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From the Lombardi Cancer Center, Georgetown University Medical Center, Washington, District of Columbia
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the hypothesis that epithelial-derived cancer cells within a tumor may be a major source of the synthesis and presentation of a protease(s) important for multiple aspects of tumor behavior, including growth and metastasis, we have isolated and characterized a membrane-bound, trypsin-like, serine protease termed matriptase and have identified its integral membrane, Kunitz-type inhibitor, called HAI-1 (hepatocyte growth factor activator inhibitior-1) from T-47D human breast cancer cells and from human milk.23-26 Matriptase is a mosaic, transmembrane, trypsin-like serine protease with two potential regulatory modules: two tandem repeats of a CUB (C1r/s, Uegf, and bone morphogenic protein-1) domain and four tandem repeats of a LDL receptor domain25 (also see updated sequence, GenBank accession no. AF118224). This protease is identical in sequence to the protease termed the membrane-type serine protease-1, MT-SP1,27 and has a high percentage of sequence identity with the mouse serine protease epithin,28 the apparent mouse homologue of matriptase. Matriptase was detected in some breast cancer cell lines and in immortalized luminal epithelial cells of the mammary gland, but not in cultured fibroblasts nor in HT1080 fibrosarcoma cells.26 Thus, it was proposed that matriptase is produced by epithelial cells in vivo. The matriptase inhibitor, an integral membrane serine protease inhibitor with two Kunitz domains separated by an LDL receptor domain, was initially identified as an inhibitor (HAI-1) of hepatocyte growth factor activator.29 This inhibitor is expressed primarily by simple columnar epithelium in multiple human tissues in vivo.30
In this study, we characterize the expression of matriptase in human primary breast cancer and in human breast cancer cell lines. Data from the breast cancer cell lines suggest that the expression of matriptase correlates with markers of an epithelial phenotype. Results from primary tumors indicate that the protease is expressed by tumors of epithelial origin. Matriptase is expressed predominately by the epithelial elements of carcinomas and not by fibroblasts. The in vivo epithelial origin of matriptase and its expression by breast cancer cells highlight the potential role of matriptase in the activation of latent growth factors and proteases at the breast epithelial cell surface and in the development, growth, invasion, and metastasis of breast cancer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All breast and ovarian cancer cell lines were obtained from the Lombardi Cancer Center Tissue Culture Shared Resources. Cells were maintained in culture by growth in Iscove’s minimal essential media (Life Technologies, Inc., Rockville, MD) supplemented with 5% fetal bovine serum at 37°C and 5% CO2, with the exception of the MCF-10A cell line, grown in Dulbecco’s modified Eagle’s medium/HAM F-12 (Life Technologies, Inc.) supplemented with 0.5% fetal bovine serum, 0.5 mg/ml hydrocortisone, 10 µg/ml insulin, and 10 ng/ml epidermal growth factor, and the 184 A1N4 human mammary epithelial cell line, grown in Iscove’s minimal essential media supplemented with 5% fetal bovine serum, 0.5 mg/ml hydrocortisone, 5 µg/ml insulin, and 20 ng/ml epidermal growth factor.
Monoclonal Antibodies (mAbs)
Anti-matriptase and anti-HAI-1 mAbs were prepared as described previously.24,26 Immunohistochemistry-competent anti-matriptase mAb S5 (IgG1) was prepared by hybridoma technology using formalin-treated matriptase, isolated from T-47D cells, as immunogen. A panel of mAbs was selected by its ability to stain paraffin-embedded breast cancer sections. Monoclonal Ab S5 was selected from these mAbs for its ability to recognize matriptase by immunoblot.
Extraction of Proteins from Frozen Human Tumors
Frozen human tumors from various sites were obtained from the Histopathology and Tissue Shared Resource at the Lombardi Cancer Center, Georgetown University. The tumor specimens were kept frozen with liquid nitrogen and ground to a fine powder by mortar and pestle. The specimens were extracted using RIPA buffer (150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), and 50 mmol/L Tris, pH 8.0). The insoluble debris was removed by centrifugation, and the protein concentration was determined by bicinchoninic acid protein assay (Pierce, Rockford, IL).
Western Blotting
Proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis, transferred overnight to polyvinylidene fluoride, and subsequently probed with mAbs as indicated. Immunoreactive polypeptides were visualized using horseradish peroxidase-labeled secondary antibodies and the ECL detection system (New England Nuclear, Boston, MA).
Northern Blotting
Total RNA was extracted from cell lines with RNAzol reagent
(Tel-Test, Inc., Friendswood, TX) according to the manufacturer’s
instructions. Ten micrograms of total RNA from each cell line was
resolved by electrophoresis on a 1.2% phosphate-buffered agarose gel
containing 2 mol/L formaldehyde. RNA was subsequently transferred to
Hybond-N nylon membranes (Amersham Pharmacia Biotech, Buckinghamshire,
UK) and hybridized to 32P-labeled riboprobes at
65°C for 36 hours, followed by three washes in 0.1x standard saline
citrate (SSC)/0.1% SDS at 80°C for 30 minutes each to remove unbound
probe. To generate the labeled riboprobes, the coding sequences of
matriptase and HAI-1 were cloned into the pcDNA3.1 vector (Invitrogen,
San Diego, CA), and were linearized with an appropriate restriction
enzyme just 5' of the coding sequences. These linearized vectors were
used in in vitro SP6 RNA polymerase reactions with
32P-UTP (3,000 Ci/mmol; New England Nuclear) to
generate labeled antisense riboprobes. To control for approximately
equal loading of RNA from each cell line, labeled riboprobes directed
against the message of the ribosomal protein 36B4 were generated in a
similar manner, except that a vector containing 
500 bp of the coding
sequence was used. Signals on hybridized membranes were visualized by
exposure to X-OMAT AR imaging film (Eastman-Kodak, Rochester, NY) for 4
to 12 hours at -80°C.
