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Int. J. Cancer (Pred. thcol.): 69,131-134 (1996)
0 1996 Wiley-Liss, Inc.
*This article is a US Government work
and, as such, is in the public domain in the United States of America.
'
Publication of the International Union Against Cancer
Publicationde I'Union Internationale Contre le Cancer
ENHANCED RNA EXPRESSION OF TISSUE INHIBITOR
OF METALL,OPROTEINASES-1 (TIMP-1) IN HUMAN BREAST CANCER
Hitoshi YOSHIJI,
Daniel E. GOMEZand Unnur P. THORGEIRSSON'
Tumor Biology and Carcinogenesis Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic
Sciences, National Cancer Institute, National Institutes of Health, Building 37, Room 2002, Bethesda, MD 20892 USA.
Tissue inhibitor of metalloproteinases-I (TIMP-I) is known to
have at least 2 distinct types of activity, i.e., as a regulator of
collagenolyticactivity, and erythroid potentiatingactivity (EPA).
In this study, we examined the expression of TIMP- I in human
mammary carcinomas, non-malignant breast tissues and benign
breast tumors. A total of 53 samples were subjectedto Northernblot analysis, including 23 of primary breast cancer, 26 of
non-malignant breast tissues, and 4 of benign tumors. Of the 53
samples, I0 were paired malignant and non-malignant breasttissue samples from the same patient. TIMP-I RNA expression
was significantly higher in the malignant tumor tissues than in
the non-malignant counterpart. Similar differences were obsewed in the level of TIMP- I protein expression in the paired
breast samples examined. Moreover, breast-cancer cell lines
secreted larger amounts of TIMP-I in vitro than non-neoplastic
breast epithelial lineis. The up-regulation of TIMP- I expression
in breast cancer may suggest that TIMP- I has an additional role
to that of metalloprcrteinase inhibitor.
Q
1996 Wiley-Liss,Inc *
Tissue degradation associated with malignancy has been
ascribed to an imbalance between proteolytic enzymes and
their inhibitors (Moscatelli and Rifkin, 1988). Matrix metalloproteinases (MMPs). which have been implicated in tumor
invasion, are secreted as latent proforms and, when activated,
can synergistically degrade the major components of the
extracellular matrix (ECM) (Thorgeirsson et al., 1994; Murphy
et al., 1989). Inifially, only one tissue inhibitor of metalloproteinases (TIMP) was known, now termed TIMP-1 (Welgus and
Stricklin, 1983). TIMP-2 was isolated in 1991 and emerging
evidence suggests that a gene family of TIMPs exists (StetlerStevenson et al., 1989; Pavloff et al., 1992).In vivo studies have
shown that TIMPs are capable of inhibiting tumor invasion
and metastases (Schultz et al., 1988; Montgomery et al., 1994).
It has also been suggested that TIMP-1 may serve as a
tumor-suppressor gene (Khokha et al., 1989). In addition to its
role as a proteinase inhibitor, TIMP-1 is known to possess
erythroid potentiating activity (EPA) (Gasson et al., 1985).
Less attention has been given to its action as a growth factor
for hematopoietic cells and malignant-lymphoid-tumor cell
lines (Alitalo et al., 1990; Kossakowska et al., 1993), although it
has been proposed that TIMP-1 may function also as a growth
factor in other malignancies (Hayakawa et al., 1991). In a
recent immunohistological study of MMP and TIMP-1 expression in human breast cancer, TTMP-1 was most frequently
associated with the tumor microvasculature, and was not
commonly expressed by the same cell types as the MMPs
(Polette et al. 1992). In the present report, TIMP-1 expression
is studied in paired samples of malignant and non-malignant
breast tissues from the same patient. We present here evidence that TIMP-1 RNA and protein expression is significantly increased in cancerous breast tissue.
