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INTRODUCTION |
The cadherin family of transmembrane glycoproteins mediates
Ca2+-dependent intercellular adhesion in a
homophilic manner (1). The classical cadherins, including N-, P-, and
E-cadherin are well-conserved and are structurally similar. The
cadherin structure consists of an extracellular domain with five tandem
repeats, a transmembrane domain, and a cytoplasmic domain that
interacts with the catenin family binding proteins (2). The
interepithelial binding of E-cadherin, which mediates lateral cell-cell
adhesion in secretory tissues such as the prostate and mammary gland
(3), results in the formation of desmosomes and adherens junctions that
are required for tissue morphogenesis and maintenance of the
differentiated phenotype (1). The integrity of E-cadherin binding is
dependent on its interaction with the catenin binding proteins in the
intracellular domain. E-cadherin binds to
-,
-, and
-catenin
(4), which physically associate with actin filaments and the actin
cytoskeleton (5-7). Inactivation of E-cadherin through gene mutation,
transcriptional inactivation, or promoter methylation has been
demonstrated in many adenocarcinomas (8-13). However, these mechanisms
are not likely to account for all mechanisms by which E-cadherin
becomes inactivated in cancer.
One mechanism of E-cadherin inactivation involves the proteolytic
ablation of the extracellular domain (14). The generation of a soluble
80-kDa fragment that was shed into the medium of cultured cancer cells
in response to calcium influx (15, 16) resulted from cleavage by matrix
metalloproteases (17, 18). It was also demonstrated that
metalloproteases mediated the cleavage of the extracellular domain of
VE-cadherin that was shed into cultures of human endothelial cells
(19). Additionally, caspase-3 has recently been shown to cleave
E-cadherin during the execution phase of apoptosis (20). Taken
together, these studies have demonstrated that specific proteases can
target and inactivate E-cadherin; however, the biological
ramifications of these proteolytic mechanisms remain unknown.
We have previously identified a novel 97-kDa truncated E-cadherin
species that may be important in localized prostate tumorigenesis (21).
We have since identified a novel E-cad100 species that is
generated by a calcium-dependent mechanism and may also be
important in prostate tumor progression. Due to the apparent
requirement of calcium in the generation of E-cad100, the
calpain family of cysteine proteases emerged as a likely candidate
mediating this cleavage event. There are two main isoforms of calpain
that are ubiquitously expressed in tissue and are designated µ- and
m-calpain based on the concentration of calcium required for their
respective function in vitro (22-25). Because physiological concentrations of calcium are in the range of 100-300 nM,
it is hypothesized that other mechanisms exist to lower the activation threshold of calcium required for calpain activation in vivo
(22, 24). Calpains are heterodimeric proteins consisting of a large catalytic 80-kDa subunit, and a smaller 29-kDa regulatory subunit. Calpain is involved in diverse physiologic roles including cell-cycle regulation (26, 27), signal transduction (28), and apoptosis (29-31).
Calpain proteolysis occurs in a limited fashion, producing distinct
peptide fragments without further degradation (32). Secondary structure
is likely to affect substrate specificity, since many proteins contain
consensus calpain sequences but are not susceptible to cleavage (32).
Proteins that have been identified as substrates of calpain include the
cytoskeletal proteins spectrin and members of the integrin family (33,
34).
In adenocarcinoma, the disruption of cellular adhesion appears to rise,
in part, through alterations of the E-cadherin cell-adhersion system
(35). However, proteolytic mechanisms of E-cadherin inactivation in
cancer have not been thoroughly investigated. In this study, we
investigated whether E-cadherin is a substrate for calpain, and whether
calpain-dependent proteolysis of E-cadherin was associated with prostate cancer progression.
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EXPERIMENTAL PROCEDURES |
Tissue Culture Cell Lines, Chemicals, and Transfections--
The
cell lines LNCaP, MCF-7, and SKBR3 were propagated in RPMI 1640 medium
supplemented with 10% fetal calf serum, 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin (Invitrogen). Cells were incubated at 37 °C and
subcultured weekly. The engineered stable cell lines were maintained by
adding 0.8 µg/ml puromycin or 1 mg/ml G418 to the culture media.