Immunohistochemistry
Paraffin-embedded sections of human primary breast cancers were obtained from the Histopathology and Tissue Shared Resource at the Lombardi Cancer Center, Georgetown University. Briefly, 5-µ tumor sections were heated in an oven to 56°C for 3 hours and then dewaxed in xylene. Slides were then rehydrated by immersion in a decreasing gradient of ethanol in water. Endogenous peroxidase activity was quenched by immersion in 1.5% H2O2/methanol for 10 minutes, followed by washes in water and phosphate-buffered saline (PBS). Sections were blocked for 30 minutes in blocking buffer (2% goat serum/5% bovine serum albumin in PBS) before incubation with the primary antibody. Sections were incubated in the presence of the matriptase-specific mAb clone S5 (IgG1) at a concentration of 1 µg/ml, the HAI-1-specific mAb clone M58 (IgG1) at a concentration of 5 µg/ml or mouse IgG1 at a concentration of 5 µg/ml in blocking buffer for 1 hour at room temperature. After incubation in the primary antibody, sections were washed in PBS to remove unbound antibody, and then were incubated with a biotinylated goat anti-mouse secondary antibody. After washes in PBS, the staining was completed by incubation with streptavidin-horseradish peroxidase and diaminobenzidine colorimetric reagents from the BioGenex immunohistochemistry kit (San Ramon, CA) according to the manufacturer’s protocol. As a control, nonrelevant mouse IgG1 was used in place of the specific mAbs at the equivalent dilutions. The colorimetric reaction for the control slides was developed for the same amount of time as the experimental slides, and did not show any development of the color reagent.
In Situ Hybridization
Probes for use in in situ hybridization were prepared by generating digoxigenin-labeled sense and antisense RNA riboprobes using the Dig-RNA labeling kit (Boehringer-Mannheim, Mannheim, Germany) according to a modified manufacturer’s protocol. Briefly, a 650-bp BamHI-SacII fragment of the matriptase sequence corresponding to the 5' end of the matriptase cDNA was cloned into the pBluescript SK-vector (Stratagene, La Jolla, CA). This vector was subsequently linearized with SacII or BamHI and used as a template for the synthesis of sense and antisense digoxigenin-labeled riboprobes, respectively, with T7 or T3 RNA polymerase (Life Technologies, Inc.), according to the manufacturer’s protocol, using digoxigenin-11-UTP. Synthesized probes were purified by G50 column chromatography to remove unincorporated nucleotides, including unincorporated digoxigenin-11-UTP. The concentration of the labeled riboprobes was determined spectrophotometrically. The accuracy of the concentration assignment was confirmed by analysis of the riboprobes by 1% agarose/2 mol/L formaldehyde gel electrophoresis, followed by ethidium bromide staining. The equal efficiency of digoxigenin incorporation into sense and antisense probes was confirmed by dot-blotting of equal amounts of probe onto Hybond-N nylon membranes, followed by detection of labeled riboprobe with an alkaline-phosphatase-conjugated anti-digoxigenin antibody and colorimetric substrate (not shown). In addition, the efficiency of digoxigenin incorporation was confirmed by dot blotting equal amounts of denatured double-stranded vector containing the full-length sequence of matriptase and probing these blots with digoxigenin-labeled sense or antisense probes for matriptase. Equal signals were observed for equal amounts of sense or antisense probe used in the hybridization to membrane-bound plasmid (not shown). For detection of matriptase mRNA in paraffin-embedded breast cancer sections, 20 ng of labeled sense or antisense riboprobe was used in a standard protocol provided by Boehringer Mannheim. Briefly, 5-µm paraffin-embedded breast cancer tissue sections were deparaffinized, rehydrated, treated with 0.2 mol/L HCl, permeabilized with proteinase K, and postfixed with 4% paraformaldehyde before prehybridization in 50% foramide/1x SSC at 65°C and hybridization at 65°C in hybridization buffer for 12 hours in a humidified chamber. After hybridization, unbound probe was removed by two washes in 2x SSC, two washes in 1x SSC, and two washes in 0.1x SSC at 42°C. Bound probe was detected by use of an alkaline-phosphatase-conjugated anti-digoxigenin antibody that produces an insoluble blue precipitate in the presence of a nitrotetrazolium blue/X-phosphate color solution. Sense and antisense probes were hybridized and washed under identical conditions, and the colorimetric reactions were stopped at the same time for sense and antisense hybridized sections.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the expression of matriptase and HAI-1 in breast
cancer cell lines, Northern blotting was performed using RNA from a
panel of breast cancer cell lines (Figure 1)
. Expression of matriptase and HAI-1 in
human breast cancer cell lines was completely concordant; matriptase
and HAI-1 were found in four of four estrogen receptor-positive (ER+)
and three of nine estrogen receptor-negative (ER-) breast cancer cell
lines. Both were expressed in two of two ER- immortalized breast
epithelial cell lines tested. Neither was expressed by a primary breast
fibroblast cell line (data not shown). Matriptase and HAI-1 were
detected in one of three human ovarian cancer cell lines tested (Figure 1)
. The expression of matriptase and HAI-1, or lack thereof, by all of
the cell lines was confirmed at the protein level by Western blot
analysis (data not shown).