M.4TERIAL AND METHODS
Human breast-tissue samples
A total of 53 samples was obtained from the National
Disease Interchange/Cooperative Human Tissue Network,
Philadelphia, PA. These included: 23 primary breast carcinomas (18 infiltrating ductal carcinomas, 4 infiltrating lobular
carcinomas, 1 mucinous carcinoma); 4 benign tumors (3
fibroadenomas, 1 lactating adenoma); and 26 samples of
non-neoplastic breast tissue, 10 of which were matched samples
taken from tumor-adjacent tissue of the breast-cancer cases.
The samples were Bash-frozen in liquid nitrogen immediately
after surgical removal and stored at -80°C.
Cell cicltures
The following human breast-derived cell lines were purchased from the (ATCC) (Rockville, MD) and grown in
ATCC-recommended media: estrogen-receptor-positive cancer cell lines (MCF-7, T-47D); estrogen-receptor-negative
cancer cell lines (MDA-MB-231, MDA-MB-468); normal
breast cell lines (HBL-100, HS-578Bst). At sub-confluence,
the cell monolayers were rinsed 3 times in PBS and incubated
in serum-free medium for 48 hr. Culture supernatants were
collected, and cell debris was removed by centrifugation.
Northern- and Westem-blot analyses
Total RNA was extracted from the breast tissues using
RNAzol kit (TEL-TEST, Friendswood, TX) according to the
procedure recommended by the supplier. Northern-blot analysis was performed using 15 pg of total RNA. After electrophoresis, the RNA was immobilized onto nylon membrane (Schleicher and Schuell, Keene, NH) and hybridized with 1 x lo6
cpm/ml of nick-translated, 32P-labelled cDNA probes of
TIMP-1 and p-actin, which were obtained as described by
Mackay et al. (1994). Densitometric analysis of gene expression
was performed by measuring optical density with a scanning
densitometer (Scanmaster 3, Hudson, NH) and interpreted
with Quantity One software (Protein Database, Huntington
Station, NY). The level of TIMP-1 expression was calculated
after normalization of RNA with the p-actin control. The
statistical significance of intergroup differences was determined by the paired Student t-test and Welch t-test.
Lysates from 12 samples (including 3 paired cases) of
malignant and non-neoplastic breast tissues were prepared as
described by Ballin et al. (1991). Samples ( 5 Fg) of the
breast-tissue lysates and 30-fold-concentrated (Centricon) cellculture supernatants (5 kg) were analyzed by Western blot,
using a specific TIMP-1 primary antibody (1:200) and a
secondary alkaline-phosphatase-conjugated anti-rabbit antibody (1:1,000).
RESULTS
Northem-blot analysis
A total of 53 non-malignant and malignant breast-tissue
samples was subjected to Northern-blot analysis. Expression of
a 0.9-kb TIMP-1 transcript is shown in Figure 1 in 7 of the
paired breast-carcinoma samples: the carcinomas displayed
much higher levels of TIMP-1 expression than the adjacent
non-neoplastic breast tissue. Results from the densitometric
'To whom correspondence and reprint requests should be addressed. Fax: (301) 402-0153.
Received: October 2,1995 and in revised form December 5,1995.
132
YOSHIJI E T A L
TABLE I - SUMMARY OF TIMP-1 GENE EXPRESSION IN PAIRED SAMPLES FROM 10 CASES
Sample
numher
Histological t y p ~
Age
Grade
51
45
73
49
44
46
64
43
57
51
1
2
3
4
5
6
7
LN
metastases]
TIMP-I RNA expression'
Tumor
4.211
I1
(+I
Ductal carcinoma
Ductal carcinoma
I11
3.965
Ductal carcinoma
I1
2.346
Ductal carcinoma
High
11.545
Ductal carcinoma
111
3.755
Lobular carcinoma
High
3.943
Ductal carcinoma
I1
3.910
8
Ductal carcinoma
111
3.680
9
Ductal carcinoma
I1
3.469
10
3.974
Ductal carcinoma
111
(+
'LN, lymph node.-*TIMP-1expression presented after normalization with P-actin.
