Inhibition of matrix metalloproteinases by over-expression of tissue inhibitor of metalloproteinase-2 inhibits the growth of experimental hemangiomas.

Inhibitors of proteases prevent tumor-associated matrix degradation, affecting tumor growth, angiogenesis and metastasis. Our study was designed to investigate the effect of inhibition of matrix metalloproteinases (MMPs) on the growth of experimental hemangiomas, using the model of murine endothelioma eEnd.1 cells. In nude mice, these cells generate hemangiomas, consisting mostly of host-recruited endothelial cells, whose growth requires the activity of MMPs. In vitro, eEnd.1 cells produce factors that recruit endothelial cells and stimulate them to release MMPs. Over-expression of TIMP-2, following retrovirus-mediated gene transfer, decreased tumor growth in vivo. The infected clone CR1, which produces high levels of TIMP-2 (as assessed by Northern blot, ELISA and reverse zymography), formed slow-growing tumors that did not grow beyond 0.4 g, while clone 1H, which produces little TIMP-2, grew not dissimilarly to mock-infected cells and parental e.End.1 cells. Histologically, control tumors presented the features of cavernous hemangiomas, while CR1 tumors had a more solid pattern, showing foci of apoptotic cells. In vitro, TIMP-2 over-expression had no autocrine anti-proliferative effect on endothelioma cells but reduced their ability to recruit endothelial cells. CR1 cells lacked the capacity of mock-infected or parental eEnd.1 cells to stimulate endothelial cell motility and invasiveness. Antibodies against TIMP-2 restored the ability of CR1 to induce endothelial cell invasion. We conclude that, in this model, genetic increase of TIMP-2 inhibits tumor growth, apparently by affecting the recruitment and organization of host endothelial cells by the transformed cells.

BAY 12-9566, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity.

Matrix metalloproteinases (MMPs) have been implicated in tumor cell invasion, metastasis, and angiogenesis. BAY 12-9566, a novel, non-peptidic biphenyl MMP inhibitor, has shown preclinical activity on a broad range of tumor models and is currently in clinical development. The purpose of this study was to investigate the antiangiogenic activity of BAY 12-9566. In vitro, BAY 12-9566 prevented matrix invasion by endothelial cells in a concentration-dependent manner (IC50 = 8.4x10(-7) M), without affecting cell proliferation. In vivo, oral daily administration of BAY 12-9566 (50-200 mg/kg) inhibited angiogenesis induced by basic fibroblast growth factor in the Matrigel plug assay, reducing the hemoglobin content of the pellets. Histological analysis showed a reduction in the amount of functional vessels within the Matrigel. We conclude that the MMP inhibitor BAY 12-9566 inhibits angiogenesis, a property that further supports its clinical development as an antimetastatic agent.

Synergistic cooperation between c-Myc and Bcl-2 in lymph node progression of T1 human breast carcinomas.

The overexpression of Bcl-2, an anti-apoptotic oncogene, identifies human T1 breast cancer patients who have an increased risk of lymph-node metastasis. We examined in these patients (n = 142) whether the c-Myc oncogene influences metastatic progression in conjunction or not with Bcl-2 expression and the loss of apoptosis in tumors. The association between Bcl-2 and lymph-node metastasis was only significant when c-Myc was concomitantly expressed (chi2 test, p = 0.008). Moreover, very large associations (pOR = 6.4) between c-Myc and lymph-node metastasis were observed among Bcl-2 positive tumors and tumors with loss of apoptosis (pOR = 8.4). In contrast, the metastatic advantage linked to Bcl-2 was decreased (pOR = 2) when c-Myc was not coexpressed. It is concluded that the synergism between Bcl-2 and c-Myc oncogenes may promote metastasis in breast tumors, linked to loss of apoptosis.

The microtubule-affecting drug paclitaxel has antiangiogenic activity.