TPA1 (Alexis Biochemicals)
was dissolved in 100% ethanol, aliquoted, and used at a final
concentration of 10 nM. Ionomycin, calpain inhibitor I,
calpain inhibitor II, calpain inhibitor III, and calpeptin were all
obtained from Calbiochem. Cycloheximide was obtained from Sigma and
used at a final concentration of 50 µM. Cells were
treated with cycloheximide 30 min prior to TPA treatment. SK-Ecad was
generated by transfection of a plasmid that contained wild-type human
E-cadherin in the pLK-pac vector with a 2×-Myc epitope added to the
last codon (gift from Dr. Margaret Wheelock) and was selected with
puromycin. Transfection was done with Lipofectin (Invitrogen) as
instructed. E-cadherin mutants were generated from wild-type human
E-cadherin cDNA cloned into the pcDNA3 vector that contained a
2×-Myc epitope added to the last codon. Transfections with
pcDNA3-based vectors were done with the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) and selected with G418.
Western Analysis and Immunoprecipitations--
Protein lysates
were prepared from tissue and cultured cells in the following buffer:
50 mM Tris, pH 7.5, 120 mM NaCl, 0.5% Nonidet
p-40, 40 µM PMSF (phenylmethylsulfonyl fluoride), 50 µg/ml leupeptin, 50 µg/ml aprotinin, 200 µM
orthovanadate, 1 mM EGTA. Tissue was homogenized prior to
lysing. Cells were allowed to lyse 1 h on ice, centrifuged,
supernatants extracted, and quantitated using a Bradford assay. Lysates
were separated by 6% Novex gels and analyzed using chemiluminescence
(Amersham Biosciences) as described previously (36). Membranes were
stripped with the following stripping solution: 62.5 mM
Tris pH 6.8, 2% SDS, 100 mM
-mercaptoethanol. Following
ECL and washing, the membrane was incubated with the stripping solution
for 30 min at 55 °C, with gentle shaking every 5 min. Densitometry
analysis was performed as previously described (21).
For immunoprecipitations, cell lysates were prepared and quantitated as
described above. The lysates were pre-cleared for 30 min at 4 °C
with the appropriate Sepharose beads (Zymed Laboratories) conjugated
with protein G or A prior to immunoprecipitation reactions. All beads
were diluted and mixed for 1 h at room temperature with an equal
volume of Tris-buffered saline (50 mM Tris, pH 7.5, 120 mM NaCl) + 2.5% milk prior to use. Following pre-clearing,
lysates were aliquoted into microcentrifuge tubes, where an equal
volume of lysate was used for each immunoprecipitation reaction. The volumes of each reaction were equalized to 500 µl with lysis buffer, 5 µg of primary antibody was added, and the reaction was carried out
at 4 °C rotating end-over-end. The following day, the appropriate Sepharose beads were added and the reaction was allowed to continue to
rotate end-over-end at 4 °C for 90 min. The beads were then pelleted
and washed three times with lysis buffer (+ protease inhibitors).
Reducing sample buffer was added to each pellet, boiled for 5 min,
centrifuged, and the supernatants loaded onto 6% NOVEX Tris-Gly
(Invitrogen) gels for Western analysis.
Ionomycin and Calpain Inhibitor Experiments--
2.0 × 105 LNCaP or MCF-7 cells were plated in 6-well plates.
72 h later, cells were pre-treated with 20 µM of the
calpain inhibitors in fresh serum-containing media for 15 min. Cells
were then treated with 10 µM ionomycin for 20 min. Cells
were harvested, lysed, quantitated, and 30 µg of protein per sample
were loaded onto 6% NOVEX gels for Western analysis.
Antibodies--
Antibodies used for the detection of E-cadherin
were HECD-1 (Zymed Laboratories), E-9 (16), 4A2 (37), C20820
(Transduction Laboratories), E2 (38), and SC-1499 (Santa Cruz
Biotechnology). Antibodies used for co-immunoprecipitation experiments
were p120 (Transduction Laboratories),
-catenin (Zymed Laboratories,
CAT-5H10), and
-catenin (Santa Cruz Biotechnology). µ-Calpain
antibody was purchased from Calbiochem and
-spectrin was purchased
from Chemicon. The normal goat IgG antibody was obtained from Santa
Cruz Biotechnology. The appropriate horseradish peroxidase-conjugated
anti-mouse, anti-rat, or anti-goat secondary antibodies were obtained
from Amresco and Jackson ImmunoResearch Laboratories, Inc.
