- Email: [email protected]
Matrix Metalloproteinases and Their Inhibitors in Gestational Trophoblastic Diseases and Normal Placenta
Gynecologic Oncology 75, 248 –253 (1999) Article ID gyno.1999.5564, available online at http://www.idealibrary.com on
Matrix Metalloproteinases and Their Inhibitors in Gestational Trophoblastic Diseases and Normal Placenta Gyorgy L. Vegh, M.D.,* Z. Selcuk Tuncer, M.D.,† Vilmos Fulop, M.D.,* David R. Genest, M.D.,‡ Samuel C. Mok, Ph.D.,† and Ross S. Berkowitz, M.D.† †Laboratory of Gynecologic Oncology, Division of Gynecologic Oncology, Department of Obstetrics, Gynecology, and Reproductive Biology, ‡Division of Women’s and Perinatal Pathology, Department of Pathology, New England Trophoblastic Disease Center, Gillette Center for Women’s Cancer, Brigham and Women’s Hospital, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115; and *Department Obstetrics and Gynecology, Haynal Imre University of Health Sciences, Budapest, Hungary Received April 28, 1999
INTRODUCTION
Objective. Our purpose was to investigate the expression of matrix metalloproteinases (MMPs) in gestational trophoblastic diseases and normal first-trimester placenta. Methods. Paraffin sections of 16 partial moles, 25 complete moles, 10 gestational choriocarcinomas, and 11 normal first-trimester placentas were studied immunohistochemically for expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and tissue inhibitor of metalloproteinase-1 (TIMP-1). Results. Nine (90.0%) of the choriocarcinoma cases showed strong intensity of staining for MMP-1. Choriocarcinoma exhibited significantly stronger staining for MMP-1 than syncytiotrophoblast in normal placenta (P < 0.01), partial mole (P < 0.01), and complete mole (P < 0.01). Choriocarcinoma also showed significantly stronger staining for MMP-1 than the extravillous trophoblast in placenta (P < 0.05). MMP-2 was expressed only in syncytio- and extravillous trophoblasts in normal placenta, partial mole, and complete mole. Choriocarcinoma and the extravillous trophoblast in partial mole and complete mole had significantly stronger staining for MMP-2 than the extravillous trophoblast in placenta (P < 0.05, P < 0.01, P < 0.01, respectively). Choriocarcinoma also exhibited significantly stronger staining for MMP-2 than syncytiotrophoblasts in placenta (P < 0.01), partial mole (P 5 0.05), and complete mole (P < 0.01). The expression of MMP-3, MMP-9, and MMP-13 was similar in all four tissues with the predominance of syncytiotrophoblast for MMP-3 and MMP-13 and cytotrophoblast for MMP-9. While 8 (73.0%) placentas, 14 (87.5%) partial moles, and 19 (76.0%) complete moles showed strong immunoreactivity for TIMP-1 in syncytiotrophoblasts, no strong staining was found in choriocarcinomas (P < 0.01, P < 0.01, P < 0.01, respectively). Conclusion. The extravillous trophoblast of first-trimester placenta has significantly less expression of MMP-1 than choriocarcinoma and significantly less expression of MMP-2 than choriocarcinoma and extravillous trophoblast of partial and complete mole. The expression of TIMP-1 was significantly less in choriocarcinoma than the syncytiotrophoblast of normal placenta, partial mole, and complete mole. MMPs and their inhibitors may play a role in the pathogenesis of gestational trophoblastic diseases. © 1999 Academic Press Key Words: MMP; TIMP; trophoblastic disease; placenta. 0090-8258/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
248
Gestational trophoblastic diseases comprise a spectrum of chorionic diseases that include partial and complete hydatidiform moles and gestational choriocarcinoma [1]. These tumors have varying propensities for local uterine invasion and spread and can be highly cured even in the presence of widespread metastases [2, 3]. Placental tissue contains a heterogeneous population of cells, including villous syncytiotrophoblast and cytotrophoblast, as well as extravillous trophoblast. Whereas villous trophoblast does not exhibit invasive behavior, the invasive capacity of the extravillous trophoblast appears to have similarities with the role of malignant cells during tumor invasion [4, 5]. However, trophoblast invasion of the endometrium is tightly regulated during the first trimester of normal pregnancy [4]. Extravillous trophoblast cells migrate from the basement membrane of anchoring villi and invade deeply, reaching the myometrium. The process of trophoblast invasion involves the enzymatic degradation of the extracellular matrix [6, 7]. Different families of matrix-degrading proteases have been found to be involved in trophoblastic invasion into the maternal tissues, including serine proteases and matrix metalloproteinases (MMPs) [6 – 8]. MMPs are a family of zinc-containing endopeptidases that degrade a wide range of components of the extracellular matrix; MMPs are also thought to play an important role in tumor progression and metastasis [5– 8]. To date at least 20 members of the MMP family have been reported and divided into four main groups according to their substrate specificities: collagenases, gelatinases, stromelysins, and membrane-type MMPs [9, 10]. All MMPs are secreted in inactive form. The activity of the MMPs is regulated by several biological modulators including tissue inhibitors of metalloproteinases (TIMPs). These secreted inhibitory proteins bind the active forms of MMPs and inhibit their individual proteolytic activity in tissue [9, 11, 12]. The potential interactions amongst MMPs are currently under active investigation and the specific
MATRIX METALLOPROTEINASES AND TROPHOBLASTIC DISEASES
