Tanja Rothhammer

Functional implication of BMP4 expression on angiogenesis in malignant melanoma

Analyses of malignant melanomas revealed a strong expression of bone morphogenic proteins (BMPs) and their autocrine effect in promoting cell invasion and migration. Here, we report a paracrine effect of BMPs on the vascular network. Both BMP2 and BMP4 induced tube formation as well as the migratory efficiency of microvascular endothelial cells. Melanoma cells with reduced BMP activity attracted less endothelial cells in invasion assays than control cells. Furthermore, reduction of BMPs in melanoma cells had a strong effect on vasculogenic mimicry. Tube formation on matrigel was analysed for melanoma cells as well as in co-cultures of endothelial and melanoma cells. Melanoma cells with reduced BMP activity were not capable of forming cord-like structures by themselves and additionally inhibited tube formation of the endothelial cells. Genes involved in angiogenesis turned out to be strongly downregulated in these cell clones. Tumors derived from cells with impaired BMP activity showed reduced tumor growth or large necrotic areas owing to lack of angiogenesis in in vivo analyses.

MicroRNA miR-196a is a central regulator of HOX-B7 and BMP4 expression in malignant melanoma

Since bone morphogenetic proteins (BMPs) play an important role in melanoma progression, we aimed to determine the molecular mechanisms leading to overexpression of BMP4 in melanoma cells compared to normal melanocytes. With our experimental approach we revealed that loss of expression of a microRNA represents the starting point for a signaling cascade finally resulting in overexpression of BMP4 in melanoma cells. In detail, strongly reduced expression of the microRNA miR-196a in melanoma cells compared to healthy melanocytes leads to enhanced HOX-B7 mRNA and protein levels, which subsequently raise Ets-1 activity by inducing basic fibroblast growth factor (bFGF). Ets-1 finally accounts for induction of BMP4 expression. We were furthermore able to demonstrate that bFGF-mediated induction of migration is achieved via activation of BMP4, thus determining BMP4 as major modulator of migration in melanoma. In summary, our study provides insights into the early steps of melanoma progression and might thereby harbor therapeutic relevance.

The Ets-1 transcription factor is involved in the development and invasion of malignant melanoma

The Ets-1 transcription factor plays a role in tumor vascularization and invasion by regulating expression of matrix-degrading proteases in endothelial cells and fibroblasts in the tumor stroma. During early embryogenesis, Ets-1 is expressed in migrating neural crest cells from which melanocytes arise. In the present study, we analyzed Ets-1 expression in various melanocytic lesions and investigated its functional importance in malignant melanomas. We found that Ets-1 was upregulated both in vivo and in vitro in malignant melanoma, compared to benign melanocytic lesions and to primary melanocytes. Assessment of DNA-binding and transactivation assays documented a strong Ets activity in melanoma cells. Using an antisense strategy, the expression and activity of Ets-1 were reduced in the melanoma cell line Mel Im. This correlated with a diminished expression of several Ets-1 target genes known to be involved in invasion, such as MMP1, MMP3, uPA and integrin β3. In line with these findings, the invasive potential of the melanoma cells measured in a Boyden Chamber model was reduced up to 60% after Ets-1 blockade. This can be attributed to the role of Ets-1 in transcriptional regulation of factors involved in invasion of melanoma cells. We conclude that over-expression of Ets-1 during melanoma development contributes to the malignant phenotype.

Bone morphogenetic protein 7 (BMP7) expression is a potential novel prognostic marker for recurrence in patients with primary melanoma1

The purpose of this study was to investigate whether protein expression of bone morphogenetic protein 7 (BMP7) is associated with clinico-pathologic characteristics in benign and malignant melanocytic skin tumors. Tissue microarrays (TMAs) were used to analyze BMP7 expression and the Ki-67 labeling index immunohistochemically. Expression was scored semi quantitatively (0-2+). BMP7 protein expression of any intensity (1+-2+) was detected in 50.2% (153/305) of informative cases. In general, BMP7 expression was significantly induced in malignant melanomas (P 5% suggesting that induction of BMP7 expression is associated with proliferation (P=0.028). None of the other clinical and histological factors analyzed was significantly related to BMP7 expression. Interestingly, lymph node metastases demonstrated a significantly higher BMP7 expression compared to skin metastases (P<0.01). Strong BMP7 expression (score 2+) was significantly associated with shorter tumor recurrence (P< 0.05). In summary, induction of BMP7 expression is frequent in melanomas and may serve as a novel prognostic marker of progression in melanoma patients.

Bone morphogenetic proteins induce expression of metalloproteinases in melanoma cells and fibroblasts.

Bone morphogenetic proteins are secreted growth factors which belong to the TGFbeta super family. In recent studies, we showed that the expression of BMP-4 and -7 is induced in melanoma cells in comparison to normal melanocytes. Functional analyses revealed that BMPs are inevitable factors for migration and invasion processes of melanoma cells; however, the role of BMPs in degradation and remodelling of the extracellular matrix remained unknown. We discovered that melanoma cell clones with reduced BMP activity, generated by stable transfection with an antisense BMP-4 construct or with the BMP inhibitor chordin, showed reduced expression of MMP-1, -2, -3 and -9. Moreover, BMPs displayed paracrine effects on stromal fibroblasts. Treatment of fibroblasts with BMP-2 or -4 led to increased MMP-1, -2, -3 and -13 expression. These data show that BMPs play an important role in dissemination of tumour cells from the primary tumour, either by enhancing the matrix degrading capacity of melanoma cells themselves or by stimulating tumour surrounding fibroblasts to induce expression of matrix metalloproteinases.