Sprouty was discovered and initially described by Hacohen et al. in 1998 as an inhibitor of fibroblast growth factor (FGF)-mediated tracheal branching (hence its name) in Drosophila melanogaster (Hacohen et al. 1998). Later, they further defined it as a common antagonist of FGF and epidermal growth factor (EGF) signaling pathways (Kramer et al. 1999). Along with the discovery of the Drosophila gene, or dSpry, they performed a search of the expressed sequence tag (EST) database and identified three human homologs of dSpry which were designated hSpry1, hSpry2, and hSpry3 (Hacohen et al. 1998). The fourth mammalian homolog (Spry4) was discovered later in mice (de Maximy et al. 1999) and humans (Leeksma et al. 2002). Since then, an expanding body of evidence has continued to support the crucial role of Sprouty in the regulation of key cellular processes and biological events, mainly as a modulator of receptor tyrosine kinase (RTK) signaling (Mason et al. 2006; Cabrita and Christofori 2008).
Gene and Protein Family
hSpry1, hSpry2, hSpry3 and hSpry4 genes are located on 4q26.1, 13q31.1, XqPAR2 (pseudoautosomal region 2), and 5q31.3, respectively, and encode the corresponding members of the human Sprouty protein family. This protein family and their regulatory relationship with RTK signaling are evolutionarily conserved. The biological functions of the Sprouty proteins have been attributed to their conserved motifs. These mainly include the N-terminal canonical Casitas B-lineage lymphoma (c-Cbl) binding domain (CBD) that contains a key tyrosine residue, the serine-rich motif (SRM), and the C-terminal cysteine-rich domain (CRD)- also known as the Sprouty (or translocation) domain. Among the Sprouty isoforms, Spry2 exhibits the highest conservation, with the human Spry2 (hSpry2) showing 97%, 85% and 51% sequence homology in the CRD domain to the mouse, chick and Drosophila homolog, respectively (Minowada et al. 1999). Although Spry2 appears to be ubiquitously expressed in embryonic and adult tissues, the expression of other isoforms shows organ/tissue specificity (Minowada et al. 1999; Leeksma et al. 2002; Ding et al. 2004).
Sprouty and MAPK/ERK Signaling
Mitogen-activated protein kinase (MAPK) signaling pathways are among the most widespread regulatory mechanisms of the eukaryotic cell biology. The first mammalian MAPK pathway to be identified and entirely mapped is extracellular signal-regulated kinase (ERK). ERK orchestrates a signal transduction from cell membrane molecules to the transcriptional machinery to promote cell growth, differentiation, and survival. As with other MAPKs, ERK represents a three-tiered kinase cascade composed of the sequentially acting kinases. ERK is activated by a wide range of extracellular signals including growth factors, cytokines, hormones, and neurotransmitters. Signal transduction is initiated when a ligand binds its transmembrane receptor tyrosine kinase (RTK) and thereby activates Ras, a small G protein anchored to the plasma membrane. Ras subsequently recruits (from the cytosol to the cell membrane) and activates Raf serine-/threonine-specific kinases of MAPK-kinase kinase (MAP3K) family. Through serine/threonine phosphorylation, Raf in turn activates a family of dual specificity kinases known as MAPK kinases (MAP2K) or MAPK/ERK kinases (MEKs). By concomitant tyrosine and threonine phosphorylation, MEKs eventually activate MAPK/Erk, which results in the expression of target genes by direct and indirect targeting of transcription factors. For a biologically coordinated functioning, ERK and its core modules are under tight, multilayered control of positive and negative regulators, including Sprouty (Cabrita and Christofori 2008). As a downstream modulator, Sprouty has been shown to inhibit activation of ERK in response to a wide range of trophic factors, including EGF, FGF (Hacohen et al. 1998; Kramer et al. 1999), platelet-derived growth factor (PDGF) (Gross et al. 2001), vascular endothelial growth factor (VEGF) (Impagnatiello et al. 2001), nerve growth factor (NGF) (Wong et al. 2002), brain-derived neurotrophic factor (BDNF) (Gross et al. 2007), and glial cell line-derived neurotrophic factor (GDNF) (Ishida et al. 2007).
