Goos N.P. van Muijen

Cancer/testis-associated genes: identification, expression profile, and putative function.

Cancer/testis‐associated genes (CTAs) are a subgroup of tumor antigens with a restricted expression in testis and malignancies. During the last decade, many of these immunotherapy candidate genes have been discovered using various approaches. Most of these genes are localized on the X‐chromosome, often as multigene families. Methylation status seems to be the main, but not the only regulator of their specific expression pattern. In testis, CTAs are exclusively present in cells of the germ cell lineage, though there is a lot of variation in the moment of expression during different stages of sperm development. Likewise, there is also a lot of heterogeneity in the expression of CTAs in melanoma samples. Clues regarding functionality of CTAs for many of these proteins point to a role in cell cycle regulation or transcriptional control. Better insights in the function of these genes may shed light on the link between spermatogenesis and tumor growth and could be of use in anti‐tumor therapies. This review outlines the CTA family and focuses on their expression and putative function during male germ cell development and melanocytic tumor progression.

Activated Leukocyte Cell Adhesion Molecule/CD166, a marker of tumor progression in primary malignant melanoma of the skin

Expression of activated leukocyte cell adhesion molecule (ALCAM)/CD166 correlates with the aggregation and metastatic capacity of human melanoma cell lines (Am J Pathol 1998, 152:805-813). Immunohistochemistry on a series of human melanocytic lesions reveals that ALCAM expression correlates with melanoma progression. Most nevi (34/38) and all thin melanomas studied (Clark levels I and II) did not express ALCAM. In contrast, immunoreactivity was detected in the invasive, vertical growth phase of 2 of the 13 Clark level III lesions tested. The fraction of positive lesions further increased in Clark level IV (13/19) and in Clark level V (4/4) lesions. ALCAM expression was exclusively detectable in the vertical growth phase of the primary tumor. In melanoma metastases, approximately half of the lesions tested (13/28) were ALCAM positive. According to the Breslow-thickness, ALCAM expression was observed in less than 10% of the lesions that were thinner than 1.5 mm and in over 70% of the lesions that were thicker than 1.5 mm. Our results strongly suggest that ALCAM plays an important role in melanocytic tumor progression and depict it as a new molecular marker for neoplastic progression of primary human melanoma.

Differentiation-related changes of cytokeratin expression in cultured keratinocytes and in fetal, newborn, and adult epidermis

Cytokeratin expression in differentiating cultured foreskin keratinocytes was studied using chain-specific anti-cytokeratin monoclonal antibodies directed against cytokeratins 4, 8, 10, 13, 18, and 19, respectively. Keratinocytes were cultured at low Ca2+ concentration (0.06 mM) to repress differentiation. At confluency, the cells were switched to high Ca2+ concentration (1.6 mM) to induce differentiation. Cells were harvested 0, 3, 8, 16, 24, 48, and 72 h after the switch. Keratinocytes cultured throughout at high Ca2+ concentration were also harvested. Immunoblots of cytokeratin preparations isolated from these cultures showed that cytokeratins 4, 13, and 19 were not present in nondifferentiating keratinocytes but could be detected from about 16 h after the Ca2+ switch. Immunohistochemical studies were performed on frozen sections of cell sheets incubated with anti-cytokeratin and anti-vimentin. Expression of cytokeratins 4, 13, and 19 was seen in superficial cells. Cytokeratin 10 was locally present in suprabasal and superficial cells. Vimentin was present in 40-70% of the basal cells and in only a few differentiating keratinocytes. Expression of cytokeratins 8 and 18 could not be detected. The same antibodies were also used to stain sections from fetal (15, 20, and 29 weeks), newborn (40 weeks), and mature (5 and 75 years) epidermis. In the 15-week-old epidermis, basal cells were positive for cytokeratins 8 and 19 and locally for cytokeratin 4; intermediate cells expressed cytokeratins 4, 10, 13, and 19; and the periderm contained cytokeratins 4, 8, 13, 18, and 19. In the 20-week-old epidermis, cytokeratin 4 had disappeared from the basal cell layer and cytokeratin 19 was present only locally; in the intermediate cell layer, cytokeratins 4 and 19 had disappeared; and in the periderm, the expression of the cytokeratins studied was the same as that in the 15-week-old epidermis. The basal cells of the 29-week-old fetal epidermis, the newborn epidermis, and the mature epidermis are negative with all antibodies tested, except for some scattered cells in the fetal and newborn skin, presumably Merkel cells, that were positive for cytokeratins 8, 18, and 19. Suprabasal cells in all specimens were positive only for cytokeratin 10. With respect to the cytokeratins studied, our results show that cultured differentiating keratinocytes resemble the suprabasal cells of early fetal epidermis. Basal cells of cultured keratinocytes resemble the basal cells of late fetal, newborn, and adult epidermis and therefore support previous observations.