In Vitro Calpain and Caspase-3 Assay--
E-cadherin was
immunoprecipitated (as described above) from LNCaP cell lysates with
the C20820 antibody. After the third wash with lysis buffer (+ protease
inhibitors), the supernatants were removed, and the reactions placed on
ice. The Sepharose beads were then re-suspended in an appropriate
volume of lysis buffer and divided equally into five Eppendorf tubes
for five separate reactions. They were then centrifuged at 6,000 rpm
for 3 min at 4 °C, the supernatants removed, and tubes placed on
ice. For the calpain assay, the following proteolysis buffer was used
at 25 µl per reaction: 100 mM HEPES, 10 mM
dithiothreitol, 10% glycerol (v/v), 1 mM
CaCl2. A separate buffer that contained 10 mM
EGTA was also used. Reactions were started by adding 0.4 units of
µ-calpain, 0.95 units of m-calpain, or 50 units of caspase-3 (all
from Calbiochem) and were carried out at room temperature. For the
caspase-3 assay, the following proteolysis buffer was used at 25 µl
per reaction: 100 mM HEPES, 10 mM
dithiothreitol, 10% glycerol (v/v), 1 mM EGTA. Reducing
sample buffer was added to each tube to stop the reaction at each time
point. The reactions were boiled for 5 min and the supernatants were
loaded onto 6% NOVEX Tris/Gly (Invitrogen) gels for Western analysis.
Mutagenesis--
E-cadherin deletion mutants were generated
using the Quikchange kit (Stratagene). The parental plasmid was
wild-type full-length human E-cadherin cloned into the pcDNA3
vector (Invitrogen) with a 2× c-Myc tag cloned in-frame at the
C terminus. The primers used were as follows: 791
5, forward:
5'-GACGTTGCACCAACCCGGTATCTTCC-3', reverse:
5'-GGAAGATACCGGGTTGGTGCAACGTC-3'; 795
5, forward:
5'-CTCATGAGTGTCCGCCCTGCCAATC- 3', reverse:
5'-GATTGGCAGGGCGGACACTCATGAG-3'; 782
6, forward: 5'-GACGCTCGGCCTGAAGCACCAACCCTCATG-3', reverse:
5'-CATGAGGGTTGGTGCTTCAGGCCGAGCGTC-3'. All mutants were verified
by DNA sequencing at the University of Michigan sequencing Core facility.
cDNA Microarray Analysis--
Analysis of samples were
performed as previously described (39). In brief, cDNA generated
from prostate samples (i.e. prostate cancer) and benign
prostate tissue (reference) samples were labeled with distinguishable
fluorescent dyes and hybridized to the cDNA microarray (10,000 clones). The cDNA microarray was analyzed using a scanner, and
fluorescence ratios were determined for each gene. Color intensities
were converted into ratios of gene expression. These ratios are
imported into a data base for further analysis. To test for significant
differences in the mean cDNA expression between all tissue types,
we performed a one-way ANOVA test. To determine differences between all
pairs (e.g. localized prostate cancer versus
benign), we performed a post-hoc analysis using the Scheffé
method. The mean expression scores for all examined cases were
presented in a graphical format using error bars with 95% confidence
intervals. The results were considered to be significant when
p < 0.05.
Tissue Procurement--
Patients with localized prostate cancer
provided written informed consent in an Institutional Review Board
(IRB)- approved study at the University of Michigan to have their
prostate tissue evaluated in experimental studies. These fresh radical
prostatectomy specimens were obtained through the University of
Michigan prostate cancer SPORE tissue bank and were frozen in liquid
nitrogen within 30 min after surgical excision. Histological
confirmation of both tumor and normal regions of each prostate gland
were confirmed by a genitourinary pathologist at the University of
Michigan, and the corresponding tumor or normal region was excised
before processing for protein analysis (21).
A Rapid Autopsy Protocol has been previously described (40). Permission
for an autopsy was granted from patients with hormone-refractory prostate cancer to participate in an IRB-approved tissue donor program
at the University of Michigan. Metastases from prostate cancer were
immediately frozen in liquid nitrogen. The metastases were confirmed as
prostate cancer in origin by a genitourinary pathologist at the
University of Michigan.
