Elisabeth A. Seftor

Reprogramming metastatic tumour cells with embryonic microenvironments

Aggressive tumour cells share many characteristics with embryonic progenitors, contributing to the conundrum of tumour cell plasticity. Recent studies using embryonic models of human stem cells, the zebrafish and the chick have shown the reversion of the metastatic phenotype of aggressive melanoma cells, and revealed the convergence of embryonic and tumorigenic signalling pathways, which may help to identify new targets for therapeutic intervention. This Review will summarize the embryonic models used to reverse the metastatic melanoma phenotype, and highlight the prominent signalling pathways that have emerged as noteworthy targets for future consideration

Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells.

Embryonic stem cells sustain a microenvironment that facilitates a balance of self-renewal and differentiation. Aggressive cancer cells, expressing a multipotent, embryonic cell-like phenotype, engage in a dynamic reciprocity with a microenvironment that promotes plasticity and tumorigenicity. However, the cancer-associated milieu lacks the appropriate regulatory mechanisms to maintain a normal cellular phenotype. Previous work from our laboratory reported that aggressive melanoma and breast carcinoma express the embryonic morphogen Nodal, which is essential for human embryonic stem cell (hESC) pluripotency. Based on the aberrant expression of this embryonic plasticity gene by tumor cells, this current study tested whether these cells could respond to regulatory cues controlling the Nodal signaling pathway, which might be sequestered within the microenvironment of hESCs, resulting in the suppression of the tumorigenic phenotype. Specifically, we discovered that metastatic tumor cells do not express the inhibitor to Nodal, Lefty, allowing them to overexpress this embryonic morphogen in an unregulated manner. However, exposure of the tumor cells to a hESC microenvironment (containing Lefty) leads to a dramatic down-regulation in their Nodal expression concomitant with a reduction in clonogenicity and tumorigenesis accompanied by an increase in apoptosis. Furthermore, this ability to suppress the tumorigenic phenotype is directly associated with the secretion of Lefty, exclusive to hESCs, because it is not detected in other stem cell types, normal cell types, or trophoblasts. The tumor-suppressive effects of the hESC microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored therapeutic modalities for cancer treatment.

Lysyl Oxidase Regulates Breast Cancer Cell Migration and Adhesion through a Hydrogen Peroxide–Mediated Mechanism

We have previously shown that lysyl oxidase (LOX) mRNA is up-regulated in invasive breast cancer cells and that catalytically active LOX facilitates in vitro cell invasion. Here we validate our in vitro studies by showing that LOX expression is up-regulated in distant metastatic breast cancer tissues compared with primary cancer tissues. To elucidate the mechanism by which LOX facilitates cell invasion, we show that catalytically active LOX regulates in vitro motility/migration and cell-matrix adhesion formation. Treatment of the invasive breast cancer cell lines, Hs578T and MDA-MB-231, with beta-aminopropionitrile (betaAPN), an irreversible inhibitor of LOX catalytic activity, leads to a significant decrease in cell motility/migration and adhesion formation. Conversely, poorly invasive MCF-7 cells expressing LOX (MCF-7/LOX32-His) showed an increase in migration and adhesion that was reversible with the addition of betaAPN. Moreover, a decrease in activated focal adhesion kinase (FAK) and Src kinase, key proteins involved in adhesion complex turnover, was observed when invasive breast cancer cells were treated with betaAPN. Additionally, FAK and Src activation was increased in MCF-7/LOX32-His cells, which was reversible on betaAPN treatment. Hydrogen peroxide was produced as a by-product of LOX activity and the removal of hydrogen peroxide by catalase treatment in invasive breast cancer cells led to a dose-dependent loss in Src activation. These results suggest that LOX facilitates migration and cell-matrix adhesion formation in invasive breast cancer cells through a hydrogen peroxide-mediated mechanism involving the FAK/Src signaling pathway. These data show the need to target LOX for treatment of aggressive breast cancer.

The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation.

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.

Molecular Pathways: Vasculogenic Mimicry in Tumor Cells: Diagnostic and Therapeutic Implications

Tumor cell vasculogenic mimicry (VM) describes the functional plasticity of aggressive cancer cells forming de novo vascular networks, thereby providing a perfusion pathway for rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with endothelial-lined vasculature. The underlying induction of VM seems to be related to hypoxia, which may also promote the plastic, transendothelial phenotype of tumor cells capable of VM. Since its introduction in 1999 as a novel paradigm for melanoma tumor perfusion, many studies have contributed new insights into the underlying molecular pathways supporting VM in a variety of tumors, including melanoma, glioblastoma, carcinomas, and sarcomas. In particular, critical VM-modulating genes are associated with vascular (VE-cadherin, EphA2, VEGF receptor 1), embryonic and/or stem cell (Nodal, Notch4), and hypoxia-related (hypoxia-inducible factor, Twist1) signaling pathways. Each of these pathways warrants serious scrutiny as potential therapeutic, vascular targets, and diagnostic indicators of plasticity, drug resistance, and the aggressive metastatic phenotype. Clin Cancer Res; 18(10); 2726–32. ©2012 AACR.