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and never with
vimentin.34-36
The protease and inhibitor were found in
all ER+ cell lines and in a smaller number of ER- cell lines tested.
However, this trend toward expression in ER+ tumor cells and absence in
ER- cells was not observed in our studies of primary breast tumors
(see below). In primary breast tumors, the expression of both have been
found in both ER+ and ER- tumors, with no trend toward either an ER+
or ER- status. Matriptase and HAI-1 do not seem to be regulated at the
transcriptional level by estrogen or progesterone, as no change in mRNA
levels for matriptase or HAI-1 were observed in estrogen-depleted MCF-7
cells when treated with 100 nmol/L 17ß-estradiol or with 100 nmol/L
17ß-estradiol plus 100 nmol/L progesterone (data not shown).
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The correlation between matriptase/HAI-1 expression and epithelial markers in cultured cells suggests that the matriptase/HAI-1 system could also be expressed in vivo by normal epithelial cells and by epithelium-derived cancer cells. To test this hypothesis, we examined various epithelium-derived and nonepithelium-derived frozen human tumor specimens and the surrounding tissues of these tumors by protein immunoblotting. The epithelium-derived tumors included 10 breast, nine ovarian, four uterine, and eight colon carcinomas. Noncancerous tissues surrounding these carcinomas were also included in the lysates tested. Nonepithelial tumors included three stroma-derived ovarian tumors and 10 sarcomas with various origins and histological grades.
Breast Tumors
Expression of matriptase in 10 infiltrating human breast
carcinomas (nine ductal carcinomas and one colloidal carcinoma; five
were ER+ specimens and five ER-) was examined by Western blot and
compared to three samples of noncancerous breast tissue surrounding a
tumor. In these surrounding normal tissues, matriptase was detected at
very low levels or below the detection sensitivity (Figure 2A
, lanes 13 to 15). In contrast, higher
levels of expression of matriptase was observed in all 10 of the
primary breast carcinomas examined (Figure 2A
, lanes 3 to 12),
consistent with the higher epithelial cell-derived component of these
specimens. The expression of HAI-1 was also observed in these 10 human
breast specimens, but under the detection sensitivity by Western blot
in the surrounding noncancerous tissue, which is composed primarily of
stromal (nonepithelial) elements. Expression of HAI-1 fluctuated widely
among these specimens (Figure 2B)
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We further investigated the expression of matriptase in
gynecological tumors (Figure 3)
. There
are more than 25 major types of ovarian neoplasms. These are classified
into three groups based on cell of origin: those of the germinal
surface epithelium, the gonadal stroma, and germ cells. We examined
ovarian tumors both of epithelial origin and of stromal origin. Among
the nine tumors of epithelial origin, matriptase was detected at
moderate to high levels (Figure 3A
, lanes 3 to 11). In contrast,
matriptase was not detected in three sex cord/stromal tumors, including
a granulosa cell tumor (Figure 3A
, lane 14), and two fibrothecomas
(Figure 3A
, lanes 11 and 12). The negative results in sex cord/stromal
tumors again suggest that expression of matriptase is restricted to
tumors of an epithelial origin. Expression of HAI-1 varied widely among
these ovarian carcinomas (Figure 3B
, lanes 3 to 11). In one of these
specimens, HAI-1 was below the detection sensitivity (Figure 3B
, lane
10), whereas matriptase was detected at a high level (Figure 3A
, lane
10). HAI-1 was not detected in the three sex cord/stromal tumors. A
minor, nonspecific band with a migration similar to that of HAI-1 could
not be depleted with anti-HAI-1 mAb; this band was also observed in
both matriptase and HAI-1 Western blots (Figure 3, A and B
; lanes 12 to
14).
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; lanes 3, 5, 7, and 8) and in two
patient-matched normal tissues (Figure 3, C and D
; lanes 4 and 6).
Expression of matriptase was observed strongly in three of four (Figure 3C
; lanes 3, 5, and 7), and weakly in one of four (Figure 3C
, lane 8)
cancer specimens, whereas the two normal tissues were below the
detection limit (Figure 3C
, lanes 4 and 6). HAI-1 expression was
observed at a high level in one specimen (Figure 3D
, lane 5) and at low
levels in the other three specimens (Figure 3C
; lanes 3, 7, and 8). Colon Tumors
Eight colon carcinoma specimens (Figure 4A
, lanes 3 to 10) and five normal colon
specimens (Figure 4A
, lanes 11 to 14) were also examined. Expression of
matriptase fluctuated among colon carcinomas as well as among normal
colon tissues. In contrast to breast and gynecological carcinomas,
expression of matriptase in some normal colon tissues was as high as
that seen in cancer specimens, consistent with the high percentage of
epithelial cells present in normal colon tissues relative to that of
normal breast and ovarian tissue. Expression of HAI-1 also varied among
these specimens (Figure 4B)
, but tended to correlate with matriptase
expression.
