Heterogeneous expression and functional relevance of the ubiquitin carboxyl-terminal hydrolase L1 in melanoma.

The expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is deregulated in human cancer cells with tumor inhibiting or promoting functions. Due to less knowledge on the role of UCHL1 in melanoma progression, the expression pattern and function of UCHL1 as well as the deregulated signaling pathways were characterized. A large number of melanoma cell lines, tissue microarrays of melanoma lesions and control tissues were analyzed for UCHL1 expression using PCR, Western blot and/or immunohistochemistry. The analysis revealed that melanocyte cultures, 24 of 331 melanoma lesions, two of 18 short-term cultures and two of 19 melanoma cell lines tested, respectively, heterogeneously expressed UCHL1. The low frequency of UCHL1 expression in melanoma cells was due to gene silencing by promoter DNA hypermethylation. Using different transfection models an enzyme activity-dependent growth promoting function of UCHL1 via the activation of the mitogen-activated protein kinase signaling pathway was found in melanoma cells. Under oxygen stress a dose-dependent effect of UCHL1 was detected, which was mediated by a dynamic modification of the PI3K-Akt signaling. Thus, the aberrant UCHL1 expression in melanoma cells is linked to dynamic changes in growth properties and signal transduction cascades suggesting that UCHL1 provides a novel marker and/or therapeutic target at least for a subset of melanoma patients.

HER-2/neu-mediated down-regulation of biglycan associated with altered growth properties.

The extracellular matrix protein biglycan (Bgn) is a leucine-rich proteoglycan that is involved in the matrix assembly, cellular migration and adhesion, cell growth, and apoptosis. Although a distinct expression of Bgn was found in a number of human tumors, the role of this protein in the initiation and/or maintenance of neoplastic transformation has not been studied in detail. Using an in vitro model of oncogenic transformation, a down-regulation of Bgn expression as well as an altered secretion of different Bgn isoforms was found both in murine and human HER-2/neu oncogene-transformed cells when compared with HER-2/neu(-) cells. This was associated with a reduced growth, wound closure, and migration capacity. Vice versa, silencing of Bgn in HER-2/neu(-) fibroblasts increased the growth rate and migration capacity of these cells. Bgn expression was neither modulated in HER-2/neu(+) cells by transforming growth factor-ß(1) nor by inhibition of the phosphoinositol 3-kinase and MAP kinase pathways. In contrast, inhibition of the protein kinase C (PKC) pathway led to the reconstitution of Bgn expression. In particular, the PKC target protein cAMP response element binding protein (CREB) is a major regulator of Bgn expression as the silencing of CREB by RNA interference was accompanied by ∼5000-fold increase in Bgn-mRNA expression in HER-2/neu(+) cells. Thus, Bgn inhibits the major properties of HER-2/neu-transformed cells, which is inversely modulated by the PKC signaling cascade.

Promoter methylation of aminopeptidase N/CD13 in malignant melanoma.

Aminopeptidase N (APN)/CD13 as ubiquitously expressed membrane peptidase exerts important functions in diverse cellular processes, such as proliferation, migration and differentiation. Previously, a role of APN in the invasiveness of melanoma cells has been demonstrated, but the underlying molecular mechanisms controlling APN expression are not understood. The present study demonstrates that lack of APN expression in primary and established melanoma cells was directly associated with a high-grade DNA methylation status of the myeloid APN promoter. Demethylation by 5-aza-2'-desoxycytidine not only induced constitutive and cytokine-regulated APN protein expression but also resulted in an increased APN-dependent migration of melanoma cells. Furthermore, its heterogeneous expression was inversely correlated to the expression of melanocytic marker proteins in established as well as in short-term cultured human melanoma cells. Staining of tissue microarrays generated from a large series of melanoma samples and control tissues demonstrated a higher APN expression in primary melanoma lesions when compared with nevi and metastases, which was neither associated with clinico-pathological parameters nor with the patients' outcome. Thus, the heterogeneous APN expression pattern in melanoma cells is epigenetically controlled and directly associated with an altered migration capacity but not of clinical significance in our study group.

