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Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. It is the leading cause of skin cancer deaths with a median overall survival for advanced-stage metastatic disease of <6 months. Despite advances in the field with conventional and targeted therapies, the heterogeneity of melanoma poses the greatest ongoing challenge, ultimately leading to relapse and progression to a more drug-resistant tumor in most patients. Particularly noteworthy are recent findings, indicating that these therapies exert selective pressure on tumors resulting in the activation of pathways associated with cancer stem cells that are unresponsive to current therapy. Our previous studies have shown how Nodal, an embryonic morphogen of the transforming growth factor-beta superfamily, is one of these critical factors that is reactivated in aggressive melanoma and resistant to conventional chemotherapy, such as dacarbazine. In the current study, we sought to determine whether BRAF inhibitor (BRAFi) therapy targeted Nodal-expressing tumor cells in uniquely matched unresectable stage III and IV melanoma patient samples before and after therapy that preceded their eventual death due to disease. The results demonstrate that BRAFi treatment failed to affect Nodal levels in melanoma tissues. Accompanying experiments in soft agar and in nude mice showed the advantage of using combinatorial treatment with BRAFi plus anti-Nodal monoclonal antibody to suppress tumor growth and metastasis. These data provide a promising new approach using front-line therapy combined with targeting a cancer stem cell-associated molecule—producing a more efficacious response than monotherapy.

Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. It is the leading cause of skin cancer deaths with a median overall survival for advanced-stage metastatic disease of <6 months. Despite advances in the field with conventional and targeted therapies, the heterogeneity of melanoma poses the greatest ongoing challenge, ultimately leading to relapse and progression to a more drug-resistant tumor in most patients. Particularly noteworthy are recent findings, indicating that these therapies exert selective pressure on tumors resulting in the activation of pathways associated with cancer stem cells that are unresponsive to current therapy. Our previous studies have shown how Nodal, an embryonic morphogen of the transforming growth factor-beta superfamily, is one of these critical factors that is reactivated in aggressive melanoma and resistant to conventional chemotherapy, such as dacarbazine. In the current study, we sought to determine whether BRAF inhibitor (BRAFi) therapy targeted Nodal-expressing tumor cells in uniquely matched unresectable stage III and IV melanoma patient samples before and after therapy that preceded their eventual death due to disease. The results demonstrate that BRAFi treatment failed to affect Nodal levels in melanoma tissues. Accompanying experiments in soft agar and in nude mice showed the advantage of using combinatorial treatment with BRAFi plus anti-Nodal monoclonal antibody to suppress tumor growth and metastasis. These data provide a promising new approach using front-line therapy combined with targeting a cancer stem cell-associated molecule—producing a more efficacious response than monotherapy.

ABSTRACTTriple-negative breast cancer (TNBC) represents an aggressive cancer subtype characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The independence of TNBC from these growth promoting factors eliminates the efficacy of therapies which specifically target them, and limits TNBC patients to traditional systemic neo/adjuvant chemotherapy. To better understand the growth advantage of TNBC – in the absence of ER, PR and HER2, we focused on the embryonic morphogen Nodal (associated with the cancer stem cell phenotype), which is re-expressed in aggressive breast cancers. Most notably, our previous data demonstrated that inhibition of Nodal signaling in breast cancer cells reduces their tumorigenic capacity. Furthermore, inhibiting Nodal in other cancers has resulted in improved effects of chemotherapy, although the mechanisms for this remain unknown. Thus, we hypothesized that targeting Nodal in TNBC cells in combination with conventional chemotherapy may improve efficacy and represent a potential new strategy. Our preliminary data demonstrate that Nodal is highly expressed in TNBC when compared to invasive hormone receptor positive samples. Treatment of Nodal expressing TNBC cell lines with a neutralizing anti-Nodal antibody reduces the viability of cells that had previously survived treatment with the anthracycline doxorubicin. We show that inhibiting Nodal may alter response mechanisms employed by cancer cells undergoing DNA damage. These data suggest that development of therapies which target Nodal in TNBC may lead to additional treatment options in conjunction with chemotherapy regimens – by altering signaling pathways critical to cellular survival.

As the frequency of melanoma increases, current treatment strategies are struggling to significantly impact patient survival. One of the critical issues in designing efficient therapies is understanding the composition of heterogeneous melanoma tumors in order to target cancer stem cells (CSCs) and drug-resistant subpopulations. In this review, we summarize recent findings pertinent to the reemergence of the embryonic Nodal signaling pathway in melanoma and its significance as a prognostic biomarker and therapeutic target. In addition, we offer a novel molecular approach to studying the functional relevance of Nodal-expressing subpopulations and their CSC phenotype.

In 1999, The American Journal of Pathologypublished an article entitled “Vascular Channel Formation by Human Melanoma Cells in Vivoand in Vitro: Vasculogenic Mimicry,” by Maniotis and colleagues, which ignited a spirited debate for several years and earned distinction as a citation classic. Tumor cell vasculogenic mimicry (VM) refers to the plasticity of aggressive cancer cells forming de novovascular networks, which thereby contribute to perfusion of rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with the constitutional endothelial-lined vasculature. The tumor cells capable of VM share a plastic, transendothelial phenotype, which may be induced by hypoxia. Since VM was introduced as a novel paradigm for melanoma tumor perfusion, many studies have contributed new findings illuminating the underlying molecular pathways supporting VM in a variety of tumors, including carcinomas, sarcomas, glioblastomas, astrocytomas, and melanomas. Facilitating the functional plasticity of tumor cell VM are key proteins associated with vascular, stem cell, and hypoxia-related signaling pathways, each deserving serious consideration as potential therapeutic targets and diagnostic indicators of the aggressive, metastatic phenotype.