substrates of MMPs that have been identified are summarized in recent review articles [13, 14]. For example, both MMP-2 and MMP-9 degrade Type IV collagen, which is a major component of the basement membrane and constitutes an important barrier to tumor cell invasion. In contrast, MMP-1 degrades Type II and III collagen. In the present study we investigated the expression of MMPs and TIMP-1 in normal first-trimester placenta, partial and complete mole, and choriocarcinoma by immunohistochemical analysis. We also evaluated whether the expression of MMPs by complete molar pregnancy was associated with the development of postmolar gestational trophoblastic tumor. MATERIALS AND METHODS Collection of Tissues and Clinical Data Archival tissues in paraffin blocks from 62 cases were collected including 16 partial moles, 25 complete moles, 10 gestational choriocarcinomas, and 11 normal first-trimester placentas as control specimens. The slides from all 62 cases were reviewed by one of the authors (DRG), who is a gynecologic pathologist with recognized special interest and expertise in the pathology of gestational trophoblastic disease. The diagnosis of partial and complete mole was made using standard published histopathologic criteria [2, 3]. All placental sections originated from therapeutic abortions of first-trimester normal pregnancies between the gestational age of 8 and 12 weeks (mean gestational age was 9.5 weeks). The mean gestational age was 8.4 weeks for partial and 9.8 weeks for complete mole. Written consent for the collection of tissue samples was obtained according to a protocol approved by the Human Subject Committee of Brigham and Women’s Hospital. All patients were diagnosed and treated at the Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School (Boston, MA). Eight (32.0%) of the patients with complete mole developed persistent postmolar gestational trophoblastic tumor. A total of 11 cycles of chemotherapy were administered to these patients. Of the choriocarcinoma cases, 8 (80.0%) had stage I (World Health Organization Score, mean 2.0, range 1– 4) and 2 (20.0%) had stage III disease (World Health Organization Score—3,8); a total of 28 cycles of chemotherapy were administered to these patients. The follow-up and management protocol for gestational trophoblastic diseases has previously been published from our center and was followed in each case [15, 16]. In brief, patients with nonmetastatic disease (Stage I) and low-risk Stage III disease (pulmonary metastases) were treated with methotrexate and citrovorum factor. The patient with high-risk Stage III disease was treated with primary EMA–CO chemotherapy (etoposide, methotrexate, actinomycin D– cyclophosphamide and Oncovin). All patients achieved complete sustained gonadotropin remission. None of the patients who developed persistent postmolar gestational trophoblastic tumor underwent hysterectomy. Fur-
249
thermore, the two patients with choriocarcinoma and pulmonary metastases did not undergo surgical resection of metastases. None of the cases of gestational trophoblastic disease had fresh tissue available for investigation. Immunohistochemical Analysis The avitin– biotin complex peroxidase method was performed on 5-mm-thick sections from formalin-fixed, paraffinembedded tissues. Sections were deparaffinized in xylene and hydrated with graded ethanol concentrations and distilled water. For both MMP-1 and MMP-3, the slides underwent pretreatment boiling in 0.01 M citric acid buffer (pH 6.0) for 15 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 30 min. After blocking nonspecific antigens with normal horse serum, the sections were incubated overnight at 4°C with primary antibodies in a moist chamber. The primary antibodies in this study (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and TIMP-1) were all mouse monoclonal antibodies and were all purchased from Oncogene Research Products (Cambridge, MA). The dilutions that were utilized with these antibodies were as follows: MMP-2, MMP-3, and MMP-13—1:400; MMP-1, MMP-9, and TIMP-1—1:800. The positive control tissues for the primary antibodies were as follows: MMP-1 and MMP-13— breast cancer; MMP-2 and MMP-3— ovarian cancer; MMP-9 —rectal cancer; and TIMP-1—normal ovary. The primary antibody was replaced by normal mouse serum for a negative control. Staining was achieved using a biotinylated antimouse secondary antibody and the ABC horseradish–peroxidase (Vectastain Elite ABC Kit; Vector, Burlingame, CA). The reaction product was visualized by 3,39 diaminobenzidine tetrahydrochloride (Vector) as chromogen. Finally, the sections were dehydrated in ethanol, cleared in xylene, and mounted. In this study 51 gestational trophoblastic disease cases and 11 normal first-trimester placentas were investigated for expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and their main inhibitor TIMP-1 by immunohistochemistry. Cytoplasmic staining of syncytiotrophoblast, cytotrophoblast, extravillous trophoblast, and trophoblastic tumor cells in choriocarcinoma sections was categorized as negative, weakly positive, or strongly positive. A strongly positive result was recorded when more than 50% of the cells exhibited strong staining. Statistical Analysis The immunohistochemical data and the postevacuation clinical outcome of complete molar cases were analyzed by StatView 4.5 package. x 2 and Fisher’s exact x 2 tests were used. Statistical significance was considered at the 0.05 level. RESULTS Expression of MMP-1 was predominantly strong in cytotrophoblasts and extravillous trophoblasts in normal placenta,
250
VEGH ET AL.
TABLE 1 Immunostaining of Placentas, Partial Moles, and Complete Moles Placenta (n 5 11)
MMP-1
MMP-2
MMP-3
MMP-9
MMP-13
TIMP-1
ST CT EVT ST CT EVT ST CT EVT ST CT EVT ST CT EVT ST CT EVT
Partial mole (n 5 16)
Complete mole (n 5 25)
Negative (%)
Weak (%)
Strong (%)
Negative (%)
Weak (%)
Strong (%)
Negative (%)
Weak (%)
Strong (%)
— — 1 (9.0) — 11 (100.0) — — 9 (82.0) 5 (45.5) — 1 (9.0) 4 (36.0) — 11 (100.0) 5 (45.0) — — —
11 (100.0) 2 (18.0) 6 (55.0) 11 (100.0) — 9 (82.0) 7 (64.0) 2 (18.0) 5 (45.5) 9 (82.0) 9 (82.0) 5 (45.0) 6 (55.0) — 6 (55.0) 3 (27.0) 11 (100.0) 10 (91.0)
— 9 (82.0) 4 (36.0) — — 2 (18.0) 4 (36.0) — 1 (9.0) 2 (18.0) 1 (18.0) 2 (18.0) 5 (45.0) — — 8 (73.0) — 1 (9.0)
— — — — 16 (100.0) — — 12 (75.0) 4 (25.0) — 1 (6.2) 6 (37.6) — 14 (87.5) 7 (43.8) — 6 (37.5) 2 (12.5)
16 (100.0) 1 (6.2) 6 (37.5) 10 (62.5) — 6 (37.5) 4 (25.0) 4 (25.0) 11 (68.8) 9 (56.2) 8 (50.0) 5 (31.2) 9 (56.2) 2 (12.5) 9 (56.2) 2 (12.5) 10 (62.5) 12 (75.0)
— 15 (93.8) 10 (62.5) 6 (37.5) — 10 (62.5) 12 (75.0) — 1 (6.2) 7 (43.8) 7 (43.8) 5 (31.2) 7 (43.8) — — 14 (87.5) — 2 (12.5)
— — — — 25 (100.0) — — 20 (80.0) 6 (24.0) — — 3 (12.0) — 22 (88.0) 3 (12.0) — 6 (24.0) 6 (24.0)
21 (84.0) 9 (43.8) 10 (40.0) 22 (88.0) — 2 (8.0) 15 (60.0) 5 (20.0) 19 (76.0) 10 (40.0) 14 (56.0) 18 (72.0) 14 (56.0) 3 (12.0) 20 (80.0) 6 (24.0) 19 (76.0) 19 (76.0)
4 (16.0) 16 (56.2) 15 (60.0) 3 (12.0) — 23 (92.0) 10 (40.0) — — 15 (60.0) 11 (44.0) 4 (16.0) 11 (44.0) — 2 (8.0) 19 (76.0) — —
Note. ST, syncytiotrophoblast; CT, cytotrophoblast; EVT, extravillous trophoblast.
