Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

Abstract

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.

Conclusion: 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.

Figure 1

Color map of Xmrk-specific gene expression. Eleven highly regulated genes are itemized. RNA was extracted at time 0 (no hEGF) and at indicated time points after hEGF addition. The color code shows differences in the expression of the eleven genes regulated > 4-fold in the range of +3 (red) to -3 (green). Sos1, son of sevenless homolog 1; Ube2i, ubiquitin-conjugating enzyme E2I; Cyr61, cysteine rich angiogenic inducer 61; Egr1, early growth response 1; Dusp4, dual specificity phosphatase 4; Igfbp3, insulin-like growth factor binding protein 3; Fosl1, FOS-like antigen 1; Emp1, epithelial membrane protein 1; Opn, osteopontin; Taal6, tumor-associated antigen L6. NA: not annotated.

Figure 2

Validation of microarray results of ten candidate genes by quantitative realtime PCR. RNA was extracted at indicated time points after HERmrk stimulation with hEGF. Expression of candidate genes was compared to unstimulated cells. The fold change of transcript, referred to the unstimulated control, which is set as 1, is indicated on the y axis. Mouse β-actin served as reference gene. Significant regulation (p < 0.05) is marked by an asterisk; two asterisks indicate highly significant regulation (p < 0.001) (Student's t-test, paired, one-tailed).

Figure 3

Expression of candidate genes in human melanoma cell lines. a, Realtime PCR was performed to determine FOSL1, EGR1, OPN, IGFBP3, DUSP4 and TAAL6 expression in human melanoma cell lines compared to NHEM cells. Fold change of transcript, referred to NHEM cells, is indicated. β-actin was used as reference gene. Significant (p < 0.05) or highly significant (p < 0.001) regulation are marked by one or two asterisks, respectively (Student's t-test, paired, one-tailed). b, Western blot analysis of the candidate genes in human melanoma cell lines. Expression of ERK2 served as loading control. Please note that the order of cell lines differs from A. On the OPN blot, the lower band represents the OPN protein. For TAAL6, the upper band corresponds to the protein of the expected size.

Figure 4

Expression of candidate genes in response to stimulation of human EGFR. Realtime PCR was performed to determine expression levels of FOSL1 and EGR1 in the human melanoma cell line A375 (a) or expression levels of Fosl1, Egr1, Opn, Dusp4, and Taal6 in HER-transgenic melan-a cells (b), stimulated with EGF for the indicated times. The fold change of transcript, referred to the unstimulated controls, is indicated on the y axis. Murine and human β-actin were used as reference genes. Significant (p < 0.05) or highly significant (p < 0.001) regulation are marked by one or two asterisks, respectively (Student's t-test, paired, two-tailed).

Figure 5

MAPK-pathway dependent expression of FOSL1 and effects of FOSL1 knockdown on proliferation and migration of melanoma cells A375 and Mel Juso. a, Western blot analysis of FOSL1 levels in murine melanocytes (left) and human melanocytes and melanoma cell lines (right). Murine melan-a and melan-a HERmrk cells were starved for 2 days before EGF and U0126 were added for 24 hours. Human melanoma cells were treated with U0126 for 24 hours. P-ERK1/2 levels were used to verify MEK inhibition, ERK2 was used as reference. b, Realtime PCR analysis (left) and Western blot analysis (right) of FOSL1 expression levels after treatment of the indicated cell lines with control siRNA or FOSL1-specific siRNA. For realtime PCR analysis, control siRNA-treated FOSL1 levels were set as 1. For the Western blot, β-actin was used as reference. c, BrdU incorporation of A375 and Mel Juso melanoma cells transfected with control siRNA or FOSL1-specific siRNA. d, Migration of A375 and Mel Juso cells treated with FOSL1-specific siRNA relative to control siRNA-treated cells. Significant (p < 0.05) or highly significant (p < 0.001) differences between control and FOSL1 siRNA treated cells are marked by one or two asterisks, respectively (Student's t-test, paired, two-tailed).