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.

HLA-G expression in melanoma: A way for tumor cells to escape from immunosurveillance

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library

Cancer/testis (CT) antigens—immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types—are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5′ end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.

Antibody-mediated inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host

Many insects feed on blood or tissue from mammalian hosts. One potential strategy for the control of these insects is to vaccinate the host with antigens derived from the insect. The larvae of the fly Lucilia cuprina feed on ovine tissue and tissue fluids causing a cutaneous myiasis associated with considerable host morbidity and mortality. A candidate vaccine antigen, peritrophin 95, was purified from the peritrophic membrane, which lines the gut of these larvae. Serum from sheep vaccinated with peritrophin 95 inhibited growth of first-instar L. cuprina larvae that fed on this serum. Growth inhibition was probably caused by antibody-mediated blockage of the normally semipermeable peritrophic membrane and the subsequent development of an impervious layer of undefined composition on the gut lumen side of the peritrophic membrane that restricted access of nutrients to the larvae. The amino acid sequence of peritrophin 95 was determined by cloning the DNA complementary to its mRNA. The deduced amino acid sequence codes for a secreted protein containing a distinct Cys-rich domain of 317 amino acids followed by a mucin-like domain of 139 amino acids. The Cys-rich domain may be involved in binding chitin. This report describes a novel immunological strategy for the potential control of L. cuprina larvae that may have general application to the control of other insect pests.

Comparison of fusion phage libraries displaying VH or single-chain Fv antibody fragments derived from the antibody repertoire of a vaccinated melanoma patient as a source of melanoma-specific targeting molecules

A single-chain Fv (scFv) fusion phage library derived from random combinations of VH and VL (variable heavy and light chains) domains in the antibody repertoire of a vaccinated melanoma patient was previously used to isolate clones that bind specifically to melanoma cells. An unexpected finding was that one of the clones encoded a truncated scFv molecule with most of the VL domain deleted, indicating that a VH domain alone can exhibit tumor-specific binding. In this report a VH fusion phage library containing VH domains unassociated with VL domains was compared with a scFv fusion phage library as a source of melanoma-specific clones; both libraries contained the same VH domains from the vaccinated melanoma patient. The results demonstrate that the clones can be isolated from both libraries, and that both libraries should be used to optimize the chance of isolating clones binding to different epitopes. Although this strategy has been tested only for melanoma, it is also applicable to other cancers. Because of their small size, human origin and specificity for cell surface tumor antigens, the VH and scFv molecules have significant advantages as tumor-targeting molecules for diagnostic and therapeutic procedures and can also serve as probes for identifying the cognate tumor antigens.

Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma

We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte–macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.

A CDKN2-like polymorphism in Xiphophorus LG V is associated with UV-B-induced melanoma formation in platyfish–swordtail hybrids

The genetic basis of spontaneous melanoma formation in spotted dorsal (Sd) Xiphophorus platyfish–swordtail hybrids has been studied for decades, and is adequately explained by a two-gene inheritance model involving a sex-linked oncogene, Xmrk, and an autosomal tumor suppressor, DIFF. The Xmrk oncogene encodes a receptor tyrosine kinase related to EGFR; the nature of the DIFF tumor suppressor gene is unknown. We analyzed the genetic basis of UV-B-induced melanoma formation in closely related, spotted side platyfish–swordtail hybrids, which carry a different sex-linked pigment pattern locus, Sp. We UV-irradiated spotted side Xiphophorus platyfish–swordtail backcross hybrids to induce melanomas at frequencies 6-fold higher than occur spontaneously in unirradiated control animals. To identify genetic determinants of melanoma susceptibility in this UV-inducible Xiphophorus model, we genotyped individual animals from control and UV-irradiated experimental regimes using allozyme and DNA restriction fragment length polymorphisms and tested for joint segregation of genetic markers with pigmentation phenotype and UV-induced melanoma formation. Joint segregation results show linkage of a CDKN2-like DNA polymorphism with UV-B-induced melanoma formation in these hybrids. The CDKN2-like polymorphism maps to Xiphophorus linkage group V and exhibits recombination fractions with ES1 and MDH2 allozyme markers consistent with previous localization of the DIFF tumor suppressor locus. Our results indicate that the CDKN2-like sequence we have cloned and mapped is a candidate for the DIFF tumor suppressor gene.