partial mole, and complete mole (Table 1). Nine (90.0%) of the choriocarcinoma cases showed strong intensity of staining for MMP-1 (Table 2). Choriocarcinoma exhibited significantly stronger staining for MMP-1 than syncytiotrophoblast in normal placenta (P , 0.01), partial mole (P , 0.01), and complete mole (P , 0.01). Choriocarcinoma also showed significantly stronger staining for MMP-1 than the extravillous trophoblast in placenta (P , 0.05). Strong staining for MMP-1 was observed in cytotrophoblasts in 9 (82.0%) placentas, 15 (93.8%) partial moles, and 16 (56.2%) complete moles (Fig. 1A). MMP-2 was expressed only in syncytio- and extravillous trophoblasts in normal placenta, partial mole, and complete mole. Choriocarcinoma and the extravillous trophoblast in partial mole and complete mole had significantly stronger staining for MMP-2 than the extravillous trophoblast in placenta (P , 0.05, P , 0.01, P , 0.01, respectively). Choriocarcinoma also exhibited significantly stronger staining for MMP-2 than syncytiotrophoblasts in placenta, partial mole, and complete mole (P , 0.01, P 5 0.05, P , 0.01, respectively), (Figs. 1B and 1C). TABLE 2 Immunostaining of Choriocarcinoma Cases (n 5 10) Antibody
Negative (%)
Weak (1) (%)
Strong (1) (%)
MMP-1 MMP-2 MMP-3 MMP-9 MMP-13 TIMP-1
— — 4 (40.0) — — 2 (20.0)
1 (10.0) 2 (20.0) 6 (60.0) 6 (60.0) 8 (80.0) 8 (80.0)
9 (90.0) 8 (80.0) — 4 (40.0) 2 (20.0) —
Strong MMP-3 immunoreactivity was found almost exclusively in syncytiotrophoblasts of placentas (36.0%), partial moles (75.0%), and complete moles (40.0%). Cytotrophoblast, extravillous trophoblast, and choriocarcinoma cells showed mostly weakly positive or negative staining for MMP-3. MMP-9 expression was observed in all three trophoblastic cell types in placenta, partial mole, and complete mole. Syncytiotrophoblast in complete mole had significantly greater expression of MMP-9 than in placenta (P , 0.05). There were no other significant differences in expression of MMP-9 amongst placenta and gestational trophoblastic disease tissues. MMP-13 expression was found to be similar in all four tissues and all trophoblastic cell types. Cytotrophoblast was mainly negative for MMP-13 staining, while syncytiotrophoblasts, extravillous trophoblasts, and tumor cells were mostly weakly positive. Expression of TIMP-1 was found in all three trophoblastic cell types in placenta, partial mole, and complete mole, with predominance of the syncytiotrophoblast. Cytotrophoblasts and extravillous trophoblasts stained weakly in almost all cases. While 8 (73.0%) placentas, 14 (87.5%) partial moles, and 19 (76.0%) complete moles showed strong immunoreactivity for TIMP-1 in syncytiotrophoblasts, no strong staining was found in choriocarcinomas (P , 0.01, P , 0.01, P , 0.01, respectively), (Figs. 1D and 1E). MMPs and TIMP-1 expression in complete moles was compared to the risk of persistent postmolar gestational trophoblastic tumor (Table 3). Strong staining was compared with both weak and negative staining. Expression of MMPs and their inhibitor, TIMP-1, in complete moles was not associated with the development of postmolar gestational trophoblastic tumor.
MATRIX METALLOPROTEINASES AND TROPHOBLASTIC DISEASES
251
FIG. 1. Immunohistochemical analysis of expression of MMP-1, MMP-2, and TIMP-1 in gestational trophoblastic diseases. (A) Strongly positive MMP-1 immunoreactivity in choriocarcinoma cells (magnification: 1003). (B) Strongly positive MMP-2 immunoreactivity in choriocarcinoma cells (magnification: 1003). (C) Strongly positive MMP-2 immunoreactivity in extravillous trophoblasts of complete mole (magnification: 1003). (D) Strongly positive TIMP-1 immunoreactivity in syncytiotrophoblasts in partial mole (magnification: 1003). (E) No immunoreactivity for TIMP-1 in choriocarcinoma cells (magnification: 2003).
DISCUSSION Because matrix metalloproteinases degrade a wide range of components of the extracellular matrix, MMPs have been suggested to play a vital role in tumor invasion and metastasis [17–19]. The expression and activity of MMPs have been investigated in several human malignancies. MMP-1 and MMP-2 have been reported to be overexpressed in a majority of intestinal cancers [11, 20, 21], and MMP-2 has been implicated in the tumorigenicity of prostate, cervical, and endometrial cancers [22–27]. Davidson et al. and Tamakoshi et al. showed strong expression of MMP-2 and MMP-9 in cervical cancer which correlated with tumor invasion and progression [24, 25, 27]. During normal pregnancy, extravillous trophoblasts locally invade maternal tissues and this process of invasion involves enzymatic degradation of the extracellular matrix. MMPs along with other proteases have been reported to be involved in trophoblastic invasion of maternal tissues [4, 6]. The current study was undertaken to investigate the expression of MMPs in gestational trophoblastic diseases including
partial mole, complete mole, and choriocarcinoma. Gestational trophoblastic diseases are well recognized to have varying propensities for local invasion and metastasis [1–3]. Importantly choriocarcinoma exhibited significantly stronger expression for both MMP-1 and MMP-2 than syncytiotrophoblast in normal placenta, partial mole and complete mole, and extravillous trophoblast in placenta. Furthermore the extravillous trophoblast in partial and complete mole had significantly stronger staining for MMP-2 than the extravillous trophoblast in normal placenta. The increased expression of MMP-2 in partial and complete mole compared to normal placenta may contribute to their risk for local myometrial invasion. While choriocarcinoma had significantly increased expression of MMP-1 and MMP-2 compared to placenta, partial mole, and complete mole, choriocarcinoma had significantly less expression of the tissue inhibitor of metalloproteinase-1 than placenta, partial mole, and complete mole. The increased expression of MMP-1 and MMP-2 and decreased expression of TIMP-1 in choriocarcinoma may contribute to the invasiveness of choriocarcinoma cells.
252
VEGH ET AL.
TABLE 3 Risk of Persistent Postmolar Gestational Disease after Complete Moles MMP-1
ST Spon. remission: n 5 17 Persistent GTD: n 5 8 CT Spon. remission: n 5 17 Persistent GTD: n 5 8 EVT Spon. remission: n 5 17 Persistent GTD: n 5 8
MMP-2
Weak 1
Strong 1
P
Weak 1
Strong 1
P
Weak 1
Strong 1
P
14 7
3 1
NS
15 7
2 1
NS
12 3
5 5
NS
5 4
12 4
NS
— —
— —
—
17 8
— —
—
5 5
12 3
NS
1 1
16 7
NS
17 8
— —
—
MMP-9
ST Spon. remission: n 5 17 Persistent GTD: n 5 8 CT Spon. remission: n 5 17 Persistent GTD: n 5 8 EVT Spon. remission: n 5 17 Persistent GTD: n 5 8
MMP-3
MMP-13
TIMP-1
Weak 1
Strong 1
P
Weak 1
Strong 1
P
Weak 1
Strong 1
P
7 3
10 5
NS
11 3
6 5
NS
4 2
13 6
NS
11 3
6 5
NS
17 8
— —
—
17 8
— —
—
14 7
3 1
NS
16 7
1 1
NS
17 8
— —
—
Note. ST, syncytiotrophoblast; CT, cytotrophoblast; EVT, extravillous trophoblast; GTD, gestational trophoblastic disease.
