Prostate-specific Antigen, a Serine Protease, Facilitates ... · prevention and intervention in prostate cancer. Introduction Prostate cancer is the most common cancer in adult men
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Vol. 1, 1089-1094, October 1995 Clinical Cancer Research 1089
Advances in Brief
Prostate-specific Antigen, a Serine Protease, Facilitates Human
Prostate Cancer Cell Invasion1
Mukta M. Webber,2 Anuradha Waghray,
and Diana Bello
Departments of Medicine and Zoology, Michigan State University,
East Lansing, Michigan 48824-i3i2
Abstract
Human prostatic epithelial cells constitutively secrete
prostate-specific antigen (PSA), a kallikrein-like serine pro-
tease, which is a normal component of the seminal plasma.
PSA is currently used as a specific diagnostic marker for the
early detection of prostate cancer. We demonstrate that PSA
degrades extracellular matrix glycoproteins fibronectin and
laminin and, thus, may facilitate invasion by prostate cancer
cells. Blocking PSA proteolytic activity with PSA-specific
mAb results in a dose-dependent decrease in vitro in the
invasion of the reconstituted basement membrane Matrigel
by LNCaP human prostate carcinoma cells which secrete
high levels of PSA. A novel PSA-SDS-PAGE zymography
method for the detection of matrix degrading ability of PSA
is also described. We propose that: (a) because of the dys-
plastic cellular disorganization in early neoplastic lesions
called prostatic intraepithelial neoplasia (PIN), PSA may be
secreted not only at the luminal end but also, abnormally, at
the cell-basement membrane interface, causing matrix deg-
radation and facilitating invasion; and (b) PSA, along with
urokinase, another serine protease secreted by prostatic
epithelium, may be involved in the proteolytic cascade dur-
ing prostate cancer invasion and metastasis. The discovery
of the extracellular matrix degrading ability of PSA not only
makes it a marker for early detection but also a target for
prevention and intervention in prostate cancer.
Introduction
Prostate cancer is the most common cancer in adult men in
the United States. An estimated 244,000 new cases and 40,400
deaths from prostate cancer will occur in the United States in
1995 (1). The incidence increases with age, and about 80% of
prostate cancers are diagnosed after the age of 65 years (2).
Because of the increasing life span and an aging American male
population, prostate cancer is a major health concern. The most
threatening and primary cause of death from prostate cancer is
invasion and metastasis. One of the first events in progression to
malignancy is the degradation of the BM3 and ECM, followed
by invasion, a critical early step in the metastatic cascade.
Proteases intervene at the transition from in situ to invasive
carcinoma where local dissolution of the BM occurs. A corre-
lation between an increase in the secretion of matrix-degrading
serine proteases and metalloproteases, and invasion, metastasis,
and aggressiveness of cancer has been demonstrated (3). We
have shown a direct relationship between the level of secreted
urokinase (u-PA) and the ability of the DU145 human prostate
carcinoma cell line, not producing PSA, to degrade and invade
ECM (4, 5). We now show that PSA, which is abundantly
secreted by prostatic epithelial cells, may also play a role in
prostate cancer invasion.
PSA is a single-chain, 240-amino acid glycoprotein with
Mr �33,000 and a primary structure showing considerable ho-
mology to kallikrein (6). The human PSA gene on chromosome
19 has been cloned (7). PSA has the His-Asp-Ser triad in its
catalytic domain, a characteristic of serine proteases. It has
chymotrypsin-like activity, does not hydrolyze synthetic sub-
strates for plasmin, and displays a weak interaction with apro-
tinin, a plasmin inhibitor (8). This suggests that PSA primarily
acts independently as a protease in protein degradation, and not
via plasmin, as does u-PA. PSA is organ-specific, is character-
istically expressed in prostatic epithelial cells, and its expression
is regulated by androgens (9). The androgen-responsive LNCaP
human prostatic carcinoma cells (10) respond by increased PSA
expression (1 1). Following surgical or hormonal castration for
prostate cancer, serum PSA levels in patients decline. A subse-
quent increase in serum PSA level indicates cancer recurrence
(12). Although elevated levels of PSA in the serum of prostate
cancer patients were observed over 25 years ago, PSA measure-
ment has come into wide use only recently for early detection of
prostate cancer, for monitoring patients following radical pros-
tatectomy, and for identifying metastatic tumors of unknown
origin (13). Serum PSA measurement in combination with dig-
ital rectal examination and transrectal ultrasonography has
greatly increased the ability to detect prostate-confined cancer
(12, 13).
PSA is an important component and one of the most
abundant serine proteases in the seminal plasma, where it is
found at an average concentration of about 1.0 mg/ml (9).
Immediately after ejaculation, the seminal plasma coagulates
into a viscous gel which liquefies within about 20 mm. PSA
mediates this liquefaction (14). An understanding of the lique-
faction process provides clues to the mechanism of matrix
degradation by PSA in prostate cancer invasion. The seminal
coagulum is formed by the two most abundant, large molecular
Received 3/23/95; revised 5/16/95; accepted 5/30/95.
I This work was supported by the Biotechnology Research Center and
Research Initiation Grant (Michigan State University).
2 To whom requests for reprints should be addressed, at 5-350 Plant
Biology Building, Michigan State University, East Lansing, MI 48824-
1312.