The greatest health threat from malignant melanoma is death due to metastatic disease. Consequently, the identification of markers predictive of metastatic disease is essential for identifying new therapeutic targets. EphA2, a protein tyrosine kinase receptor commonly expressed in epithelial cells, has been found to be over-expressed and constitutively active in melanoma tumor cells having a metastatic phenotype as characterized by increased invasion, proliferation, and vasculogenic mimicry (VM). Based on this observation, we hypothesized that increased expression of EphA2 by melanoma tumor cells could promote these characteristics of a metastatic phenotype in addition to promoting tumorigenicity as a whole. We analyzed a panel of human melanoma tumor cell lines derived from patient tissues classified as primary (either radial growth phase or vertical growth phase) and/or metastatic for the expression of EphA2 and found a correlation between increased EphA2 expression and metastatic potential. Experiments using the most metastatic of the human melanoma cell lines demonstrated that down-regulation of EphA2 results in a significant decrease in invasion, proliferation, clonogenicity, and VM in vitro, in addition to suppressed tumorigenicity in an orthotopic mouse model. Lastly, utilization of a human phospho-kinase array revealed increased phosphorylation of several different protein kinases involved in mediating various aspects of cellular proliferation. To the best of our knowledge these results provide the first direct in vivo evidence demonstrating a role for EphA2 in promoting melanoma tumorigenicity and suggest EphA2 as a significant molecular target for the therapeutic intervention of malignant melanoma.

We have previously shown that the microenvironment of human embryonic stem cells (hESCs) is able to change and reprogram aggressive cancer cells to a less aggressive state. Some mechanisms implicated in the phenotypic changes observed after this exposure are mainly associated with the Nodal signaling pathway, which plays a key role in tumor cell plasticity. However, several other molecular mechanisms might be related directly and/or indirectly to these changes, including microRNA (miRNA) regulation and DNA methylation. Aim:To further explore the epigenetic mechanisms potentially underlying the phenotypic changes that occur after exposing metastatic melanoma cells to a hESC microenvironment. Materials & Methods:A total of 365 miRNAs were screened using the TaqMan®®Low Density Arrays. We also evaluated whether DNA methylation could be one of the factors regulating the expression of the inhibitor of Nodal, Lefty, in hESCs (where it is highly expressed) vs melanoma cells (where it is not expressed). Results:Using these experimental approaches, we identified miRNAs that are up- and down-regulated in melanoma cells exposed to a hESC microenvironment, such as miR-302a and miR-27b, respectively. We also demonstrate that Notch4 is one of the targets of miR-302a, which is upstream of Nodal. Additionally, one of the mechanisms that might explain the absence of the inhibitor of Nodal, Lefty, in cancer cells is silencing by DNA methylation, which provides new insights into the unregulated expression of Nodal in melanoma. Conclusion:These findings suggest that epigenetic changes such as DNA methylation and regulation by microRNAs might play a significant role in tumor cell plasticity and the metastatic phenotype.

The incidence of melanoma has increased dramatically over the last 50?yr, and although melanoma accounts for only 10 of all skin cancers, it is responsible for over 80 of skin cancer deaths. Recent studies have uncovered critical molecular events underlying melanocytic transformation and melanomagenesis. Among these noteworthy observations are the acquisition of stem cell-associated proteins, such as the Notch receptors and Nodal, which have also been implicated in melanoma progression. For example, we have demonstrated that Nodal expression is limited to invasive vertical growth phase and metastatic melanoma lesions, and that inhibition of Nodal signaling promotes the reversion of metastatic melanoma cells toward a more differentiated, less invasive non-tumorigenic phenotype. In addition, molecular cross-talk exists between the Notch and Nodal signaling pathways. Interestingly, the acquisition of stem cell-associated plasticity is often acquired via epigenetic mechanisms, and is therefore receptive to reprogramming in response to embryonic microenvironments. Here, we review the concept of melanoma plasticity, with an emphasis on the emerging role of Nodal as a regulator of melanoma tumorigenesis and progression, and present findings related to epigenetic reprogramming.

Metastatic melanoma continues to be a significantly deadly cancer with a cure rate of < 20% and a median survival of 6 – 9 months. The aggressiveness associated with metastatic melanoma is largely attributable to its inherent plasticity, a property that is mediated by the secretion of Nodal, a stem-cell associated protein belonging to the transforming growth factor- superfamily. This is supported by the observations that Nodal expression is limited to invasive vertical growth phase and metastatic melanoma lesions, and that inhibition of Nodal signaling promotes the reversion of metastatic melanoma cells toward a more differentiated, less invasive non-tumorigenic phenotype. Hence, due to its restricted expression pattern and function as a melanoma-tumor-promoter, Nodal (and its signaling partners) present unique targets for both immunologic and pharmacologic therapies.