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RESULTS |
Ionomycin Induces a Calpain-dependent Cleavage of
E-cadherin to 100 kDa--
Previous studies from our laboratory have
shown that PKC activation induces a signal transduction pathway that
leads to the truncation of E-cadherin in prostate and mammary
epithelial cells (41, 42). Activation of PKC has a number of
physiological ramifications in the cell, including cell proliferation
and differentiation, changes in gene expression, and transmembrane ion
transport (43). We hypothesized that E-cadherin cleavage may be due to
the influx of calcium ions initiated by PKC activation. To specifically
address the role of calcium in the cleavage of E-cadherin, we treated LNCaP cells with the calcium ionophore, ionomycin, and observed the
rapid accumulation of a novel 100-kDa fragment of E-cadherin that was
designated E-cad100 (Fig.
1A, top panel).
Ionomycin did not result in the accumulation of the previously
described E-cadherin species, E-cad97. Due to the apparent
role of calcium in E-cadherin cleavage, we speculated that the
calcium-dependent protease calpain may mediate the cleavage
of E-cadherin. E-cadherin cleavage and the accumulation of
E-cad100 was dramatically inhibited in cells pretreated
with the calpain inhibitors calpain inhibitor I, calpain inhibitor II
or calpeptin (Fig. 1A, top panel). These
inhibitors had no effect on the generation of the 97-kDa species ruling
out the possibility that calpain generates both fragments. E-cadherin
cleavage to 100 kDa was also prevented by calpain inhibitor I following
TPA treatment (data not shown).

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Fig. 1.
Calpain inhibitors prevent induction of
E-cad100, -spectrin cleavage, and
µ-calpain activation. LNCaP cells (A) or MCF-7 cells
(B) were pretreated with 20 µM calpain
inhibitors for 15 min, then treated with 10 µM ionomycin
for 20 min. The inhibitors used were calpain inhibitor I, calpain
inhibitor II, calpain inhibitor III, or calpeptin. 30 µg of protein
extracts were resolved on a 6% SDS-PAGE gel. Each of the membranes
were immunoblotted with HECD-1 antibody (top panels),
stripped and re-probed with an -spectrin (middle panels),
and a µ-calpain antibody (bottom panels).
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To help confirm that ionomycin induced a calpain-mediated cleavage of
E-cadherin, we re-probed the membranes with an
-spectrin antibody.
-Spectrin is a well documented calpain substrate with a molecular
mass of 280 kDa that is cleaved by calpain to produce a 145/150-kDa
doublet (33). The resulting
-spectrin immunoblot revealed that
spectrin cleavage was induced by ionomycin, and that this induction was
effectively inhibited by calpain inhibitors (Fig. 1A,
middle panel). In the inactive state, calpain exists as
pro-enzyme heterodimer of 80-29 kDa, and following calcium binding
undergoes autolysis to an active 78-18 kDa heterodimer (32). Again,
using the same membrane, we assayed for the autolysis of the large
subunit of calpain by immunoblot (Fig. 1A, bottom panel). Although faint, immunoblot analysis revealed a clear
conversion of the 80-kDa large subunit to the 78-kDa active monomer
following the addition of ionomycin. Pretreatment of LNCaP cells with
calpain inhibitor I, calpain inhibitor II, and calpeptin prevented this autolysis.
A similar experiment was performed using MCF-7 cells (Fig.
1B). MCF-7 cells were pretreated with calpain inhibitor III
and calpeptin prior to ionomycin treatment. Fig. 1B shows
that these inhibitors effectively blocked E-cadherin cleavage by
ionomycin. Re-probing the membrane for
-spectrin shows that calpain
inhibitor III and calpeptin blocked calpain-mediated spectrin cleavage
(Fig 1B, middle panel). Although partial,
re-probing with µ-calpain antibody showed some inhibition of
autolysis of the µ-calpain large subunit (Fig. 1B,
bottom panel). We observed that calpain inhibitor 3 slightly
reduced the amount of calpain; this may be an indirect effect of the
inhibitor on calpain stability. Taken together, these data show that
ionomycin treatment of LNCaP and MCF-7 cells induces a
calpain-dependent truncation of E-cadherin to the 100-kDa
form that was significantly inhibited by calpain inhibitors.