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Ten human sarcomas were examined for the expression of matriptase (data not shown). These included three high-grade osteosarcomas, three well-differentiated (low grade) liposarcomas, two malignant fibrous histiocytomas that were clinically metastatic, one dermatofibrosarcoma protuberance (a low-grade sarcoma), and one high-grade leiomyosarcoma that was most likely of uterine origin. Matriptase was below the detection limit or barely detectable for all of the sarcomas. HAI-1 was below the detection limit for all 10 sarcomas.
From this preliminary screening, matriptase was detected in all 31 human carcinomas tested. In contrast, expression of matriptase and HAI-1 was negligible or not detected in all of the 13 nonepithelial tumors tested. These results suggest that matriptase is selectively expressed in epithelium-derived tumors in vivo, consistent with the observation that matriptase was detected in cultured cells that tended to express epithelial markers, but not in cells with a mesenchymal marker. Matriptase and HAI-1 are found at higher levels in breast and gynecological cancer tissue when compared to normal surrounding tissue; however, this is likely because of the increased epithelial cellularity of cancer tissue versus normal tissue. This observation is supported by the fact that matriptase was detected at high levels in some normal colon tissues for which the epithelial element represents a major portion of normal colon tissue.
Matriptase Protein and mRNA Are Detected in Normal and Cancerous Epithelial Cells in Human Breast Tumor Sections
To further determine which cell types express matriptase protein
and mRNA in primary tumor specimens, immunohistochemistry and in
situ hybridization using digoxigenin-labeled riboprobes were
performed using formalin-fixed, paraffin-embedded human breast
carcinomas. Matriptase protein was detected in breast cancer cells
(Figure 5
; A, B, and C) as well as in
surrounding normal breast epithelial cells with comparable intensity
(Figure 6, A and B)
. Within normal breast
epithelium and in surrounding hyperplastic ducts, the ducts stained
intensely, whereas mild or no staining was observed in surrounding
terminal duct lobular units (TDLUs). The localization of matriptase to
the breast cancer cell component of the breast tumors was confirmed by
in situ hybridization using digoxigenin-labeled riboprobes
(Figure 7)
. The localization of HAI-1
protein was also determined by immunohistochemistry in the primary
breast tumors and in surrounding normal breast tissue. The inhibitor
co-localized with that of matriptase in the tumor cell compartment
(Figure 5
; D, E, and F). Within surrounding normal breast tissue, focal
staining was observed for HAI-1 in the TDLUs, with no staining seen
in myoepithelial cells (Figure 6, C and D)
. Normal ducts surrounding
TDLUs show variable staining for HAI-1. These results are consistent
with the expression of HAI-1 by epithelial elements of breast and other
tissues as found by others.30
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Tissue Concentration of Matriptase in Breast Carcinomas
The tissue concentrations of matriptase in six breast
carcinomas and two surrounding noncancerous breast tissues were
determined by immunoblot (Figure 8)
. The
concentration of matriptase in the six carcinomas ranged from 13 to 24
ng/mg tissue protein, with the exception of a colloidal carcinoma
specimen, which contained 7 ng/mg tissue protein. The relatively low
matriptase protein level in the colloidal carcinoma may be explained by
the high proportion of nonepithelial elements in this tumor type. The
concentration of matriptase in the two normal surrounding breast tissue
specimens was 2 and 3 ng/mg tissue protein, respectively. Again,
this lower value in normal breast tissue is consistent with the lower
epithelial representation of this tissue relative to breast carcinomas.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Matriptase was initially identified by gelatin zymography only in ER+ hormone-dependent breast cancer cells, including T-47D, MCF-7, and ZR-75-1 and BT474,23 but not in the ER- hormone-independent cell lines MDA-MB-231, MDA-MB-435, MDA-MB-436, and BT-549.23 To test the hypothesis that matriptase may be expressed exclusively by ER+ breast cancer cell lines, we screened additional ER+ and ER- cell lines for expression of matriptase and HAI-1 by Western and Northern blotting. We found that both matriptase and HAI-1 protein and mRNA are expressed in three ER- cell lines, SKBR3, MDA-MB-453, and MDA-MB-468, but not in numerous other ER- cell lines. Thus, the expression of matriptase and HAI-1 does not always correlate with the expression of the estrogen receptor in cultured breast cancer cells. The expression of matriptase and HAI-1 in vivo in primary breast tumors differs from the expression pattern in cultured cells in that all ER- tumors examined to date have been found to express matriptase and HAI-1, as have all ER+ tumors. Therefore, matriptase and HAI-1 expression does not seem to rely on the expression status of the estrogen receptor either in vitro or in vivo. Furthermore, both genes do not seem to be transcriptionally regulated by estrogen or progesterone, as neither is induced by the exposure of estrogen-stripped MCF-7 cells to 17ß-estradiol nor to 17ß-estradiol plus progesterone.