3 The abbreviations used are: BM, basement membrane; ECM, extra-cellular matrix; PIN, prostatic intraepithelial neoplasia; PSA, prostate-
specific antigen; PSA-Ab, antibody to PSA; u-PA, urokinase-type plas-
Fig. 1 A, an immunoblot of affinity-purified PSA derived from human
seminal plasma. Lanes I and 2, a PSA sample. Lane 1, stained with mAb
to PSA (1:100). A series of bands at Mr �33,0(X) represent PSA. Lane
2, stained with polyclonal antibody to urokinase (1:500). B, an SDS-
PAGE zymogram (10% gel) for the detection of trace urokinase activityin the PSA sample. Lanes I and 2, received a sample of PSA. Lane 1,
faint zone of lysis at Mr 54,(K)O represents urokinase. Lane 2, PSAsample was blocked with antibody to u-PA. C, SDS-PAGE zymogram(12% gel) to determine whether PSA has protease activity and candegrade fibronectin. Three mg fibronectin were incorporated into the gel
as a substrate for 6 jig PSA loaded into the lane. Lysis caused by PSA
is indicated (arrowhead) at a level lower than the Mr 31,00() marker. D,
SDS-PAGE zymogram (12% gel) to determine whether the antibody toPSA has anticatalytic ability. The gel contained 5 mg fibronectin sub-
strate for PSA. Lane I, 10 jig PSA. Two zones of lysis at Mr �31,000
and a lower molecular weight (arrowhead) are seen. Lane 2, 10 p.g PSAwere incubated with 0.75 jig antibody (lgG) to PSA for 17 h at 37#{176}C
prior to SDS-PAGE.
with PSA antibody (IgG) at 6.25, 12.5, 25, 50, and 100 ngjml in
the final cell suspension. A suspension of one or two million
LNCaP cells/ml of serum-free RPMI 1640 medium containing
0.1% BSA was incubated with the antibody for 2 h before
performing the assay. A nonspecific immunoglobulin (IgG) was
used as a control. Nine such separate experiments were con-
ducted.
Results and Discussion
To attribute degradation of fibronectin and laminin to PSA,
it was necessary to first ascertain whether any u-PA activity was
present in the PSA sample to be used to assess its potential for
matrix degradation. Both PSA and urokinase are normally se-
creted by the prostate and constitute the prostate’s contribution
to the seminal plasma, from which PSA was purified. As stated
earlier, urokinase is also involved in matrix degradation (4, 5).
In the immunoblot in Fig. 1A, both lanes received a PSA
sample. Lane 1 was stained with human PSA-specific and Lane
2 with u-PA-specific antibody. In Lane 1, a series of bands at Mr
25,000-33,000 represent PSA. Variations in prostatic PSA mo-
lecular weight (20,000-35,000) due to glycosylation and alter-
native splicing have been reported (6, 18). Absence of staining
in Lane 2 indicates that u-PA was not detected in the PSA
Fig. 2 Degradation of fibronectin and laminin by PSA is demonstrated
by Western blot analysis. Samples were resolved on 4-15% gradientgels using SDS-PAGE and transferred, and blots were stained with the
respective antibodies. A, immunoblot to show fibronectin degradation
by PSA. Lane 1, 2.5-jig sample offibronectin only (control); Lane 2, 2.5
�i.g fibronectin were incubated with PSA prepared as described in
‘ ‘ Materials and Methods. ‘ ‘ fibronectin degradation products are shown.
Blot was stained with mouse antihuman primary antibody against fi-
bronectin (1:5(X)). B, immunoblot for laminin degradation by PSA. Lane1, 2.5 jig of reduced laminin only; Lane 2, 2.5 jig laminin were
incubated with PSA prepared as described above. Laminin sample was
reduced before loading into the gel. Blot was stained with mouse anti-human primary antibody against laminin (1:5(X)).
sample using immunoblot analysis. A more sensitive zymo-
graphic assay, which can detect as little as 10- “ mol u-PA (19),
was then used to detect trace u-PA activity in the PSA sample by
SDS-PAGE in which the gel also contained plasminogen, a
substrate for urokinase (4). The faint zone of lysis at Mr
-54,000 (Fig. 1B, Lane 1) represents the high molecular weight
u-PA, indicating the presence of trace u-PA activity in the PSA
sample. This activity could be completely blocked by urokinase-
specific antibody (Fig. 1B, Lane 2). Even traces of urokinase are
sufficient to generate plasmin in the presence of plasminogen.
Plasmin, also a serine protease, is formed by urokinase-medi-
ated catalysis of plasminogen to plasmin, and it can degrade
both fibronectin and laminin. The zone of lysis in the PSA
zymogram in Fig. 1C (6 jig PSA), which used fibronectin as a
substrate, shows that PSA has protease activity and it can
degrade fibronectin. In Fig. 11) (Lane 1), when a larger amount
(10 jig) of PSA was run in a similar gel, two zones of lysis, one
at Mr �3l,000 and the other at a lower molecular weight
(-25,000) were observed. This lysis could be blocked by pre-
incubation of PSA with PSA-Ab (Fig. 1D, Lane 2), indicating
that the antibody to PSA used has anticatalytic ability.
To further determine whether PSA alone can degrade fi-
bronectin and laminin, samples of PSA were incubated with
antibody to urokinase to block u-PA activity. Although the
absence of other lysis bands in the zymogram (Fig. !B, Lane 1)
excluded the presence of plasmin, #{128}-aminocaproic acid, a plas-
mm inhibitor, was also incubated with PSA concurrently to
block any trace plasmin activity. PSA samples prepared in this
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1995;1:1089-1094. Clin Cancer Res M M Webber, A Waghray and D Bello prostate cancer cell invasion.Prostate-specific antigen, a serine protease, facilitates human