As our understanding of embryonic stem cell biology becomes more sophisticated, the similarities between multipotent cancer cells and these totipotent precursors are increasingly striking. Both multipotent cancer cells and embryonic stem cells possess the ability to self-renew, epigenetically alter their neighboring cellular architecture, and populate a tissue mass with a phenotypically heterogeneous composition of cells. While the molecular signature of these cell types continues to be elucidated, new insights are emerging related to the convergence of embryonic and tumorigenic signaling pathways. Understanding the molecular underpinnings of these two stem cell phenotypes may lead to new therapeutic targets for the elusive cancer cell. While still in its infancy, the potential of adapting embryonic stem cells, and more specifically the factors they produce, is enormous for clinical application. Here we outline evidence that demonstrates the inductive influence of embryonic stem cells and their microenvironment to reprogram cancer cells to exhibit a more benign phenotype, with profound implications for differentiation therapy.

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 co-localized 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 co-localization 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.

A dynamic, complex relationship exists between tumor cells and their microenvironment, which plays a pivotal role in cancer progression, yet remains poorly understood. Particularly perplexing is the finding that aggressive melanoma cells express genes associated with multiple cellular phenotypes, in addition to their ability to form vasculogenic‐like networks in three‐dimensional matrix ‐ called vasculogenic mimicry, which is illustrative of tumor cells plasticity. This study addressed the unique epigenetic effect of the microenvironment of aggressive melanoma cells on the behavior of poorly aggressive melanoma cells exposed to it. The data show significant changes in the global gene expression of the cells exposed to 3‐D matrices preconditioned by aggressive melanoma cells, including the acquisition of a vasculogenic cell phenotype, upregulation of ECM remodeling genes, and increased invasive ability ‐ indicative of an epigenetic, microenvironment‐induced reprogramming of poorly aggressive melanoma cells. However, this epigenetic effect was completely abrogated when a highly cross‐linked collagen matrix was used, which could not be remodeled by the aggressive melanoma cells. These findings offer an unique perspective of the inductive properties associated with an aggressive melanoma microenvironment that might provide new insights into the epigenetic regulation of tumor cell plasticity and differentiation, as well as mechanisms that could be targeted for novel therapeutic strategies.

Certain aggressive melanoma cell lines exhibit a dedifferentiated phenotype, expressing genes that are characteristic of various cell types including endothelial, neural, and stem cells. Moreover, we have shown that aggressive melanoma cells can participate in neovascularization in vivo and vasculogenic mimicry in vitro, demonstrating that these cells respond to microenvironmental cues and manifest developmental plasticity. To explore this plasticity further, we transplanted human metastatic melanoma cells into zebrafish blastula‐stage embryos and monitored their behavior post‐transplantation. The data show that human metastatic melanoma cells placed in the zebrafish embryo survive, exhibit motility, and divide. The melanoma cells do not form tumors nor integrate into host organs, but instead become scattered throughout the embryo in interstitial spaces, reflecting the dedifferentiated state of the cancer cells. In contrast to the fate of melanoma cells, human melanocytes transplanted into zebrafish embryos most frequently become distributed to their normal microenvironment of the skin, revealing that the zebrafish embryo contains possible homing cues that can be interpreted by normal human cells. Finally, we show that within the zebrafish embryo, metastatic melanoma cells retain their dedifferentiated phenotype. These results demonstrate the utility of the zebrafish embryonic model for the study of tumor cell plasticity and suggest that this experimental paradigm can be a powerful one in which to investigate tumor‐microenvironment interactions. Developmental Dynamics 233:1560–1570, 2005. © 2005 Wiley‐Liss, Inc.

Previous studies from our laboratory and others have demonstrated that treatment of breast cancer cells with exogenous maspin led to a significant decrease in cell motility, and an increase in cell adhesion to human fibronectin. However, the signaling mechanisms by which maspin, a putative tumor suppressor gene, might regulate cell motility and adhesion have not been previously addressed. In this study, we hypothesized that maspin could inhibit cell motility through the Rho GTPase pathway, specifically by affecting Rac activity. To test this intriguing hypothesis we utilized an experimental approach where invasive and metastatic MDA-MB-231 breast cancer cells were either treated exogenously with recombinant maspin protein, or stably transfected with maspin. The data revealed decreased Rac1 activity within 4 h, and a decrease in the Rac1 effector, PAK1, within 12 h. In addition, an increase in PI3K and ERK1/2 activities within 1 h of recombinant maspin (rMaspin) treatment was observed, which returned to baseline level after 12 h. ERK activity was shown to be downstream of PI3K, as pretreatment with the PI3K inhibitor, LY294002, inhibited the stimulation of ERK activity by rMaspin. Furthermore, rMaspin-treated cells displayed approximately a 30% increase in cell adhesion which was abrogated by pretreatment with LY294002. Increased focal adhesions and stress fibers were observed after 12 h of rMaspin treatment, when the cells were least motile and had reverted to a more epithelial-like phenotype. These data suggest that maspin may inhibit cell motility by regulating Rac1 and subsequently PAK1 activity, and promote cell adhesion via PI3K/ERK pathways. This study provides new insights into the diverse signaling pathways affected by maspin to suppress the metastatic phenotype, and could contribute to novel therapeutic approaches for the treatment of invasive and metastatic breast cancer.

