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Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is activated by integrin clustering. There are limited data regarding the functional role of FAK in ovarian cancer migration and invasion. In the current study, FAK expression was evaluated in ovarian cell lines (nontransformed and cancer), 12 benign ovarian samples, and in 79 invasive epithelial ovarian cancers. All three ovarian cancer cell lines overexpressed FAK compared to the nontransformed cells. The dominant-negative construct called FAK-related nonkinase (FRNK) was introduced into two ovarian cancer cell lines (SKOV3 and 222). FRNK promoted FAK dephosphorylation without changing total FAK levels in these cell lines. Furthermore, FRNK decreased the in vitro invasive ability of ovarian cancer cells by 56 to 85% and decreased migration by 52 to 68%. FRNK-transfected cells also displayed poor cell spreading. Immunohistochemical analysis revealed that the surface epithelium from all benign ovarian samples had weak FAK expression. In contrast, 68% of invasive ovarian cancers overexpressed FAK. FAK overexpression was significantly associated with high tumor stage, high tumor grade, positive lymph nodes and presence of distant metastasis (all P values 1 cm were independent predictors of poor survival. These data indicate that FAK is overexpressed in most invasive ovarian cancers and plays a functionally significant role in ovarian cancer migration and invasion. Thus, FAK may be an important therapeutic target in ovarian carcinoma.

The maspin gene functions as a tumor suppressor in human breasts, and its expression is frequently lost during breast cancer progression. In vitro models of human breast cancer indicate that the loss of maspin expression is closely linked to aberrant methylation of the maspin promoter. We conducted a study on 30 archival ductal carcinoma in situ (DCIS) specimens to determine if aberrant methylation of the maspin promoter occurred in vivo, and whether it occurred early in breast cancer evolution. Healthy tissue obtained from reduction mammoplasty was used as normal control. Results from immunohistochemical analysis indicate that maspin expression is lost in a substantial fraction of DCIS specimens (57%). Bisulfite sequencing of DNA isolated from laser capture-microdissected normal and neoplastic ducts showed that loss of maspin expression was often, but not always, linked to aberrant methylation of the maspin promoter, suggesting that other mechanisms, in addition to aberrant methylation, participate and/or cooperate to silence maspin gene expression. Taken together, these results indicate that aberrant methylation of the maspin promoter is an early event in human breast cancer.

Objective We previously demonstrated that aggressive ovarian cancer cells are able to display in vitro vasculogenic mimicry, which is reflected by their ability to form vasculogenic-like networks in 3-dimensional cultures and to express vascular cell-associated markers. The goal of this study was to examine the functional role of specific matrix metalloproteinases in the formation of vasculogenic-like networks and extracellular matrix remodeling in vitro. We also investigated the clinical relevance of matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase in human ovarian cancers with evidence of tumor cell-lined vasculature. Study design Ovarian cancer cells (A2780-PAR, SKOV3, and EG) were seeded onto separate 3-dimensional cultures that contained either Matrigel or type I collagen, in the absence of endothelial cells or fibroblasts. These cultures were treated with either chemically modified tetracycline-3 (general matrix metalloproteinase inhibitor), recombinant tissue inhibitor of metalloproteinase-1 or -2, or function-blocking antibodies to matrix metalloproteinase-2 or -9 or membrane type 1-matrix metalloproteinase. In addition, 78 invasive epithelial ovarian cancers were evaluated for expression of matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase and correlated with various clinical parameters. Results The aggressive ovarian cancer cells (SKOV3 and EG) were able to form in vitro vasculogenic-like networks and contract 3-dimensional collagen I gels, whereas the poorly aggressive A2780-PAR cell line did not. Chemically modified tetracycline-3 completely blocked the network formation. Blocking antibodies to matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase inhibited the formation of the vasculogenic-like networks and collagen gel contraction, but the antibody to matrix metalloproteinase-9 had no effect on network formation and minimal effect on gel contraction. Treatment of 3-dimensional cultures with tissue inhibitor of metalloproteinase-2 retarded the network formation and only small, partially developed structures were noted that did not form network connections. Tissue inhibitor of metalloproteinase-1 had no appreciable effect on the extent or efficiency of network formation. Human invasive ovarian cancers with evidence of tumor cell-lined vasculature were significantly more likely to have strong epithelial and stromal matrix metalloproteinase-2 and -9 and membrane type 1-matrix metalloproteinase expression (all probability values were Conclusion Matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase appear to play a key role in the development of vasculogenic-like networks and matrix remodeling by aggressive ovarian cancer cells. Human ovarian cancers with matrix metalloproteinase overexpression are more likely to have tumor cell-lined vasculature. These results may offer new insights for consideration in ovarian cancer treatment strategies.

