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.

The role of MHC class I glycoproteins in the regulation of induction of cell death in immunocytes by malignant melanoma cells

A deranged expression of MHC class I glycoproteins, characteristic of a variety of malignancies, contributes to the ability of cancer to avoid destruction by T cell-mediated immunity. An abrogation of the metastatic capacity of B16 melanoma cells has been achieved by transfecting an MHC class I-encoding vector into class I-deficient B16 melanoma clones [Gorelik, E., Kim, M., Duty, L. & Galili, U. (1993) Clin. Exp. Metastasis 11, 439–452]. We report here that the deranged expression of class I molecules by B16 melanoma cells is more than a mere acquisition of the capacity to escape immune recognition. Namely, cells of the B16 melanoma prompted splenic lymphocytes to commit death after coculture. However, a class I-expressing and nonmetastatic CL8-2 clone was found to be less potent as an inducer of apoptosis than class I-deficient and metastatic BL9 and BL12 clones. Both Thy1.2+ and Thy1.2− splenocytes underwent cell death when exposed to the class I-deficient BL9 clone. A proportion of CD4+ and CD8+ cells among splenocytes exposed to the BL9 clone was lower than that observed in a coculture with cells of the CL8-2 clone. Consistently, none of the melanoma clones studied produced a ligand to the FAS receptor (FAS-L). Thus, our results provide evidence that (i) the production of FAS-L may not be the sole mechanism by which malignant cells induce apoptosis in immunocytes, and (ii) absence of MHC class I glycoproteins plays an important role in preventing the elimination of potential effector immunocytes by tumor cells.

Molecular cloning of a human melanoma-associated chondroitin sulfate proteoglycan.

A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA encoding the entire core protein of human MCSP and provide its deduced amino acid sequence. This core protein contains an open reading frame of 2322 aa, encompassing a large extracellular domain, a hydrophobic transmembrane region, and a relatively short cytoplasmic tail. Northern blot analysis indicated that MCSP cDNA probes detect a single 8.0-kb RNA species expressed in human melanoma cell lines. In situ hybridization experiments with a segment of the MCSP coding sequence localized MCSP mRNA in biopsies prepared from melanoma skin metastases. Multiple human Northern blots with an MCSP-specific probe revealed a strong hybridization signal only with melanoma cells and not with other human cancer cells or a variety of human fetal and adult tissues. These data indicate that MCSP represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The availability of cDNAs encoding MCSP should facilitate studies designed to establish correlations between structure and function of this molecule and help to establish its role in the progression of human malignant melanoma.

A splice variant of alpha 6 integrin is associated with malignant conversion in mouse skin tumorigenesis.

The epithelial-specific integrin alpha 6 beta 4 is suprabasally expressed in benign skin tumors (papillomas) and is diffusely expressed in carcinomas associated with an increase in the proliferating compartment. Analysis of RNA samples by reverse transcriptase-PCR and DNA sequencing revealed that chemically or oncogenically induced papillomas (n = 8) expressed a single transcript of the alpha 6 subunit, identified as the alpha 6 A splice variant. In contrast, carcinomas (n = 13) expressed both alpha 6A and an alternatively spliced form, alpha 6B. Primary keratinocytes and a number of keratinocyte cell lines that vary in biological potential from normal skin, to benign papillomas, to well-differentiated slowly growing carcinomas exclusively expressed alpha 6A. However, I7, an oncogene-induced cell line that produces highly invasive carcinomas, expressed both alpha 6A and alpha 6B transcript and protein. The expression of alpha 6B in I7 cells was associated with increased attachment to a laminin matrix compared to cell lines exclusively expressing alpha 6A. Furthermore, introduction of an alpha 6B expression vector into a papilloma cell line expressing alpha 6A increased laminin attachment. When a papilloma cell line was converted to an invasive carcinoma by introduction of the v-fos oncogene, the malignant cells expressed both alpha 6A and alpha 6B, while the parent cell line and cells transduced with v-jun or c-myc, which retained the papilloma phenotype, expressed only alpha 6A. Comparative analysis of alpha 6B expression in cell lines and their derived tumors indicate that alpha 6B transcripts are more abundant in tumors than cell lines, and alpha 6B is expressed to a greater extent in poorly differentiated tumors. These results establish a link between malignant conversion and invasion of squamous tumor cells and the regulation of transcript processing of the alpha 6 beta 4 integrin.

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.

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.

A subset of skin tumor modifier loci determines survival time of tumor-bearing mice

Studies of mouse models of human cancer have established the existence of multiple tumor modifiers that influence parameters of cancer susceptibility such as tumor multiplicity, tumor size, or the probability of malignant progression. We have carried out an analysis of skin tumor susceptibility in interspecific Mus musculus/Mus spretus hybrid mice and have identified another seven loci showing either significant (six loci) or suggestive (one locus) linkage to tumor susceptibility or resistance. A specific search was carried out for skin tumor modifier loci associated with time of survival after development of a malignant tumor. A combination of resistance alleles at three markers [D6Mit15 (Skts12), D7Mit12 (Skts2), and D17Mit7 (Skts10)], all of which are close to or the same as loci associated with carcinoma incidence and/or papilloma multiplicity, is significantly associated with increased survival of mice with carcinomas, whereas the reverse combination of susceptibility alleles is significantly linked to early mortality caused by rapid carcinoma growth (χ2 = 25.22; P = 5.1 × 10−8). These data indicate that host genetic factors may be used to predict carcinoma growth rate and/or survival of individual backcross mice exposed to the same carcinogenic stimulus and suggest that mouse models may provide an approach to the identification of genetic modifiers of cancer survival in humans.