A necessity for development and tumor progression is a blood supply formed by vasculogenic andor angiogenic events, involving the cooperative interactions of cells with their microenvironment. Based on the recent characterization of vasculogenic mimicry by aggressive melanoma cells, particularly their ability to express VE (vascular endothelial)-cadherin, TIE-1, and EphA2, current studies have focused on the molecular signals deposited by these cells as they remodel their microenvironment. The experimental approach utilizes unique three-dimensional collagen matrices preconditioned by metastatic melanoma cells, which contain laminin 5 ?2 chain-enriched tracks with promigratory cleavage fragments produced by cooperative interactions with specific matrix metalloproteinases (MMPs). The results demonstrate that the collagen matrices preconditioned by the metastatic cells induce poorly aggressive melanoma cells to express, for the first time, key angiogenicvasculogenicmatrix-remodeling genes. Treatment of aggressive melanoma cells with an MMP inhibitor resulted in the inhibition of vasculogenic mimicry-associated genes in these tumor cells and abrogation of the inductive effects of the preconditioned matrix on poorly aggressive melanoma cells. These observations illustrate the remarkable influence of the microenvironment on the phenotype of melanoma cells and may provide new perspectives on tumor cell plasticity and unique treatment strategies.

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.

In vitro morphogenesis of epithelial cells to form tube-like structures is regulated by hepatocyte growth factor-scatter factor (HGF/SF). The placenta is a rich source of HGF/SF, and its absence in mice has been shown to lead to impaired placental growth and embryonic death. There is no information in the literature regarding in vitro morphogenesis of human cytotrophoblasts or the effect of HGF/SF on this process. In this study, cytotrophoblasts were isolated from human placentae obtained from all three trimesters of gestation and cultured on the recombinant basement membrane matrix (Matrigel). Under these conditions, cytotrophoblasts participated in morphogenetic events including formation of spheroid-like structures, radial linear processes with branching, and invaded Matrigel and formed large, tube-like structures. The presence of a developing lumen was documented in the linear projections arising from spheroids and in the tube-like structures by both confocal and transmission electron microscopy. Immunohistochemistry was used to characterize the phenotype of the cells, and staining with anti-cytokeratin and anti-E-cadherin antibodies confirmed the presence of cytotrophoblasts in both the spheroids and tube-like structures. Recombinant HGF (rHGF) significantly increased the invasive activity of cytotrophoblasts isolated from the first and second (P< 0.001) and third trimesters (P< 0.01). In addition, rHGF significantly increased the percentage of spheroids with branching processes in the first and second trimesters (P< 0.05). Anti-HGF antibody inhibited both these effects in a dose-dependent manner, indicating the specificity of the above findings. This study provides new evidence indicating that HGF/SF regulates invasion and branching morphogenesis of cytotrophoblasts throughout gestation, with maximum effects in the first and second trimester. These findings may help to elucidate the importance of the reduced expression of HGF/SF identified in placentae from women with preeclampsia or intrauterine growth restriction and suggest that HGF/SF may serve as an important candidate in therapeutic intervention strategies.

To investigate the possible role of human parvovirus B19 as an etiologic agent in rheumatoid arthritis (RA), with particular emphasis on its ability to induce invasiveness in human synovial fibroblasts. We established an experimental in vitro system in which normal primary human synovial fibroblasts were treated with or without parvovirus B19–containing human sera for 7 days. The fibroblasts were then tested for their ability to degrade reconstituted cartilage matrix using a well-characterized cartilage invasion assay system. Incubation with parvovirus B19–containing serum induced an invasive phenotype in normal human synovial fibroblasts. B19 serum–treated synovial fibroblasts exhibited an increase in invasion of up to 248% compared with the activity of fibroblasts in media alone, in contrast to B19-negative sera–treated synovial fibroblasts, which exhibited no significant change compared with that in media alone. In addition, preincubation of viremic serum with a neutralizing antibody to B19 abrogated the observed effect. These results provide direct evidence regarding the ability of parvovirus B19 to induce invasive properties in normal human synovial fibroblasts. Parvovirus B19 has been proposed as an etiologic agent of RA, and our data provide the first biologic link between exposure to B19 and phenotypic changes in normal human synovial fibroblasts.

During development, the formation and remodeling of primary vascular networks occurs by vasculogenesis and angiogenesis. Recently, the term “vasculogenic mimicry” has been used by our laboratory and collaborators to reflect the embryonic-like ability of aggressive, but not nonaggressive, melanoma tumor cells to form a pattern of matrix-rich networks (containing channels) surrounding spheroids of tumor cells in three-dimensional culture, concomitant with their expression of vascular cell markers. Ovarian cancer is usually diagnosed as advanced stage disease in most patients when widespread metastases have already been established within the peritoneal cavity. In this study, we explored whether invasive ovarian carcinoma cells could engage in molecular vasculogenic mimicry reflected by their plasticity, compared with their normal cell counterparts. The data revealed that the invasive ovarian cancer cells, but not normal ovarian surface epithelial cells, formed patterned networks containing solid and hollow matrix channels when grown in three-dimensional cultures containing Matrigel or type I collagen, in the absence of endothelial cells or fibroblasts. Immunohistochemical analysis showed that matrix metalloproteinases (MMP)-1, -2, and -9, and MT1-MMP were discretely localized to these networks, and the formation of the networks was inhibited by treatment with MMP inhibitors. Furthermore, the RNase protection assay revealed the expression of multiple vascular cell-associated markers by the invasive ovarian cancer cells. In patient tumor sections from high-stage, high-grade ovarian cancers, 7 to 10% of channels containing red blood cells were lined by tumor cells. By comparison, all vascular areas in benign tumors and low-stage cancers were endothelial lined. These results may offer new insights and molecular markers for consideration in ovarian cancer diagnosis and treatment strategies based on molecular vascular mimicry by aggressive tumor cells.