Cutaneous melanoma has been increasing at an alarming rate over the past two decades, however, there are no acceptable histopathological markers that classify various stages of melanoma progression. Recently, the molecular analysis of cancer has contributed significantly to our understanding of the cellular and molecular underpinnings of tumor progression. The data summarized in this review describe the molecular signature of aggressive cutaneous melanoma cells as that of multiple phenotypes which may be similar to a pluripotent, embryonic-like phenotype. An example of the plasticity of this phenotype is demonstrated by the ability of aggressive melanoma cells to engage in vasculogenic mimicry and neovascularization. A review of the current data demonstrating important cellular and molecular determinants of human melanoma vasculogenic mimicry is presented. These findings should stimulate additional studies to address the biological relevance of the multiple molecular phenotypes expressed by aggressive melanoma cells which may lead to the development of new diagnostic markers and therapeutic targets for clinical intervention.

Purpose: This study was designed to (1) describe the vascular patterns of ciliary body and choroidal nevi by light microscopy, (2) compare the vascular ultrastructure of nevi with vessels of the normal uvea and uveal melanomas, and (3) compare the behavior of ciliochoroidal melanomas with and without a nevus-like vascular architecture. Methods: After delineating the vascular patterns of 23 choroidal and ciliary body nevi by light microscopy, the authors identified 49 melanomas that had the same vascular patterns as nevi from a previously published series of 234 uveal melanomas. The survival of these 49 patients who had melanomas with a nevus-like vascular architecture was compared with the 185 patients who had melanomas that lacked this vascular profile. Results: By light microscopy, the only vascular patterns identified in nevi are “normal” vessels, zones of avascularity (“silent” pattern), straight, and parallel vessels; closed vascular loops and networks were not detected in nevi. By transmission electron microscopy, the vascular basement membrane of malignant melanomas was multilaminar, fragmented, and significantly thicker than in normal eyes or nevi. None of the patients with nevi died of metastatic disease. Fourteen percent of patients whose melanomas had the same vascular profile as nevi died of metastatic disease, whereas 32% of patients whose melanomas had vascular patterns other than those seen in nevi died of metastatic melanoma ( P = 0.012). Conclusions: The microcirculation architecture marks tumor progression in uveal melanocytic lesions by light and electron microscopy. In the spectrum of these lesions, nevi are benign, melanomas that have the same vascular profile as nevi have an intermediate biologic behavior, and melanomas with vascular networks are strongly associated with death due to metastatic disease.

Background: It is possible to identify at least nine vascular patterns in melanomas of the ciliary body and choroid from histologic sections. An association between the presence of at least one closed vascular loop and death from metastases was shown in a matched-pair, case-control study of 40 patients whose eyes were removed for ciliary body or choroidal melanomas. Methods: Two independent observers who were masked to the follow-up of patients examined histologic preparations of 234 eyes removed for ciliary body or choroidal melanomas for the presence of each of the tumor vascular patterns. Statistical analyses included tests for interobserver reliability, Kaplan-Meier survival curves, and the fitting of Cox regression models. Results: The detection of each of the nine vascular patterns is highly reproducible. The Cox model indicates that the presence of vascular networks, defined as at least three back-to-back closed vascular loops, is the feature most strongly associated with death from metastatic melanoma. Other significant factors in the Cox model include (in descending order of importance) largest tumor dimension, mitoses, the parallel with cross-linking vascular pattern, age, the presence of tumor-infiltrating lymphocytes, and male gender. Conclusions: The presence of vascular networks provides the most significant association with death from metastatic melanoma of all variables tested. The presence of this pattern should be recorded on pathology reports. If it becomes possible to detect this vascular pattern clinically using a noninvasive imaging technique, then ophthalmologists may be able to determine the likely biologic behavior of a melanoma before resorting to the removal of tissue.

Nine morphologic patterns of tumor vessels were identified in eyes removed for ciliary body or choroidal melanoma by the examination of tissue sections stained with fluorescein-conjugated Ulex europaeus I using laser scanning confocal microscopy. This technique also highlights intravascular tumor invasion. Each of these nine morphologic patterns of tumor vessels also may be demonstrated by a modification of the periodic acid-Schiff reaction, viewed with a green narrow band pass filter, but this modified histochemical technique does not accurately identify intravascular tumor invasion. Most tumors have a heterogeneous distribution of vascular patterns. Melanomas in two groups of 20 tumors each were matched by tumor size and location (one group of tumors from patients who survived at least 15 years free of metastatic melanoma after enucleation and one group of tumors from patients who died of metastatic melanoma). A matched case-control analysis indicates that the presence of at least one closed vascular loop in a uveal melanoma is the most significant vascular pattern associated with death from metastatic melanoma after enucleation. Closed loops are associated with other histologic features that are predictive of an unfavorable outcome after enucleation: epithelioid cells and mitotic figures. In this preliminary study the formation of closed vascular loops is a marker of tumor progression in ciliary body and choroidal melanomas.