Calpain Cleaves E-cadherin in Vitro--
To determine if purified
calpain could generate E-cad100 in vitro, we
incubated E-cadherin immunopurified from untreated LNCaP cells with
either µ- or m-calpain. In the presence of both isozymes, full-length
E-cadherin was rapidly cleaved to the 100-kDa fragment, and this
truncation could be completely inhibited by addition of the calcium
chelator EGTA (Fig. 2, A and
B). In the absence of calpain, the 100-kDa product was not
detected. Over the course of 20 min, some general degradation of
E-cadherin was observed, which was unrelated to the effects of calpain
cleavage of E-cadherin. These results provide compelling evidence that
both µ- and m-calpain can specifically cleave mature E-cadherin to
the 100-kDa fragment in vitro.

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Fig. 2.
Calpain cleaves E-cadherin in
vitro. E-cadherin, immunoprecipitated from LNCaP cells was
pretreated with EGTA prior to the addition of µ-calpain
(A) or m-calpain (B) for the indicated times. All
cleavage reactions were resolved by 6% SDS-PAGE and immunoblotted with
HECD-1 antibody.
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Examination of the cytoplasmic domain of E-cadherin revealed a
potential caspase-3 cleavage site within residues 747DTRD.
Caspase-3 is a cysteine protease that is involved in proteolytic breakdown during the execution of apoptosis and has been implicated in
the cleavage of E-cadherin (20). We wanted to determine if E-cad100 was generated by caspase-3. A similar in
vitro assay was performed using purified caspase-3, which failed
to demonstrate cleavage of E-cadherin to E-cad100 (data not shown).
PKC Activation Induces E-cad100--
We utilized the
E-cad
/
mammary epithelial cell line SKBR3 (44) to exogenously
express E-cadherin. We selected stable clones that expressed moderate
to high levels of the 2×-Myc-tagged E-cadherin protein, which migrates
at a slightly higher molecular weight of 130 kDa (37). Activation of
PKC in these E-cad-expressing SKBR3 cells (termed SK-Ecad) revealed the
rapid accumulation of E-cad100 over a course of 12 h
(Fig. 3). PKC activation by TPA treatment did not induce expression of any endogenous E-cadherin protein in
untransfected SKBR3 cells. We also noticed a dramatic increase in the
expression levels of E-cadherin in the transfectants following TPA-treatment; however, the mechanism through which this up-regulation occurs is currently unclear, as the E-cadherin cDNA is driven by a
cytomegalovirus promoter and does not contain any phorbol-responsive elements. To address whether increased expression of E-cadherin was
required for cleavage, LNCaP cells were pretreated with cycloheximide prior to TPA treatment. Fig. 4
demonstrates that blocking E-cadherin synthesis with cycloheximide did
not prevent cleavage. Thus, protein synthesis is not required for
calpain-dependent cleavage of E-cadherin. In order to
determine what proportion of E-cadherin is cleaved in cell culture, we
performed densitometry on multiple E-cadherin Western blots in which
E-cad100 has been induced with ionomycin or TPA. On
average, the amount of total cellular E-cadherin cleaved was ~30%
(data not shown).

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Fig. 3.
Expression and truncation of exogenous
E-cadherin in SKBR3 cells. A, Protein extracts from
SKBR3 parental cells (lanes 1-2) and SK-Ecad cells
(lanes 3-6), treated with TPA at the indicated times, were
immunoblotted with the HECD-1 antibody.
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Fig. 4.
E-cadherin synthesis is not required for
cleavage. LNCaP cells were pretreated with 50 µM
cycloheximide 30 min prior to TPA treatment. 50 µg of protein
extracts from cells that were harvested at the indicated times were
resolved by 6% SDS-PAGE and immunoblotted with HECD-1 antibody.
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Epitope and Functional Mapping of
E-cad100--
TPA-treated SK-Ecad cells were utilized to
map the cleavage site on E-cadherin. The specific antibody epitopes
that were used are shown in Fig. 5.
