A genome-wide survey of RAS transformation targets.

An important aspect of multi-step tumorigenesis is the mutational activation of genes of the RAS family, particularly in sporadic cancers of the pancreas, colon, lung and myeloid system. RAS genes encode small GTP-binding proteins that affect gene expression in a global way by acting as major switches in signal transduction processes, coupling extracellular signals with transcription factors. Oncogenic forms of RAS are locked in their active state and transduce signals essential for transformation, angiogenesis, invasion and metastasis via downstream pathways involving the RAF/MEK/ERK cascade of cytoplasmic kinases, the small GTP-binding proteins RAC and RHO, phosphatidylinositol 3-kinase and others. We have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, to contrast differential gene expression profiles in immortalized, non-tumorigenic rat embryo fibroblasts and in HRAS- transformed cells. Sequence and expression analysis of more than 1,200 subtracted cDNA fragments revealed transcriptional stimulation or repression of 104 ESTs, 45 novel sequences and 244 known genes in HRAS- transformed cells compared with normal cells. Furthermore, we identified common and distinct targets in cells transformed by mutant HRAS, KRAS and NRAS, as well as 61 putative target genes controlled by the RAF/MEK/ERK pathway in reverted cells treated with the MEK-specific inhibitor PD 98059.

Growth-inhibitory activity and downregulation of the class II tumor-suppressor gene H-rev107 in tumor cell lines and experimental tumors.

The H-rev107 gene is a new class II tumor suppressor, as defined by its reversible downregulation and growth-inhibiting capacity in HRAS transformed cell lines. Overexpression of the H-rev107 cDNA in HRAS-transformed ANR4 hepatoma cells or in FE-8 fibroblasts resulted in 75% reduction of colony formation. Cell populations of H-rev107 transfectants showed an attenuated tumor formation in nude mice. Cells explanted from tumors or maintained in cell culture for an extended period of time no longer exhibited detectable levels of the H-rev107 protein, suggesting strong selection against H-rev107 expression in vitro and in vivo. Expression of the truncated form of H-rev107 lacking the COOH-terminal membrane associated domain of 25 amino acids, had a weaker inhibitory effect on proliferation in vitro and was unable to attenuate tumor growth in nude mice. The H-rev107 mRNA is expressed in most adult rat tissues, and immunohistochemical analysis showed expression of the protein in differentiated epithelial cells of stomach, of colon and small intestine, in kidney, bladder, esophagus, and in tracheal and bronchial epithelium. H-rev107 gene transcription is downregulated in rat cell lines derived from liver, kidney, and pancreatic tumors and also in experimental mammary tumors expressing a RAS transgene. In colon carcinoma cell lines only minute amounts of protein were detectable. Thus, downregulation of H-rev107 expression may occur at the level of mRNA or protein.

Oncogenic HRAS suppresses clusterin expression through promoter hypermethylation.

Silencing of gene expression by methylation of CpG islands in regulatory elements is frequently observed in cancer. However, an influence of the most common oncogenic signalling pathways onto DNA methylation has not yet been investigated thoroughly. To address this issue, we identified genes suppressed in HRAS-transformed rat fibroblasts but upregulated after treatment with the demethylating agent 5-Aza-2-deoxycytidine and with the MEK1,2 inhibitor U0126. Analysis of gene expression by microarray and Northern blot analysis revealed the MEK/ERK target genes clusterin, matrix metalloproteinase 2 (Mmp2), peptidylpropyl isomerase C-associated protein, syndecan 4, Timp2 and Thbs1 to be repressed in the HRAS-transformed FE-8 cells in a MEK/ERK- and methylation-dependent manner. Hypermethylation of putative regulatory elements in HRAS-transformed cells as compared to immortalized fibroblasts was detected within a CpG island 14.5 kb upstream of clusterin, within the clusterin promoter and within a CpG island of the Mmp2 promoter by bisulphite sequencing. Furthermore, hypermethylation of the clusterin promoter was observed 10 days after induction of HRAS in immortalized rat fibroblasts and a clear correlation between reduced clusterin expression and hypermethlyation could also be observed in distinct rat tissues. These results suggest that silencing of individual genes by DNA methylation is controlled by oncogenic signalling pathways, yet the mechanisms responsible for initial target gene suppression are variable.

MUC18, a melanoma-progression associated molecule, and its potential role in tumor vascularization and hematogenous spread.