MMPs and tissue inhibitors may play an important role in the pathogenesis of gestational trophoblastic diseases. Fortunately, gestational trophoblastic tumors are generally remarkably sensitive to chemotherapy and all patients in the current study achieved complete remission. However, infrequently, patients with drug-resistant gestational trophoblastic tumors are encountered and there is a continuing need to develop and evaluate new chemotherapeutic agents. Anti-neoplastic drugs are currently being developed that modify the activity of MMPs and their inhibitors and these agents may exhibit activity against gestational trophoblastic diseases [28, 29]. Further understanding of the role of MMPs in the biology of gestational trophoblastic diseases may therefore lead to new and novel therapies for patients with these diseases. ACKNOWLEDGMENT This research was supported by the United States–Hungarian Science and Technology Joint Fund (No. 552) and the Soros Foundation.
REFERENCES
Gestational Trophoblastic Tumors, in Knapp RC, Berkowitz RS (eds): Gynecologic Oncology. 2nd ed. New York, NY, McGraw–Hill, 1993, pp 328 –338 4. Crescimanno C, Foidart JM, Noel A, Polette M, Maquoi E, Birembaut P, Baramova E, Kaufmann P, Castellucci M: Cloning of choriocarcinoma cells shows that invasion correlates with expression and activation of gelatinase A. Exp Cell Res 227:240 –251, 1996 5. Lala PK, Graham CH: Mechanism of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. Cancer Metast Rev 9:369 –379, 1994 6. Huppertz B, Kerschanska S, Demir AY, Frank HG, Kaufmann P: Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta. Cell Tissue Res 291:133–148, 1998 7. Bishof P, Haenggli L, Campana A: Gelatinase and oncofetal fibronectin expression is dependent on integrin expression on human cytotrophoblasts. Hum Reprod 10:734 –742, 1995 8. Castellucci M, Theelen T, Pompili E, Fumagalli L, De Renzis G, Muhlhauser J: Immunohistochemical localization of serine-protease inhibitors in the human placenta. Cell Tissue Res 278:283–289, 1994 9. Leber T, Boyd R, Balkwill F: Tumour Cell–Stromal Cell Interactions: Proteases and Protease Inhibitors, in Sharp F, Blackett T, Berek J, Bast R (eds): Ovarian Cancer 5. Oxford, Isis Medical Media Ltd, 1998, pp 121–129
1. Lewis JL Jr: Diagnosis and management of gestational trophoblastic disease. Cancer 71:1639 –1647, 1993
10. Sato H, Takino T, Okada Y: A matrix metalloproteinase expressed on the surface of invasive tumor cells. Nature 370:61– 65, 1994
2. Berkowitz RS, Goldstein DP: Chorionic tumors. N Engl J Med 335:1740 – 1748, 1996
11. Murray GI, Duncan ME, Arbucle E, Melvin WT, Fothergill JE: Matrix metalloproteinases and their inhibitors in gastric cancer. Gut 43:791–797, 1998
3. Berkowitz RS, Goldstein DP: The Management of Molar Pregnancy and
MATRIX METALLOPROTEINASES AND TROPHOBLASTIC DISEASES 12. Denhardt DT, Feng B, Edwards DR: Tissue inhibitor of metalloproteinases (TIMP, akaEPA): structure, control of expression and biological functions. Pharmacol Ther 59:329 –341, 1993 13. Matrisian LM. The matrix-degrading metalloproteinases. Bioessays 14: 455– 462, 1992 14. Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. JNCI 89:1260 –1270, 1997 15. Berkowitz RS, Goldstein DP: Presentation and Management of Molar Pregnancy, in Hancock BW, Newlands ES, Berkowitz RS (eds): Gestational Trophoblastic Disease. London, Chapman & Hall, 1997, pp 127–142 16. Goldstein DP, Zanten-Przybysz IV, Bernstein MR, Berkowitz RS: Revised FIGO staging system for gestational trophoblastic tumors. Recommendations regarding therapy. J Reprod Med 43:37– 43, 1998 17. Fulop V, Mok SC, Genest DR, Szigetvari I, Cseh I, Berkowitz RS: c-myc, c-erbB-2, c-fms and bcl-2 oncoproteins. Expression in normal placenta, partial and complete mole, and choriocarcinoma. J Reprod Med 43:101– 110, 1998 18. Cottam DW, Rees RC: Regulation of matrix metalloproteinases: their role in tumor invasion and metastasis. Intl J Oncol 2:861– 872, 1993 (review) 19. Murphy G, Reynolds JJ, Hembry RM: Metalloproteinases and cancer invasion and metastasis. Int J Cancer 44:757–760, 1989 20. Murray GI, Duncan ME, O’Neil P: Matrix metalloproteinase-1 is associated with poor prognosis in colorectal cancer. Nat Med 2:461– 462, 1996 21. Murray GI, Duncan ME, O’Neil P: Matrix metalloproteinase-1 is associated with poor prognosis in oesophageal cancer. J Pathol 185:256 –261, 1998 22. Montironi R, Fabris G, Lucarini G, Biagini G: Location of 72-kD metalloproteinase (type IV collagenase) in untreated prostatic adenocarcinoma. Path Res Pract 191:1140 –1146, 1995
253
23. Montironi R, Lucarini G, Castaldini C, Galluzzi CM, Biagini G, Fabris G: Immunohistochemical evaluation of type IV collagenase (72-kD metalloproteinase) in prostatic intraepithelial neoplasia. Anticancer Res 16:2057– 2062, 1996 24. Tamakoshi K, Kikkawa F, Nawa A, Ishikawa H, Mizuno K, Tamakoshi A, Yamagata S, Suganuma N, Tomoda Y: Characterization of extracellular matrix degrading proteinase and its inhibitor in gynecologic cancer tissues with clinically different metastatic form. Cancer 76:2565–2571, 1995 25. Davidson B, Goldberg I, Liokumovich P, Kopolovic J, Gotlieb WH, Lerner-Geva L, Reder I, Ben-Baruch G, Reich R: Expression of metalloproteinases and their inhibitors in adenocarcinoma of the uterine cervix. Int J Gynecol Pathol 17:295–301, 1998 26. Talvensaari-Mattila A, Apaja-Sarkkinen M, Hoyhtya M, Westerlund A, Puistola U, Turpeenniemi-Hujanen T: Matrix metalloproteinase 2 immunoreactive protein appears early in cervical epithelial dedifferentiation. Gynecol Oncol 72:306 –311, 1999 27. Davidson B, Goldberg I, Kopolovic J, Lerner-Geva L, Gotlieb WH, Weis B, Ben-Baruch G, Reich R: Expression of matrix metalloproteinase-9 in squamous cell carcinoma of the uterine cervix— clinicopathologic study using immunohistochemistry and mRNA in situ hybridization. Gynecol Oncol 72:380 –386, 1999 28. Rasmussen HS, McCann PP: Matrix metalloproteinase inhibition as a novel anticancer strategy: a review with specifial focus on batimastat and marimastat. Pharmacol Ther 75:69 –75, 1997 29. Wojtowitz-Praga S, Torri J, Johnson M, Steen V, Marchall J, Ness E, Bickson R, Sale M, Rasmussen HS, Chiodo TA, Hawkins MJ: Phase I trial Marimastat, a novel matrix metalloproteinase inhibitor, administered orally to patients with advanced lung cancer. J Clin Oncol 16:2150 –2156, 1998
