Naira V. Margaryan

Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness

Bidirectional cellular communication is integral to both cancer progression and embryological development. In addition, aggressive tumor cells are phenotypically plastic, sharing many properties with embryonic cells. Owing to the similarities between these two types of cells, the developing zebrafish can be used as a biosensor for tumor-derived signals. Using this system, we show that aggressive melanoma cells secrete Nodal (a potent embryonic morphogen) and consequently can induce ectopic formation of the embryonic axis. We further show that Nodal is present in human metastatic tumors, but not in normal skin, and thus may be involved in melanoma pathogenesis. Inhibition of Nodal signaling reduces melanoma cell invasiveness, colony formation and tumorigenicity. Nodal inhibition also promotes the reversion of melanoma cells toward a melanocytic phenotype. These data suggest that Nodal signaling has a key role in melanoma cell plasticity and tumorigenicity, thereby providing a previously unknown molecular target for regulating tumor progression.

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.

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.

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.

Mammary Serine Protease Inhibitor (Maspin) Binds Directly to Interferon Regulatory Factor 6 IDENTIFICATION OF A NOVEL SERPIN PARTNERSHIP

Since its reported discovery in 1994, maspin (mammary serine protease inhibitor) has been characterized as a class II tumor suppressor by its ability to promote apoptosis and inhibit cell invasion. Maspin is highly expressed in normal mammary epithelial cells but reduced or absent in aggressive breast carcinomas. However, despite efforts to characterize the mechanism(s) by which maspin functions as a tumor suppressor, its molecular characterization has remained somewhat elusive. Therefore, in an attempt to identify maspin-interacting proteins and thereby gain insight into the functional pathways of maspin, we employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. IRF6 belongs to the IRF family of transcription factors, which is best known for its regulation of interferon and interferon-inducible genes following a pathogenic stimulus. Although many of the IRF family members have been well characterized, IRF6 remains poorly understood. We report that IRF6 is expressed in normal mammary epithelial cells and that it directly associates with maspin in a yeast two-hybrid system and in vitro. The interaction occurs via the conserved IRF protein association domain and is regulated by phosphorylation of IRF6. We have shown that, similar to maspin, IRF6 expression is inversely correlated with breast cancer invasiveness. We further demonstrated that the transient re-expression of IRF6 in breast cancer cells results in an increase of N-cadherin and a redistribution of vimentin commensurate with changes in cell morphology, suggestive of an epithelial-to-mesenchymal transition event. Concomitantly, we showed that maspin acts as a negative regulator of this process. These findings help to elucidate the molecular mechanisms of maspin and suggest an interactive role between maspin and IRF6 in regulating cellular phenotype, the loss of which can lead to neoplastic transformation.

Hypoxia/reoxygenation: a dynamic regulator of lysyl oxidase-facilitated breast cancer migration.

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up‐regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (β‐aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX‐dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor‐1 (HIF‐1α) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF‐1α could not up‐regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co‐localization with hypoxic regions (defined by HIF‐1α expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide‐mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency. J. Cell. Biochem. 103: 1369–1378, 2008.

Reactivation of embryonic nodal signaling is associated with tumor progression and promotes the growth of prostate cancer cells.

Nodal is a member of the transforming growth factor β (TGFβ) superfamily that directs embryonic patterning and promotes the plasticity and tumorigenicity of tumor cells, but its role in the prostate is unknown. The goal of this study was to characterize the expression and function of Nodal in prostate cancer and determine whether, like other TGFβ ligands, it modulates androgen receptor (AR) activity.

Deciphering the Signaling Events that Promote Melanoma Tumor Cell Vasculogenic Mimicry and Their Link to Embryonic Vasculogenesis: Role of the Eph Receptors

During embryogenesis, the primordial microcirculation is formed through a process known as vasculogenesis. The term “vasculogenic mimicry” has been used to describe the manner in which highly aggressive, but not poorly aggressive melanoma tumor cells express endothelial and epithelial markers and form vasculogenic‐like networks similar to embryonic vasculogenesis. Vasculogenic mimicry is one example of the remarkable plasticity demonstrated by aggressive melanoma cells and suggests that these cells have acquired an embryonic‐like phenotype. Since the initial discovery of tumor cell vasculogenic mimicry by our laboratory, we have been focusing on understanding the molecular mechanisms that regulate this process. This review will highlight recent findings identifying key signal transduction events that regulate melanoma vasculogenic mimicry and their similarity to the signal transduction events responsible for promoting embryonic vasculogenesis and angiogenesis. Specifically, this review will focus on the role of the Eph receptors and ligands in embryonic vasculogenesis, angiogenesis, and vasculogenic mimicry. Developmental Dynamics 236:3283–3296, 2007.

Interferon Regulatory Factor 6 Promotes Cell Cycle Arrest and Is Regulated by the Proteasome in a Cell Cycle-Dependent Manner

ABSTRACT Interferon regulatory factor 6 (IRF6) is a novel and unique member of the IRF family of transcription factors. IRF6 has not been linked to the regulatory pathways or functions associated with other IRF family members, and the regulation and function of IRF6 remain unknown. We recently identified a protein interaction between IRF6 and the tumor suppressor maspin. To gain insight into the biological significance of the maspin-IRF6 interaction, we examined the regulation and function of IRF6 in relation to maspin in normal mammary epithelial cells. Our results demonstrate that in quiescent cells, IRF6 exists primarily in a nonphosphorylated state. However, cellular proliferation leads to rapid IRF6 phosphorylation, resulting in proteasome-dependent IRF6 degradation. These data are supported in situ by the increased expression of IRF6 in quiescent, differentiated lobuloalveolar cells of the lactating mammary gland compared to its expression in proliferating ductal and glandular epithelial cells during pregnancy. Furthermore, the reexpression of IRF6 in breast cancer cells results in cell cycle arrest, and the presence of maspin augments this response. These data support a model in which IRF6, in collaboration with maspin, promotes mammary epithelial cell differentiation by facilitating entry into the G0 phase of the cell cycle.

Nodal as a biomarker for melanoma progression and a new therapeutic target for clinical intervention.

Nodal, an embryonic morphogen belonging to the TGF-β superfamily, is an important regulator of embryonic stem cell fate. We have recently demonstrated that Nodal is expressed significantly in aggressive melanoma. Surprisingly, expression of the Nodal coreceptor, Cripto-1, was detected in only a small fraction of the melanoma tumor cell population, indicating a primary role for Cripto-1-independent signaling of Nodal in melanoma. In this review, we discuss how regulatory factors present in an embryonic environment, such as Lefty, can downregulate Nodal expression and inhibit tumorigenicity and plasticity of melanoma cells. Our translational studies show that antibodies against Nodal are capable of repressing melanoma vasculogenic mimicry and of inducing apoptosis in melanoma tumors in an in vivo lung-colonization assay. Our previous work and ongoing studies suggest that Nodal may represent a novel diagnostic marker and therapeutic target in melanoma.