Several studies have correlated escape from TGF-β-mediated cell cycle arrest with the tumorigenic phenotype. Most often, this escape from growth control has been linked to dysfunctional TGF-β receptors or defects in the TGF-β-mediated SMAD signaling pathway. In this report, we found that highly metastatic 4T1 mammary carcinoma cells express functional TGF-β receptors capable of initiating SMAD-mediated transcription, yet are not growth inhibited by TGF-β1. We further observed that TGF-β directly contributes to the metastatic behavior of this cell line. Exposure to TGF-β caused 4T1 cells to undergo morphological changes associated with the metastatic phenotype and invade more readily through collagen coated matrices. Furthermore, expression of a dominant negative truncated type II receptor diminished TGF-β signaling and significantly restricted the ability of 4T1 cells to establish distant metastases. Our results suggest that regardless of 4T1 resistance to TGF-β-mediated growth inhibition, TGF-β signaling is required for tumor invasion and metastases formation. Int. J. Cancer 91:76–82, 2001. © 2001 Wiley-Liss, Inc.

During embryogenesis, the formation of primary vascular networks occurs via the processes of vasculogenesis and angiogenesis. In uveal melanoma, vasculogenic mimicry describes the 'embryonic-like' ability of aggressive, but not nonaggressive, tumor cells to form networks surrounding spheroids of tumor cells in three-dimensional culture; these recapitulate the patterned networks seen in patients' aggressive tumors and correlates with poor prognosis. The molecular profile of these aggressive tumor cells suggests that they have a deregulated genotype, capable of expressing vascular phenotypes. Similarly, the embryonic-like phenotype expressed by the aggressive human breast cancer cells is associated with their ability to express a variety of vascular markers. These studies may offer new insights for consideration in breast cancer diagnosis and therapeutic intervention strategies.

There are fundamental issues regarding the role of integrins in human disease which remain to be elucidated. Human cutaneous melanoma is an attractive model for studying integrin involvement in tumor progression because it generally follows a sequential series of definable stages. Furthermore, the most specific marker for the transition of cells from the more benign, non-metastatic radial growth phase stage to the more malignant, metastatically competent vertical growth phase stage is associated with the onset of αvβ3integrin expression and function. This same pattern, however, does not hold true for human ocular/uveal melanomas which do not progress through these stages, but preferentially metastasize to the liver by dissemination of the cells via a direct hematogenous pathway. It is also unclear whether the αvβ3integrin is functionally involved in uveal melanoma metastasis or not. Our results show that perturbation of the αvβ3integrin on moderately invasive A375M human cutaneous melanoma cells with either specific antibodies or ligands results in an increase in the cells' ability to invade in vitrocoincident with an increase in the cells' expression and extracellular levels of matrix metalloproteinase-2 (MMP-2, gelatinase A). The highly invasive C8161 human cutaneous melanoma cells express little-to-no αvβ3integrin, but are more invasive and express higher levels of MMPs after perturbation of their α5β1integrin. This augmented invasiveness could subsequently be abrogated with a function-blocking anti-MMP-2 antibody. Primary uveal melanoma cells and cells derived from uveal metastases appear to grow in either a spindle or epithelioid morphology. The less invasive uveal melanoma cells are spindle shaped and express higher levels of the αvβ3integrin, while the more invasive cell lines are epithelioid shaped and express reduced levels of the αvβ3integrin. The apparent conflict between these results and the current model for cutaneous melanoma progression may be addressed as follows: The expression and function of the αvβ3integrin plays an important role(s) during the transition of cells from the radial growth phase stage to the vertical growth phase stage. However, further progression leading to metastases may require changes in the cells' integrins that would facilitate their ability to leave the primary tumor, and aid in their ability to invade and ultimately form metastases. It is also conceivable that the αvβ3integrin is reexpressed during various stages of metastatic dissemination, and, in particular, during tumor reestablishment.

Tissue sections from aggressive human intraocular (uveal) and metastatic cutaneous melanomas generally lack evidence of significant necrosis and contain patterned networks of interconnected loops of extracellular matrix. The matrix that forms these loops or networks may be solid or hollow. Red blood cells have been detected within the hollow channel components of this patterned matrix histologically, and these vascular channel networks have been detected in human tumors angiographically. Endothelial cells were not identified within these matrix-embedded channels by light microscopy, by transmission electron microscopy, or by using an immunohistochemical panel of endothelial cell markers (Factor VIII-related antigen, Ulex, CD31, CD34, and KDR[Flk-1]). Highly invasive primary and metastatic human melanoma cells formed patterned solid and hollow matrix channels (seen in tissue sections of aggressive primary and metastatic human melanomas) in three-dimensional cultures containing Matrigel or dilute Type I collagen, without endothelial cells or fibroblasts. These tumor cell-generated patterned channels conducted dye, highlighting looping patterns visualized angiographically in human tumors. Neither normal melanocytes nor poorly invasive melanoma cells generated these patterned channels in vitrounder identical culture conditions, even after the addition of conditioned medium from metastatic pattern-forming melanoma cells, soluble growth factors, or regimes of hypoxia. Highly invasive and metastatic human melanoma cells, but not poorly invasive melanoma cells, contracted and remodeled floating hydrated gels, providing a biomechanical explanation for the generation of microvessels in vitro. cDNA microarray analysis of highly invasive versuspoorly invasive melanoma tumor cells confirmed a genetic reversion to a pluripotent embryonic-like genotype in the highly aggressive melanoma cells. These observations strongly suggest that aggressive melanoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis.