Biological Functions and Interactions
As shown in Figs. 1 and 2, biological functions of Sprouty is resulted from, or regulated through, interactions with a number of molecules (Masoumi-Moghaddam et al. 2014). These interactions impact functionality of ERK and other signaling pathways. Sprouty binds c-Cbl and CIN85 and sequestrate c-Cbl to augment and prolong RTK signaling by inhibiting receptor endocytosis. This mechanism has been implicated in cell differentiation.
E3 ubiquitin ligase c-Cbl, on the other hand, binds and induces degradation of Sprouty to restrict ERK activation. Sprouty has also been shown to interact with different phosphatases. It increases active contents of PTEN to mediate antiproliferative actions by inhibiting Akt activation. PTEN is also phosphorylated and accumulated in the nucleus in response to the Sprouty deficiency to induce p53-mediated growth arrest independently of its phosphatase activity. It is likely that the proto-oncogenic potential of NEDD4 is resulted in part from its ability to ubiquitinate both Sprouty and PTEN, resulting in unchecked activation of Akt. Sprouty also increases PTP1B content. However, there is no evidence of direct interaction between Sprouty and PTP1B in RTK dephosphorylation. Phosphatases PP2A and SHP2 differentially regulate the Sprouty activity. While PP2A potentiates Sprouty binding to Grb2 and thus positively regulates Sprouty by serine dephosphorylation, SHP2 promotes dissociation of Sprouty from Grb2 through tyrosine dephosphorylation and checks Sprouty inhibition of ERK. Also, Sprouty is under the regulatory control of a number of kinases. DYRK1A is considered a negative regulator of the Sprouty activity by threonine phosphorylation. TESK1 interferes with Sprouty/Grb2 interaction as well as with Sprouty serine dephosphorylation by PP2A, thereby attenuating Sprouty functioning. Sprouty isoforms also exhibit differential cooperativity with Cav-1 to repress growth factor activation of ERK. At low cell density, however, Cav-1 inhibits the Sprouty function. Sprouty is a general inhibitor of PLC-dependent signaling and inhibits various PKC upstream and downstream signals, including PIP2 hydrolysis. Sprouty is an interacting partner of the Gαo/GRIN pathway.
GRIN modulates Sprouty repression of ERK by binding and sequestering Sprouty. Activated Gαo, on the other hand, promotes inhibition of ERK via interacting with GRIN and releasing Sprouty. Finally, interaction among the Sprouty isoforms is a mechanism through which oligomers with more potent activity can form (Fig. 1).
Sprouty-mediated regulation of cell migration, adhesion, and cytoskeletal rearrangement is depicted in Fig. 2. To this end, Sprouty is shown to interact with phosphatases and kinases. As such, Sprouty increases active contents of PTP1B to mediate its antimigrative action by inhibiting activation of Rac1. On the other hand, it inhibits the kinase activity of TESK1 that plays a critical role in integrin-mediated actin cytoskeletal reorganization and cell spreading. Sprouty is a general inhibitor of PLC-dependent signaling and inhibits various PKC upstream and downstream signals. Protocadherin PAPC implicated in modulating beta-catenin-independent Wnt-signaling has been suggested to mediate its regulatory effect by binding and sequestering Sprouty (Masoumi-Moghaddam et al. 2014).
Deregulation of Sprouty and Pathological Conditions
Given the critical role of Sprouty in regulating the strength and duration of RTK signaling and its influence on MAPK/ERK partner pathways, deregulated expression of this family has been reported in, and implicated in the pathogenesis of, a number of pathological conditions, including cardiovascular diseases and cancer. As a negative inhibitor of the ERK cascade in the human heart, Sprouty was shown to inhibit vascular smooth muscle cell proliferation and migration (Zhang et al. 2005) as well as VEGF-induced proliferation of the endothelial cells (Huebert et al. 2004), governing ventricular remodeling through a coordinated coupling of both myocyte and vascular alterations in response to mechanical load. On the other hand, evidence has identified Sprouty as a potential target of microRNA21 (miR21) that is up-regulated during cardiac hypertrophy and heart failure (Edwin et al. 2009). As such, miR21 was shown to suppress Spry1 and Spry2 in cardiac fibroblasts (Thum et al. 2008) and cardiomyocytes (Sayed et al. 2008) of the hypertrophic and failing heart, respectively.
