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). 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