Expression of neutral endopeptidase (NEP/CD10) on pancreatic tumor cell lines, pancreatitis and pancreatic tumor tissues.

Neutral endopeptidase (NEP/CD10) is a cell surface zinc metalloprotease cleaving peptide bounds on the amino terminus of hydrophobic amino acids and inactivating multiple physiologically active peptides. Loss or decrease in NEP/CD10 expression have been reported in many types of malignancies, but the role of NEP/CD10 in pancreatic carcinoma has not yet been identified. Using real-time RT-PCR, flow cytometry as well as immunohistochemistry, NEP/CD10 expression was quantified in both pancreatic carcinoma cell lines and in tumor specimens obtained from patients with primary pancreatic carcinomas. Three out of 8 pancreatic carcinoma cell lines exhibit heterogeneous NEP/CD10 expression levels: PATU-8988T expressed the highest NEP/CD10 levels, whereas HUP-T4 and HUP-T3 cells showed a moderate to low NEP/CD10 expression. NEP/CD10 immunoreactivity was found in 6 of 24 pancreatic ductal adenocarcinomas, but also in 3 of 6 tissues of patients with chronic pancreatitis. NEP/CD10 expression in pancreatic tumor lesions and cell lines was not associated with tumor grading and staging. Treatment of PATU-8988T cells with the histone deacetylase inhibitors sodium butyrate and valproic acid induced an increase of NEP/CD10 expression. This was accompanied by a reduced cell proliferation rate of PATU-8988T cells, which was increased by the addition of the enzyme activity inhibitors phosphoramidon and thiorphan. Thus, NEP/CD10 is differentially expressed in pancreatic tumors and might be involved in the proliferative activity of pancreatic cancer cells. However, further studies are needed to provide more detailed information of the role of NEP/CD10 under physiological and pathophysiological conditions of the pancreas.

Forced expression of deltaN-TCF-1B in colon cancer derived cell lines is accompanied by the induction of CEACAM5/6 and mesothelin.

The colon cancer cell lines HT29 and SW480 were transfected with an N-terminal beta-catenin binding site-deficient high mobility group (HMG)-box T-cell factor 1 (deltaN-TCF-1) construct to identify differentially expressed genes. Oligonucleotide HG-U133A microarray expression profiling revealed increased mRNA levels of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 5, 6 and mesothelin in transfectants positive for nuclear deltaN-TCF-1B. Increased amounts of CEACAM5 (CEA) were detectable in membrane-associated compartments, particularly in cholesterol-enriched microdomains. Similarly, mesothelin was demonstrated as an uncleaved membrane-bound constituent. The identified markers were examined in specimens of 46 colorectal carcinomas (CRC) by immunohistochemistry. Patchy areas of increased CEACAM5/6 staining were seen at the tumour-host front in all samples studied. Twenty-eight (58%) of these cases showed over-expression of mesothelin in a small fraction of tumor cells displaying dedifferentiation and dissemination at the invasion front. We conclude that forced expression of deltaN-TCF-1B in HT29 and SW480 is associated with up-regulation of GPI-anchored adhesion molecules, which were assigned to the tumour-host front in CRC patients.

Two C-terminal cysteines are necessary for proper folding of the peptidase neprilysin/CD10.

Neprilysin (NEP) consists of 749 amino acids with two conserved cysteines (734, 746) and a putative CAAX motif (residues 746-749, CRVW) at the C-terminus. To investigate the role of the C-terminal conserved cysteine residues, three NEP mutants (C734S, C746S, and double mutant C734S/C746S) were constructed by use of site-directed mutagenesis. Western blot analysis of lysates of transfected cells revealed the presence of three NEP forms in wild type and mutants with a different glycosylation pattern. Point mutations of C734 as well as C746 by serine dramatically diminished the plasma membrane association of NEP as detected by flow cytometry and laser scanning microscopy. Endoprotease enzyme activity was slightly diminished in the C746S-NEP variant and was not detectable in the C734S-form of NEP suggesting a pivotal role of the C734 in the proper folding of the enzyme. Prenylation of NEP was not detected in an in vivo assay.