Evidence shows that ERK is aberrantly activated in malignant conditions. Accordingly, Sprouty has been reported to be deregulated in different cancer types, shown to impact tumor development, progression, and metastasis. Since Sprouty-mediated regulation under physiological condition per se is complex and multifaceted, one can anticipate that the role of deregulated Sprouty in malignant conditions, where physiological homeostasis is altered in favor of neoplastic growth and progression, is fraught with intricacy and controversy. As with normal conditions, current knowledge supports the notion that Sprouty functions in a cell type- and context-dependent manner in cancer. In addition, although deregulation of the Sprouty genes can indicate a general aspect of the Sprouty status in a given cancer, this needs to be interpreted in relation to the gene expression at the protein level and pertinent functional outcomes. A rewired genetic network with the involvement of Sprouty and ERK signaling apparently promotes tumorigenesis. However, the Sprouty gene association with tumor susceptibility or resistance may not be necessarily associated with a consistent in vivo phenotype due to somatic alterations. Thus, a combination of genetic and gene expression analysis has been recommended to complement genetic association methods for identification of susceptibility or resistance factors (Quigley et al. 2011). The expression of the Sprouty proteins, on the other hand, might be variably altered during tumorigenesis based on the pathogenic mechanism involved. Therefore, the expression pattern of Sprouty might be reflecting, for instance, a response to the mutant RAS-induced hyperactivation of ERK or, on the contrary, epigenetic silencing of the Sprouty promoter. Moreover, Sprouty’s mode of action can be converted under malignant conditions. In the context of the RAS mutation, for example, Sprouty can function as an inhibitor or facilitator of the tumor development and progression. This might be resulted in part from different functionality of the RAS isoforms (Lito et al. 2008). The status and role of Sprouty in different cancers have been reviewed in detail elsewhere (Masoumi-Moghaddam et al. 2014). These include the breast, prostate, liver, lung, colon, thyroid, pituitary, endometrial, ovarian and testicular germ cell cancer, clear cell renal cell carcinomas, melanoma, sarcoma, and lymphoma.
By regulating the strength and duration of growth factor signaling, Sprouty may also play a critical role in the pathogenesis of other diseases (Edwin et al. 2009). As such, Spry2 levels are elevated in chondrocytes of mice and humans with thanatophoric dysplasia type II, a lethal form of chondrodysplasia (Guo et al. 2008). A decrease in the amount of Spry2 in prefrontal cortex of schizophrenic and bipolar patients has been reported and correlated with a decrease in brain-derived neurotrophic factor (Pillai 2008). It was also reported that electroconvulsive therapy decreases the expression of Spry2 within the prefrontal cortex of rats and augments glial cell proliferation, thereby increasing neuronal plasticity (Ongur et al. 2007).
Initially discovered as a growth factor antagonist with involvement in developmental processes, Sprouty is now recognized as a versatile modulator of ERK that also impacts other pathways to control crucial physiological processes in interaction with an increasing number of effectors, mediators, and regulators. Physiological functions of Sprouty are cell-specific and context-dependent. As such, Sprouty is differentially induced in response to different growth factors and elicits divergent cellular responses, including cell proliferation, differentiation, migration, adhesion, survival, and cytoskeletal rearrangement. Moreover, transcriptional and posttranslational regulation of the Sprouty content and activity provide spatiotemporal control of the Sprouty-mediated regulation. Given its critical role as an evolutionarily conserved regulator of key biological processes, deregulation of Sprouty in pathological conditions, in particular cancer, has been the focus of a wide variety of investigations for more than a decade. As a result, different patterns of the Sprouty deregulation have been reported in different cancers. Evidence shows that Sprouty functions in a cell specific- and context-dependent manner in cancer, hence its implication as a negative or positive regulator of the tumor progression. The presence of accompanying mutations of such oncogenes as RAS isoforms has also been shown to be an important determinant of the Sprouty’s mode of action. To evaluate the role of Sprouty in a particular cancer and its putative utilities as a biomarker or a target of targeted therapies, in-depth investigation of its expression and function in relation to the malignant behavior of cancer cells in the specific tumor microenvironment is essential.