The MUC18 protein, a member of the immunoglobulin superfamily and related to several adhesion molecules, shows an expression pattern in human malignant melanoma which is closely associated with tumor progression and the onset of metastasis. To determine the expression pattern of MUC18 in normal human tissues, immunohistochemical analysis was performed on frozen sections of a variety of normal human tissues using monoclonal antibodies against three different epitopes. This analysis showed that expression of MUC18 is limited to smooth muscle cells and to vascular endothelium. No reactivity could be observed with epithelial cells or with quiescent or activated hemopoetic cells. Smooth muscle cells in lung, skin, and in the gastrointestinal tract express MUC18 as does vascular smooth muscle, whereas myocardium or skeletal muscle appeared negative. Comparison of MUC18 staining with that of the panendothelial marker CD31 showed that MUC18 is expressed on the endothelia of a subset of blood capillaries and in tumor vessels but is absent on the endothelium of arterial vessels and large veins. The regulation of MUC18 expression was investigated in vascular smooth muscle cells and endothelial cells cultured in vitro. These studies revealed induction of the gene in endothelial cells upon proliferation. The observation that the MUC18 protein is not only present on melanoma cells but also on the endothelia of blood vessels penetrating primary and metastatic melanomas suggests a complex involvement of this potential cell adhesion molecule in tumor angiogenesis and metastasis.

Phorbol ester and cyclic AMP-mediated regulation of the melanoma-associated cell adhesion molecule MUC18/MCAM.

MUC18/MCAM is a melanoma-associated cell adhesion molecule that is also occasionally found on carcinomas and other tumor types. On melanomas, MUC18 expression increases with tumor progression and is found on more than 70% of metastatic lesions. To investigate the regulation of MUC18 expression, cell lines of diverse tissue origin were exposed to cytokines, regulators of intracellular cyclic AMP (cAMP), and to phorbol ester. MUC18 expression could not be induced in negative cell lines and could only be modulated by changes in cAMP levels or by exposure to phorbol ester in positive cells. An increase in intracellular cAMP led to an up-regulation in cell surface MUC18 that was maximal at 48 h. Increased MUC18 mRNA levels were observed as soon as 4 h and were 3-fold higher than in control cells by 48 h. Exposure of the cells to phorbol ester reduced MUC18 surface expression to background levels by 24 h. This downregulation was associated with decreased mRNA levels that were apparent at 8 h. By 24 h, steady-state levels of MUC18 mRNA had been reduced by 58%. Whereas similar changes in MUC18 surface expression were observed in MUC18-expressing glioma and carcinoma cell lines, melanoma cells were more resistant to the MUC18-modulating effects of cAMP analogues and phorbol ester. These observations suggest that the strong MUC18 expression observed in advanced melanomas may reflect disturbances in the normal regulation of this molecule.

Uncoupling of EGFR-RAS signaling and nuclear localization of YBX1 in colorectal cancer.

The transcription factor YBX1 can act as a mediator of signals transmitted via the EGFR-RAS-MAPK axis. YBX1 expression has been associated with tumor progression and prognosis in multiple types of cancer. Immunohistochemical studies have revealed dependency between YBX1 expression and individual EGFR family members. We analyzed YBX1 and EGFR family proteins in a colorectal cancer (CRC) cohort and provide functional analyses of YBX1 in the context of EGFR-RAS-MAPK signaling. Immunohistochemistry for YBX1 and EGFR family receptors with two antibodies for YBX1 and EGFR were performed and related to clinicopathological data. We employed Caco2 cells expressing an inducible KRASV12 gene to determine effects on localization and levels of YBX1. Mouse xenografts of Caco2-KRASV12 cells were used to determine YBX1 dynamics in a tissue context. The two different antibodies against YBX1 showed discordant immunohistochemical stainings in cell culture and clinical specimens. Expression of YBX1 and EGFR family members were not correlated in CRC. Analysis of Caco2 xenografts displayed again heterogeneity of YBX1 staining with both antibodies. Our results suggest that YBX1 is controlled via complex regulatory mechanisms involving tumor stroma interaction and signal transduction processes. Our study highlights that YBX1 antibodies have different specificities, advocating their use in a combined manner.

Transcriptional and translational downregulation of H-REV107, a class II tumour suppressor gene located on human chromosome 11q11-12.