Matrix Metalloproteinases and Their Inhibitors in Gestational Trophoblastic Diseases and Normal Placenta Gyorgy L. Vegh, M.D.,* Z. Selcuk Tuncer, M.D.,† Vilmos Fulop, M.D.,* David R. Genest, M.D.,‡ Samuel C. Mok, Ph.D.,† and Ross S. Berkowitz, M.D.† †Laboratory of Gynecologic Oncology, Division of Gynecologic Oncology, Department of Obstetrics, Gynecology, and Reproductive Biology, ‡Division of Women’s and Perinatal Pathology, Department of Pathology, New England Trophoblastic Disease Center, Gillette Center for Women’s Cancer, Brigham and Women’s Hospital, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115; and *Department Obstetrics and Gynecology, Haynal Imre University of Health Sciences, Budapest, Hungary Received April 28, 1999
INTRODUCTION
Objective. Our purpose was to investigate the expression of matrix metalloproteinases (MMPs) in gestational trophoblastic diseases and normal first-trimester placenta. Methods. Paraffin sections of 16 partial moles, 25 complete moles, 10 gestational choriocarcinomas, and 11 normal first-trimester placentas were studied immunohistochemically for expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and tissue inhibitor of metalloproteinase-1 (TIMP-1). Results. Nine (90.0%) of the choriocarcinoma cases showed strong intensity of staining for MMP-1. Choriocarcinoma exhibited significantly stronger staining for MMP-1 than syncytiotrophoblast in normal placenta (P < 0.01), partial mole (P < 0.01), and complete mole (P < 0.01). Choriocarcinoma also showed significantly stronger staining for MMP-1 than the extravillous trophoblast in placenta (P < 0.05). MMP-2 was expressed only in syncytio- and extravillous trophoblasts in normal placenta, partial mole, and complete mole. Choriocarcinoma and the extravillous trophoblast in partial mole and complete mole had significantly stronger staining for MMP-2 than the extravillous trophoblast in placenta (P < 0.05, P < 0.01, P < 0.01, respectively). Choriocarcinoma also exhibited significantly stronger staining for MMP-2 than syncytiotrophoblasts in placenta (P < 0.01), partial mole (P 5 0.05), and complete mole (P < 0.01). The expression of MMP-3, MMP-9, and MMP-13 was similar in all four tissues with the predominance of syncytiotrophoblast for MMP-3 and MMP-13 and cytotrophoblast for MMP-9. While 8 (73.0%) placentas, 14 (87.5%) partial moles, and 19 (76.0%) complete moles showed strong immunoreactivity for TIMP-1 in syncytiotrophoblasts, no strong staining was found in choriocarcinomas (P < 0.01, P < 0.01, P < 0.01, respectively). Conclusion. The extravillous trophoblast of first-trimester placenta has significantly less expression of MMP-1 than choriocarcinoma and significantly less expression of MMP-2 than choriocarcinoma and extravillous trophoblast of partial and complete mole. The expression of TIMP-1 was significantly less in choriocarcinoma than the syncytiotrophoblast of normal placenta, partial mole, and complete mole. MMPs and their inhibitors may play a role in the pathogenesis of gestational trophoblastic diseases. © 1999 Academic Press Key Words: MMP; TIMP; trophoblastic disease; placenta. 0090-8258/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
248
Gestational trophoblastic diseases comprise a spectrum of chorionic diseases that include partial and complete hydatidiform moles and gestational choriocarcinoma [1]. These tumors have varying propensities for local uterine invasion and spread and can be highly cured even in the presence of widespread metastases [2, 3]. Placental tissue contains a heterogeneous population of cells, including villous syncytiotrophoblast and cytotrophoblast, as well as extravillous trophoblast. Whereas villous trophoblast does not exhibit invasive behavior, the invasive capacity of the extravillous trophoblast appears to have similarities with the role of malignant cells during tumor invasion [4, 5]. However, trophoblast invasion of the endometrium is tightly regulated during the first trimester of normal pregnancy [4]. Extravillous trophoblast cells migrate from the basement membrane of anchoring villi and invade deeply, reaching the myometrium. The process of trophoblast invasion involves the enzymatic degradation of the extracellular matrix [6, 7]. Different families of matrix-degrading proteases have been found to be involved in trophoblastic invasion into the maternal tissues, including serine proteases and matrix metalloproteinases (MMPs) [6 – 8]. MMPs are a family of zinc-containing endopeptidases that degrade a wide range of components of the extracellular matrix; MMPs are also thought to play an important role in tumor progression and metastasis [5– 8]. To date at least 20 members of the MMP family have been reported and divided into four main groups according to their substrate specificities: collagenases, gelatinases, stromelysins, and membrane-type MMPs [9, 10]. All MMPs are secreted in inactive form. The activity of the MMPs is regulated by several biological modulators including tissue inhibitors of metalloproteinases (TIMPs). These secreted inhibitory proteins bind the active forms of MMPs and inhibit their individual proteolytic activity in tissue [9, 11, 12]. The potential interactions amongst MMPs are currently under active investigation and the specific
MATRIX METALLOPROTEINASES AND TROPHOBLASTIC DISEASES