The expression of matriptase did correlate with the expression of markers of an epithelial phenotype (E-cadherin- or ZO-1-positive) and did not correlate with the expression of a mesenchymal phenotype (vimentin-positive). The co-expression of matriptase and HAI-1 negatively correlates with the previously determined in vitro invasion of these cells into Matrigel.34 This data may be interpreted as indicating that matriptase is not involved in augmenting the invasive phenotype of cultured breast cancer cells. However, the significance of this observation with regard to invasion in vivo in human carcinomas is unclear, because most of the breast cancer cell lines tested originated from pleural effusions or ascites in human cancer patients with metastatic disease, and are therefore by definition invasive. Additionally, all of the cell lines tested have been cultured for many passages, allowing significant opportunity for phenotypic and genetic drift. Furthermore, we found the expression of matriptase and HAI-1 in 10 of 10 primary invasive breast cancers examined. This observation and the fact that primary invasive breast cancers rarely express mesenchymal markers such as vimentin further suggests that the lack of expression of matriptase and HAI-1 in vitro in a subset of invasive breast cancer cell lines may be a tissue culture artifact.
Although no absolute correlation exists between matriptase/HAI-1 expression and estrogen receptor expression in cultured breast cancer cells, a reverse correlation between matriptase/HAI-1 and vimentin, a mesenchymal marker, was observed among these breast cancer cells. This tendency toward lack of expression in cells that express a mesenchymal phenotype is consistent with our previous study that matriptase was not detected in cultured human fibroblasts and HT-1080 fibrosarcoma cells.26 The in vivo expression analyses for matriptase and HAI-1 seem to support this in vitro correlation. Expression of matriptase and HAI-1 was not detected, or found at negligible levels, in stromal-derived ovarian tumors and various human sarcomas. In contrast, matriptase and HAI-1 were detected in all of the human carcinoma specimens in this study.
The detection of matriptase mRNA by in situ hybridization in primary human breast tumors revealed that the matriptase/HAI-1 system is synthesized by epithelial cells and epithelium-derived cancer cells in vivo. The lack or negligible amount of matriptase in stromal-derived ovarian tumors and primary human sarcomas, including osteosarcoma, liposarcoma, leiomyosarcoma, malignant fibrous histiocytoma, and dermatofibrosarcoma protuberans, further confirms that epithelial cells, rather than mesenchymal cells, are the major sources of matriptase in vivo. These results are consistent with our earlier observation that matriptase and HAI-1 are produced in vitro by breast cancer cells and milk-derived, immortalized luminal epithelial cells of the mammary gland, but not by cultured foreskin fibroblasts nor the fibrosarcoma cell line HT1080.26
When assayed by Western blotting of tumor cell lysates and normal
tissue lysates, matriptase protein is present in tumor tissue and in
normal breast tissue. Direct comparison of matriptase protein levels
between tumor and surrounding normal tissue by Western analysis,
however, is of limited value because of cellularity issues, because
tumors tend to contain primarily epithelial cells whereas normal breast
tissue is composed primarily of stromal tissue with a smaller
epithelial component. When examined by immunohistochemistry, there is
no obvious difference in the expression of matriptase between tumor
cells and normal breast epithelial cells (Figure 6)
. This is similar to
the observation for cultured immortalized/nontumorigenic breast
epithelial cells and tumorigenic breast cancer cell lines for which no
obvious overall difference exists in the level of expression of
matriptase mRNA and protein.
These results suggest that if the catalytic activity of the serine protease matriptase is important for the growth and/or invasion of breast cancer cells in human breast tumors, then the increased activity of the protease in breast cancer is likely because of mechanisms other than a simple increase in matriptase protein or mRNA. An increase in matriptase activity could be manifested in multiple ways, for example, by an increase in the matriptase:inhibitor ratio within a tumor, tipping the balance in favor of the protease relative to the inhibitor, or by an increase in the activation of matriptase on the cell surface by proteolytic cleavage. Like many other proteases, matriptase requires proteolytic cleavage from a one-chain latent form to a two-chain active form,37 an event that is not measured by the immunohistochemistry or in situ hybridization assays presented in this paper. Future studies will include the examination of the active form of matriptase (two-chain, active matriptase). This may be done by immunocytochemistry using a mAb that recognizes only the two-chain, active form of matriptase,37 or by activity assays of tumor extracts, to assess the relative activity of matriptase in breast cancers of various stages and grades, and in normal breast tissue. Furthermore, we will asses the matriptase:HAI-1 ratio within a large series of primary human breast carcinomas of various stages and grades to determine the relevance of the protease:inhibitor ratio in breast tumors.
A significant imbalance of matriptase and HAI-1 expression was observed
in some of the tumor specimens analyzed by Western blot in this study.
