Sprouty negatively regulates receptor tyrosine kinase signals by inhibiting Ras/ERK pathways. Sprouty is down-regulated in breast, prostate and liver cancers and appears to function as a tumor suppressor. The role of Sprouty in colonic neoplasia, however, has not been investigated. Sprouty-2 protein and mRNA transcripts were significantly up-regulated in human colonic adenocarcinomas. Strikingly, the c-Met receptor was also upregulated in tumors with increased sprouty-2. To delineate a potential causal relationship between sprouty-2 and c-Met, K-ras mutant HCT-116 colon cancer cells were transduced with purified TAT-sprouty-2 protein or stably transfected with full-length human sprouty-2 gene. Sprouty-2 up-regulation significantly increased cell proliferation by accelerating cell cycle transition. Sprouty-2 transfectants demonstrated strong up-regulation of c-Met protein and mRNA transcripts and hepatocyte growth factor stimulated ERK and Akt phosphorylation and enhanced cell migration and invasion. In contrast, knockdown of c-Met by siRNA significantly decreased cell proliferation, migration and invasion in sprouty-2 transfectants. Further, knockdown of sprouty-2 by siRNA in parental HT-29 and LS-174T colon cancer cells also decreased cell invasion. Sprouty-2 transfectants formed significantly larger tumor xenografts and demonstrated increased proliferation and angiogenesis and suppressed apoptosis. Sprouty-2 tumors metastasized to liver from cecal orthotopic implants suggesting sprouty-2 might also enhance metastatic signals. Thus in colon cancer sprouty functions as an oncogene and its effects are mediated in part by c-Met up-regulation.

Sprouty family proteins are novel regulators of growth factor actions. Human Sprouty 2 (hSPRY2) inhibits the proliferation of a number of different cell types. However, the mechanisms involved in the anti-proliferative actions of hSPRY2 remain to be elucidated. Here we have demonstrated that hSPRY2 increases the amount of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and decreases its phosphorylation. The resultant increase in PTEN activity is reflected in decreased activation of Akt by epidermal growth factor and serum. Consistent with increased PTEN activity, in hSPRY2-expressing cells, the progression of cells from the G 1 to S phase is decreased. By using PTEN null primary mouse embryonic fibroblasts and their isogenic controls as well as small interfering RNA against PTEN, we demonstrated that PTEN is necessary for hSPRY2 to inhibit Akt activation by epidermal growth factor as well as cell proliferation. Overall, we concluded that hSPRY2 mediates its anti-proliferative actions by altering PTEN content and activity.Over the past few years, the Sprouty (SPRY) 2 family of proteins has emerged as an important modulator of growth factor actions. The ability of the Sprouty proteins to regulate the biological activity of growth factors has been conserved through evolution. Drosophila SPRY (dSPRY) was the first member of the family to be identified and has been shown to regulate tracheal branching in response to fibroblast growth factor (1). Later studies demonstrated that dSPRY also inhibited the actions of EGF (2). Like dSPRY, mammalian SPRY isoforms (SPRY1-4) have also been shown to modulate growth factor-mediated actions. For instance, mouse SPRY2 expression resulted in increased lung branching morphogenesis (3). These findings suggest that SPRY proteins have conserved function to modulate respiratory morphogenesis. The ability of mouse SPRY4 to inhibit angiogenesis (4) and cause pulmonary hypoplasia as well as the ability of SPRY2 and SPRY1 to decrease uteretic branching and kidney development (5, 6) also demonstrates that the SPRY proteins play a profound role in regulating tubular morphogenesis. Notably, however, SPRY proteins do not exclusively regulate tubular morphogenesis, but by opposing the actions of growth factors they may also play a role in the development of other organs, such as the brain and limbs (7-9).At the cellular level, we and others have shown that overexpression of SPRY1 (10), SPRY2 (11, 12), and SPRY4 (5) inhibits migration and proliferation of cells in response to serum and growth factors. Treatment of cells with EGF results in translocation of the human SPRY2 (hSPRY2) protein from the vicinity of microtubules to membrane ruffles (11, 13). The abrogation of co-localization of hSPRY2 with microtubules or deletion of the region that is necessary for translocation to membrane ruffles obliterates the ability of the protein to inhibit cell migration and proliferation (11). We have shown previously that hSPRY2, in part, mediates its anti-migratory action...

Receptor tyrosine kinase (RTK) signaling is spatially and temporally regulated by a number of positive and negative regulatory mechanisms. These regulatory mechanisms control the amplitude and duration of the signals initiated at the cell surface to have a normal or aberrant biological outcome in development and disease, respectively. In the past decade, the Sprouty (Spry) family of proteins has been identified as modulators of RTK signaling in normal development and disease. This review summarizes recent advances concerning the biological activities modulated by Spry family proteins, their interactions with signaling proteins, and their involvement in cardiovascular diseases and cancer. The diversity of mechanisms in the regulation of Spry expression and activity in cell systems emphasizes the crucial role of Spry proteins in development and growth across the animal kingdom.The Sprouty (Spry) protein was first described by Hacohen et al. (1998) as an inhibitor of fibroblast growth factor (FGF)-stimulated tracheal branching during Drosophila melanogaster development. Subsequent work established D. melanogaster Spry (dSpry) as a widespread inhibitor of receptor-tyrosine kinase (RTK) signaling during organogenesis. For example, spry-null flies or flies harboring loss of function mutations on spry exhibit eye and wing phenotypes indicative of uncontrolled epidermal growth factor receptor (EGFR) signaling (Minowada et al., 1999).Four mammalian spry genes have been defined based on sequence similarity with dSpry. Three homologs of dSpry were first identified in a search of the human expressed

on ubiquitin and decrease its stability. Silencing of endogenous Nedd4 increased the cellular Spry2 content and attenuated fibroblast growth factor-elicited ERK1/2 activation that was reversed when elevations in Spry2 levels were prevented by Spry2-specific small interfering RNA. Mnk2 silencing decreased Spry2-Nedd4 interactions and also augmented the ability of Spry2 to inhibit fibroblast growth factor signaling. This is the first report demonstrating the regulation of cellular Spry content and its ability to modulate receptor tyrosine kinase signaling by a HECT domain-containing E3 ubiquitin ligase.

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