substrates of MMPs that have been identified are summarized in recent review articles [13, 14]. For example, both MMP-2 and MMP-9 degrade Type IV collagen, which is a major component of the basement membrane and constitutes an important barrier to tumor cell invasion. In contrast, MMP-1 degrades Type II and III collagen. In the present study we investigated the expression of MMPs and TIMP-1 in normal first-trimester placenta, partial and complete mole, and choriocarcinoma by immunohistochemical analysis. We also evaluated whether the expression of MMPs by complete molar pregnancy was associated with the development of postmolar gestational trophoblastic tumor. MATERIALS AND METHODS Collection of Tissues and Clinical Data Archival tissues in paraffin blocks from 62 cases were collected including 16 partial moles, 25 complete moles, 10 gestational choriocarcinomas, and 11 normal first-trimester placentas as control specimens. The slides from all 62 cases were reviewed by one of the authors (DRG), who is a gynecologic pathologist with recognized special interest and expertise in the pathology of gestational trophoblastic disease. The diagnosis of partial and complete mole was made using standard published histopathologic criteria [2, 3]. All placental sections originated from therapeutic abortions of first-trimester normal pregnancies between the gestational age of 8 and 12 weeks (mean gestational age was 9.5 weeks). The mean gestational age was 8.4 weeks for partial and 9.8 weeks for complete mole. Written consent for the collection of tissue samples was obtained according to a protocol approved by the Human Subject Committee of Brigham and Women’s Hospital. All patients were diagnosed and treated at the Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School (Boston, MA). Eight (32.0%) of the patients with complete mole developed persistent postmolar gestational trophoblastic tumor. A total of 11 cycles of chemotherapy were administered to these patients. Of the choriocarcinoma cases, 8 (80.0%) had stage I (World Health Organization Score, mean 2.0, range 1– 4) and 2 (20.0%) had stage III disease (World Health Organization Score—3,8); a total of 28 cycles of chemotherapy were administered to these patients. The follow-up and management protocol for gestational trophoblastic diseases has previously been published from our center and was followed in each case [15, 16]. In brief, patients with nonmetastatic disease (Stage I) and low-risk Stage III disease (pulmonary metastases) were treated with methotrexate and citrovorum factor. The patient with high-risk Stage III disease was treated with primary EMA–CO chemotherapy (etoposide, methotrexate, actinomycin D– cyclophosphamide and Oncovin). All patients achieved complete sustained gonadotropin remission. None of the patients who developed persistent postmolar gestational trophoblastic tumor underwent hysterectomy. Fur-
249
thermore, the two patients with choriocarcinoma and pulmonary metastases did not undergo surgical resection of metastases. None of the cases of gestational trophoblastic disease had fresh tissue available for investigation. Immunohistochemical Analysis The avitin– biotin complex peroxidase method was performed on 5-mm-thick sections from formalin-fixed, paraffinembedded tissues. Sections were deparaffinized in xylene and hydrated with graded ethanol concentrations and distilled water. For both MMP-1 and MMP-3, the slides underwent pretreatment boiling in 0.01 M citric acid buffer (pH 6.0) for 15 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 30 min. After blocking nonspecific antigens with normal horse serum, the sections were incubated overnight at 4°C with primary antibodies in a moist chamber. The primary antibodies in this study (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and TIMP-1) were all mouse monoclonal antibodies and were all purchased from Oncogene Research Products (Cambridge, MA). The dilutions that were utilized with these antibodies were as follows: MMP-2, MMP-3, and MMP-13—1:400; MMP-1, MMP-9, and TIMP-1—1:800. The positive control tissues for the primary antibodies were as follows: MMP-1 and MMP-13— breast cancer; MMP-2 and MMP-3— ovarian cancer; MMP-9 —rectal cancer; and TIMP-1—normal ovary. The primary antibody was replaced by normal mouse serum for a negative control. Staining was achieved using a biotinylated antimouse secondary antibody and the ABC horseradish–peroxidase (Vectastain Elite ABC Kit; Vector, Burlingame, CA). The reaction product was visualized by 3,39 diaminobenzidine tetrahydrochloride (Vector) as chromogen. Finally, the sections were dehydrated in ethanol, cleared in xylene, and mounted. In this study 51 gestational trophoblastic disease cases and 11 normal first-trimester placentas were investigated for expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and their main inhibitor TIMP-1 by immunohistochemistry. Cytoplasmic staining of syncytiotrophoblast, cytotrophoblast, extravillous trophoblast, and trophoblastic tumor cells in choriocarcinoma sections was categorized as negative, weakly positive, or strongly positive. A strongly positive result was recorded when more than 50% of the cells exhibited strong staining. Statistical Analysis The immunohistochemical data and the postevacuation clinical outcome of complete molar cases were analyzed by StatView 4.5 package. x 2 and Fisher’s exact x 2 tests were used. Statistical significance was considered at the 0.05 level. RESULTS Expression of MMP-1 was predominantly strong in cytotrophoblasts and extravillous trophoblasts in normal placenta,
250
VEGH ET AL.
TABLE 1 Immunostaining of Placentas, Partial Moles, and Complete Moles Placenta (n 5 11)
MMP-1
MMP-2
MMP-3
MMP-9
MMP-13
TIMP-1
ST CT EVT ST CT EVT ST CT EVT ST CT EVT ST CT EVT ST CT EVT
Partial mole (n 5 16)
Complete mole (n 5 25)
Negative (%)
Weak (%)
Strong (%)
Negative (%)
Weak (%)
Strong (%)
Negative (%)
Weak (%)
Strong (%)
— — 1 (9.0) — 11 (100.0) — — 9 (82.0) 5 (45.5) — 1 (9.0) 4 (36.0) — 11 (100.0) 5 (45.0) — — —
11 (100.0) 2 (18.0) 6 (55.0) 11 (100.0) — 9 (82.0) 7 (64.0) 2 (18.0) 5 (45.5) 9 (82.0) 9 (82.0) 5 (45.0) 6 (55.0) — 6 (55.0) 3 (27.0) 11 (100.0) 10 (91.0)
— 9 (82.0) 4 (36.0) — — 2 (18.0) 4 (36.0) — 1 (9.0) 2 (18.0) 1 (18.0) 2 (18.0) 5 (45.0) — — 8 (73.0) — 1 (9.0)
— — — — 16 (100.0) — — 12 (75.0) 4 (25.0) — 1 (6.2) 6 (37.6) — 14 (87.5) 7 (43.8) — 6 (37.5) 2 (12.5)
16 (100.0) 1 (6.2) 6 (37.5) 10 (62.5) — 6 (37.5) 4 (25.0) 4 (25.0) 11 (68.8) 9 (56.2) 8 (50.0) 5 (31.2) 9 (56.2) 2 (12.5) 9 (56.2) 2 (12.5) 10 (62.5) 12 (75.0)
— 15 (93.8) 10 (62.5) 6 (37.5) — 10 (62.5) 12 (75.0) — 1 (6.2) 7 (43.8) 7 (43.8) 5 (31.2) 7 (43.8) — — 14 (87.5) — 2 (12.5)
— — — — 25 (100.0) — — 20 (80.0) 6 (24.0) — — 3 (12.0) — 22 (88.0) 3 (12.0) — 6 (24.0) 6 (24.0)
21 (84.0) 9 (43.8) 10 (40.0) 22 (88.0) — 2 (8.0) 15 (60.0) 5 (20.0) 19 (76.0) 10 (40.0) 14 (56.0) 18 (72.0) 14 (56.0) 3 (12.0) 20 (80.0) 6 (24.0) 19 (76.0) 19 (76.0)
4 (16.0) 16 (56.2) 15 (60.0) 3 (12.0) — 23 (92.0) 10 (40.0) — — 15 (60.0) 11 (44.0) 4 (16.0) 11 (44.0) — 2 (8.0) 19 (76.0) — —
Note. ST, syncytiotrophoblast; CT, cytotrophoblast; EVT, extravillous trophoblast.