We have determined the ratio of matriptase to HAI-1 expression by
comparing their intensity on Western blot after normalizing the signals
with that of the control sample from T-47D cells (lane 1 in Figures 2, 3, and 4
) (data not shown). These ratios fluctuate among the breast and
gynecological tumors. For example, two infiltrating carcinomas (Figure 2
, lanes 4 and 6) had a relatively low ratio, whereas other samples,
all invasive carcinomas, had a relatively higher ratio. Among the
gynecological tumors, a high ratio was observed in some specimens
(Figure 3, A and B
, lanes 10 and 11; and Figure 3, C and D
, lanes 3 and
7), but not in others. The matriptase to HAI-1 ratio among the colon
tumors analyzed showed a more consistent value, with one exception
(Figure 4
, lane 9). Taken together, these results suggest that the
ratio of matriptase to HAI-1 varies among tumors of breast and
gynecological origin, and warrants further study to determine whether a
trend in the matriptase to HAI-1 ratio, assessed in a much larger set
of tumors, correlates with pathological grade or stage of the tumors,
or with clinical measures of outcome such as disease-free and overall
survival or response to chemotherapy. Such studies are currently in
progress.
In summary, we have characterized the expression of matriptase and HAI-1 both in cultured human breast cancer cells and in primary human breast carcinomas and other primary human cancers. We have found that matriptase and HAI-1 are expressed concomitantly by both ER- and ER+ breast cancer cell lines. The expression of these proteins correlates with the expression of epithelial markers and not with markers of a mesenchymal phenotype in cultured breast cancer cells. In primary breast cancers and in normal breast epithelial tissue, matriptase and HAI-1 are expressed by the epithelial component of the tissue, and not by stromal elements such as fibroblasts and adipocytes. The expression pattern of matriptase and HAI-1 in primary breast cancers suggests that this protease system is an epithelial-derived system that may activate stromal-derived proteases such as uPA, and growth/motility factors such as HGF/SF, on the surface of breast cancer cells, enhancing their growth and/or invasive properties. Therefore, matriptase may represent an important link in our understanding of how stromally derived proteases and growth/motility factors may be activated on the surface of normal breast epithelial cells or breast cancer cells. Within a breast tumor, such activity may contribute to the tumorigenic and metastatic properties of breast cancer cells.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health Specialized Program of Research Excellence grant 1P50CA58158 in breast cancer and National Institutes of Health grant R21CA80897.
Accepted for publication December 21, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 T. J. Carney, S. von der Hardt, C. Sonntag, A. Amsterdam, J. Topczewski, N. Hopkins, and M. Hammerschmidt Inactivation of serine protease Matriptase1a by its inhibitor Hai1 is required for epithelial integrity of the zebrafish epidermis Development, October 1, 2007; 134(19): 3461 - 3471. [Abstract] [Full Text] [PDF]
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M.-S. Lee, I-C. Tseng, Y. Wang, K.-i. Kiyomiya, M. D. Johnson, R. B. Dickson, and C.-Y. Lin Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain Am J Physiol Cell Physiol, July 1, 2007; 293(1): C95 - C105. [Abstract] [Full Text] [PDF]
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 C. Parr, A. J. Sanders, G. Davies, T. Martin, J. Lane, M. D. Mason, R. E. Mansel, and W. G. Jiang Matriptase-2 Inhibits Breast Tumor Growth and Invasion and Correlates with Favorable Prognosis for Breast Cancer Patients Clin. Cancer Res., June 15, 2007; 13(12): 3568 - 3576. [Abstract] [Full Text] [PDF]
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S. Netzel-Arnett, B. M. Currie, R. Szabo, C.-Y. Lin, L.-M. Chen, K. X. Chai, T. M. Antalis, T. H. Bugge, and K. List Evidence for a Matriptase-Prostasin Proteolytic Cascade Regulating Terminal Epidermal Differentiation J. Biol. Chem., November 3, 2006; 281(44): 32941 - 32945. [Abstract] [Full Text] [PDF]
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 L. M. Kilpatrick, R. L. Harris, K. A. Owen, R. Bass, C. Ghorayeb, A. Bar-Or, and V. Ellis Initiation of plasminogen activation on the surface of monocytes expressing the type II transmembrane serine protease matriptase Blood, October 15, 2006; 108(8): 2616 - 2623. [Abstract] [Full Text] [PDF]
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 O. K. Hallikas, J. M. Aaltonen, H. von Koskull, L.-A. Lindberg, L. Valmu, N. Kalkkinen, T. Wahlstrom, H. Kataoka, L. Andersson, D. Lindholm, et al. Identification of Antibodies against HAI-1 and Integrin {alpha}6{beta}4 as Immunohistochemical Markers of Human Villous Cytotrophoblast J. Histochem. Cytochem., July 1, 2006; 54(7): 745 - 752. [Abstract] [Full Text] [PDF]