We have previously shown that human breast carcinoma cells demonstrating an interconverted phenotype, where keratin (epithelial marker) and vimentin (mesenchymal marker) intermediate filaments are both expressed, have an increased ability to invade a basement membrane matrix in vitro. This increase in invasive potential has been demonstrated in MDA‐MB‐231 cells, which constitutively express keratins and vimentin, and in MCF‐7 cells transfected with the mouse vimentin gene (MoVi). However, vimentin expression alone is not sufficient to confer the complete metastatic phenotype in MoVi cells, as determined by orthotopic administration. Thus, in the present study, differential display analysis was utilized to identify genes that are associated with the invasive and/or metastatic phenotype of several human breast cancer cell lines. Forty‐four of 84 PCR fragments were differentially expressed as assessed by Northern hybridization analysis of RNA isolated from MCF‐7, MoVi, and MB‐231 cell lines. Polyadenylated RNA from a panel of poorly invasive, invasive/non‐metastatic, and invasive/metastatic breast carcinoma cell lines was used to differentiate between cell‐specific gene expression and genes associated with the invasive and/or metastatic phenotype(s). We observed that lysyl oxidase and a zinc finger transcription factor were expressed only in the invasive and/or metastatic cell line; whereas, a thiol‐specific antioxidant and a heterochromatin protein were down‐regulated in these cells. In contrast, tissue factor was expressed only in breast carcinoma cell lines having the highest invasive potential. These results suggest that specific genes involved in breast cancer invasion and metastasis can be separated by differential display methodology to elucidate the molecular basis of tumor cell progression.

The light‐inducible nuclear gene coding for the small subunit of ribulose‐1,5‐bisphosphate carboxylase (Rubisco), produces a precursor protein with an amino‐terminal transit peptide which is transported into the plastids and cleaved by a specific proteinase. To test whether the promoter and transit peptide‐coding sequences of the small subunit gene can be used to direct the light‐inducible synthesis and transport of a foreign protein into chloroplasts, a chimaeric gene was constructed consisting of the promoter, first exon and intron as well as part of the second exon of the small subunit Rubisco gene fused to the amino‐terminal end of the neomycin phosphotransferase II gene, (nptII) of Tn5. Tobacco tissue, as well as whole plants, into which this chimaeric gene was introduced, were resistant to kanamycin. The transcription of the chimaeric gene as well as the NPTII activity of the resulting fusion protein were shown to be light inducible. The fusion protein is processed and located within the chloroplasts of the transformed plants.

Previously, we observed that dexamethasone decreased the extracellular activity of some matrix metalloproteinases and tissue plasminogen activator in trabecular meshwork containing organ explants and cell cultures. We have extended these studies to determine whether the decrease in extracellular tissue plasminogen activator activity is the result of a decrease in the expression of tissue plasminogen activator, a change in the extracellular level of plasminogen activator inhibitor-1, or a combination of both. We demonstrate that dexamethasone decreases the steady-state levels of tissue plasminogen activator mRNA by 75 in trabecular meshwork cells using Northern blot analysis, and show that this occurs concurrently with a 31 increase in the levels of extracellular plasminogen activator inhibitor-1 as determined by enzyme-linked immunosorbent assay analysis. A 38 increase was also measured in the extracellular plasminogen activator inhibitor-1 levels in trabecular meshwork containing organ explants after treatment. The amount of active, extracellular tissue plasminogen activator decreased by 72 in trabecular meshwork organ explant cultures, and 96 in trabecular meshwork cell cultures after treatment as determined using a parabolic rate assay for tissue plasminogen activator activity. These data suggest that dexamethasone decreases the extracellular activity of tissue plasminogen activator in trabecular meshwork containing organ and cell cultures by decreasing the expression of tissue plasminogen activator and increasing the extracellular levels of plasminogen activator inhibitor-1.

The light‐inducible nuclear gene coding for the small subunit of ribulose‐1,5‐bisphosphate carboxylase (Rubisco), produces a precursor protein with an amino‐terminal transit peptide which is transported into the plastids and cleaved by a specific proteinase. To test whether the promoter and transit peptide‐coding sequences of the small subunit gene can be used to direct the light‐inducible synthesis and transport of a foreign protein into chloroplasts, a chimaeric gene was constructed consisting of the promoter, first exon and intron as well as part of the second exon of the small subunit Rubisco gene fused to the amino‐terminal end of the neomycin phosphotransferase II gene, (nptII) of Tn5. Tobacco tissue, as well as whole plants, into which this chimaeric gene was introduced, were resistant to kanamycin. The transcription of the chimaeric gene as well as the NPTII activity of the resulting fusion protein were shown to be light inducible. The fusion protein is processed and located within the chloroplasts of the transformed plants.