The H-rev107 tumour suppressor was isolated as a gene specifically expressed in rat fibroblasts resistant toward malignant transformation by the activated HRAS gene (Sers et al., 1997; Hajnal et al., 1994). Here we describe the human homologue of the rat H-rev107 gene. The predicted rat and human proteins are highly conserved exhibiting an overall amino acid identity of 83%. The H-REV107-1 gene is ubiquitously expressed with the exception of haematopoetic cells and tissues. In contrast, H-REV107-1 mRNA was found only in eight of 27 cell lines derived from mammary carcinoma, lung carcinoma, gastric carcinoma, kidney carcinoma, melanoma, neuroblastoma and other tumours. The H-REV107-1 protein was not detectable in any of these tumour cells. Loss of H-REV107-1 expression was not restricted to cultured human tumour cell lines, but also found in primary squamous cell carcinomas. Gross structural aberrations of the H-REV107-1 gene were absent in tumorigenic cell lines. Thus, the block to H-REV107-1 expression is achieved both at the level of transcription and translation. By fluorescence in situ hybridisation the human H-REV107-1 gene was localised to chromosome 11q11-12.

Gene expression profiling of fibroblasts resistant toward oncogene-mediated transformation reveals preferential transcription of negative growth regulators.

The signal-transducing Ras proteins are important driving forces of diverse cellular processes such as proliferation, neoplastic transformation, differentiation and growth inhibition. As a step toward understanding the complex mechanisms underlying cellular responses, gene expression patterns were examined in two phenotypically normal fibroblast lines which differ in their sensitivity toward oncogene-mediated transformation. Suppression subtractive hybridization (SSH) was used to establish a subtracted cDNA library specific for the REF52 cell line which, like normal diploid fibroblasts, is refractory toward neoplastic transformation induced by mutated HRAS oncogenes. In contrast, rat 208F control cells can be efficiently transformed by HRAS. The nucleotide sequence of 549 subtracted cDNA clones ('REF52 minus 208F') was determined. We identified 93 preferentially expressed gene fragments in resistant REF52 cells as compared to 208F cells. Seventeen of the 52 known genes (32.6%) are capable of inhibiting cell proliferation or of adversely affecting oncogenic signal transduction pathways. These results suggest that the anti-oncogenic properties of resistant REF52 cells are determined by multiple negative growth regulators. The preneoplastic state expressed in 208F cells is characterized by impairment of unexpectedly redundant control mechanisms. Our results also demonstrate that SSH is a powerful method for identifying specific transcriptional patterns in closely related cell types.

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by ß-catenin activity.

Colon cancer cells frequently carry mutations that activate the ß-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/ß-catenin signals encourage ISC identity, we asked whether ß-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3ß. Similarly, transgenic expression of stabilized ß-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAF(V637E) knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/ß-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/ß-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.

A 2015 update on predictive molecular pathology and its role in targeted cancer therapy: a review focussing on clinical relevance.

In April 2013 our group published a review on predictive molecular pathology in this journal. Although only 2 years have passed many new facts and stimulating developments have happened in diagnostic molecular pathology rendering it worthwhile to present an up-date on this topic. A major technical improvement is certainly given by the introduction of next-generation sequencing (NGS; amplicon, whole exome, whole genome) and its application to formalin-fixed paraffin-embedded (FFPE) tissue in routine diagnostics. Based on this 'revolution' the analyses of numerous genetic alterations in parallel has become a routine approach opening the chance to characterize patients' malignant tumors much more deeply without increasing turn-around time and costs. In the near future this will open new strategies to apply 'off-label' targeted therapies, e.g. for rare tumors, otherwise resistant tumors etc. The clinically relevant genetic aberrations described in this review include mutation analyses of RAS (KRAS and NRAS), BRAF and PI3K in colorectal cancer, KIT or PDGFR alpha as well as BRAF, NRAS and KIT in malignant melanoma. Moreover, we present several recent advances in the molecular characterization of malignant lymphoma. Beside the well-known mutations in NSCLC (EGFR, ALK) a number of chromosomal aberrations (KRAS, ROS1, MET) have become relevant. Only very recently has the clinical need for analysis of BRCA1/2 come up and proven as a true challenge for routine diagnostics because of the genes' special structure and hot-spot-free mutational distribution. The genetic alterations are discussed in connection with their increasingly important role in companion diagnostics to apply targeted drugs as efficient as possible. As another aspect of the increasing number of druggable mutations, we discuss the challenges personalized therapies pose for the design of clinical studies to prove optimal efficacy particularly with respect to combination therapies of multiple targeted drugs and conventional chemotherapy. Such combinations would lead to an extremely high complexity that would hardly be manageable by applying conventional study designs for approval, e.g. by the FDA or EMA. Up-coming challenges such as the application of methylation assays and proteomic analyses on FFPE tissue will also be discussed briefly to open the door towards the ultimate goal of reading a patients' tissue as 'deeply' as possible. Although it is yet to be shown, which levels of biological information are most informative for predictive pathology, an integrated molecular characterization of tumors will likely offer the most comprehensive view for individualized therapy approaches. To optimize cancer treatment we need to understand tumor biology in much more detail on morphological, genetic, proteomic as well as epigenetic grounds. Finally, the complex challenges on the level of drug design, molecular diagnostics, and clinical trials make necessary a close collaboration among academic institutions, regulatory authorities and pharmaceutical companies.