Endothelial cell migration is a critical event during angiogenesis, and inhibitors of cell motility can affect the angiogenic process. Paclitaxel (Taxol(R)), a microtubule-stabilizing antineoplastic cytotoxic drug, inhibits motility and invasiveness of several cell types. The aim of this study was to investigate the effect of paclitaxel on endothelial cell functions and on angiogenesis. In vivo, paclitaxel (20-28 mg/kg i.v.) significantly inhibited the angiogenic response induced by tumor cell supernatant embedded in a pellet of reconstituted basement membrane (Matrigel) injected s.c. into C57BL/6N mice. In vitro, paclitaxel inhibited endothelial cell proliferation, motility, invasiveness, and cord formation on Matrigel in a dose-dependent manner. The antiangiogenic activity of paclitaxel was not linked to its cytotoxicity, since inhibition of endothelial cell chemotaxis and invasiveness occurred at drug concentrations which did not affect endothelial cell proliferation. Another cytotoxic drug, cisplatin, that inhibited endothelial cell proliferation in vitro, did not affect angiogenesis in vivo. These data indicate that paclitaxel has a strong antiangiogenic activity, a property that might contribute to its antineoplastic activity in vivo.

Inhibition of angiogenesis and murine hemangioma growth by batimastat, a synthetic inhibitor of matrix metalloproteinases.

BACKGROUND: The importance of matrix metalloproteinases in angiogenesis, tumor growth, and metastasis is well known. However, little is known about the role of matrix metalloproteinases in the formation of hemangiomas and about the possible therapeutic use of matrix metalloproteinase inhibitors in aggressive vascular tumors. PURPOSE: To study the role of matrix metalloproteinase in vascular tumors, we tested the antineoplastic activity of a synthetic inhibitor of matrix metalloproteinases, batimastat, on an experimental model of hemangioma, formed by murine endothelioma cells transformed by polyoma middle-T oncogene (eEnd.1). METHODS: The effect of batimastat was studied in vivo on the formation of hemorrhaging, cavernous hemangiomas by eEnd.1 endothelioma cells injected subcutaneously in nude mice and on the angiogenic response induced by an endothelioma cell supernatant embedded in a pellet of reconstituted basement membrane (Matrigel). The effect of batimastat was investigated in vitro on endothelial cell proliferation, motility, and invasion of a layer of Matrigel. RESULTS: Daily treatment with batimastat (30, 3, and 0.3 mg/kg at the site of eEnd.1 cell injection) inhibited tumor growth, with increased doubling time. The carboxamide derivative of batimastat, BB-374, a poor inhibitor of matrix metalloproteinase activity, was less active in reducing hemangioma growth. Histologic analysis of treated tumors indicated a reduction in the size of blood-filled spaces and in hemorrhage. Batimastat also inhibited the angiogenic response induced by cultured eEnd.1 endothelioma cell supernatant embedded in a pellet of Matrigel. Batimastat significantly inhibited endothelial cell invasion in vitro through a layer of Matrigel, but it showed no direct cytotoxic activity. CONCLUSIONS: Batimastat reduces in vivo growth of experimental hemangiomas, most probably by blocking endothelial cell recruitment by the transformed cells or by interfering with cell organization in vascular structures. IMPLICATIONS: These results confirm the importance of matrix metalloproteinase in endothelial cell recruitment that occurs in angiogenesis and in the formation of vascular tumors and suggest a therapeutic potential for synthetic matrix metalloproteinase inhibitors.

The pathology of low-grade adenosquamous carcinoma of the breast. An immunohistochemical study.