While a number of chemoattractants of vascular endothelial cells have now been identified in vitro, differences in methodology preclude comparisons of substances evaluated in different assays. Here, we report a standardized chemotactic assay in which the migration of calf pulmonary artery endothelial cells in a 48-well microchemotaxis chamber was determined. Nonstimulated (control) migration was remarkably constant (mean ± SD, 96 ± 14) from plate to plate, thus allowing the indexing of relative migration of stimulated cells to that of nonstimulated cells in the control wells of that plate. Based on the relative migrations observed in response to each of the substances evaluated, those proving to be stimulatory of migration were placed in rank order by potency. The growth factors epidermal growth factor, transforming growth factor-α, and basic fibroblast growth factor (followed by pentosan polysulfate, plasmin, fibronectin, fibrinogen, granulocyte-macrophage colony stimulating factor heparin, adenosine, and MgS04) were the most potent. Only the platelet factors platelet-derived growth factor-BB and platelet activating factor proved inhibitory of migration. Combining fibrinogen with other chemoattractants produced either stimulation or inhibition in comparison to the migration observed with fibrinogen one, suggesting that more than one signal transduction mechanism was, in all likelihood, invoked by the various agents. This assay will allow the rapid screening and rank ordering of additional putative chemoattractants, will facilitate the study of the biochemical mechanisms involved in endothelial cell migration, and will permit the evaluation of pharmacologic agents capable of modulating stimulated or unstimulated migration.

The expression of intermediate filament proteins is remarkably tissue-specific which suggests that the intermediate filament (IF) type(s) present in cells is somehow related to their biological function. However, in some cancers-particularly malignant melanoma and breast carcinoma, there is a strong indication that vimentin and keratin IFs are coexpressed, thus presenting as a dedifferentiated or interconverted (between epithelial and mesenchymal) phenotype. In this review, twoin vitro models are presented which recapitulate the interconverted phenotype in human melanoma and breast carcinoma, and allow, for the first time, unique observations to be made with respect to the role of IFs in cancer progression.

Tumor cells and fibroblasts were isolated from the tumor-host interface of a colon 4047 tumor growing subcutaneously in a Fischer 344 rat. The populations were co-cultured to recapitulate the tumor-host interface in vitro. The co-cultured populations grew in a predictable pattern with tumor cells forming nodules surrounded by fibroblasts. Population dynamic experiments demonstrated the fibroblasts enhanced the growth of the tumor cells but tumor inhibited and ultimately destroyed the fibroblasts. Video microscopic examination of the fibroblasts demonstrated intense membrane ruffling adjacent to the tumor nodules followed by membrane fragmentation and detachment. Immunohisto-chemical staining for gelatinase A was markedly positive within the fibroblasts surrounding the tumor nodules; but negative within the tumor and in fibroblasts when tumor was absent. This technique recapitulates many aspects of the tumor-host interface in vitro and may be a useful model for evaluating several aspects of tumor-host interaction.

Glycolysis is increased in cancer cells compared with normal cells. It has been shown that glucose enters cells via a family of five functional glucose transporters (GLUT). However, GLUT expression appears to be altered in human breast cancer, which may serve as a selective advantage and facilitate the metastatic potential of these cells. The relationship of GLUT isoform expression and breast cancer cell invasiveness has not been adequately addressed. Thus, the purpose of this study was to investigate whether an association exists between GLUT expression and human breast cancer cell invasiveness. Invasiveness of the human breast cancer lines MCF-7, MDA-MB-435 and MDA-MB-231 was measured using anin vitroassay and compared with cellular GLUT isoform expression, assessed by Western blot analysis and verified by immunohistochemistry in a poorly differentiated human ductal breast cancer. Cell surface GLUT-1 expression was associated with the invasive ability of MCF-7 (2.0 ± 0.02%), MDA-MB-435 (6.4 ±0.4%), and MDA-MB-231 (19.3 ± 2.0%). However, GLUT-2 and GLUT-5 were inversely associated with invasiveness; GLUT-3 expression was variable; and GLUT-4 was undetected. In a poorly differentiated human ductal breast cancer,in situGLUT-1 staining was intense. GLUT-1 expression was associated with the in vitro invasive ability of human breast cancer cells which was validatedin situ. If this relationship is found to exist in a larger number of human breast cancer tissues, it may be possible to develop diagnostic and therapeutic strategies based on targeted GLUT isoform expression.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of joints involving the pathological development of an invasive and destructive pannus tissue which contributes to the loss of cartilage and bone. To further analyze the process of cartilage degradation and invasion, we have developed an in vitro model composed of cartilage matrix and synoviocytes (isolated from RA pannus tissue, as well as normal synovial membrane). The matrix is derived from pig articular cartilage and contains collagen type II and proteoglycans and is similar in composition to human cartilage. Data generated from this model reveal that synoviocytes isolated from RA pannus tissue invaded cartilage matrix in a manner which directly correlated with the severity of the disease. Analysis of mechanisms associated with the invasive process demonstrate that highly invasive RA synoviocytes maintain a round morphology during attachment and spreading on cartilage matrix, compared with their normal counterparts. Furthermore, the level of secretion of matrix metalloprote-inase (MMP) activity was shown to correlate with the RA phenotype, which could be modulated with a novel MMP inhibitor. Normal synoviocytes could be “converted” to an RA phenotype by specific inflammatory cytokines, such that invasion of cartilage matrix was augmented by cultunng these cells in the presence of 5 U/ml IL-lß or 18 U/ml TGFß. Invasion was inhibited by 150 U/ml TNFα, and unaffected by 100 ng/ml PDGF. In addition, synovial fluid from RA patients induced invasion of normal synoviocytes, in a concentration dependent manner, from 150% to 460%; however, synovial fluid from another inflammatory arthritidy (Crohn’s) did not augment invasion to the same degree. Moreover, this “converted effect” appears to be specific for synoviocytes, since similar effects could not be achieved with human skin fibroblasts. This in vitro model of synoviocyte-medi-ated cartilage invasion allows for further molecular characterization of the invasive properties of the synoviocyte which contribute to RA.