Stromal cell-derived factor-1alpha promotes melanoma cell invasion across basement membranes involving stimulation of membrane-type 1 matrix metalloproteinase and Rho GTPase activities.

Tissue invasion by tumor cells involves their migration across basement membranes through activation of extracellular matrix degradation and cell motility mechanisms. Chemokines binding to their receptors provide chemotactic cues guiding cells to specific tissues and organs; they therefore could potentially participate in tumor cell dissemination. Melanoma cells express CXCR4, the receptor for the chemokine stromal cell-derived factor-1α (SDF-1α). Using Matrigel as a model, we show that SDF-1α promotes invasion of melanoma cells across basement membranes. Stimulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) activity by SDF-1α was necessary for invasion, involving at least up-regulation in the expression of this metalloproteinase, as detected in the highly metastatic BLM melanoma cell line. Moreover, SDF-1α triggered the activation of the GTPases RhoA, Rac1, and Cdc42 on BLM cells, and expression of dominant-negative forms of RhoA and Rac1, but not Cdc42, substantially impaired the invasion of transfectants in response to SDF-1α, as well as the increase in MT1-MMP expression. Furthermore, CXCR4 expression on melanoma cells was notably augmented by transforming growth factor-β1, a Matrigel component, whereas anti-transforming growth factor-β antibodies inhibited increases in CXCR4 expression and melanoma cell invasion toward SDF-1α. The identification of SDF-1α as a potential stimulatory molecule for MT1-MMP as well as for RhoA and Rac1 activities during melanoma cell invasion, associated with an up-regulation in CXCR4 expression by interaction with basement membrane factors, could contribute to better knowledge of mechanisms stimulating melanoma cell dissemination.

Expression of integrin αvβ3 correlates with activation of membrane-type matrix metalloproteinase-1 (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) in human melanoma cells in vitro and in vivo

Activation of matrix metalloproteinase‐2 (MMP‐2) is mediated by binding to the complex of membrane‐type matrix metalloproteinase‐1 (MT1‐MMP) with tissue inhibitor of MMP‐2 (TIMP‐2) on the cell surface. Binding of MMP‐2 to integrin αvβ3 has been implicated in presenting activated MMP‐2 on the cell surface of invasive cells, but interactions with the MT1‐MMP‐TIMP‐2 system have not been considered. Therefore, we studied the expression and interaction of MT1‐MMP, MMP‐2 and TIMP‐2 in the αvβ3‐negative melanoma cell line BLM and in its β3‐transfected, αvβ3‐expressing counterpart BLM‐β3, both on cell lines and in xenografts. Total expression levels of MMP‐2, MT1‐MMP and TIMP‐2 did not differ markedly between the αvβ3‐negative and αvβ3‐positive cells. Remarkable differences, however, exist in the presence of active MMP‐2 and MT1‐MMP. Zymography on cell lysates revealed that active MMP‐2 was restricted to αvβ3‐positive cell line and clearly accumulated in xenografts derived from the BLM‐β3 cells, confirming the relevance of this integrin for MMP‐2 function. Western blotting of cell lysates showed that processing of proMT1‐MMP to the activated form was enhanced in BLM‐β3. The ratio of active and inactive MT1‐MMP was 3‐fold higher in the β3‐transfectants. Immunofluorescence double‐labeling followed by confocal laser microscopy showed co‐localization of MT1‐MMP and αvβ3 on BLM‐β3 cells. In xenografts from BLM‐β3 cells, active MT1‐MMP was markedly increased. Our results demonstrate that expression of αvβ3 in cell lines and xenografts was accompanied by an accumulation of active MT1‐MMP and MMP‐2. Furthermore, MT1‐MMP and αvβ3 are co‐localized on the cell membrane of tumor cells. These findings suggest that activated MT1‐MMP co‐localized with αvβ3 may be involved in activation of αvβ3‐bound MMP‐2. Int. J. Cancer 87:12–19, 2000.

