Melanoma tumor antigen p97 or melanotransferrin (MTf) is an iron (Fe)-binding protein with high homology to serum transferrin. MTf is expressed at very low levels in normal tissues and in high amounts in melanoma cells. The over-expression of MTf in tumor cells was hypothesized to assist rapidly proliferating neoplastic cells with their increased Fe requirements. However, our recent characterization of the MTf knockout (MTf -/-) mouse demonstrated that MTf did not have an essential role in Fe metabolism. To understand the function of MTf, we utilized whole-genome microarray analysis to examine the gene expression profile of five models after modulating MTf expression. These models included two new stably transfected MTf hyper-expression models (SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type (SK-Mel-28 melanoma) where MTf was down-regulated by post-transcriptional gene silencing. These findings were compared to alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the gene array data were also assessed in a new model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased cellular proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and up-regulation, we identified three genes modulated by MTf expression. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and transcription factor 4 (Tcf4) were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and cell proliferation/survival, respectively. This study identifies novel molecular targets directly or indirectly regulated by MTf and potential pathways involved in its function. These molecular targets could be involved, at least in part, to the role of MTf in modulating proliferation.
Keywords: Melanotransferrin, hyperexpression cell lines, comparative genomic hybridization
Overall design: Hyper-expression constructs of the mouse MTf cDNA was generated using the pCI-neo (Promega, Madison, WI) expression vector. The inserts were sequenced and confirmed against mouse (Genbank Accession: NM_013900) using the public NCBI database. Transfections of pCI-neo transgenes/vectors into murine LMTK- fibroblasts was performed with Lipofectin® reagent (Invitrogen, Melbourne, Australia) according to the manufacturer’s protocol. Total RNA was isolated from the cells using TRIzol Reagent® (Sigma-Aldrich) according to the manufacturer’s protocol. Total RNA from the stably-transfected mouse LMTK- cell lines were prepared and hybridized onto Mouse Genome 430 2.0 Array. The mouse GeneChip® 430 2.0 consists of greater than 39,000 transcripts and variants from over 34,000 well characterized mouse genes (Affymetrix, Santa Clara, CA).
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