Severe feto-placental abnormalities precede the onset of hypertension and proteinuria in a mouse model of preeclampsia.

Preeclampsia is a prevalent and potentially devastating disorder of pregnancy. Characterized by a sudden spike in blood pressure and urinary protein levels, it is associated with significant obstetric complications. BPH/5 is an inbred mouse model of preeclampsia with borderline hypertension before pregnancy. BPH/5 mice develop hypertension, proteinuria, and endothelial dysfunction during late gestation (after E14.5). We hypothesized that BPH/5 mice might exhibit early feto-placental abnormalities before the onset of maternal disease. All placental cell lineages were present in BPH/5 mice. However, the fetal and placental weights were reduced, with abnormalities in all the placental zones observed starting early in gestation (E9.5-E12.5). The fractional area occupied by the junctional zone was significantly reduced at all gestational timepoints. Markedly fewer CDKN1C-stained trophoblasts were seen invading the proximal decidual zone, and this was accompanied by reductions in Cdkn1c gene expression. Trophoblast giant cell morphology and cytokeratin staining were not altered, although the mRNA levels of several giant cell-specific markers were significantly downregulated. The labyrinth layer displayed decreased branching morphogenesis of endothelial cells, with electron microscopy evidence of attenuated trophoblast layers. The maternal decidual arteries showed increased wall-to-lumen ratios with persistence of actin-positive smooth muscle cells. These changes translated into dramatically increased vascular resistance in the uterine arteries, as measured by pulse-wave Doppler. Collectively, these results support the hypothesis that defects at the maternal-fetal interface are primary causal events in preeclampsia, and further suggest the BPH/5 model is important for investigations of the underlying pathogenic mechanisms in preeclampsia.

The clinical relevance of stromal matrix metalloproteinase expression in ovarian cancer.

PURPOSE: Matrix metalloproteinases (MMP) are proteolytic enzymes implicated in cancer progression and metastasis. We sought to determine the role of epithelial (tumor cell-derived) and stromal (host-derived) expression of MMPs in predicting the clinical outcome of patients with epithelial ovarian cancer (EOC). EXPERIMENTAL DESIGN: MMP-2, MMP-9, and membrane type 1 (MT1)-MMP expression was evaluated using immunohistochemistry in 90 invasive EOCs, and samples were scored for epithelial and stromal staining. Results were correlated with clinicopathologic characteristics using univariate and multivariate analyses. RESULTS: High expression of MMP-2, MMP-9, and MT1-MMP in tumor epithelium was detected in 54%, 97%, and 100% of cases, and in stromal compartments, in 38%, 70%, and 38% of cases, respectively. High stromal expression of MMP-2, MMP-9, and MT1-MMP was significantly associated with aggressive features such as high stage, high grade ascites, and positive lymph node status. Kaplan-Meier analysis showed that high epithelial and stromal expression of MMP-2, MMP-9, and MT1-MMP were each significantly associated with shorter disease-specific survival (DSS; P < 0.01). On tree-structured survival analysis, patients with strong epithelial MT1-MMP expression had the shortest DSS, whereas patients with moderate epithelial MT1-MMP and low stromal MMP-9 expression had the longest DSS (P < 0.01). On multivariate analysis, high stromal expression of MMP-9 (P = 0.01) and MT1-MMP (P = 0.04), strong epithelial MT1-MMP (P = 0.01) and high stage (P = 0.04) were independent predictors of poor DSS. CONCLUSIONS: Overexpression of stromal MMP-9 and MT1-MMP is independently associated with shorter DSS in EOC. Thus, host-derived MMPs are valuable predictors of clinical outcome in EOC.

VE-cadherin regulates EphA2 in aggressive melanoma cells through a novel signaling pathway: implications for vasculogenic mimicry.