Novel Ras antagonist blocks human melanoma growth

During past decades, knowledge of melanoma biology has increased considerably. Numerous therapeutic modalities based on this knowledge are currently under investigation. Advanced melanoma, nevertheless, remains a prime example of poor treatment response that may, in part, be the consequence of activated N-Ras oncoproteins. Besides oncogenic Ras, wild-type Ras gene products also play a key role in receptor tyrosine kinase growth factor signaling, known to be of importance in oncogenesis and tumor progression of a variety of human neoplasms, including malignant melanoma; therefore, it is reasonable to speculate that a pharmacological approach that curtails Ras activity may represent a sensible approach to inhibit melanoma growth. To test this concept, the antitumor activity of S-trans, trans-farnesylthiosalicylic acid (FTS), a recently discovered Ras antagonist that dislodges Ras from its membrane-anchoring sites, was evaluated. The antitumor activity of FTS was assessed both in vitro and in vivo in two independent SCID mouse xenotransplantation models of human melanoma expressing either wild-type Ras (cell line 518A2) or activated Ras (cell line 607B). We show that FTS (5–50 μM) reduces the amounts of activated N-Ras and wild-type Ras isoforms both in human melanoma cells and Rat-1 fibroblasts, interrupts the Ras-dependent extracellular signal-regulated kinase in melanoma cells, inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells in vitro and reverses their transformed phenotype. FTS also causes a profound and statistically significant inhibition of 518A2 (82%) and 607B (90%) human melanoma growth in SCID mice without evidence of drug-related toxicity. Our findings stress the notion that FTS may qualify as a novel and rational treatment approach for human melanoma and possibly other tumors that either carry activated ras genes or rely on Ras signal transduction more heavily than nonmalignant cells.

Natural killer cell lines kill autologous β2-microglobulin-deficient melanoma cells: Implications for cancer immunotherapy

Cancer vaccines used to generate specific cytotoxic T lymphocytes are not effective against tumor cells that have lost or suppressed expression of their class I major histocompatibility complex proteins. This loss is common in some cancers and particularly in metastatic lesions. We show that β2-microglobulin-deficient class I-negative melanoma variants derived from patients undergoing specific T cell therapy are lysed by heterologous as well as autologous natural killer (NK) lines and clones, but not by specific T cells. Moreover, the minor NK cell fraction but not the major T cell fraction derived from heterologous lymphokine activated killer cells kills those tumor cell lines. ICAM-1 expression by the different class I protein deficient tumors was correlated with their sensitivity to lysis by NK cells. Adoptive autologous NK therapy may be an important supplement to consider in the design of new cancer immunotherapies.

Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: A projected basis for immunotherapy

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase–PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Δ1b and Δ1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Δ1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Δ1d reached 4.0% as compared with 0.8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Δ1b and Δ1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.

Inhibition of Rho Is Required for cAMP-induced Melanoma Cell Differentiation

Up-regulation of the cAMP pathway by forskolin or α-melanocyte stimulating hormone induces melanocyte and melanoma cell differentiation characterized by stimulation of melanin synthesis and dendrite development. Here we show that forskolin-induced dendricity is associated to a disassembly of actin stress fibers. Since Rho controls actin organization, we studied the role of this guanosine triphosphate (GTP)-binding protein in cAMP-induced dendrite formation. Clostridium botulinum C3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia coli toxin cytotoxic necrotizing factor-1 (CNF-1), which activates Rho and the expression of a constitutively active Rho mutant, blocked forskolin-induced dendrite outgrowth. In addition, overexpression of a constitutively active form of the Rho target p160 Rho-kinase (P160ROCK) prevented the dendritogenic effects of cAMP. Our results suggest that inhibition of Rho and of its target p160ROCK are required events for cAMP-induced dendrite outgrowth in B16 cells. Furthermore, we present evidence that Rho is involved in the regulation of melanogenesis. Indeed, Rho inactivation enhanced the cAMP stimulation of tyrosinase gene transcription and protein expression, while Rho constitutive activation impaired these cAMP-induced effects. This reveals that, in addition to controlling dendricity, Rho also participates in the regulation of melanin synthesis by cAMP.

Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen MatricesV⃞

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including β1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both β1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only β1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-β1 and anti-α2 integrin mAbs, whereas mAbs blocking CD44, α3, α5, α6, or αv integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of β1 integrins was not restored via CD44. Because α2β1-mediated migration was neither synergized nor replaced by CD44–HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.

Subcellular Localization of Myosin-V in the B16 Melanoma Cells, a Wild-type Cell Line for the dilute Gene

The discovery that the dilute gene encodes a class V myosin led to the hypothesis that this molecular motor is involved in melanosome transport and/or dendrite outgrowth in mammalian melanocytes. The present studies were undertaken to gain insight into the subcellular distribution of myosin-V in the melanoma cell line B16-F10, which is wild-type for the dilute gene. Immunofluorescence studies showed some degree of superimposed labeling of myosin-V with melanosomes that predominated at the cell periphery. A subcellular fraction highly enriched in melanosomes was also enriched in myosin-V based on Western blot analysis. Immunoelectron microscopy showed myosin-V labeling associated with melanosomes and other organelles. The stimulation of B16 cells with the α-melanocyte-stimulating hormone led to a significant increase in myosin-V expression. This is the first evidence that a cAMP signaling pathway might regulate the dilute gene expression. Immunofluorescence also showed an intense labeling of myosin-V independent of melanosomes that was observed within the dendrites and at the perinuclear region. Although the results presented herein are consistent with the hypothesis that myosin-V might act as a motor for melanosome translocation, they also suggest a broader cytoplasmic function for myosin-V, acting on other types of organelles or in cytoskeletal dynamics.

An autologous oral DNA vaccine protects against murine melanoma

We demonstrated that peripheral T cell tolerance toward murine melanoma self-antigens gp100 and TRP-2 can be broken by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to minigenes encoding peptide epitopes gp10025–33 and TRP-2181–188. These epitopes contain dominant anchor residues for MHC class I antigen alleles H-2Db and H-2Kb, respectively. The DNA vaccine was delivered by oral gavage by using an attenuated strain of Salmonella typhimurium as carrier. Tumor-protective immunity was mediated by MHC class I antigen-restricted CD8+ T cells that secreted TH1 cytokine IFN-γ and induced tumor rejection and growth suppression after a lethal challenge with B16G3.26 murine melanoma cells. Importantly, the protective immunity induced by this autologous DNA vaccine against murine melanoma cells was at least equal to that achieved through xenoimmunization with the human gp10025–33 peptide, which differs in its three NH2-terminal amino acid residues from its murine counterpart and was previously reported to be clearly superior to an autologous vaccine in inducing protective immunity. The presence of ubiquitin upstream of the minigene proved to be essential for achieving this tumor-protective immunity, suggesting that effective antigen processing and presentation may make it possible to break peripheral T cell tolerance to a self-antigen. This vaccine design might prove useful for future rational designs of other recombinant DNA vaccines targeting tissue differentiation antigens expressed by tumors.

Melanoma angiogenesis and metastasis modulated by ribozyme targeting of the secreted growth factor pleiotrophin

Clinical and experimental evidence suggests that spreading of malignant cells from a localized tumor (metastasis) is directly related to the number of microvessels in the primary tumor. This tumor angiogenesis is thought to be mediated by tumor-cell-derived growth factors. However, most tumor cells express a multitude of candidate angiogenesis factors and it is difficult to decipher which of these are rate-limiting factors in vivo. Herein we use ribozyme targeting of pleiotrophin (PTN) in metastatic human melanoma cells to assess the significance of this secreted growth factor for angiogenesis and metastasis. As a model we used human melanoma cells (1205LU) that express high levels of PTN and metastasize from subcutaneous tumors to the lungs of experimental animals. In these melanoma cells, we reduced PTN mRNA and growth factor activity by transfection with PTN-targeted ribozymes and generated cell lines expressing different levels of PTN. We found that the reduction of PTN does not affect growth of the melanoma cells in vitro. In nude mice, however, tumor growth and angiogenesis were decreased in parallel with the reduced PTN levels and apoptosis in the tumors was increased. Concomitantly, the metastatic spread of the tumors from the subcutaneous site to the lungs was prevented. These studies support a direct link between tumor angiogenesis and metastasis through a secreted growth factor and identify PTN as a candidate factor that may be rate-limiting for human melanoma metastasis.