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K.-i. Kiyomiya, M.-S. Lee, I-C. Tseng, H. Zuo, R. J. Barndt, M. D. Johnson, R. B. Dickson, and C.-Y. Lin Matriptase activation and shedding with HAI-1 is induced by steroid sex hormones in human prostate cancer cells, but not in breast cancer cells Am J Physiol Cell Physiol, July 1, 2006; 291(1): C40 - C49. [Abstract] [Full Text] [PDF]
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 F. Le Naour, M. Andre, C. Greco, M. Billard, B. Sordat, J.-F. Emile, F. Lanza, C. Boucheix, and E. Rubinstein Profiling of the Tetraspanin Web of Human Colon Cancer Cells Mol. Cell. Proteomics, May 1, 2006; 5(5): 845 - 857. [Abstract] [Full Text] [PDF]
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 Y. Ito, A. Akinaga, K. Yamanaka, T. Nakagawa, A. Kondo, R. B. Dickson, C.-Y. Lin, A. Miyauchi, N. Taniguchi, and E. Miyoshi Co-expression of matriptase and N-acetylglucosaminyltransferase V in thyroid cancer tissues--its possible role in prolonged stability in vivo by aberrant glycosylation Glycobiology, May 1, 2006; 16(5): 368 - 374. [Abstract] [Full Text] [PDF]
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K. List, R. Szabo, A. Molinolo, B. S. Nielsen, and T. H. Bugge Delineation of Matriptase Protein Expression by Enzymatic Gene Trapping Suggests Diverging Roles in Barrier Function, Hair Formation, and Squamous Cell Carcinogenesis Am. J. Pathol., May 1, 2006; 168(5): 1513 - 1525. [Abstract] [Full Text] [PDF]
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W. Ge, H. Hu, K. Ding, L. Sun, and S. Zheng Protein Interaction Analysis of ST14 Domains and Their Point and Deletion Mutants J. Biol. Chem., March 17, 2006; 281(11): 7406 - 7412. [Abstract] [Full Text] [PDF]
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 M. Saleem, V. M. Adhami, W. Zhong, B. J. Longley, C.-Y. Lin, R. B. Dickson, S. Reagan-Shaw, D. F. Jarrard, and H. Mukhtar A novel biomarker for staging human prostate adenocarcinoma: overexpression of matriptase with concomitant loss of its inhibitor, hepatocyte growth factor activator inhibitor-1. Cancer Epidemiol. Biomarkers Prev., February 1, 2006; 15(2): 217 - 227. [Abstract] [Full Text] [PDF]
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 J.-S. Jin, A. Chen, D.-S. Hsieh, C.-W. Yao, M.-F. Cheng, and Y.-F. Lin Expression of Serine Protease Matriptase in Renal Cell Carcinoma: Correlation of Tissue Microarray Immunohistochemical Expression Analysis Results with Clinicopathological Parameters International Journal of Surgical Pathology, January 1, 2006; 14(1): 65 - 72. [Abstract] [PDF]
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 K. List, R. Szabo, A. Molinolo, V. Sriuranpong, V. Redeye, T. Murdock, B. Burke, B. S. Nielsen, J. S. Gutkind, and T. H. Bugge Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation Genes & Dev., August 15, 2005; 19(16): 1934 - 1950. [Abstract] [Full Text] [PDF]
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M. D. Oberst, L.-Y. L. Chen, K.-I. Kiyomiya, C. A. Williams, M.-S. Lee, M. D. Johnson, R. B. Dickson, and C.-Y. Lin HAI-1 regulates activation and expression of matriptase, a membrane-bound serine protease Am J Physiol Cell Physiol, August 1, 2005; 289(2): C462 - C470. [Abstract] [Full Text] [PDF]
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B. S. Knudsen, J. M. Lucas, L. Fazli, S. Hawley, S. Falcon, I. M. Coleman, D. B. Martin, C. Xu, L. D. True, M. E. Gleave, et al. Regulation of Hepatocyte Activator Inhibitor-1 Expression by Androgen and Oncogenic Transformation in the Prostate Am. J. Pathol., July 1, 2005; 167(1): 255 - 266. [Abstract] [Full Text] [PDF]
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 T. X. Pedersen, C. J. Pennington, K. Almholt, I. J. Christensen, B. S. Nielsen, D. R. Edwards, J. Romer, K. Dano, and M. Johnsen Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas Carcinogenesis, July 1, 2005; 26(7): 1233 - 1240. [Abstract] [Full Text] [PDF]
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 M Lacroix, R-A Toillon, and G Leclercq Stable 'portrait' of breast tumors during progression: data from biology, pathology and genetics Endocr. Relat. Cancer, September 1, 2004; 11(3): 497 - 522. [Abstract] [Full Text] [PDF]
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 A Noel, C Maillard, N Rocks, M Jost, V Chabottaux, N E Sounni, E Maquoi, D Cataldo, and J M Foidart Membrane associated proteases and their inhibitors in tumour angiogenesis J. Clin. Pathol., June 1, 2004; 57(6): 577 - 584. [Abstract] [Full Text] [PDF]
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 G. U. Di Maria and P. Comba Malignant Pleural Mesothelioma: The Puzzling Role of Gene-Environment Interaction Chest, May 1, 2004; 125(5): 1604 - 1607. [Full Text] [PDF]
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C. D. Hoang, J. D'Cunha, M. G. Kratzke, C. E. Casmey, S. P. Frizelle, M. A. Maddaus, and R. A. Kratzke Gene Expression Profiling Identifies Matriptase Overexpression in Malignant Mesothelioma Chest, May 1, 2004; 125(5): 1843 - 1852. [Abstract] [Full Text] [PDF]