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.

Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1

It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G1/S checkpoint and maintain high levels of p27KIP1 mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27KIP1 is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin α2β1, which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.

Structure of melanoma inhibitory activity protein, a member of a recently identified family of secreted proteins

Melanoma inhibitory activity (MIA) is a 12-kDa protein that is secreted from both chondrocytes and malignant melanoma cells. MIA has been reported to have effects on cell growth and adhesion, and it may play a role in melanoma metastasis and cartilage development. We report the 1.4-Å crystal structure of human MIA, which consists of an Src homology 3 (SH3)-like domain with N- and C-terminal extensions of about 20 aa each. The N- and C-terminal extensions add additional structural elements to the SH3 domain, forming a previously undescribed fold. MIA is a representative of a recently identified family of proteins and is the first structure of a secreted protein with an SH3 subdomain. The structure also suggests a likely protein interaction site and suggests that, unlike conventional SH3 domains, MIA does not recognize polyproline helices.

Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and 2B) was partially inhibited by rapamycin, a blocker of some of the signals mediated by IL-2R, but not by cyclosporin A, FK-506, and prednisone, which suppress signals mediated by the T-cell receptor. All the cell lines expressed on their surface the high-affinity IL-2R (alpha, beta, and gamma c chains). They showed basal, constitutive phosphorylation, and coassociation of Jak3, STAT5, and STAT3. Weak basal phosphorylation of IL-2R gamma c was also detected. In regard to SzS, peripheral blood mononuclear cells from 10 of 14 patients showed basal phosphorylation of Jak3, accompanied by phosphorylation of STAT5 in 9 patients, and STAT3 in 4 patients. However, in vitro overnight culture of SzS cells without exogenous cytokines resulted in markedly decreased Jak3 and STAT5 phosphorylation, which could be reversed by stimulation with IL-2. This indicates that the basal phosphorylation of Jak3 and STAT5 in freshly isolated SzS cells is induced rather than constitutive. The basal activation of the Jak/STAT pathway involved in IL-2R signal transduction in ALCL and SzS cells reported here suggests that this pathway may play a role in the pathogenesis of cutaneous T-cell lymphomas, although the mechanism (induced versus constitutive) may vary between different lymphoma types.

The melanoma differentiation associated gene mda-7 suppresses cancer cell growth.

Cancer is a disease characterized by defects in growth control, and tumor cells often display abnormal patterns of cellular differentiation. The combination of recombinant human fibroblast interferon and the antileukemic agent mezerein corrects these abnormalities in cultured human melanoma cells resulting in irreversible growth arrest and terminal differentiation. Subtraction hybridization identifies a melanoma differentiation associated gene (mda-7) with elevated expression in growth arrested and terminally differentiated human melanoma cells. Colony formation decreases when mda-7 is transfected into human tumor cells of diverse origin and with multiple genetic defects. In contrast, the effects of mda-7 on growth and colony formation in transient transfection assays with normal cells, including human mammary epithelial, human skin fibroblast, and rat embryo fibroblast, is quantitatively less than that found with cancer cells. Tumor cells expressing elevated mda-7 display suppression in monolayer growth and anchorage independence. Infection with a recombinant type 5 adenovirus expressing antisense mda-7 eliminates mda-7 suppression of the in vitro growth and transformed phenotype. The ability of mda-7 to suppress growth in cancer cells not expressing or containing defects in both the retinoblastoma (RB) and p53 genes indicates a lack of involvement of these critical tumor suppressor elements in mediating mda-7-induced growth inhibition. The lack of protein homology of mda-7 with previously described growth suppressing genes and the differential effect of this gene on normal versus cancer cells suggests that mda-7 may represent a new class of cancer growth suppressing genes with antitumor activity.

A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.

Changes in expression of putative antigens encoded by pigment genes in mouse melanomas at different stages of malignant progression.

Cutaneous melanomas of Tyr-SV40E transgenic mice (mice whose transgene consists of the tyrosinase promoter fused to the coding regions of simian virus 40 early genes) strikingly resemble human melanomas in their development and progression. Unlike human melanomas, the mouse tumors all arise in genetically identical individuals, thereby better enabling expression of specific genes to be characterized in relation to advancing malignancy. The products of pigment genes are of particular interest because peptides derived from these proteins have been reported to function as autoantigens with immunotherapeutic potential in some melanoma patients. However, the diminished pigmentation characteristic of many advanced melanomas raises the possibility that some of the relevant products may no longer be expressed in the most malignant cells. We have therefore investigated the contributions of several pigment genes in melanotic vs. relatively amelanotic components of primary and metastatic mouse melanomas. The analyses reveal marked differences within and among tumors in levels of mRNAs and proteins encoded by the wild-type alleles at the albino, brown, slaty, and silver loci. Tyrosinase (the protein encoded by the albino locus) was most often either absent or undetectable as melanization declined. The protein encoded by the slaty locus (tyrosinase-related protein 2) was the only one of those tested that was clearly present in all the tumor samples. These results suggest that sole reliance on targeting tyrosinase-based antigens might selectively favor survival of more malignant cells, whereas targeting the ensemble of the antigens tested might contribute toward a more inclusive and effective antimelanoma strategy.

A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.