Oncogenic transformation increases the sensitivity for apoptosis induction by inhibitors of macromolecular synthesis.

Inhibition of RNA or protein synthesis causes apoptosis in fibroblasts. This points to the constitutive expression of a long-lived apoptosis machinery which is controlled by shortlived negative regulatory proteins, termed endogenous survival factors. The length of time between addition of the inhibitor of macromolecular synthesis and the onset of apoptosis can be used as an estimation of the effective survival factor concentration. Transformation of rat fibroblasts by a constitutively expressed src oncogene or an inducible ras oncogene increases the sensitivity for apoptosis induction by inhibitors of macromolecular synthesis, indicating that their endogenous survival factor pool has been decreased.

The progression associated antigen MUC18: a unique member of the immunoglobulin supergene family.

The cell surface glycoprotein MUC18 was originally identified as a progression associated antigen in melanoma. MUC18 is expressed most strongly on metastatic lesions and advanced primary tumours and is only rarely detected in benign lesions. cDNA cloning revealed MUC18 to be a novel member of the immunoglobulin superfamily with sequence similarity to a number of cell adhesion molecules. Cloning of both the human and mouse MUC18 genes indicate that their predicted protein structures are very similar with an overall amino acid identity of 75%. Like its human counterpart, murine MUC18 is also expressed by transformed melanocytes. Analysis of the promoter region of the human gene has provided evidence for regulatory elements found in smooth muscle specific genes and in both human and mouse: this is the normal site of MUC18 expression. The presence of putative binding sites for the transcriptional factors AP-1, AP-2 and CREB, suggest that MUC18 gene expression can also be modulated by external factors.

Predictive molecular pathology and its role in targeted cancer therapy: a review focussing on clinical relevance.

The increasing importance of targeting drugs in the treatment of several tumor entities (breast, colon, lung, malignant melanoma (MM), lymphoma, and so on) and the necessity of a companion diagnostic (human epidermal growth factor receptor 2, Kirsten rat sarcoma viral oncogene, epidermal growth factor receptor (EGFR), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), and so on) is leading to new challenges for surgical pathology. As all the biomarkers to be specifically detected are tissue based, a precise and reliable diagnostic is absolutely crucial. To meet this challenge, surgical pathology has adapted a number of molecular methods (semi-quantitative immunohistochemistry, fluorescence in situ hybridization), PCR and its multiple variants, (pyro/Sanger) sequencing, next-generation sequencing, DNA-arrays, methylation analyses, and so on) to be applicable for formalin-fixed paraffin-embedded (FFPE) tissue. To read a patients' tissue as 'deeply' as possible and to obtain information on morphological, genetic, proteomic as well as epigenetic background is the actual task of pathologists and molecular biologists in order to provide the clinicians with information relevant for individualized medicine. The intensified cooperation of clinicians and pathologists will provide the basis of improved clinical drug selection as well as guide development of new cancer gene therapies and molecularly targeted drugs by research units and the pharmaceutical industry. This review will give some information on (1) biomarker detection methods adapted to FFPE tissue, (2) the potency of predictive pathology in tumor detection and treatment and (3) the implications of pathology on the development of new drugs in molecularly targeted and gene therapies.

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) has potential tumour-suppressive activity in human lung cancer.

