNA PSA probe. Upon sequencing, the cDNA polymerase chain reaction product was found to have 100% homology with cDNA from prostatic tissue. PSA production by breast carcinoma cell lines was achieved after in vitro stimulation with norethindrone and ethinylestradiol. Our data suggest that PSA can no longer be regarded as a specific prostatic protein because it is produced by breast tumors with good prognosis and by normal breast tissue after steroid hormone stimulation.
Oral contraceptives are among the most widely used drugs, but
the genes that are regulated by these agents are currently unknown. No
genes have as yet been found which are specifically regulated by oral
contraceptives in the female breast. We have recently found that about
30% of female breast tumors overexpress prostate-specific antigen
(PSA)()(1) , a protein that was thought to be
produced exclusively by the epithelial cells of the prostate gland (2, 3) . PSA overexpression in breast cancer is
associated with the presence of steroid hormone receptors(4) .
The mechanism of such overexpression in breast cancer is currently
obscure. It has been hypothesized that either ovarian or adrenal
steroids or tumor-derived molecules derepress the PSA gene through the
steroid hormone receptors or that the PSA gene is constitutively
overexpressed because of poststeroid hormone-receptor complex defects,
loss of physiological repressors, or because of defects in the hormone
response elements. We have observed a significant advantage in both
overall and disease-free survival of breast cancer patients who are
PSA-positive. (
)As PSA was found to be associated with more
benign breast tumors, we speculated that it could also be expressed by
normal breasts either under physiological circumstances or after
steroid hormone stimulation. In this paper we demonstrate that the
breasts of one woman who was receiving a progestin-based oral
contraceptive contained high levels of PSA. This protein was absent
from the breasts of eight other women who were not receiving any
medication. These data strongly suggest that PSA is not a
prostate-specific protein and that it can be produced by the female
breast either in cases of malignancy or after stimulation by steroidal
compounds. The biological role of PSA in either normal or malignant
breast is currently under investigation.
After electrophoresis the PSA cDNA PCR products were transferred onto Hybond nylon membranes (Amersham) and hybridized with a PSA cDNA probe (kindly provided by Dr. Jose Moreno). Southern blotting, probe radiolabeling, hybridization, and autoradiography were performed by standard techniques(10) . A second PCR amplification was performed on the remaining PCR reaction to provide sufficient material for subsequent analysis. Finally, the fragments were purified by ion exchange columns (Qiagen, Dusseldorf, Germany) according to the instructions of the manufacturer.
We have prepared cytosolic extracts from 18 normal breast tissues removed from nine women (left and right breasts) during cosmetic breast reduction surgery. PSA immunoreactivity was measured in these extracts using a highly specific and sensitive immunofluorometric technique (6) and by two widely used commercial PSA assays. Breast extracts from eight of the nine women were found to contain <0.03 ng of PSA/mg of total protein and were considered negative for PSA. Two breast extracts from the same woman (left and right breasts) had a relatively high concentrations of PSA (0.11 and 1.53 ng/mg). None of the eight PSA-negative women was receiving oral contraceptives or other medications. The woman with PSA-positive breasts was receiving only one medication, Brevicon. The PSA-positive and -negative results in the breast extracts by the immunofluorometric procedure were verified by using two widely used commercial PSA methods: the IMx from Abbott Laboratories and the Tandem-E kit from Hybritech Inc. Additionally, the highly positive breast extract was serially diluted in female serum from 2- to 32-fold and analyzed by immunofluorometry and the IMx assay. Excellent agreement between results was obtained. The serum from the patient on oral contraceptives and sera from another 10 women on oral contraceptives were analyzed for PSA and found to contain <0.02 µg/l PSA.
The highly positive breast extract
was subjected to HPLC (Fig. 1), and fractions were analyzed by
two immunofluorometric procedures that measure either total PSA (free
PSA plus PSA bound to -antichymotrypsin) or
specifically the PSA-
-antichymotrypsin (ACT)
complex(6) . More than 80% of the total PSA in normal breast
was in the free, 33-kDa form; a small proportion was present as
ACT
PSA complex (100 kDa). Another minor species, containing PSA
and ACT, was also detected (660 kDa), but its identity is unknown. The
presence of PSA in the highly positive breast extract was further
confirmed by Western blot analysis (Fig. 2). The 33-kDa form of
PSA, shown here to be present in normal breasts stimulated by oral
contraceptives, is similar to the form of PSA found in breast
tumors(1) . In male serum, the majority of PSA is present as
ACT
PSA complex with a molecular mass of 100 kDa (data not
shown)(6) .
Figure 1:
HPLC with a gel
filtration column. Details of the method are given in (6) .