TPA-treated SK-Ecad cell lysates were immunoblotted with the different
antibodies that recognized epitopes in either the extracellular or
cytoplasmic domains of E-cadherin. E-cad100 was recognized
by the N-terminal antibody E-9, as well as by the cytoplasmic antibody
4A2, which has been mapped to residues 757-778 and by another
cytoplasmic antibody C20820, which has been mapped to residues 773-791
(45). The antibody SC-1499 (46) that maps to the C terminus of
E-cadherin failed to recognize E-cad100 (Fig. 5). Although
three antibodies had variable sensitivities for E-cad100,
the immunoblot data indicated that the cleavage site was in the
cytoplasmic domain of E-cadherin located between the C20820 and SC-1499
epitopes.

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Fig. 5.
Epitope mapping of
E-cad100. A schematic of full-length E-cadherin
and a table of the antibodies and epitopes to which they are mapped are
shown. TPA-treated SK-Ecad protein extracts were resolved by SDS-PAGE
and immunoblotted with E-9 (site A), 4A2 (site
B), C20820 (site C), and SC-1499 (site D)
antibodies. Lysates were resolved by 6% SDS-PAGE.
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To further verify the antibody mapping and to functionally map the
truncation, we employed co-immunoprecipitations utilizing the catenin
binding proteins to determine if the
-catenin,
-catenin, and p120
binding domains are present in E-cad100. The
-catenin and
-catenin binding domains have been mapped to
residues 815-839 of the cytoplasmic domain of E-cadherin (47). The
p120 binding site has been mapped to residues 758-773 (48). We
predicted that p120 would retain adhesiveness to E-cad100,
while
-catenin and
-catenin would not. Co-immunoprecipitations from TPA-treated LNCaP lysates revealed that only full-length E-cadherin is bound to these proteins (Fig.
6). As a positive control, E-cadherin was
also immunoprecipitated using a cytoplasmic antibody that recognizes
E-cad100. The co-immunoprecipitation data demonstrate that
E-cad100 has lost the
- and
-catenin binding
domains.

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Fig. 6.
E-cad100 does not associate with
the catenin complex. TPA-treated LNCaP protein extracts were
immunoprecipitated with E-cadherin antibody 4A2, -catenin, p120, and
-catenin antibodies. A control immunoprecipitation was also
performed using a normal goat IgG antibody and an immunoprecipation
using protein A Sepharose beads alone. Immunoprecipitations were
resolved by 6% SDS-PAGE and immunoblotted with HECD-1 antibody.
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Mutation of the E-cadherin Cytoplasmic Domain Inhibits Truncation
of E-cadherin in Vivo--
Based on the data from the immunoblot and
co-immunoprecipitation experiments, the calpain cleavage site appeared
to reside between the C20820 antibody epitope and the
-catenin
binding domain. Examination of the amino acid sequence of E-cadherin
between residues 773 and 815 revealed three potential calpain cleavage sites (Fig. 7A). To identify
the calpain cleavage site within the E-cadherin cytoplasmic domain
in vivo, we generated three E-cadherin deletion mutants
targeted within the proposed region in the E-cadherin cytoplasmic
domain (Fig. 7A). In a pcDNA3 vector harboring the
wild-type human E-cadherin cDNA, we engineered in-frame deletion
mutants of 5-6 amino acids spanning the three potential calpain
cleavage sites and transfected the constructs into SKBR3 cells. Stable
pools were selected, and PKC was activated by TPA over a 24-h time
course. Two of the E-cadherin deletion mutants, SK-791
5 and
SK-795
5 were truncated in an identical fashion as the wild type
control, while deletion mutant SK-782
6 exhibited complete inhibition
of truncation (Fig. 7B). These data indicate that amino acid
residues 782-787 are essential for calpain cleavage as SK-782
6
failed to be truncated in vivo.

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Fig. 7.
E-cadherin mutagenesis inhibits truncation
in vivo. A, E-cadherin amino acid sequence, from
771-810, is shown with the targeted mutation sites. B, the
corresponding mutant constructs containing in-frame internal deletions
were stably transfected into SKBR3 cells and protein extracts harvested
at the indicated times following TPA treatment. Lysates from SK-791 5
cells (lanes 1-3), SK-795 5 cells (lanes
4-6), and SK-782 6 (lanes 7-9) were resolved by 6%
SDS-PAGE and immunoblotted with HECD-1 antibody. SKBR3-Ecad wild type
cells are also shown (lane 10).