The expression of the metalloproteinase stromelysin correlates with the progression of chemically induced squamous cell carcinomas. We demonstrate that the expression of activated stromelysin in papilloma-derived cells enhances in vitro cell invasion. We also demonstrate that the Ha-ras oncogene induces the transcription of the stromelysin gene through an AP-1 dependent pathway. The hypothesis is that alterations in oncogenes and suppressor genes influence stromelysin expression and thus influence subsequent steps of tumor invasion and metastasis.

As a consequence of poor perfusion and elevated acid production, the extracellular pH (pHex) of tumors is generally acidic. Despite this, most in vitroexperiments are still performed at the relatively alkaline pHexof 7.4. This is significant, because slight changes in pHexcan have profound effects on cell phenotype. In this study we examined the effects of mildly acidic conditions on the in vitroinvasive potential of two human melanoma cell lines: the highly invasive C8161, and poorly invasive A375P. We observed that culturing of either cell line at acidic pH (6.8) caused dramatic increases in both migration and invasion, as measured with the Membrane Invasion Culture System (MICS). This was not due to a direct effect of pH on the invasive machinery, since cells cultured at normal pH (7.4) and tested at acidic pH did not exhibit increased invasive potential. Similarly, cells cultured at acidic pH were more aggressive than control cells when tested at the same medium pH. These data indicate that culturing of cells at mildly acidic pH induces them to become more invasive. Since acid pH will affect the intracellular pH (pHin) and intracellular calcium ([Ca2+]in), we examined the effect of these parameters on invasion. While changes in [Ca2+]inwere not consistent with invasive potential, the changes in pHinwere. While these conditions decrease the overall amount of gelatinases A and B secreted by these cells, there is a consistent and significant increase in the proportion of the activated form of gelatinase B.

Cellular heterogeneity of neoplasia is well demonstrated in the Dunning R-3327 rat prostate adenocarcinoma. In this study, we measured the differential expression of invasive and metastatic properties of this prostate model by cloning from a heterogeneous parental cell line. Four cell clones were derived and characterized by morphological studies, E-cadherin expression, and invasive and metastatic potential. Three of the clones (clones 5′A, 5′C, and 5′D) demonstrated a fibroblastic morphology and were anchored to the substrate by loose microvillous processes. The fourth clone (clone 5′B) grew in tight clusters and displayed many closely spaced microvilli, long overlapping cytoplasmic regions with well-defined junctional complexes. The parental line (R3327-5′) demonstrated a combination of both these growth patterns. E-cadherin expression was absent in clones 5′A, 5′C, and 5′D and very prominent in clone 5′B, when compared to the parental line. The absence of E-cadherin expression correlated with increased invasiveness, as measured in an in vitro invasion assay. Subcutaneous injections of clones 5′A, 5′C, and 5′D yielded lung metastases and no primary tumors at the site of inoculation while clone 5′B was tumorigenic and produced fewer lung metastases in vivo. These clones, therefore, provide a potential for studying a variety of molecules involved in prostate cancer invasion and metastasis, especially for the direct testing of the significance of E-cadherin expresssion in prostate cancer progression.

No abstract.

Background: Several protein markers, including vimentin, have been used to diagnose human melanoma. Because melanoma often has metastasized by the time of diagnosis, early markers prognostic for metastatic potential need to be identified. Commonly, vimentin is found in mesenchymal cells, and keratins are present in epithelial cells, but recent studies report coexpression of vimentin and keratin(s) in epithelial and nonepithelial neoplasms, including some melanomas. Purpose: Our purpose was to determine whether coexpression of vimentin and keratin(s) is correlated with tumor cell invasion and metastatic behavior. Methods: We evaluated nine human melanoma cell lines expressing vimentin and other markers of aggressive tumor behavior (HMB-45, S-100, HLA-ABC class I and HLA-DR class II histocompatibility antigens, and K8 and K18 keratins). Levels of K8 and K18 keratins were determined in the highly metastatic C8161 cell line, the poorly metastatic A375P line, and the moderately metastatic A375M line. To determine whether the presence of keratin affects migratory ability, we altered the conformational structure of keratin filaments in C8161 cells by transfection with a mutant K18 complementary DNA. We also determined messenger RNA levels of human type IV collagenase, an enzyme marker for invasion and metastasis. Results: In A375P cells, two-dimensional electrophoresis with Coomassie-stained gels, immunoblott-ing, and immunofluorescence staining showed no detectable levels of K8 or K18. A375M cells showed low levels of K8 and K18 by Western and Northern blotting, with a distinctive fluorescent subpopulation of cells. In comparison, K8 and K18 levels in C8161 cells were high in all cells. Type IV collagenase messenger RNA levels were lowest in A375P cells and highest in C8161 cells, correlating with invasive ability in vitro and metastatic potential in athymic nude mice. The transfectant clones C1070–10 and C1070–14 derived from the C8161 parent line showed dramatic morphological changes, disrupted keratin filaments, and decreased invasive and metastatic potential directly correlated with a reduction in migratory activity. Conclusion: These findings show a correlation between the coexpression of vimentin with K8 and K18 keratins and the invasive and metastatic behavior of three representative human melanoma cell lines. [J Natl Cancer Inst 84: 165–174, 1992]