Each HPLC fraction (0.5 ml) was analyzed with an assay that measures
free and -antichymotrypsin-bound PSA (ACT
PSA)
(
) or an assay that measures only ACT
PSA (
). The
response of the latter assay is in arbitrary fluorescence units since
no ACT
PSA standard exists. Panel A, injection of
purified seminal PSA, which elutes at fraction 39 corresponding to a
molecular mass of 33 kDa. No ACT
PSA is detected. Panel
B, injection of a breast extract from the woman receiving the oral
contraceptive Brevicon. The PSA assay detects two peaks: one at
fraction 39 (free PSA, major peak) and one at fraction 30 (100-kDa
minor peak). The latter peak is ACT
PSA as confirmed by the
ACT
PSA assay. The identity of the minor peak at fraction 21 (650
kDa) is unknown. These data confirm that more than 80% of the breast
tissue PSA is in the free, 33-kDa form. The HPLC column was calibrated
with molecular mass standards eluting at fraction 21 (660 kDa), 28 (160
kDa), 37 (44 kDa), 42 (17 kDa), and 49 (1.4
kDa).
Figure 2: Western blot analysis. Samples were electrophoresed on 8-16% gradient polyacrylamide minigels under reducing conditions, electrotransferred to nitrocellulose membranes, and probed with a rabbit polyclonal anti-PSA antibody. Detection was achieved by using a horseradish peroxidase-conjugated goat anti-rabbit antibody and chemiluminescence. Lane 1, molecular mass markers. Lane 2, purified seminal PSA dissolved in bovine serum albumin; the PSA band appears at 33 kDa (just above the marker at 31 kDa). Lane 3, supernatant from a prostatic carcinoma cell line (LNCaP) producing PSA. Lane 4, PSA-positive normal breast extract from the woman receiving Brevicon, containing a band at 33 kDa. Lane 5, another normal breast extract tested negative for PSA by the immunofluorometric procedure. Lane 6, an amniotic fluid tested for comparison; the identity of the band at 28 kDa, present in PSA-positive and PSA-negative normal breast extracts and in amniotic fluids, is unknown(16) .
To study the oral contraceptive-induced PSA
production further, we cultured T-47D and MCF-7 breast carcinoma cell
lines in the absence of any steroid hormones or in the presence of
norethindrone or ethinylestradiol at various concentrations (Fig. 3). No PSA was detected in the tissue culture supernatants
in the absence of steroid hormones after 11 days of confluent cultures.
Ethinylestradiol stimulated low levels of PSA production at
concentrations 10
M. Norethindrone was
effective in mediating intense PSA gene expression at concentrations as
low as 10
M. Other progestins were also
effective in mediating PSA gene expression (data not shown). The
identity of PSA in the tissue culture supernatants was characterized
further by HPLC and Western blot analysis (data not shown).
Figure 3:
Production of PSA by the breast carcinoma
cell line MCF-7. Cells were grown to confluence and then stimulated
with varying concentrations of either norethindrone (1) or
ethinylestradiol (2) at the final concentrations indicated, in
the absence of fetal calf serum from the culture medium. PSA was
measured in the culture supernatant 10 days poststimulation. No PSA was
detected in cell cultures grown identically but either nonstimulated or
stimulated with the solvent alone (ethyl alcohol). Norethindrone
stimulates PSA production at concentrations as low as 10M.
Two RNA
samples isolated from normal human breast tissue obtained at reduction
mammoplasty were analyzed for PSA gene expression by PCR. Southern blot
hybridization of the PCR products with a PSA cDNA probe detected a PSA
band of the expected size (571 bp) in the sample obtained from the
woman who was receiving oral contraceptives. The other sample, obtained
from a woman not receiving any medication, was PSA-negative (Fig. 4). To determine whether the PSA mRNA detected in breast
tissue is identical to the mRNA present in prostate tissue, we
performed DNA sequence analysis on the PSA cDNA fragment isolated from
breast tissue and compared it with the published PSA cDNA sequence.
Partial results are shown in Fig. 5. DNA sequence analysis of
the breast tissue PSA cDNA fragment showed 100% identity with the PSA
cDNA sequence data from normal prostate tissue recently obtained by
Monne and Croce ()(GenBank accession number U17040). No
mutations were identified in the 571-bp fragment that was sequenced.
Figure 4:
Top panel, reverse transcription-PCR of
RNA isolated from normal breast tissue and detected by Southern
blotting and hybridization of the filter with a radiolabeled PSA cDNA
probe. A PSA hybridization band of 571 bp is detected in lane
2, but the band is absent in lane 1. Bottom
panel, ethidium bromide-stained agarose gel of -actin reverse
transcription-PCR products. Lane 1 represents RNA isolated
from breast tissue negative for PSA protein immunoreactivity. Lane
2 represents RNA isolated from the breast tissue of the woman
receiving Brevicon. This breast tissue was positive for PSA protein
immunoreactivity.