Low-grade adenosquamous carcinoma of the breast is a variant of metaplastic mammary carcinoma characterized by a locally invasive growth pattern and a low risk for metastases. In this study none of the carcinomas exhibited greater than 5 percent nuclear immunoreactivity for estrogen or progesterone receptors, and as a result they were classified as negative for these receptors. Reactivity for cathepsin D was found in 39 percent of the tumors, largely limited to areas of epidermoid differentiation. Membrane immunoreactivity for HER-2/neu oncogenes was present in glandular components of 46 percent of the carcinomas. Immunoreactivity for p53 (greater than 10 percent of nuclei) was present in 13 percent of the tumors, also in glandular elements. Six different patterns of coexpression of p53, HER-2/neu and cathepsin D were found, the most frequent being the following: HER-2/neu(+), p53(-), cathepsin D(-) (9 cases, 39%); cathepsin D(+), p53(-), HER-2/neu(-) (5 cases, 22%); and the three markers negative (5 cases, 22 percent). Coexpression of the two oncogenes was found in only one tumor which was also positive for cathepsin D. These results indicate that the expression of various immunohistochemical prognostic markers may be heterogeneous and that there may not be a specific pattern of marker coexpression within a carefully defined histologic subtype of mammary carcinoma. Furthermore, characteristics reported to be associated with an unfavorable prognosis (negative hormone receptors, presence of cathepsin D, and expression of oncogenes such as HER-2/neu) may be found in a substantial proportion of tumors that comprise this clinically and histologically low-grade variant of mammary carcinoma. This disassociation between expected prognosis based on expression of current prognostic markers and observed prognosis occurs in other forms of mammary carcinoma. Medullary carcinoma, when diagnosed on the basis of rigorously defined criteria, has an excellent prognosis despite the fact that these tumors are characterized by absence of estrogen and progesterone receptors and a high proliferative rate. The histological classification of mammary carcinomas is itself an important prognostic variable that may take precedence over selected biochemical markers.

Low-grade adenosquamous carcinoma of the breast. A clinocopathologic study of 32 cases with ultrastructural analysis.

Low-grade adenosquamous carcinoma of the breast is an uncommon neoplasm of uncertain pathogenesis, clinical behavior, and malignant potential. This report describes the clinical and pathologic features of 32 cases of low-grade adenosquamous carcinoma. All patients presented with palpable tumors ranging from 0.6 to 8.6 cm (mean, 2.8 cm). Origin from an intraductal papillary tumor was found in 12 cases, including three with adenomyoepitheliomatous features. Electron microscopy disclosed glandular and squamous differentiation; the squamous cells often lined ducts that were structurally analogous to the acrosyringium of the eccrine sweat gland. Treatment consisted of mastectomy (13 patients) or excisional biopsy (19 patients). A single lymph node in one patient with a 3.5-cm primary carcinoma harbored metastatic adenocarcinoma. Axillary dissection revealed no metastases in 11 other patients. Another patient with an 8.0-cm breast tumor had metastatic adenosquamous carcinoma in the lung at initial diagnosis. After follow-up of 12 to 124 months, 20 of 25 patients had no recurrence. Five women treated by excisional biopsy had local recurrences in the breast. In one patient, the local recurrence was ultimately fatal due to invasion of the hemithorax. Estrogen and progesterone receptor studies were negative in 13 of 15 cases studied by biochemical analysis. The two tumors that were hormone-receptor positive were histologically associated with a papilloma and an adenomyoepithelioma, respectively. In the latter case, immunohistochemical studies showed the carcinoma to be hormone-receptor negative. Hormone receptor activity was limited to the adenomyoepitheliomatous component. This study confirms the largely indolent, but locally aggressive, clinical course of low-grade adenosquamous carcinoma of the breast. Although complete limited excision of small lesions may be curative, tumors greater than 3.0 cm may require more aggressive therapy.

P53 and rb alterations in primary breast-carcinoma - correlation with hormone receptor expression and lymph-node metastases.

Expression of Rb and mutant p53 nuclear phosphoproteins was analyzed immunohistochemically in 69 breast cancer patients. Results were correlated with hormone receptor ( ER and PR ) and lymph node ( LN ) status. There was a significant association between the immunohistochemical evidence of p53 and/or Rb alterations and loss of hormone receptor expression. Mutations in p53 and/or low level Rb expression were not associated with the presence of axillary lymph node metastases. However, in patients with hormone receptor positive tumors, there was statistically significant correlation between altered expression of p53 and/or Rb and the presence of LN metastases. These results indicate that: (i) loss of steroid hormone receptor expression (and thus loss of hormonal growth control) is accompanied by somatic inactivation of the p53 or Rb tumor suppressor genes; and (ii) in tumors that remain under hormonal growth regulation inactivation of p53 and/or Rb may play a role in tumor progression.