Dysregulation of receptor tyrosine kinase-induced pathways is a critical step driving the oncogenic potential of brain cancer. In this study, we investigated the role of two members of the Sprouty (Spry) family in brain cancer-derived cell lines. Using immunoblot analyses we found essential differences in the pattern of endogenous Spry3 and Spry4 expression. While Spry4 expression was mitogen-dependent and repressed in a number of cells from higher malignant brain cancers, Spry3 levels neither fluctuated in response to serum withdrawal nor were repressed in glioblastoma (GBM)-derived cell lines. In accordance to the well-known inhibitory role of Spry proteins in fibroblast growth factor (FGF)-mediated signaling, both Spry proteins were able to interfere with FGF-induced activation of the MAPK pathway although to a different extent. In response to serum solely, Spry4 exerts its role as a negative regulator of MAPK activation. Ectopic expression of Spry4 inhibited proliferation and migration of GBM-originated cells, positioning it as a tumor suppressor in brain cancer. In contrast, elevated Spry3 levels accelerated both proliferation and migration of these cell lines, while repression of Spry3 levels using shRNA caused a significant diminished growth and migration velocity rate of a GBM-derived cell line. This argues for a tumor-promoting function of Spry3 in GBMs. Based on these data we conclude that Spry3 and Spry4 fulfill different if not opposing roles within the cancerogenesis of brain malignancies.

Ab st rac t . Sp rout y p rot ei ns a r e mo du lat or s of mitogen-induced signalling processes and are therefore hypothesized to affect malignant diseases. As a member of the Sprouty family, Sprouty4 has been previously shown to function as a tumour suppressor in lung and breast cancer. The present study analysed the expression of two known Sprouty4 splice variants in cells established from malignant and normal lung and breast tissues using semi-quantitative reverse transcription-polymerase chain reaction and immunoblotting. The results indicated that the expression of the two messenger RNA (mRNA) variants was reduced in the cells derived from malignant tissue in comparison to the normal counterparts. Although the expression of the two splice variants were associated in both tissue types, on average, the relative expression of the longer variant was slightly increased in malignant cells compared with normal tissues. Notably, the protein levels reflected the expression observed at the mRNA level only in breast-derived cells. Contrarily, with regards to the measured mRNA levels, Sprouty4 protein was disproportional augmented in lung cells known to harbour the mutated K-Ras gene. IntroductionThe alteration of important molecules involved in signal transduction is a common characteristic of all malignant cells (1). The mitogen activated protein kinase (MAPK) pathway, which is crucially involved in balancing cell death and differentiation on one side and proliferation and migration on the other, is one of the best described signalling routes that is deregulated in cancer (2). Due to the vital role of the MAPK pathway, numerous feedback loops are present to allow a fine-tuning of signal intensity and duration (3). Certain molecules acting in such a regulatory loop are the members of the Sprouty (Spry) protein family. First discovered in Drosophila (4,5), the Spry proteins counteract processes induced by the presence of various growth factors. In humans, 4 Spry family members are known; all of which share a conserved Spry domain at the C-terminus and 2 homolog N-terminal boxes (6). As indicated by knockout studies in mice, the functions of Sprouty proteins overlap but are not redundant (7-10). Although Spry1, Spry2 and Spry4 can be detected in all organs, their expression pattern in the various cell types is clearly distinguishable (11). Like in Drosophila, mammalian Spry proteins interfere with signal transduction, particularly in the MAPK pathway, in response to various mitogens, such as the members of the fibroblast growth factors (6). In contrast to Drosophila, in mammalian systems, signalling activated by epidermal growth factor is increased with augmented Spry expression. Therefore, the Spry proteins in humans are generally considered as modulators of signal transduction (12). Accordingly, dependent on the tumour type, Spry proteins are found to fulfil multiple roles in cancer. For example, Spry2 has been indicated to function as an oncogene in colon cancer (13,14) and Spry1 has a tumour promoting role in rhabd...

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