The formation of matrix-rich, vasculogenic-like networks, termed vasculogenic mimicry (VM), is a unique process characteristic of highly aggressive melanoma cells found to express genes previously thought to be exclusively associated with endothelial and epithelial cells. This study contributes new observations demonstrating that VE-cadherin can regulate the expression of EphA2 at the cell membrane by mediating its ability to become phosphorylated through interactions with its membrane bound ligand, ephrin-A1. VE-cadherin and EphA2 were also found to be colocalized in cell-cell adhesion junctions, both in vitro and in vivo. Immunoprecipitation studies revealed that EphA2 and VE-cadherin could interact, directly and/or indirectly, during VM. Furthermore, there was no change in the colocalization of EphA2 and VE-cadherin at cell-cell adhesion sites when EphA2 was phosphorylated on tyrosine residues. Although transient knockout of EphA2 expression did not alter VE-cadherin localization, transient knockout of VE-cadherin expression resulted in the reorganization of EphA2 on the cells' surface, an accumulation of EphA2 in the cytoplasm, and subsequent dephosphorylation of EphA2. Collectively, these results suggest that VE-cadherin and EphA2 act in a coordinated manner as a key regulatory element in the process of melanoma VM and illuminate a novel signaling pathway that could be potentially exploited for therapeutic intervention.

Effects of angiogenesis inhibitors on vascular network formation by human endothelial and melanoma cells.

Endothelial cells involved in vasculogenesis and angiogenesis are key targets in cancer therapy. Recent evidence suggests that tumor cells can express some genes typically expressed by endothelial cells and form extracellular matrix-rich tubular networks, phenomena known as vasculogenic mimicry. We examined the effects of three angiogenesis inhibitors (i.e., anginex, TNP-470, and endostatin) on vasculogenic mimicry in human melanoma MUM-2B and C8161 cells and compared them with their effects in human endothelial HMEC-1 and HUVEC cells. Anginex, TNP-470, and endostatin markedly inhibited vascular cord and tube formation by HMEC-1 and HUVEC cells in vitro, whereas tubular network formation by MUM-2B and C8161 cells was relatively unaffected. Endothelial cells expressed higher mRNA and protein levels for two putative endostatin receptors, alpha5 integrin and heparin sulfate proteoglycan 2, than melanoma cells, suggesting a mechanistic basis for the differential response of the two cell types to angiogenesis inhibitors. These findings may contribute to the development of new antivascular therapeutic agents that target both angiogenesis and tumor cell vasculogenic mimicry.

Biological significance of focal adhesion kinase in ovarian cancer: role in migration and invasion.

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 <0.05). FAK overexpression was also associated with shorter overall survival (P = 0.008). Multivariate analysis revealed that FAK overexpression and residual disease >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.

Aberrant methylation of the maspin promoter is an early event in human breast cancer.

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.

Functional role of matrix metalloproteinases in ovarian tumor cell plasticity.

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 <.05). 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.

Regulating the tumor suppressor gene maspin in breast cancer cells: a potential mechanism for the anticancer properties of tamoxifen.

PURPOSE: Mammary epithelial cells and the majority of breast cancer tumors require estrogen for continued growth. Antiestrogen therapy alone, or in combination with other drugs, has long been a common procedure for breast cancer treatment and prophylaxis. Thus, there is a critical need to elucidate the mechanism(s) of action of antiestrogen treatment, especially for patients who are at risk of breast cancer development or who are currently receiving hormone therapy. In this study, we examined the ability of hormones to regulate the expression of a tumor suppressor gene, maspin, which is a serine protease inhibitor (serpin) that plays an important role in mammary gland development and is silenced during breast cancer progression. Specifically, our hypothesis tested the clinical efficacy of tamoxifen to regulate maspin expression. EXPERIMENTAL DESIGN: We used maspin promoter luciferase reporter plasmids that were transfected into normal human mammary epithelial (HMEC1331) and MCF-7 breast cancer cells, followed by determination of the effect of hormones and their antagonists on maspin promoter activity. At the protein level, cytosolic fractions from both cell types before and after hormone treatment were subjected to Western blot analysis to determine maspin level. RESULTS AND CONCLUSIONS: Our studies revealed that the antiestrogen tamoxifen induces maspin promoter activity. Interestingly, antiandrogen flutamide could also induce maspin in both cell lines tested. These observations were further confirmed in patient tissues. These novel findings provide a new mechanism of action for tamoxifen under normal and pathological conditions. More significantly, these findings could have a potential impact on future therapeutic intervention strategies for breast cancer.

Differential role of tissue factor pathway inhibitors 1 and 2 in melanoma vasculogenic mimicry.