Biological functions of maspin.

Maspin (Mammary Serine Protease Inhibitor) was first reported in 1994 as a serpin with tumor suppressive properties. Maspin was initially isolated through subtractive hybridization and differential display analysis as a 42‐kDa protein that is expressed in normal mammary epithelial cells but reduced or absent in breast carcinomas (Zou et al., 1994 ). Further research led to maspins characterization as a class II tumor suppressor based on its ability to inhibit cell invasion, promote apoptosis, and inhibit angiogenesis (Sheng et al., 1996 ; Zhang et al., 2000b ; Jiang et al., 2002 ). Since then, efforts have been made to characterize maspins tumor suppressive mechanisms. In particular, researchers have studied maspin localization, the regulation of maspin expression, and more recently, maspin protein interactions. By elucidating these mechanisms, researchers are beginning to understand the complex, pleiotropic nature of maspin and the pathways through which maspin exerts its tumor suppressive properties. These new findings not only further enhance our understanding of cancer biology but also provide an avenue to develop maspins potential as a diagnostic marker for cancer progression, and as a potentially powerful therapeutic agent in the fight against breast cancer. J. Cell. Physiol. 209: 617–624, 2006.

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 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.

Focal Adhesion Kinase Promotes the Aggressive Melanoma Phenotype

Malignant melanoma continues to remain a significant health threat, with death often occurring as a result of metastasis. The metastatic phenotype typically is characterized by augmented tumor cell invasion and migration in addition to tumor cell plasticity as shown by vasculogenic mimicry. Therefore, understanding the molecular mechanisms that promote an aggressive phenotype is essential to predicting the likelihood of metastasis at a stage when intervention may be possible. This study focuses on the role of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase important for many cellular processes, including cell survival, invasion, and migration. We found FAK to be phosphorylated on its key tyrosine residues, Tyr397 and Tyr576, in only aggressive uveal and cutaneous melanoma cells, which correlates with their increased invasion, migration, and vasculogenic mimicry plasticity. Additionally, we confirmed the presence of FAK phosphorylated on Tyr397 and Tyr576 in both cutaneous and uveal melanoma tumors in situ. Examination of a functional role for FAK in aggressive melanoma revealed that disruption of FAK-mediated signal transduction pathways, through the expression of FAK-related nonkinase (FRNK), results in a decrease in melanoma cell invasion, migration, and inhibition of vasculogenic mimicry. Moreover, we found that FRNK expression resulted in a down-regulation of Erk1/2 phosphorylation resulting in a decrease in urokinase activity. Collectively, these data suggest a new mechanism involved in promoting the aggressive melanoma phenotype through FAK-mediated signal transduction pathways, thus providing new insights into possible therapeutic intervention strategies.

Influence of the microenvironment on melanoma cell fate determination and phenotype

Tumor cells communicate bidirectionally with the surrounding microenvironment, sending and receiving topographical and molecular cues that direct diverse cellular phenomena, including differentiation, growth, and invasion. The microenvironment has long been acknowledged as a facilitator of melanoma progression, and recent studies have illuminated tumor-associated factors, including hypoxia and the extracellular matrix, as important mediators of melanocyte transformation and transdifferentiation. Although these findings portray the microenvironment as a perilous obstacle to the successful treatment of advanced melanomas, it is important to note that certain molecular milieus may be capitalized on as potential treatment modalities. Indeed, our group and others have elucidated the unique ability of embryonic microenvironments to normalize aggressive melanoma cells toward a more benign melanocytic phenotype. The microenvironment therefore presents a novel target for the treatment and ultimately the prevention of melanoma progression and metastasis.

Regulation of the embryonic morphogen Nodal by Notch4 facilitates manifestation of the aggressive melanoma phenotype.

Metastatic melanoma is an aggressive skin cancer associated with poor prognosis. The reactivation of the embryonic morphogen Nodal in metastatic melanoma has previously been shown to regulate the aggressive behavior of these tumor cells. During the establishment of left-right asymmetry in early vertebrate development, Nodal expression is specifically regulated by a Notch signaling pathway. We hypothesize that a similar relationship between Notch and Nodal may be reestablished in melanoma. In this study, we investigate whether cross talk between the Notch and Nodal pathways can explain the reactivation of Nodal in aggressive metastatic melanoma cells. We show a molecular link between Notch and Nodal signaling in the aggressive melanoma cell line MV3 via the activity of an RBPJ-dependent Nodal enhancer element. We show a precise correlation between Notch4 and Nodal expression in multiple aggressive cell lines but not poorly aggressive cell lines. Surprisingly, Notch4 is specifically required for expression of Nodal in aggressive cells and plays a vital role both in the balance of cell growth and in the regulation of the aggressive phenotype. In addition, Notch4 function in vasculogenic mimicry and anchorage-independent growth in vitro is due in part to Notch4 regulation of Nodal. This study identifies an important role for cross talk between Notch4 and Nodal in metastatic melanoma, placing Notch4 upstream of Nodal, and offers a potential molecular target for melanoma therapy.