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Calpain Is Up-regulated in Localized and Metastatic Prostate
Tumors--
Both epigenetic and genetic mechanisms of E-cadherin
inactivation have been demonstrated in adenocarcinoma (49). We explored the possibility that calpain activity and expression may be associated with prostate tumorigenesis, and that E-cadherin cleavage may represent
a novel mechanism of E-cadherin inactivation in cancer. We hypothesized
that the loss of the catenin binding domain ablated the adhesive
function of E-cadherin, and that this loss of function might support
the malignant transformation of prostate epithelium. Using a cDNA
microarray (39), benign prostate (n = 26), localized prostate cancer (n = 49), and metastatic prostate
cancer (n = 19) demonstrated mean cDNA expression
for m-calpain of 1.0 (95% confidence interval (CI) 0.9-1.1), 1.2 (95% CI 1.1-1.2), and 1.4 (95% CI 1.3-1.4), respectively (one-way
ANOVA, p < 0.001) (Fig. 8). Pair-wise comparisons demonstrated
significant differences in cDNA expression levels between
clinically localized prostate cancer and metastatic prostate cancer
with respect to benign prostate tissue (post-hoc analysis using
Scheffé method, p < 0.01). Immunoblot analysis
of E-cad100 expression in matched normal and tumor prostate
specimens revealed the appearance of E-cad100 in the tumor
aspect of the prostate gland compared with the normal (Fig.
9A). Of 16 normal/tumor-matched pairs examined, 6 showed an increase in
E-cad100 in the tumor aspect of the prostate gland compared
with normal; two representative matched pairs are shown in Fig. 9. The
E-cad100 species was also observed in prostate cancer
metastases (Fig. 9B). Multiple metastatic samples were
harvested from the liver, diaphragm, soft tissue, peritoneum, adrenal
gland, and paratracheal lymph node. The samples were analyzed by
Western blot for E-cadherin, and each of the metastases demonstrated
the presence of E-cad100 (Fig. 9B). Of over 40 prostate cancer metastases examined, virtually 100% contained the
E-cad100 species (data not shown). The abundance at which
E-cad100 was detected in localized and metastatic prostate
cancer compared with normal prostate tissue correlated with the
increase in m-calpain expression observed in the cDNA microarray.
These data demonstrate that increased expression of calpain in prostate
tumors is associated with the generation of the 100-kDa fragment in
localized and metastatic disease.

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Fig. 8.
m-Calpain is up-regulated in localized and
metastatic prostate tumors. cDNA microarray analysis was
performed using 26 benign prostate, 49 localized prostate cancer, and
19 metastatic prostate cancer samples. m-Calpain transcript levels were
found to be significantly up-regulated in localized prostate and
metastatic cancer samples compared with benign prostate.
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Fig. 9.
E-cad100 is detected in localized
and metastatic prostate cancer. A, protein extracts (30 µg) of prostate tissue from two different patients were analyzed for
E-cadherin expression. Extracts from both the normal and tumor aspect
of the prostate gland were run on a 6% SDS-PAGE gel and immunoblotted
with HECD-1 antibody. B, protein extracts were prepared from
metastatic lesions removed from the following sites: 1,
liver; 2, diaphragm; 3, soft tissue;
4, peritoneum; 5, adrenal gland; 6,
paratracheal lymph node. 30 µg of protein were resolved on a 6%
SDS-PAGE gel and immunoblotted with HECD-1 antibody.
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DISCUSSION |
Results from the current study demonstrate that calpain induces a
specific, inactivating proteolytic cleavage within the cytoplasmic domain of E-cadherin in prostate and mammary epithelial cells. Initial
experiments indicated that cleavage of E-cadherin to the 100-kDa
fragment was a calcium-dependent mechanism that implicated a number of calcium-regulated proteases. Pretreatment of LNCaP and
MCF-7 cells with calpain inhibitors blocked the generation of
ionomycin-induced E-cad100, strongly implicating calpain in
this process.