CaseNo
1
2
n
n
NCa
NCa
4
n
NCa
5
n
GI
11
I'
;I
6
n
NCa
NCa
7
n
r
NCa
9
-
NCa
TIMP-1
409kb
I
p-actin
FIGURE1 - Northern-blot analysis of TIMP-1 RNA expression
in paired malignant and non-malignant human breast-tissue
samples. Total RNA was extracted from breast-cancer (Ca) tissue
and adjacent non-malignant tissue (N) in each patient, transferred
onto nylon membranes and hybridized with 32P-labelledprobes for
TIMP-1 and p-actin as a control. Each lane was loaded with 15 kg
of total RNA.
p c 0.001
p c 0.01
Ca
(n=23)
N
(n=26)
Total Cases
Ca
N
(%lo)
Paired Cases
Ca
(n=13)
N
(n=l6)
Non-Paired Cases
FIGURE2 - Quantitative comparison of TIMP-1 RNA expression in malignant and non-malignant human breast samples. The
TIMP-1 levels are presented as total, paired or non-paired
samples of malignant (Ca) and non-malignant (N) breast tissues.
Columns, mean; bars, SD.
analysis of the 10 paired samples is shown in Table I.
Quantitation of the TIMP-1 signal of the paired samples after
normalization with p-actin revealed a mean optical density of
4.480 (range 2.346-11.545) for the carcinomas and 1.554
(range 1.100-3.072) for the non-malignant breast tissue (Table
I, Fig. 2). For the non-paired breast-tissue samples, the mean
density for the carcinomas was 4.593 (range 3.642-8.212) and
for the non-malignant breast tissues 1.058 (range 0.425-3.072).
By combining the results from the paired and non-paired
breast samples, the mean density for the total number of
carcinomas was 4.528 (range 2.346-11.545) and for the nonmalignant breast samples 1.010 (range 0.425-3.072) (Fig. 2).
Normal
1.366
1.165
3.072
2.629
1.310
1.310
1.329
1.110
1.163
1.100
The difference in TIMP-1 RNA levels between malignant and
non-malignant breast tissues was statistically significant, for
the paired ( p < 0.01), non-paired ( p < 0.001) and the total
( p < 0.001) samples.
Only 4 cases of benign breast tumors were available for
Northern-blot analysis. They exhibited variable levels of
TIMP-1 RNA expression. The densitometric values for the 3
fibroadenomas were 0.586, 2.010 and 5.242 and for one
lactating adenoma 4.312. The number of cases was too low to
permit statistical analysis, but these results suggest that the
level of TIMP-1 expression of fibroadenomas was in between
that of the carcinomas and that of the non-malignant breast
tissues.
Western-blot analysis
In only 6 of the malignant tumor samples was sufficient
material available for both RNA and protein extraction. Tissue
lysates were prepared from the 6 tumor samples and 6
non-neoplastic breast samples which included 3 of the pairs of
samples from 3 patients. The samples were equalized for
protein concentration and subjected to Western-blot analysis,
using a TIMP-1-specific antibody. An immunoreactive band of
approximately 30 kDa, consistent with TIMP-1, was prominent
in all the tumor samples, but was either very faint or absent in
the non-neoplastic breast tissues. The elevated levels of the
TIMP-1 protein in the tumors did correspond to the findings
obtained by Northern-blot analysis. The results from the
Western-blot analysis of the paired breast samples are shown
in Figure 3a.
We examined whether the difference in TIMP-1 protein
expression was also present in malignant and non-neoplastic
breast epithelial cell lines. Western-blot analysis was carried
out on concentrated serum-free culture supernatants from
estrogen-receptor(ER)-negative (MDA-MB-231, MDA-MB468) and ER-positive(MCF-7, T-47D) breast-carcinoma cell
lines, as well as from non-malignant (HS-578Bst, HBL-100)
breast epithelial lines. A TIMP-1-specific immunoreactive
band was observed in the supernatants of the 4 breastcarcinoma cell lines, but not those of the non-malignant lines
(Fig. 3b). There appeared to be no relationship between the
intensity of the TIMP-1-immunoreactive bands and the E R
status of the breast-carcinoma cell lines. In the supernatant of
the MDA-MB-231 line, we observed a second immunoreactive
band of higher molecular weight, which may represent a
differently glycosilated form of TIMP-1.