Expression of gp100, MART-1, tyrosinase, and S100 in paraffin-embedded primary melanomas and locoregional, lymph node, and visceral metastases: implications for diagnosis and immunotherapy. A study conducted by the EORTC Melanoma Cooperative Group.

With the recent availability of novel antibodies against melanoma antigens tyrosinase and MART‐1, it is important to validate their usefulness in pathology practice and in screening patients for immunotherapy treatment. In the present study conducted by the Melanoma Cooperative Group of the European Organization for Research and Treatment of Cancer (EORTC‐MCG), immunohistochemical staining for gp100 (antibodies NKI‐beteb and HMB‐45), MART‐1 (A103), tyrosinase (T311), and S100 (S100) was compared on formalin‐fixed and paraffin‐embedded tumour lesions from 80 patients with 130 malignant melanoma lesions, comprising 44 primary tumours, 18 locoregional metastases, 41 lymph node metastases, and 27 visceral metastases from the lung, liver, and brain. A score between 0 and 5 was allocated to each immunohistochemically stained section. These scores were evaluated in a statistical analysis. S100 was by far the most sensitive marker in all four types of lesions tested. Apart from a significantly better performance for T311 in primary melanomas compared with HMB‐45, no significant differences were observed between the four remaining antigens tested. Three settings were next investigated to determine whether the expression of melanoma antigens decreases with tumour progression. First, within the primary melanomas, only NKI‐beteb and A103 staining showed a nearly significant negative correlation with Clarks level of invasion and a similar tendency was observed for these antibodies with Breslow thickness. Second, when comparing primary melanoma–metastasis pairs from the same patient, lymph node metastases showed less staining with NKI‐beteb, HMB‐45, A103, and T311, at a level near significance. This difference was not significant when comparing the primary tumour with visceral metastases, probably due to the lower numbers of pairs. Third, regarding tumour progression from primary melanoma to locoregional, to lymph node, to visceral metastasis, a significant decrease with progression was found only for T311. The apparently stable expression of most of the melanoma antigens, and the small contribution of decreased expression in melanoma tumour progression, supports the rationale for immunotherapy based on the melanoma immunogens gp100, MART‐1, and tyrosinase Copyright

TM7XN1, A NOVEL HUMAN EGF-TM7-LIKE CDNA, DETECTED WITH MRNA DIFFERENTIAL DISPLAY USING HUMAN MELANOMA CELL LINES WITH DIFFERENT METASTATIC POTENTIAL

We have identified a novel 3845 bp cDNA differentially expressed in a human melanoma metastasis model. Northern blot analysis showed expression in the poorly and intermediately metastasizing cell lines and a marked downregulation in the highly metastatic cell lines. Using RT‐PCR expression was also seen in several other tumor cell lines and normal cell types of human origin. cDNA sequence analysis revealed an ORF of 687 amino acids containing seven putative transmembrane domains C‐terminally and a long N‐terminus. The gene was mapped to 16q13. Highest homology was observed with members of the EGF‐TM7 subfamily of the secretin/calcitonin receptor family. We propose the delineation of a subfamily of TM7 proteins, LN‐TM7, containing seven transmembrane proteins with a long N‐terminal extracellular part.