In vitro experiments also supported the hypothesis that
E-cadherin is a substrate for calpain. Both µ- and m-calpain
effectively cleaved full-length E-cadherin in vitro to 100 kDa. We proceeded to characterize this truncation using the mammary
epithelial cell line SKBR3, which does not express E-cadherin protein
(44). We showed that the truncation of exogenous E-cadherin to 100-kDa was rapidly induced by PKC activation in SKBR3 cells transfected with
wild-type E-cadherin. We found that this truncation occurred in the
cytoplasmic domain of E-cadherin, and that this truncated species of
E-cadherin is unable to bind to
-catenin,
-catenin, and p120,
which are essential for the adhesive and cell signaling functions of
E-cadherin (50). In our study E-cad100 does not
associate with p120; however, an association would be predicted from
previous studies that have mapped the p120 binding site on
E-cadherin (48). One explanation is that cleavage of E-cadherin
changes the protein conformation near the juxtamembrane domain,
resulting in the loss of the p120 binding site.
An examination of the amino acid sequence of the cytoplasmic domain of
E-cadherin revealed potential calpain cleavage sites that were targeted
for mutation. The E-cadherin 782
6 mutant failed to generate
E-cad100 when transfected into cells, while other
constructs encoding adjacent mutant sites were truncated in the same
fashion as wild-type E-cadherin. While the calpain cleavage site may
reside within residues 782-787 in the E-cadherin cytoplasmic domain,
it is possible that the 782
6 mutation has merely disrupted secondary
structure or a calpain binding site that is required for cleavage.
Based on our mutagenic analysis, we hypothesize that the residues
782-787 are either critical for calpain binding or represent the
actual cleavage site.
Calpain has been implicated in the cleavage of other membrane-proteins
involved adhesion, most notably members of the integrin family (34).
However, the precise function of calpain in cellular adhesion remains
to be elucidated. Caspases are a well-studied family of cysteine
proteases that act as the executers of apoptosis (51). Caspase-3 has
been shown to cleave E-cadherin during apoptosis (20). However, we
found that caspase-3, was not responsible for the specific cleavage of
E-cadherin to generate the 100-kDa species (data not shown).
In a variety of adenocarcinomas, aberrations in E-cadherin expression
have been noted in malignant transformation of epithelium, metastatic
progression, and decreased patient survival (9, 52-54). The
development of metastatic prostate cancer is a multistep process in
which the disruption of cellular adhesion promotes both local invasion
and distant dissemination. In prostate cancer the loss of E-cadherin
function does not appear to result from allelic loss or gene mutation
to any great extent but rather to other mechanisms, such as decreased
transcription due to the aberrant methylation of its promoter (13).
Although important in the etiology of some cancers, genetic alteration
and promoter methylation do not account for all of the physiologic
mechanisms by which E-cadherin function is abrogated during tumorigenesis.
The first reports of E-cadherin proteolysis emerged nearly two decades
ago when it was discovered that a novel, 80-kDa fragment of E-cadherin
was released into the culture media of mouse and human mammary tumor
cells (16). This proteolytic fragment, which was derived from the
amino-terminal, extracellular domain of E-cadherin was generated by a
number of stress stimuli, including serum deprivation and high calcium
loading and was particularly intriguing due to its ability to disrupt
the lateral adhesion of mammary epithelium. These findings were also of
interest due to the fact this fragment was elevated in the serum of
patients suffering from a variety of adenocarcinomas (55). This
laboratory has been involved in the characterization of proteolytic
mechanisms that target and inactivate E-cadherin during prostate and
mammary tumorigenesis and had previously demonstrated the generation of
a novel, membrane bound fragment of E-cadherin that migrated at 97-kDa
(E-cad97) in prostate and mammary epithelial cells (41).
This fragment resulted from the truncation of E-cadherin in the
cytosolic domain and had lost its ability to bind
-catenin thus
abolishing the adherent function of the molecule. In a later study, we
observed that the accumulation of E-cad97 was significantly
associated with localized prostate cancer as compared with adjacent
normal tissue derived from the same prostate specimen (21). We have
since observed the presence of E-cad100, that while not as
prominent as E-cad97 in localized prostate cancer, appears
to be present in all metastatic prostate tumors examined. We have also
found that calpain is up-regulated in localized and metastatic prostate
tumors and this is the first report associating calpain with prostate
tumorigenesis. Based on these observations, we hypothesized that
specific proteolytic cleavage events, such as that mediated by calpain,
target and inactivate E-cadherin. Such a loss of E-cadherin
function may result in the net reduction of interepithelial adhesion
and promote the malignant transformation of prostate epithelial cells.