DISCUSSION
Although it is generally accepted that TIMP-1 is an important regulator of matrix metalloproteinases, there is emerging
evidence to suggest a far more complex role for TIMP-1 in
tumor progression. In experimental models, TIMP-1 and
TIMP-2 have been shown to inhibit tumor invasion and
TIMP-1 EXPRESSION IN HUMAN BREAST CANCER
133
FIGURE3 - Western-blot analysis of TIMP-1 expression in paired samples from human breast-cancer and culture supernatants of
breast carcinoma and non-malignant breast epithelial cell lines. (a) Three pairs malignant (Ca) and non-malignant breast-tissue (N)
samples. (6) Culture: supernatants from estrogen-receptor-positive(MCF-7, T-47D) and estrogen-receptor-negative cancer cell lines
(MDA-MB-231, MDIA-MB-468)and non-neoplastic breast cell lines (HBL-100, HS-578Bst).
metastasis (Schultz: et al., 1988; Montgomery et al., 1994).
Conversely, over-ex.pression of TIMP-1 in human lymphomas
has been associated with more aggressive behavior (Kossakowska et al., 1993). Moreover, a steroid hormone stimulating
testicular protein was found to he identical to TIMP-1 (Boujrad et al., 1995). These paradoxical findings raise interesting
questions about TIMP-1 as a multifunctional protein.
Our findings showed significant elevation of TIMP-1 transcripts in human breast carcinoma as compared with nonmalignant breast tissue, both in paired and non-paired cases.
TIMP-1 protein secretion was also found to be higher in
limited numbers of the breast-cancer cases studied, as well as
in malignant and non-neoplastic breast epithelial cell lines.
Elevated TIMP-1 expression has been observed in mammary tumors of transgenic mice, expressing H-ras or c-myc
oncogenes (Li et al., 1994). In these tumors, high TIMP-1 levels
were found in undifferentiated and metastatic H-ras-induced
tumors, whereas TIMP-1 was not detected in well-differentiated and non-metastatic tumors induced by c-myc.
In an immunohisitochemica1 study of human breast samples,
TIMP-1 was detected in 7 out of 30 benign lesions and 55 out
of 79 carcinomas (Polette et al., 1992). The TIMP-1 transcripts
were localized to well-differentiated tumor cells, both in
invasive and in non-invasive human breast carcinomas (Polette
et al., 19936). Further evidence of TIMP-1 association with
malignancy comes from studies of head-and-neck cancer
(Polette et al., 1993a), colon cancer (Lu et al., 1991) and
non-Hodgkin’s lymphoma (Kossakowska et al., 1991). In paired
samples of human colon cancer, higher levels of TIMP-1
protein were found in the tumors than in the adjacent normal
mucosa (Luetal., 1991).
In accordance with our findings, an in situ hybridization
study of paired normal and malignant breast samples has
localized enhanced TIMP-1 mRNA expression to the cancer
cells and the tumor-stromal interface (Dr. C. Lindsay, personal
communication).
The significance of the increased TIMP-1 expression in breast
carcinomas is unclear. A conventional explanation would be
that TIMP-1 is needed to halt tumor invasion, mediated by
matrix-degrading MMPs. Alternatively, TIMP-1 may contribute to tumor progression through growth-promoting activity,
as has been demonstrated for hematological cells (Gasson et
al., 1985) and a range of other cell types, including a human
breast-cancer cell line (MCF-7) (Hayakawa et al., 1991).
In summary, we have demonstrated in the present study of
paired human breast samples from the same patient that
TIMP-1 R N A and protein expression is significantly increased
in the cancerous portion of the breast. Further studies are
ongoing to characterize the role of TIMP-1 in breast-cancer
progression.
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