Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade, a pathway known to be frequently deregulated in human non -small cell lung cancer (NSCLC). In this study, we show a consistently lowered Spry2 expression in NSCLC when compared with the corresponding normal lung epithelium. Based on these findings, we investigated the influence of Spry2 expression on the malignant phenotype of NSCLC cells. Ectopic expression of Spry2 antagonized mitogen-activated protein kinase activity and inhibited cell migration in cell lines homozygous for K-Ras wild type, whereas in NSCLC cells expressing mutated K-Ras, Spry2 failed to diminish extracellular signal-regulated kinase (ERK) phosphorylation. Nonetheless, Spry2 significantly reduced cell proliferation in all investigated cell lines and blocked tumor formation in mice. Accordingly, a Spry2 mutant unable to inhibit ERK phosphorylation reduced cell proliferation significantly but less pronounced compared with the wild-type protein. Therefore, we conclude that Spry2 interferes with ERK phosphorylation and another yet unidentified pathway. Our results suggest that Spry2 plays a role as tumor suppressor in NSCLC by antagonizing receptor tyrosine kinase -induced signaling at different levels, indicating feasibility for the usage of Spry in targeted gene therapy of NSCLC. (Mol Cancer Res 2007;5(5):509 -20)

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.

Sprouty (Spry) proteins are well-known negative regulators of receptor tyrosine kinase-mediated signalling. Their expression is controlled by mitogens, implying a negative feedback loop. Correspondingly, the different members of the family fulfil important roles during organogenesis by adjustment of growth factor-induced processes. In addition, Spry4, one member of this protein family, has been shown to regulate angiogenesis by inhibiting vascular endothelial cell growth factor-induced extracellular signalling-regulated kinase (ERK) activation. Because oxygen is an important regulator of angiogenesis, we investigated Spry4 expression patterns under hypoxic conditions. Our data demonstrate that both hypoxia and desferrioxamine (DFO) treatment increased Spry4 expression. Following iron depletion, elevated Spry4 levels were detected in several cell types independent of tissue origin, presence of mitogens, cell differentiation and malignancy. Evaluation of the underlying regulative mechanisms revealed that augmented transcription and increased mRNA stability enhance mRNA levels of Spry4 in response to DFO. This study unveils a growth factor-independent regulation mechanism of Spry4 expression. Because increased Spry4 levels are accompanied by disappearing ERK phosphorylation, Spry4 might be involved in the timely restriction of MAPK signals under hypoxic conditions, similar to its role in mitogen-regulated processes. However, the functional significance of the observed upregulation of Spry4 during iron depletion remains to be clarified.

Sprouty1 protein belongs to a family of receptor tyrosine kinase-mediated signaling inhibitors, whose members are usually regulated by growth factors to form a negative feedback loop. Correspondingly fluctuations of Sprouty1 mRNA in response to single growth factors have been observed. In this report, we investigate Sprouty1 protein levels and show that in non-small cell lung carcinoma-derived cells, the expression levels are unaffected by the serum content in the cellular environment. Although cells harboring K-Ras mutations express insignificant higher Sprouty1 levels, ectopic expression of constitutive active Ras in normal human lung fibroblasts fails to augment Sprouty1 protein content. Furthermore, serum starvation for three days has no influence on Sprouty1 expression and addition of serum or of singular growth factors leaves Sprouty protein levels unchanged. Cell cycle analysis reveals that Sprouty1 levels remain constant throughout the whole cell cycle. These data demonstrate that Sprouty1 expression is not connected with mitogenic signaling and cell proliferation.