Figure 5: Representative chromatogram of a 60-nucleotide region of the PSA cDNA sequence from normal breast tissue. Total RNA was isolated from a breast tissue specimen obtained at reduction mammoplasty from a woman receiving Brevicon. RNA was reversed transcribed to cDNA, amplified by PCR, purified, and directly sequenced. We obtained a 100% identity between the PSA cDNA sequence from breast tissue (bottom) and the PSA cDNA published sequence (top).
PSA is a serine protease found at very high concentrations in
sperm. The molecular mass of the glycosylated and nonglycosylated PSA
is 28.430 and 26.079 kDa, respectively(12) . However, with
polyacrylamide gel electrophoresis, PSA runs as a 32-33-kDa
protein. It has been suggested that PSA is involved in semen
liquefaction postejaculation. PSA is considered a highly specific
biochemical marker of the prostate gland and is currently used for
prostate cancer diagnosis, population screening, and postsurgical
monitoring of patients with prostate cancer(2, 3) .
Recently, we have demonstrated that PSA is produced by 30% of breast
tumors and provided evidence that this marker may be a new favorable
prognostic indicator of the disease(1) . Patients whose tumors
produce PSA live longer and relapse less frequently in comparison with
patients with PSA-negative tumors.
In this paper we have
examined if PSA could also be produced by normal breasts either under
physiological conditions or under conditions of stimulation by
exogenously administered steroid hormones. Eight patients who received
no medication had breast PSA levels below 0.03 ng/mg protein and were
considered negative for PSA. One patient who was receiving a
progestin-containing oral contraceptive was found to have breast PSA
levels of 0.11 and 1.53 ng/mgf total protein (left and right breast,
respectively). This immunoreactive PSA molecule was measurable by the
immunofluorometric assay (6) as well as by commercial PSA
assays that are used widely for prostate cancer diagnosis. We have
verified, using HPLC (Fig. 1), that the immunoreactive PSA
species in breast is present in two molecular forms: as a 33-kDa
protein, corresponding to the seminal form of prostatic PSA; and as a
100-kDa protein, corresponding to PSA complexed with
-antichymotrypsin. The latter form, which is
predominantly found in the serum of prostate cancer patients, was
present in the breast extract at relatively low levels (<10% of
total PSA); the major form was comprised of the 33-kDa protein (Fig. 1B). We have further characterized PSA in the
positive breast extract using Western blot analysis. This data have
shown that the PSA-positive breast extract, but not a PSA-negative
breast extract, contained an immunoreactive band with a molecular mass
of 33 kDa (Fig. 2). PSA was not elevated in the serum of
patients receiving oral contraceptives. The additional band on Western
blots, at 28 kDa, present in PSA-positive and PSA-negative breast
extracts and in amniotic fluid, may represent a PSA isoform or a PSA
fragment.
We have further molecularly characterized the presence of PSA in the normal breast at the mRNA level. For this analysis, we extracted total RNA from the PSA-positive and a PSA-negative breast tissue and amplified it, after reverse transcription, with specific primers derived from the known PSA gene sequence. We found that amplification occurred only with RNA from the PSA-positive breast tissue. The PCR product was hybridized with a specific cDNA probe for PSA and revealed the expected 571-bp fragment (Fig. 4). Furthermore, sequencing of the PCR product derived from breast tissue has shown that the sequence was identical to the sequence of the cDNA for PSA derived from prostatic tissue. No mutations were identified (Fig. 5).
The data presented support the notion that the PSA
gene is expressed in normal breast tissue under conditions of
stimulation by steroid hormones. To reproduce the phenomenon in
vitro we cultured T-47D and MCF-7 cells, two breast cancer cell
lines that are both positive for steroid hormone receptors. Tissue
culture supernatants from the nonstimulated cell lines contained no
detectable PSA. However, upon stimulation with norethindrone or
ethinylestradiol, the two components of the contraceptive used by the
patient whose breast was positive for PSA, the cell lines produced PSA.
Notably, norethindrone was active at concentrations as low as
10M (Fig. 3).
The physiological
relevance of our finding is currently unknown. However, it has recently
been suggested (13, 14) that PSA, a serine protease,
is a new potential growth factor regulator, enzymatically digesting
insulin growth factor-binding protein-3 to release insulin growth
factor-I or enzymatically activating latent human transforming growth
factor- and proteolytically modulating cell adhesion
receptors(15) . Our previous data on PSA gene expression in
breast cancer that has good prognosis (1, 5) and the
demonstration here that the PSA gene is regulated by oral
contraceptives in normal breast suggest that this enzyme, until
recently thought to be associated only with male prostatic tissue, may
have important, previously unrecognized extraprostatic functions
related to breast and other tissue growth and possibly breast cancer.
This suggestion is further supported by our finding of the PSA presence
in amniotic fluid during gestational weeks 13-21 (16) and
in the milk of lactating women(5) . The role of PSA in
embryonal life and its involvement in growth factor regulation are
currently under investigation.