The expression of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is decreased in various tumours, but the role of IGFBP-rP1 in lung cancer is not yet clear. In this study, IGFBP-rP1 expression in lung cancer cell lines was evaluated and reduced expression of IGFBP-rP1 was found. In tissue microarrays containing 138 primary tumours and 20 normal lung tissues analysed by immunohistochemistry, 58 tumours (42%) exhibited no expression of IGFBP-rP1, while all 20 normal lung tissues showed high expression. In squamous cell lung cancer, low expression of IGFBP-rP1 was significantly linked to high-grade tumours. Treatment with 5-aza-2'-deoxycytidine restored the expression of IGFBP-rP1 in three of four lung cancer cell lines. Sequencing of PCR products of sodium bisulphite-treated genomic DNA from the three lung cancer cell lines revealed a heterogeneous methylation pattern in the region of exon 1 and intron 1. Stable transfection of IGFBP-rP1 full-length cDNA into the H2170 lung cancer cell line led to increased expression of IGFBP-rP1 protein. IGFBP-rP1-positive transfectants exhibited remarkably reduced colony-forming ability in soft agar, suppression of tumour growth rate in nude mice, and increased apoptotic cell number as well as activated caspase-3 expression level. The data suggest that IGFBP-rP1 is a tumour suppressor inactivated by DNA methylation in human lung cancer.

Receptor activator of nuclear factor kappaB ligand is expressed in resident and inflammatory cells in aseptic and septic prosthesis loosening.

OBJECTIVE: The pathogenesis of periprosthetic bone loss in aseptic and septic prosthesis loosening is unclear. There is considerable evidence that macrophages and osteoclasts play a key role in focal bone erosion and osteolysis around the prosthesis. RANKL (receptor activator of nuclear factor kappaB ligand) was shown to be a potent osteoclastogenic factor, and to be involved in bone destruction of myeloma and rheumatoid arthritis patients. Osteoprotegerin (OPG) is the natural RANKL inhibitor and may prevent periprosthetic bone loss. METHODS: The presence and distribution of RANKL, its receptor RANK and OPG in the periprosthetic interface of septically (n = 5) and aseptically (n = 6) loosened prostheses was examined by immunohistochemistry and immunoblotting. Additionally, the immunophenotype of the inflammatory infiltrate was determined [CD3, CD68, Ki-67, tartrate-resistant acid posphatase (TRAP)]. RESULTS: Aseptic and septic cases revealed a different histopathologic pattern. However, in all cases RANKL and RANK could be demonstrated in macrophages and giant cells. In addition, RANKL detected by immunoblot analysis proved to have the same molecular weight as a recombinant RANKL used as a control (31 kD and approximately 48 kD). OPG was detected in aseptic loosening, where macrophages showed a strong staining, but multinucleated giant cells were only weakly stained. A weak OPG staining was also observed in septic loosening. CONCLUSION: The pathogenesis of bone loss in septic loosening remains unclear, because the septic membrane bears few macrophages and giant cells, and half of them express OPG. In aseptic loosening, macrophages might not be stimulated by RANKL as a result of OPG expression. But multinucleated giant cells may be activated, as they hardly express OPG. They might be responsible for periprosthetic bone loss in aseptic loosening as a result of their RANKL and RANK expression.

Genomic organization of the melanoma-associated glycoprotein MUC18: implications for the evolution of the immunoglobulin domains.

The cell surface glycoprotein MUC18, a member of the immunoglobulin superfamily and homologous to several cell adhesion molecules, is associated with tumor progression and the development of metastasis in human malignant melanoma. Immunohistochemical and Northern blot analysis revealed that expression of the antigen is restricted to advanced primary and metastatic melanomas and to cell lines of the neuroectodermal lineage. The genomic sequence encoding the cell surface antigen spans approximately 14 kb and consists of 16 exons. The organization of the gene, which is related to that of the neural cell adhesion molecule N-CAM, shows a structure where each immunoglobulin-related domain is encoded by more than one exon. Sequencing of the putative MUC18 promoter region revealed a G + C-rich promoter lacking conventional TATA and CAAT boxes. Several motifs for binding of transcription factor Sp1 are present in the regulatory region, and only a single transcription start site within a presumed initiator sequence was identified. Sequence elements which might confer melanocyte-specific expression were not detected. Instead, recognition sequences for the transcription factors CREB, AP-2, and c-Myb, as well as CArG-box motifs, were observed. These elements may contribute to the differential regulation of the MUC18 gene in normal and malignant tissues and suggest a role for this putative adhesion molecule in neural crest cells during embryonic development.

ras oncogene expression determines sensitivity for intercellular induction of apoptosis.