Vasculogenic mimicry (VM), the formation of matrix-rich vascular-like networks in three-dimensional culture corresponding with the expression of vascular cell-associated genes, and the lining of matrix-rich networks in situ, has been observed in highly aggressive and malignant melanoma. However, little is known about the molecular underpinnings of this phenomenon. On the basis of gene profiling, protein detection, and immunohistochemistry, aggressive relative to poorly aggressive melanoma showed up-regulation of tissue factor (TF), TF pathway inhibitor 1 (TFPI-1) and 2 (TFPI-2), critical genes that initiate and regulate the coagulation pathways. The procoagulant function of TF on highly aggressive melanoma is shown to be regulated by TFPI-1 but not by TFPI-2. Thus, aggressive melanoma exhibits endothelial cell-like anticoagulant mechanisms that may contribute to the fluid-conducting potential of melanoma cell-lined networks, as studied by correlative in vivo Doppler flow measurements. Antibody inhibition experiments reveal that TFPI-2 is required for VM in vitro, but plasmin is an unlikely target protease of TFPI-2. Blockade of TFPI-2 suppressed matrix metalloproteinase-2 activation, and, therefore, TFPI-2 appears to regulate an essential pathway of VM. TFPI-2 is synthesized by endothelial and tumor cells, which deposit TFPI-2 into extracellular matrices. Culturing poorly aggressive melanoma cells on three-dimensional matrix containing recombinant TFPI-2 produces some of the phenotypic changes associated with aggressive, vasculogenic melanoma cells. Thus, TFPI-2 contributes to VM plasticity, whereas TFPI-1 has anticoagulant functions of relevance for perfusion of VM channels formed by TF-expressing melanoma cells.

Elevated focal adhesion kinase expression facilitates oral tumor cell invasion.

BACKGROUND: Understanding the molecular mechanisms of metastasis is critical with respect to oral tumorigenesis. The focal adhesion kinase (FAK) is an intracellular tyrosine kinase associated with the regulation of cell growth, migration, and survival. The purpose of the current study was to determine whether elevated FAK expression in oral malignancies was associated with increased invasiveness and oral carcinoma. METHODS: Immunohistochemical analysis was used to assess levels of FAK expression in archived oral carcinoma tissue samples. Invasion assays after transfections were used to assess the effect of increased FAK expression on invasive potential of oral tumor cells. RESULTS: The human oral carcinoma cell line SCC25 was significantly more invasive (P < 0.05) and expressed higher levels of FAK compared with the less invasive human oral carcinoma cell line SCC15. FAK expression was 3.0-fold higher in the SCC15 cell line and 5.0-fold higher in the SCC25 cell line compared with normal epithelial cells. In the highly invasive SCC25 cell line, FAK expression was 1.5-fold higher compared with the less invasive SCC15 cell line. FAK immunostaining in oral tumors was significantly more intense compared with the immunostaining in surrounding normal epithelium or chronic mucositis. Overexpression of FAK in low-invading SCC15 cells resulted in a 4.5-fold increase in the rate of invasion compared with untransfected or neotransfected control SCC15 cell lines and a nearly 1.5-fold greater rate compared with the highly invasive untransfected SCC25 cell line. CONCLUSIONS: The current results suggest that enhanced expression of FAK in oral carcinoma cells may lead to a selective growth advantage and increased invasive potential of the primary oral tumor.

Expression of multiple molecular phenotypes by aggressive melanoma tumor cells: role in vasculogenic mimicry.

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.

The paradoxical expression of maspin in ovarian carcinoma.

Maspin is a noninhibitory member of the serpin family that is down-regulated in breast carcinoma but overexpressed in pancreatic carcinoma. There are no published data regarding the role of maspin in ovarian carcinoma, which is the focus of the present study. Ovarian cell lines (normal and cancer) and tumors (80 invasive, 14 benign, and 10 low malignant potential) were evaluated for maspin expression and localization. Normal ovarian surface epithelial cells had low levels of maspin. Two of four ovarian cancer cell lines (OVCAR3 and SKOV3) expressed maspin, whereas the cell line EG had weak expression, and 222 had no detectable maspin. Subcellular fractionation studies revealed that the two maspin-positive ovarian cancer cell lines contained maspin in both the nuclear and cytosolic compartments. Wild-type maspin was transfected into the aggressive ovarian cancer cell lines SKOV3 and 222. The in vitro invasive activity of the maspin-transfected cell lines was 44-68% lower than respective controls. The histopathology analysis revealed that among the ovarian tumors examined, 57 (71%) were ranked positive for maspin. Thirty (37%) of the invasive tumors overexpressed maspin. Invasive cancers were more likely to have predominantly cytoplasmic staining compared with benign and low-malignant-potential tumors. Maspin overexpression was significantly associated with a high tumor grade (P = 0.004), the presence of ascites (P = 0.02), a lower likelihood of optimal surgical cytoreduction (P = 0.04), and a shorter duration of overall survival (median survival, 6.33 versus 2.67 years; P = 0.003). The Cox proportional hazards multivariate model revealed that maspin overexpression and high stage were independent predictors of survival. Thus, maspin was found to be overexpressed in a substantial proportion of ovarian tumors, which may serve as an adverse prognostic factor; however, its localization may provide new clues as to its activity and function. These paradoxical results may offer new insights regarding the role of maspin in ovarian cancer progression that may also impact diagnosis and treatment strategies.

