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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 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.

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.

Bisindolylmaleimides (Bis) were originally described as protein kinase C inhibitors. Several studies have shown that Bis potentiate tumor necrosis factor (TNF) receptor family-mediated apoptosis in lymphoid and dendritic cells, but the inhibition of protein kinase C cannot account for these effects (Zhou, T., Song, L., Yang, P., Wang, Z., Lui, D., and Jope, R. S. (1999) Nat. Med.5, 42–48). We investigated the effect of four Bis derivatives (I, II, VIII, and IX) in human prostatic carcinoma cell lines and found that Bis IX was the most potent inducer of apoptosis under simultaneous treatment with TNF-α, agonistic anti-Fas monoclonal antibody, and TNF-related apoptosis-inducing ligand (TRAIL). Bis IX synergistically induced caspase activity in combination with apoptosis-inducing ligands and converted the phenotype of cell lines from apoptosis-resistant to -sensitive. Bis IX induced p53 accumulation in LNCaP (lymph node carcinoma of prostate), which expresses wild-type p53 that was not accompanied by the induction of p53-responsive genes, p21/WAF1, and Mdm2. Moreover, the induction of p21/WAF1 and Mdm2 by doxorubicin was abrogated by simultaneous treatment with Bis IX. These effects apparently result from general inhibition of transcription by Bis IX. We have shown by Northern blot analysis that the transcription activity of the hygromycin gene after transient transfection of pcDNA3.1-Hygro plasmid in 293 and HeLa cells was inhibited by Bis IX in a dose-dependent manner. Moreover, DNA binding activity of Bis IX was prevented by actinomycin D, suggesting that actinomycin D and Bis IX have similar mechanisms of interaction with DNA. In addition, we found that actinomycin D and Bis IX induced caspase activity to the same extent during TRAIL-mediated apoptosis. In summary, these results suggest that Bis IX potentiates TNF receptor family-mediated cell death in part as an inhibitor of transcription.

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.

Mucin is the major component of mucus and can be used as a marker for mucus secretion. The purpose of this study was to develop an in vitro method to evaluate the regulation of mucin secretion. To do this, we used a sandwiched enzyme–linked lectin assay to measure mucin secretion from isolated ferret tracheal segments. This assay entailed coating microtiter plate wells with dolichos biflorus agglutinin and detecting the bound mucin that was secreted into a buffer solution by the tracheal segments. We used this method to evaluate the secretory response to four secretagogues: prostaglandin F 2α (PGF 2α ), adenosine triphosphate (ATP), methacholine, and human neutrophil elastase (HNE). Each agent stimulated mucin secretion above baseline secretion (ATP [ p = 0.022], PGF 2α [ p = 0.009], and HNE [ p < 0.05]), and the relative potency of these secretagogues was PGF 2α ≤ ATP < MCh < HNE. We also demonstrated that there is an anatomic gradient for both constitutive and stimulated mucin secretion, with the distal tracheal segments secreting more mucin per gram of weight than the proximal segments. This fairly simple and reproducible technique can be used to evaluate the regulation of mucin secretion in the airway and to assess the efficacy of agents that might alter the secretory response. (Otolaryngol Head Neck Surg 1997;117:480-6.)

Steroid-induced glaucoma is believed to result from increased aqueous outflow resistance and evidence suggests that this is the result of an excess accumulation of extracellular matrix components. This accumulation could result from an imbalance in the natural turnover of these components.