Mary J.C. Hendrix

Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry.

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 vitro under 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 versus poorly 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.

Acidic pH enhances the invasive behavior of human melanoma cells

As a consequence of poor perfusion and elevated acid production, the extracellular pH (pHex) of tumors is generally acidic. Despite this, most in vitro experiments are still performed at the relatively alkaline pHex of 7.4. This is significant, because slight changes in pHex can have profound effects on cell phenotype. In this study we examined the effects of mildly acidic conditions on the in vitro invasive 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 pHin were. 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.

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

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.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

A SIMPLE QUANTITATIVE ASSAY FOR STUDYING THE INVASIVE POTENTIAL OF HIGH AND LOW HUMAN METASTATIC VARIANTS

This paper presents a more reliable model for studying the extent of tumor cell migration and invasion in vitro. Polycarbonate filters were uniformly coated with a reconstituted basement membrane material and allowed to dry; each filter measured 0.035 mm in thickness when hydrated with media. Subsequently, the membrane-coated filters were suspended in Membrane Invasion Culture System (MICS) chambers, and high (A375M) and low (A375P) metastatic variants of human melanoma cells were seeded onto the membranes and allowed to incubate for 3 days. At the end of this period, cells were examined morphologically, and the invasive cells of both metastatic variants were collected, stained and counted microscopically. The tumor cells could be seen attached to the reconstituted basement membrane, buried within it, and forming colony-like aggregates in the matrix. It was determined that approximately twice as many A375M cells invaded the artificial biological matrix compared with the A375P cells (P less than 0.0005). Substantially more cells from each variant invaded uncoated polycarbonate filters, thus indicating the selective barrier imposed by the Matrigel. The utilization of such a reconstituted matrix for in vitro invasion studies allows one the opportunity to examine tumor cell attachment, migration and invasion of a uniform matrix over an extended period of time.

Role of intermediate filaments in migration, invasion and metastasis

SummaryThe 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.These studies have provided direct evidence linking overexpression of keratin IFs in human melanoma with increased migratory and invasive activityin vitro, which can be down-regulated by substituting dominant-negative keratin mutants. Overexpression of vimentin IFs in the breast carcinoma model leads to augmentation of motility and invasivenessin vitro, which can be transiently down-regulated by treatment with antisense oligonucleotides to vimentin. Additional experimental evidence suggests that the mechanism(s) responsible for the differential expression of metastatic properties associated with the interconverted phenotype rest(s) in the unique interaction, either direct or indirect, of IFs with specific integrins interacting with the extracellular matrix.In this review, we discuss the observations derived from the human melanoma and breast carcinoma models to address the hypothesis that the ability to coexpress vimentin and keratins confers a selective advantage to tumor cells in their interpretation of and response to signaling cues from the extracellular matrix. The ramifications of these observations are discussed with respect to the patholophysiology of the respectivein situ tumors.

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.