partial mole, and complete mole (Table 1). Nine (90.0%) of the choriocarcinoma cases showed strong intensity of staining for MMP-1 (Table 2). Choriocarcinoma exhibited significantly stronger staining for MMP-1 than syncytiotrophoblast in normal placenta (P , 0.01), partial mole (P , 0.01), and complete mole (P , 0.01). Choriocarcinoma also showed significantly stronger staining for MMP-1 than the extravillous trophoblast in placenta (P , 0.05). Strong staining for MMP-1 was observed in cytotrophoblasts in 9 (82.0%) placentas, 15 (93.8%) partial moles, and 16 (56.2%) complete moles (Fig. 1A). MMP-2 was expressed only in syncytio- and extravillous trophoblasts in normal placenta, partial mole, and complete mole. Choriocarcinoma and the extravillous trophoblast in partial mole and complete mole had significantly stronger staining for MMP-2 than the extravillous trophoblast in placenta (P , 0.05, P , 0.01, P , 0.01, respectively). Choriocarcinoma also exhibited significantly stronger staining for MMP-2 than syncytiotrophoblasts in placenta, partial mole, and complete mole (P , 0.01, P 5 0.05, P , 0.01, respectively), (Figs. 1B and 1C). TABLE 2 Immunostaining of Choriocarcinoma Cases (n 5 10) Antibody
Negative (%)
Weak (1) (%)
Strong (1) (%)
MMP-1 MMP-2 MMP-3 MMP-9 MMP-13 TIMP-1
— — 4 (40.0) — — 2 (20.0)
1 (10.0) 2 (20.0) 6 (60.0) 6 (60.0) 8 (80.0) 8 (80.0)
9 (90.0) 8 (80.0) — 4 (40.0) 2 (20.0) —
Strong MMP-3 immunoreactivity was found almost exclusively in syncytiotrophoblasts of placentas (36.0%), partial moles (75.0%), and complete moles (40.0%). Cytotrophoblast, extravillous trophoblast, and choriocarcinoma cells showed mostly weakly positive or negative staining for MMP-3. MMP-9 expression was observed in all three trophoblastic cell types in placenta, partial mole, and complete mole. Syncytiotrophoblast in complete mole had significantly greater expression of MMP-9 than in placenta (P , 0.05). There were no other significant differences in expression of MMP-9 amongst placenta and gestational trophoblastic disease tissues. MMP-13 expression was found to be similar in all four tissues and all trophoblastic cell types. Cytotrophoblast was mainly negative for MMP-13 staining, while syncytiotrophoblasts, extravillous trophoblasts, and tumor cells were mostly weakly positive. Expression of TIMP-1 was found in all three trophoblastic cell types in placenta, partial mole, and complete mole, with predominance of the syncytiotrophoblast. Cytotrophoblasts and extravillous trophoblasts stained weakly in almost all cases. While 8 (73.0%) placentas, 14 (87.5%) partial moles, and 19 (76.0%) complete moles showed strong immunoreactivity for TIMP-1 in syncytiotrophoblasts, no strong staining was found in choriocarcinomas (P , 0.01, P , 0.01, P , 0.01, respectively), (Figs. 1D and 1E). MMPs and TIMP-1 expression in complete moles was compared to the risk of persistent postmolar gestational trophoblastic tumor (Table 3). Strong staining was compared with both weak and negative staining. Expression of MMPs and their inhibitor, TIMP-1, in complete moles was not associated with the development of postmolar gestational trophoblastic tumor.
MATRIX METALLOPROTEINASES AND TROPHOBLASTIC DISEASES
251
FIG. 1. Immunohistochemical analysis of expression of MMP-1, MMP-2, and TIMP-1 in gestational trophoblastic diseases. (A) Strongly positive MMP-1 immunoreactivity in choriocarcinoma cells (magnification: 1003). (B) Strongly positive MMP-2 immunoreactivity in choriocarcinoma cells (magnification: 1003). (C) Strongly positive MMP-2 immunoreactivity in extravillous trophoblasts of complete mole (magnification: 1003). (D) Strongly positive TIMP-1 immunoreactivity in syncytiotrophoblasts in partial mole (magnification: 1003). (E) No immunoreactivity for TIMP-1 in choriocarcinoma cells (magnification: 2003).
DISCUSSION Because matrix metalloproteinases degrade a wide range of components of the extracellular matrix, MMPs have been suggested to play a vital role in tumor invasion and metastasis [17–19]. The expression and activity of MMPs have been investigated in several human malignancies. MMP-1 and MMP-2 have been reported to be overexpressed in a majority of intestinal cancers [11, 20, 21], and MMP-2 has been implicated in the tumorigenicity of prostate, cervical, and endometrial cancers [22–27]. Davidson et al. and Tamakoshi et al. showed strong expression of MMP-2 and MMP-9 in cervical cancer which correlated with tumor invasion and progression [24, 25, 27]. During normal pregnancy, extravillous trophoblasts locally invade maternal tissues and this process of invasion involves enzymatic degradation of the extracellular matrix. MMPs along with other proteases have been reported to be involved in trophoblastic invasion of maternal tissues [4, 6]. The current study was undertaken to investigate the expression of MMPs in gestational trophoblastic diseases including
partial mole, complete mole, and choriocarcinoma. Gestational trophoblastic diseases are well recognized to have varying propensities for local invasion and metastasis [1–3]. Importantly choriocarcinoma exhibited significantly stronger expression for both MMP-1 and MMP-2 than syncytiotrophoblast in normal placenta, partial mole and complete mole, and extravillous trophoblast in placenta. Furthermore the extravillous trophoblast in partial and complete mole had significantly stronger staining for MMP-2 than the extravillous trophoblast in normal placenta. The increased expression of MMP-2 in partial and complete mole compared to normal placenta may contribute to their risk for local myometrial invasion. While choriocarcinoma had significantly increased expression of MMP-1 and MMP-2 compared to placenta, partial mole, and complete mole, choriocarcinoma had significantly less expression of the tissue inhibitor of metalloproteinase-1 than placenta, partial mole, and complete mole. The increased expression of MMP-1 and MMP-2 and decreased expression of TIMP-1 in choriocarcinoma may contribute to the invasiveness of choriocarcinoma cells.
252
VEGH ET AL.
TABLE 3 Risk of Persistent Postmolar Gestational Disease after Complete Moles MMP-1
ST Spon. remission: n 5 17 Persistent GTD: n 5 8 CT Spon. remission: n 5 17 Persistent GTD: n 5 8 EVT Spon. remission: n 5 17 Persistent GTD: n 5 8
MMP-2
Weak 1
Strong 1
P
Weak 1
Strong 1
P
Weak 1
Strong 1
P
14 7
3 1
NS
15 7
2 1
NS
12 3
5 5
NS
5 4
12 4
NS
— —
— —
—
17 8
— —
—
5 5
12 3
NS
1 1
16 7
NS
17 8
— —
—
MMP-9
ST Spon. remission: n 5 17 Persistent GTD: n 5 8 CT Spon. remission: n 5 17 Persistent GTD: n 5 8 EVT Spon. remission: n 5 17 Persistent GTD: n 5 8
MMP-3
MMP-13
TIMP-1
Weak 1
Strong 1
P
Weak 1
Strong 1
P
Weak 1
Strong 1
P
7 3
10 5
NS
11 3
6 5
NS
4 2
13 6
NS
11 3
6 5
NS
17 8
— —
—
17 8
— —
—
14 7
3 1
NS
16 7
1 1
NS
17 8
— —
—
Note. ST, syncytiotrophoblast; CT, cytotrophoblast; EVT, extravillous trophoblast; GTD, gestational trophoblastic disease.