The XAGE family of cancer/testis-associated genes: alignment and expression profile in normal tissues, melanoma lesions and Ewing's sarcoma.

The existence of XAGE genes was first reported after database homology searches for PAGE‐like sequences identified 3 XAGE EST clusters. One of these clusters, XAGE‐1, has in later studies been identified as a cancer/testis‐associated gene. Here, we report the expression profiles of all 3 reported XAGE genes, as well as several splice variants of XAGE‐1, in normal human tissues, Ewings sarcoma and melanocytic tumors. We also provide the genetic structure of the corresponding genes. Moreover, by searching the databases for XAGE homologues, we identified 3 additional GAGE‐like genes. RT‐PCR studies showed frequent expression in melanoma metastases and Ewings sarcoma for 2 XAGE‐1‐derived transcripts. XAGE‐2 was expressed at lower frequency in these tissues, while XAGE‐3 was seen only in normal placenta. Due to a frameshift, the largest XAGE‐1 putative protein is far less homologous to GAGE‐like proteins than the other XAGEs. Interestingly, all GAGE‐like genes contain a large secondary open reading frame, coding for putative proteins homologues to the XAGE‐1 primary protein. The XAGE family of cancer/testis‐associated genes is located on chromosome Xp11.21‐Xp11.22. The data outline a superfamily of GAGE‐like cancer/testis antigens, consisting of at least 19 genes.

Intermediate filaments in merkel cell tumors

A series of ten Merkel cell tumors is described, with special emphasis on intermediate filament expression. The presence of cytoskeletal proteins was studied with a polyclonal antiserum directed against cytokeratin and with monoclonal antibodies against cytokeratin, neurofilament, and vimentin by the immunoperoxidase technique. Cytokeratin was demonstrated in nine of ten tumors. Neurofilament was observed in the two snap-frozen tissues tested and in three of the eight formalin-fixed, paraffin-embedded tissues. No reactivity for vimentin was found. By electron microscopy desmosomes were found to be present in all cases, while tonofilaments were found in only a few cases. neurosecretory granules, although seen in all tumors, were generally present in low numbers. The results of this study indicate that the Merkel cell tumor is a poorly differentiated small cell carcinoma that has the ability to express some neuroendocrine features.

Characterization of XAGE-1b, a short major transcript of cancer/testis-associated gene XAGE-1, induced in melanoma metastasis.

Suppression subtractive hybridization, comparing mRNA expression profiles of common nevocellular nevi and melanoma metastases, was used to identify potential markers of melanoma progression. From the metastases we isolated XAGE‐1b, a 470 bp transcript of the XAGE‐1 gene. In general, expression of XAGE‐1b was much more prominent than expression of the longer XAGE‐1 transcript, isolated from Ewings sarcoma. The XAGE‐1b open‐reading frame codes for a putative protein of 81 amino acids, harboring a functional bipartite nuclear localization signal and a C‐terminal acidic transcription‐activation‐like domain. On the nucleotide level, XAGE‐1b has a 50% homology with members of the GAGE family. However, homology between the corresponding proteins is weak. Expression of XAGE‐1b in normal tissues was mainly restricted to testis, while placenta and brain were sporadically positive. In human tumor cell lines as well as in human tumor lesions, expression was most frequently found in melanocytic tumors and Ewings sarcoma. In the different stages of melanocytic tumor progression, expression was exclusively seen in melanoma metastases (38%; n = 61), while all tested common and atypical nevi (n = 10) as well as primary melanomas (n = 8) were negative. Upregulation of expression after treatment with demethylating agent 5‐aza‐2′‐deoxycytidine was detected in 1 of 4 human melanoma cell lines tested. The XAGE‐1 gene consists of 4 exons and is located on chromosome Xp11.21–Xp11.22. After transfection into COS cells, the corresponding protein can direct the coupled fluorescent protein to the nucleus, showing a distinct speckled staining aspect. Our data imply the nuclear cancer/testis‐associated XAGE‐1b to be a marker for late melanocytic tumor progression.