p54nrb is a new regulator of progression of malignant melanoma

Susanne Schiffnery 0 1 Nicole Zimaray 0 1 Rainer Schmidy 0 1 Anja-Katrin Bosserhoff 0 1 To whom correspondence should be addressed. Tel: 0 1 Fax: 0 1 Email: 0 1 0 Snap-frozen tissue samples of primary tumors (TB62 , TB71, TB72, TB97) and metastatic melanomas (TB12, TB43, TB100: all skin metastasis; TB50: lung metastasis; TB69: brain metastasis) were obtained from the tissue collection of the Institute of Pathology, University of Regensburg, Germany. Sampling and handling of human tissue material was carried out in accordance with the ethical principle of the Declaration of Helsinki 1 University of Regensburg Medical School, Institute of Pathology, Molecular Pathology , D-93053 Regensburg , Germany Nuclear RNA-binding protein p54nrb and its murine homolog NonO are known to be involved in a variety of nuclear processes including transcription and RNA processing. Melanoma inhibitory activity (MIA) has been shown to play an essential role in the progression of malignant melanoma and to influence melanomaassociated molecules and pathways in the early tumor formation steps. Interestingly, recent studies suggest that MIA is a regulator of p54nrb. Here, we show that p54nrb is strongly expressed and localized in the nucleus of both melanoma cell lines and melanoma tissue samples compared with normal human melanocytes or normal skin, respectively. Furthermore, all tested melanoma cell lines revealed strong p54nrb promoter activity. Treatment with MIAspecific small interfering RNAs showed an influence of MIA on p54nrb expression on both messenger RNA (mRNA) and protein level. Knockdown of p54nrb protein in melanoma cell lines led to reduced proliferation rates and to a strong decrease in their migratory potential. In addition, attachment to laminin and poly-Llysine was significantly increased. We could identify Connexin-43 (Cx-43) as a downstream target molecule of p54nrb as knockdown of p54nrb resulted in enhanced Cx-43 mRNA and protein levels. As a confirmation of these findings, melanoma cell lines showed very low Cx-43 expression levels compared with melanocytes. Our results demonstrate that p54nrb is highly expressed in malignant melanoma and, as a MIA target molecule, it seems to be involved in the development and progression of malignant melanoma. Introduction According to the World Health Organization, malignant melanoma is the most dangerous variant of skin cancer. Patients can be cured by early surgical excision of the non-metastatic primary tumor, whereas prognosis of patients suffering from metastatic melanoma is poor. One of the key molecules regulating melanoma progression is the protein melanoma inhibitory activity (MIA) which is strongly expressed in malignant melanoma but absent in normal human melanocytes (1). Several studies suggest an important role for MIA also in the early tumor formation steps by regulating melanoma-related pathways and molecules (1). Recent investigations in mesenchymal stem cells hinted to paraspeckle protein p54nrb as one of the MIA-regulated proteins (2). The nuclear p54nrb protein is a RNA-binding molecule of 54 kDa containing two RNA recognition motifs. It was first purified from HeLa cells in 1993 (3). p54nrb is able to bind double-stranded DNA, single-stranded DNA and RNA, allowing the conclusion that p54nrb Abbreviations: AEC, 3-amino-9-ethylcarbazole; DEPC, diethylpyrocarbonate; DMEM, Dulbeccos modified Eagles medium; dsDNA, double-stranded DNA; ECM, extracellular matrix; MIA, melanoma inhibitory activity; mRNA, messenger RNA; NHEM, normal human epidermal melanocytes; PARP, poly ADP-ribose polymerase; PBS, phosphate-buffered saline; POD, peroxidase; qRTPCR, quantitative real-time polymerase chain reaction; RIPA, radioimmunoprecipitation; siMIA, cells treated with specific MIA siRNA; siRNAs, small interfering RNAs; ssDNA, single-stranded DNA; XTT, 2,3-bis-(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. yThese authors contributed equally to this work. has dual roles in transcription and splicing (4). Moreover, it retains Ato-I-hyperedited messenger RNA (mRNA) in the nucleus and therefore influences gene expression and differentiation of stem cells (5). p54nrb was found to play an important role during chondrogenesis by directly interacting with the transcription factor Sox9 in paraspeckles. This results in the enhanced transcriptional activation of the collagen type II a1 (Col2a1) promoter through Sox9 (6). Paraspeckles are nucleoplasmic compartments containing mainly four different RNA-binding proteins: paraspeckle protein 1, paraspeckle protein 2, polypyrimidine tract-binding protein-associated splicing factor and p54nrb [also called NonO (Non-POU-domain-containing octamerbinding protein)]. All human cell types contain 1020 paraspeckles (5,7,8). Non-coding RNAs of a certain length are arranged in close contact to paraspeckle proteins, especially the nuclear-enriched autosomal transcript non-coding RNA 1 (5,8). These RNA-protein interactions are important for maintaining the structure of paraspeckle compartiments (5,8). Within the paraspeckles, p54nrb can either form heterodimers with protein-associated splicing factor or act as a monomer (9). Recent studies in chondrocytes suggest that MIA is able to regulate the transcription of p54nrb (2) and that p54nrb plays an important role in modification mRNA expression (6). We performed this study to reveal whether there is also a correlation between MIA and p54nrb in malignant melanoma. Therefore, we analyzed the expression and regulation of p54nrb in melanoma cell lines and melanoma tissue samples and examined the functional role of p54nrb for the development and progression of malignant melanoma. Materials and methods Cell lines and culture conditions The melanoma cell lines Mel Ei, Mel Wei, Mel Ho, Mel Juso (derived from primary cutaneous melanomas), Mel Ju, SK-MEL-3, SK-MEL-28, Mel Im and HMB2 (derived from metastases of malignant melanomas) have been described previously (10,11). The MIA-deficient HMB2 cell clones HMB2-MIA 5 and HMB2-MIA 8 have been generated through antisense technology and described previously in detail (12). As a mock control, b-galactosidase-expressing HMB2 lacZ cells were used. For tissue culture, cells were maintained in Dulbeccos modified Eagles medium (DMEM) (PAA; Pasching, Austria) supplemented with penicillin (400 U/ml), streptomycin (50 lg/ml) (both PAA) and 10% fetal calf serum (Sigma, Deisenhofen, Germany). Stable-transfected HMB2 cell clones were cultured in selection medium, containing 50 lg/ml G418 (SigmaAldrich, Taufkirchen, Germany). Normal human epidermal melanocytes (NHEM) derived from normal skin of different donors were cultivated in melanocyte growth medium (Promocell, Heidelberg, Germany). All cells were incubated in humidified atmosphere containing 8% CO2 at 37 C. Tissue samples RNA isolation and reverse transcription Total cellular RNA was isolated from cultured cells using the e.Z.N.A. MicroElute Total RNA Kit (...truncated)