MMPs and tissue inhibitors may play an important role in the pathogenesis of gestational trophoblastic diseases. Fortunately, gestational trophoblastic tumors are generally remarkably sensitive to chemotherapy and all patients in the current study achieved complete remission. However, infrequently, patients with drug-resistant gestational trophoblastic tumors are encountered and there is a continuing need to develop and evaluate new chemotherapeutic agents. Anti-neoplastic drugs are currently being developed that modify the activity of MMPs and their inhibitors and these agents may exhibit activity against gestational trophoblastic diseases [28, 29]. Further understanding of the role of MMPs in the biology of gestational trophoblastic diseases may therefore lead to new and novel therapies for patients with these diseases. ACKNOWLEDGMENT This research was supported by the United States–Hungarian Science and Technology Joint Fund (No. 552) and the Soros Foundation.
REFERENCES
Gestational Trophoblastic Tumors, in Knapp RC, Berkowitz RS (eds): Gynecologic Oncology. 2nd ed. New York, NY, McGraw–Hill, 1993, pp 328 –338 4. Crescimanno C, Foidart JM, Noel A, Polette M, Maquoi E, Birembaut P, Baramova E, Kaufmann P, Castellucci M: Cloning of choriocarcinoma cells shows that invasion correlates with expression and activation of gelatinase A. Exp Cell Res 227:240 –251, 1996 5. Lala PK, Graham CH: Mechanism of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. Cancer Metast Rev 9:369 –379, 1994 6. Huppertz B, Kerschanska S, Demir AY, Frank HG, Kaufmann P: Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta. Cell Tissue Res 291:133–148, 1998 7. Bishof P, Haenggli L, Campana A: Gelatinase and oncofetal fibronectin expression is dependent on integrin expression on human cytotrophoblasts. Hum Reprod 10:734 –742, 1995 8. Castellucci M, Theelen T, Pompili E, Fumagalli L, De Renzis G, Muhlhauser J: Immunohistochemical localization of serine-protease inhibitors in the human placenta. Cell Tissue Res 278:283–289, 1994 9. Leber T, Boyd R, Balkwill F: Tumour Cell–Stromal Cell Interactions: Proteases and Protease Inhibitors, in Sharp F, Blackett T, Berek J, Bast R (eds): Ovarian Cancer 5. Oxford, Isis Medical Media Ltd, 1998, pp 121–129
1. Lewis JL Jr: Diagnosis and management of gestational trophoblastic disease. Cancer 71:1639 –1647, 1993
10. Sato H, Takino T, Okada Y: A matrix metalloproteinase expressed on the surface of invasive tumor cells. Nature 370:61– 65, 1994
2. Berkowitz RS, Goldstein DP: Chorionic tumors. N Engl J Med 335:1740 – 1748, 1996
11. Murray GI, Duncan ME, Arbucle E, Melvin WT, Fothergill JE: Matrix metalloproteinases and their inhibitors in gastric cancer. Gut 43:791–797, 1998
3. Berkowitz RS, Goldstein DP: The Management of Molar Pregnancy and
MATRIX METALLOPROTEINASES AND TROPHOBLASTIC DISEASES 12. Denhardt DT, Feng B, Edwards DR: Tissue inhibitor of metalloproteinases (TIMP, akaEPA): structure, control of expression and biological functions. Pharmacol Ther 59:329 –341, 1993 13. Matrisian LM. The matrix-degrading metalloproteinases. Bioessays 14: 455– 462, 1992 14. Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. JNCI 89:1260 –1270, 1997 15. Berkowitz RS, Goldstein DP: Presentation and Management of Molar Pregnancy, in Hancock BW, Newlands ES, Berkowitz RS (eds): Gestational Trophoblastic Disease. London, Chapman & Hall, 1997, pp 127–142 16. Goldstein DP, Zanten-Przybysz IV, Bernstein MR, Berkowitz RS: Revised FIGO staging system for gestational trophoblastic tumors. Recommendations regarding therapy. J Reprod Med 43:37– 43, 1998 17. Fulop V, Mok SC, Genest DR, Szigetvari I, Cseh I, Berkowitz RS: c-myc, c-erbB-2, c-fms and bcl-2 oncoproteins. Expression in normal placenta, partial and complete mole, and choriocarcinoma. J Reprod Med 43:101– 110, 1998 18. Cottam DW, Rees RC: Regulation of matrix metalloproteinases: their role in tumor invasion and metastasis. Intl J Oncol 2:861– 872, 1993 (review) 19. Murphy G, Reynolds JJ, Hembry RM: Metalloproteinases and cancer invasion and metastasis. Int J Cancer 44:757–760, 1989 20. Murray GI, Duncan ME, O’Neil P: Matrix metalloproteinase-1 is associated with poor prognosis in colorectal cancer. Nat Med 2:461– 462, 1996 21. Murray GI, Duncan ME, O’Neil P: Matrix metalloproteinase-1 is associated with poor prognosis in oesophageal cancer. J Pathol 185:256 –261, 1998 22. Montironi R, Fabris G, Lucarini G, Biagini G: Location of 72-kD metalloproteinase (type IV collagenase) in untreated prostatic adenocarcinoma. Path Res Pract 191:1140 –1146, 1995
253
23. Montironi R, Lucarini G, Castaldini C, Galluzzi CM, Biagini G, Fabris G: Immunohistochemical evaluation of type IV collagenase (72-kD metalloproteinase) in prostatic intraepithelial neoplasia. Anticancer Res 16:2057– 2062, 1996 24. Tamakoshi K, Kikkawa F, Nawa A, Ishikawa H, Mizuno K, Tamakoshi A, Yamagata S, Suganuma N, Tomoda Y: Characterization of extracellular matrix degrading proteinase and its inhibitor in gynecologic cancer tissues with clinically different metastatic form. Cancer 76:2565–2571, 1995 25. Davidson B, Goldberg I, Liokumovich P, Kopolovic J, Gotlieb WH, Lerner-Geva L, Reder I, Ben-Baruch G, Reich R: Expression of metalloproteinases and their inhibitors in adenocarcinoma of the uterine cervix. Int J Gynecol Pathol 17:295–301, 1998 26. Talvensaari-Mattila A, Apaja-Sarkkinen M, Hoyhtya M, Westerlund A, Puistola U, Turpeenniemi-Hujanen T: Matrix metalloproteinase 2 immunoreactive protein appears early in cervical epithelial dedifferentiation. Gynecol Oncol 72:306 –311, 1999 27. Davidson B, Goldberg I, Kopolovic J, Lerner-Geva L, Gotlieb WH, Weis B, Ben-Baruch G, Reich R: Expression of matrix metalloproteinase-9 in squamous cell carcinoma of the uterine cervix— clinicopathologic study using immunohistochemistry and mRNA in situ hybridization. Gynecol Oncol 72:380 –386, 1999 28. Rasmussen HS, McCann PP: Matrix metalloproteinase inhibition as a novel anticancer strategy: a review with specifial focus on batimastat and marimastat. Pharmacol Ther 75:69 –75, 1997 29. Wojtowitz-Praga S, Torri J, Johnson M, Steen V, Marchall J, Ness E, Bickson R, Sale M, Rasmussen HS, Chiodo TA, Hawkins MJ: Phase I trial Marimastat, a novel matrix metalloproteinase inhibitor, administered orally to patients with advanced lung cancer. J Clin Oncol 16:2150 –2156, 1998