Transendothelial function of human metastatic melanoma cells: role of the microenvironment in cell-fate determination.

On the basis of the ability of aggressive melanoma cells to participate in vasculogenic mimicry, particularly their expression of endothelial-associated genes, we examined the plasticity of human metastatic cutaneous melanoma cells with respect to vascular function. Fluorescently labeled metastatic melanoma cells were challenged to an ischemic microenvironment surgically induced in the hind limbs of nude mice. The data reveal the capability of these melanoma cells to express cell-fate determination molecules, normally expressed during embryonic vasculogenesis, and to participate in the neovascularization of circulation-deficient muscle. These results demonstrate the powerful influence of the microenvironment on the transendothelial differentiation of aggressive melanoma cells, and may provide new perspectives on tumor cell plasticity that could be exploited for novel therapeutic strategies.

Prostatic tumor cell plasticity involves cooperative interactions of distinct phenotypic subpopulations: role in vasculogenic mimicry.

BACKGROUND: Tumor cell plasticity represents a significant clinical challenge in that the fate and function of tumor cells can be elusive until a tumor mass is evident. A remarkable example of plasticity is tumor cell vasculogenic mimicry, recently described in aggressive uveal and cutaneous melanoma, in addition to ovarian carcinoma, whereby tumor cells express endothelial-associated genes and form de novo vasculogenic-like networks in three-dimensional (3-D) culture. In the current investigation, we examined whether there is evidence for vasculogenic mimicry in heterogeneous prostatic neoplasms. METHODS: Dunning rat and human prostate cancer cell lines (comprised of epithelial- and fibroblastic-like tumor subpopulations) were tested for their ability to express selected endothelial-associated genes, laminin, the alpha6beta1 laminin-binding integrin, and for their potential to form perfusable tubular networks in 3-D culture. Simultaneous morphological analysis of tumor-lined channels in rat and human tumors was also performed. RESULTS: Green fluorescent protein labeling of prostatic clonal subpopulations revealed unique cooperative interactions of epithelial- and fibroblastic-like tumor cells in the formation of perfusable vasculogenic-like networks. Furthermore, while these cell lines were shown to express various vascular markers, prostatic tumor cell-lined channels were also detected in vivo in high grade tumors, and occurred in some cases in close proximity to conventional endothelial-lined vasculature. CONCLUSIONS: A multidisciplinary approach to assess vasculogenic mimicry by prostatic tumor cells has revealed supportive evidence that it occurs in invasive, heterogeneous prostate cancer cell lines, and circumstantially in aggressive rat and human tumors. These results reflect the plasticity of aggressive prostatic tumor cells and may provide new prognostic markers for clinical diagnosis and new therapeutic intervention strategies.

The clinical significance of tumor cell-lined vasculature in ovarian carcinoma: implications for anti-vasculogenic therapy.

Vasculogenic mimicry reflects the plasticity of aggressive tumor cells that express vascular cell markers and line tumor vasculature; such has been demonstrated in aggressive ovarian carcinoma. This study measured the clinical significance of tumor cell-lined vasculature in ovarian carcinomas (n=77), which was detected in 23 (29.8%) tumors. The data show that tumor cell-lined vasculature was associated with aggressive tumor features and with shorter overall survival (p<0.001). Cox proportional hazards model revealed that tumor cell-lined vasculature (p=0.002) was independently associated with poor survival. This is the first study demonstrating the clinical implications of tumor cell-lined vasculature in ovarian carcinoma.