BACKGROUND The actions of the extracellular‐matrix degrading enzymes, matrix metalloproteinases (MMPs), are implicated in tumorigenesis. The cellular localization of MMP‐2, MMP‐9, membrane type 1 (MT1)‐MMP, tissue inhibitors of metalloproteinases (TIMPs) 1‐3, and the presence of active gelatinases were investigated in endometrial carcinoma. METHODS Endometrial carcinomas were grouped according to histologic grade (Grades 1‐3), depth of myometrial invasion (0, < 50%, > 50%) and the presence of vascular/lymphatic invasion. Twenty‐nine endometrial carcinoma biopsies were investigated immunohistochemically to determine the tissue localization of MMP‐2 (gelatinase A), MMP‐9 (gelatinase B), MT1‐MMP, and TIMPs 1‐3. In situ hybridization was performed to localize MMP‐2 and MMP‐9 mRNA. The presence of active gelatinases was assessed using in situ zymography. RESULTS Epithelial tumor cells were the main site of MMP‐2, MMP‐9, and MT1‐MMP protein. Variable stromal cell localization was also observed, particularly in areas adjacent to tumor nests. Semiquantitative analysis revealed increases in MMP‐9 and MMP‐2 but not MT1‐MMP staining scores in tumor epithelial cells in the transition from histologic Grade 1 to Grades 2 and 3. Matrix metalloproteinase‐9 and MT1‐MMP staining scores in tumor cells were significantly associated with the presence of myometrial invasion and vascular/lymphatic invasion, while MMP‐2 did not correlate with these factors. In addition, MT1‐MMP was co‐localized with MMP‐2, supporting its role in the activation of proMMP‐2. Tumor cells from all histologic grades stained intensely for TIMP‐2 and TIMP‐3 proteins, while variable stromal staining was observed. In Grade 1 carcinomas TIMP‐1 was predominantly immunolocalized to the stromal compartment with variable tumor cell localization being observed in Grades 2 and 3 carcinomas. Matrix metalloproteinase‐9 and MMP‐2 mRNAs were predominantly observed in tumor epithelial cells as well as in the stroma to varying degrees. In situ zymography revealed active forms of gelatinases at the cellular surface and in association with tumor epithelial cells within endometrial carcinoma tissues. CONCLUSIONS These data suggest that increasing expression of MMPs and endometrial carcinoma progression are closely related. Active gelatinases are present in endometrial carcinoma, resulting in alterations to the microenvironment that promote tumor invasion and metastasis. Cancer 2002;94:1466–75. © 2002 American Cancer Society. DOI 10.1002/cncr.10355

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The mammalian HtrA family consists of serine proteases with distinct domains homologous to the bacterial high temperature requirement factor (HtrA). Three human HtrA members have been reported: HtrA1 (PRSS11 or L56), HtrA2 (OMI) and HtrA3 (PRSP). The function of HtrA1 is not well characterised, but it has been shown to be downregulated in malignant tissues (1–3) indicating that the downregulation of HtrA1 is associated with cancer progression. HtrA2 regulates apoptosis by interacting with X-linked inhibitors of apoptosis (XIAP) thus preventing the caspase-inhibitory function of XIAP (4). The function of newly identified HtrA3 is not known, however it shares a high degree of sequence and domain homologies with HtrA1 and may therefore share a functional similarity with HtrA1 (5). Endometrial cancer (EC) is a prevalent gynaecological cancer, commonly affecting women after menopause. In this study we examined the expression of HtrA1, 2 and 3 in EC. Reverse transcriptase-PCR (semi-quantitative) analysis showed decreased mRNA expression of both HtrA1 and HtrA3, but no significant change for HtrA2, in EC tissue samples compared to normal endometrium. We then determined the protein level of expression and the cellular localisation of all three HtrA members in EC progression using immunohistochemistry. HtrA1 and HtrA3 showed a similar pattern of expression and both decreased dramatically with the progression of cancer from grade 1 through to 3. Surprisingly, HtrA2 protein expression was also decreased with cancer progression, but the decline was not as dramatic as that for HtrA1 and HtrA3. Interestingly, considerably less staining was observed for all three HtrA proteins in grade 3 cancer tissues. These data suggest that decreased expression of HtrA proteins, particularly HtrA1 and HtrA3, is associated with the progression of endometrial cancer. (1) Nie, G., Hampton, A., Li, Y., Findlay, J., Salamonsen, L.A. (2003) Identification and cloning of two isoforms of human high-temperature requirement factor A3 (HtrA3), characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2. Biochem. J. 371, 39–48. (2) van Loo, G., van Gurp, M., Depuydt, B., Srinivasula, S.M., Rodriguez, I., Alnemri, E.S., Gevaert, K., Vandekerckhove, J., Declercq, W., Vandenabeele, P. (2002) The serine protease OMI/HtrA2 is released from mitochondria during apoptosis. OMI interacts with caspase-inhibitor XIAP and induces enhanced caspase activity. Cell Death Diff. 9, 20–26. (3) Chien, J., Staub, J., Hu, S., Erickson-Johnson, M.R., Couch, F.J., Smith, D.I., Crowl, R.M., Kaufmann, S., Shridhar, V. (2004) A candidate tumour supressor HtrA1 is down-regulated in ovarian cancer. Oncogene 23, 1636–1644. (4) Shridhar, V., Sen, A., Chien, J., Staub, J., Avula, R., Kovats, S., Lee, J., Lillie, J., Smith, D.I. (2002) Identification of underexpressed genes in early- and late-stage primary ovarian tumours by suppression subtraction hybridization. Cancer Res. 62, 262–270. (5) Baldi, A., De Luca, A., Morini, M., Battista, T., Felsani, A., Baldi, F., Catricala, C., Amantea, A., Noonan, D. M., Albini, A., Ciorgio, P., Lombardi, D., Paggi, M. G. (2002) The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells. Oncogene 21, 6684–6688.