Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins.
BACKGROUND
Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation.


METHODS
Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated.


RESULTS
Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration.


CONCLUSIONS
Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.
Teutschbein
Janka
J
Department of Physiological Chemistry I, Biocenter, University of Wurzburg, Wurzburg, Germany.
Haydn
Johannes M
JM
Samans
Birgit
B
Krause
Michael
M
Eilers
Martin
M
Schartl
Manfred
M
Meierjohann
Svenja
S
D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D004789:Enzyme Activation; D020869:Gene Expression Profiling; D006801:Humans; D008544:Melanocytes / Q000201:enzymology* / Q000473:pathology; D008545:Melanoma / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D016760:Proto-Oncogene Proteins c-fos / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D000099169:Fos-Related Antigen 1
D014408:Biomarkers, Tumor; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020794:Receptor Protein-Tyrosine Kinases; D000099169:Fos-Related Antigen 1