Fibroblasts carrying an inducible ras oncogene acquire the transformed phenotype after oncogene induction. As a consequence, the transformed cells become sensitive to intercellular induction of apoptosis, a novel regulatory process directed by non-transformed fibroblasts against their transformed descendants. The causal relationship between oncogene expression and sensitivity to intercellular induction of apoptosis is based on extracellular superoxide anion production by oncogene-expressing cells. Superoxide anions (after dismutation to hydrogen peroxide) thereby foster HOCl synthesis and at the same time direct the selectivity of apoptosis induction through hydroxyl generation from HOCl. In parallel, ras expression enhances the sensitivity of fibroblasts for apoptosis-inducing stimuli like cycloheximide, ceramide and mitomycin C. This sensitization seems to be based on a decreased concentration of short lived endogenous apoptosis inhibitors. TGF-beta, like ras induction, decreases the concentration of endogenous apoptosis inhibitors, but does not induce the transformed phenotype. Therefore, TGF-beta treatment alone is not sufficient to render fibroblasts sensitive for intercellular induction of apoptosis, but TGF-beta treatment in parallel with ras activation enhances intercellular induction of apoptosis. Our findings demonstrate that Ras-mediated superoxide anion production determines sensitivity to intercellular induction of apoptosis, whereas the parallel decrease in endogenous apoptosis inhibitors modulates the kinetics of apoptosis induction.

Functional aspects of three molecules associated with metastasis development in human malignant melanoma.

To identify molecules which may be functionally associated with the development of metastasis in human melanoma, monoclonal antibodies which discriminate between benign and malignant melanocytic lesions in situ were selected. Biochemical studies and cDNA cloning identified the antigens HLA-DR, ICAM-1 and MUC18 which showed an expression pattern on primary tumors correlating with vertical tumor thickness, the most predictive parameter for the development of metastasis in melanoma. ICAM-1 and MUC18 show sequence similarity to a family of cell adhesion molecules which include the neural cell adhesion molecule NCAM. Both HLA-DR and ICAM-1 can be induced on melanoma cells by lymphokines, suggesting a role of the mononuclear cell infiltrate in the control of tumor cell phenotype. Knowledge of the normal function of these molecules allows one to hypothesize how they may contribute to the successful development of metastases.

Down-regulation of caveolin-1, a candidate tumor suppressor gene, in sarcomas.

Caveolae are plasma membrane microdomains that have been implicated in the regulation of several intracellular signaling pathways. Previous studies suggest that caveolin-1, the main structural protein of caveolae, could function as a tumor suppressor. Caveolin-1 is highly expressed in terminally differentiated mesenchymal cells including adipocytes, endothelial cells, and smooth muscle cells. To study whether caveolin-1 is a possible tumor suppressor in human mesenchymal tumors, we have analyzed the expression using immunohistochemistry in normal mesenchymal tissues, 22 benign and 79 malignant mesenchymal tumors. Caveolin-1 was found to be expressed in fibromatoses, leiomyomas, hemangiomas, and lipomas at high levels comparable to normal mesenchymal tissues. The expression of caveolin-1 was slightly reduced in four of six well-differentiated liposarcomas and strongly reduced or lost in three of three fibrosarcomas, 17 of 20 leiomyosarcomas, 16 of 16 myxoid/round cell/pleomorphic liposarcomas, five of eight angiosarcomas, 15 of 18 malignant fibrous histiocytomas, and eight of eight synovial sarcomas. The immunohistochemical findings were confirmed by Western blot analysis in a number of tumors. High levels of both the 24-kd [alpha]- and the 21-kd [beta]-isoform of caveolin-1 were detected in the nontumorigenic human fibroblast cell line IMR-90. In contrast, in HT-1080 human fibrosarcoma cells, caveolin-1 is strongly down-regulated. We show that the [alpha]-isoform of caveolin-1 is potently up-regulated in HT-1080 cells by inhibition of the mitogen-activated protein kinase-signaling pathway with the specific inhibitor PD 98059, whereas the specific inhibitor of DNA methylation 5-aza-2'-deoxycytidine only marginally up-regulates caveolin-1. In addition, re-expression of caveolin-1 in HT-1080 fibrosarcoma cells potently inhibited colony formation. From these we conclude that caveolin-1 is likely to act as a tumor suppressor gene in human sarcomas.