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R.-J. Hung, I.-W. J. Hsu, J. L. Dreiling, M.-J. Lee, C. A. Williams, M. D. Oberst, R. B. Dickson, and C.-Y. Lin Assembly of adherens junctions is required for sphingosine 1-phosphate-induced matriptase accumulation and activation at mammary epithelial cell-cell contacts Am J Physiol Cell Physiol, May 1, 2004; 286(5): C1159 - C1169. [Abstract] [Full Text] [PDF]
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M. Suzuki, H. Kobayashi, N. Kanayama, Y. Saga, M. Suzuki, C.-Y. Lin, R. B. Dickson, and T. Terao Inhibition of Tumor Invasion by Genomic Down-regulation of Matriptase through Suppression of Activation of Receptor-bound Pro-urokinase J. Biol. Chem., April 9, 2004; 279(15): 14899 - 14908. [Abstract] [Full Text] [PDF]
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D. B. Martin, D. R. Gifford, M. E. Wright, A. Keller, E. Yi, D. R. Goodlett, R. Aebersold, and P. S. Nelson Quantitative Proteomic Analysis of Proteins Released by Neoplastic Prostate Epithelium Cancer Res., January 1, 2004; 64(1): 347 - 355. [Abstract] [Full Text] [PDF]
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C. Parr, G. Watkins, R. E. Mansel, and W. G. Jiang The Hepatocyte Growth Factor Regulatory Factors in Human Breast Cancer Clin. Cancer Res., January 1, 2004; 10(1): 202 - 211. [Abstract] [Full Text] [PDF]
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 K. List, R. Szabo, P. W. Wertz, J. Segre, C. C. Haudenschild, S.-Y. Kim, and T. H. Bugge Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1 J. Cell Biol., November 24, 2003; 163(4): 901 - 910. [Abstract] [Full Text] [PDF]
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D. Kirchhofer, M. Peek, W. Li, J. Stamos, C. Eigenbrot, S. Kadkhodayan, J. M. Elliott, R. T. Corpuz, R. A. Lazarus, and P. Moran Tissue Expression, Protease Specificity, and Kunitz Domain Functions of Hepatocyte Growth Factor Activator Inhibitor-1B (HAI-1B), a New Splice Variant of HAI-1 J. Biol. Chem., September 19, 2003; 278(38): 36341 - 36349. [Abstract] [Full Text] [PDF]
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M. D. Oberst, B. Singh, M. Ozdemirli, R. B. Dickson, M. D. Johnson, and C.-Y. Lin Characterization of Matriptase Expression in Normal Human Tissues J. Histochem. Cytochem., August 1, 2003; 51(8): 1017 - 1025. [Abstract] [Full Text] [PDF]
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M. D. Oberst, C. A. Williams, R. B. Dickson, M. D. Johnson, and C.-Y. Lin The Activation of Matriptase Requires Its Noncatalytic Domains, Serine Protease Domain, and Its Cognate Inhibitor J. Biol. Chem., July 11, 2003; 278(29): 26773 - 26779. [Abstract] [Full Text] [PDF]
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J. Y. Kang, M. Dolled-Filhart, I. T. Ocal, B. Singh, C.-Y. Lin, R. B. Dickson, D. L. Rimm, and R. L. Camp Tissue Microarray Analysis of Hepatocyte Growth Factor/Met Pathway Components Reveals a Role for Met, Matriptase, and Hepatocyte Growth Factor Activator Inhibitor 1 in the Progression of Node-negative Breast Cancer Cancer Res., March 1, 2003; 63(5): 1101 - 1105. [Abstract] [Full Text] [PDF]
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G. Velasco, S. Cal, V. Quesada, L. M. Sanchez, and C. Lopez-Otin Matriptase-2, a Membrane-bound Mosaic Serine Proteinase Predominantly Expressed in Human Liver and Showing Degrading Activity against Extracellular Matrix Proteins J. Biol. Chem., September 27, 2002; 277(40): 37637 - 37646. [Abstract] [Full Text] [PDF]
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 M. D. Hollenberg and S. J. Compton International Union of Pharmacology. XXVIII. Proteinase-Activated Receptors Pharmacol. Rev., June 1, 2002; 54(2): 203 - 217. [Abstract] [Full Text] [PDF]
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M. D. Oberst, M. D. Johnson, R. B. Dickson, C.-Y. Lin, B. Singh, M. Stewart, A. Williams, A. al-Nafussi, J. F. Smyth, H. Gabra, et al. Expression of the Serine Protease Matriptase and Its Inhibitor HAI-1 in Epithelial Ovarian Cancer: Correlation with Clinical Outcome and Tumor Clinicopathological Parameters Clin. Cancer Res., April 1, 2002; 8(4): 1101 - 1107. [Abstract] [Full Text] [PDF]
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C. Benaud, M. Oberst, J. P. Hobson, S. Spiegel, R. B. Dickson, and C.-Y. Lin Sphingosine 1-Phosphate, Present in Serum-derived Lipoproteins, Activates Matriptase J. Biol. Chem., March 15, 2002; 277(12): 10539 - 10546. [Abstract] [Full Text] [PDF]
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R. Friedrich, P. Fuentes-Prior, E. Ong, G. Coombs, M. Hunter, R. Oehler, D. Pierson, R. Gonzalez, R. Huber, W. Bode, et al. Catalytic Domain Structures of MT-SP1/Matriptase, a Matrix-degrading Transmembrane Serine Proteinase J. Biol. Chem., January 11, 2002; 277(3): 2160 - 2168. [Abstract] [Full Text] [PDF]
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