Gene name - wingless
Synonyms - Dint-1
Cytological map position - 28A1-28A3
Function - secreted signaling protein
Symbol - wg
Genetic map position - 2-
Classification - WNT family
Cellular location - extracellular and cytoplasmic
|Recent literature||Li, X., Wu, Y., Shen, C., Belenkaya, T. Y., Ray, L. and Lin, X. (2015). Drosophila p24 and Sec22 regulate Wingless trafficking in the early secretory pathway. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 26002470
The Wnt signaling pathway is crucial for development and disease. The regulation of Wnt protein trafficking is one of the pivotal issues in the Wnt research field. A genetic screen was performed in Drosophila melanogaster for genes involved in Wingless/Wnt secretion, and the p24 protein family members Baiser, CHOp24, Eclair and a v-SNARE protein Sec22, were identified that are involved in the early secretory pathway of Wingless/Wnt. Genetic evidence is provided demonstrating that loss of p24 proteins or Sec22 impedes Wingless (Wg) secretion in Drosophila wing imaginal discs. Baiser cannot replace other p24 proteins (CHOp24 or Eclair) in escorting Wg, and only Baiser and CHOp24 interact with Wg. Moreover, it was shown that the v-SNARE protein Sec22 and Wg are packaged together with p24 proteins. Taken together, these data provide important insights into the early secretory pathway of Wg/Wnt.
|Kumar, S. R., Patel, H. and Tomlinson, A. (2015). Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye. Dev Biol [Epub ahead of print]. PubMed ID: 26428511
Morphogen gradients play pervasive roles in development, and understanding how they are established and decoded is a major goal of contemporary developmental biology. This study examined how a Wingless (Wg) morphogen gradient patterns the peripheral specialization of the fly eye. The outermost specialization is the pigment rim; a thick band of pigment cells that circumscribes the eye and optically insulates the sides of the retina. It results from the coalescence of pigment cells that survive the death of the outermost row of developing ommatidia. This study investigated here how the Wg target genes expressed in the moribund ommatidia direct the intercellular signaling, the morphogenetic movements, and ultimately the ommatidial death. A salient feature of this process is the secondary expression of the Wg morphogen elicited in the ommatidia by the primary Wg signal. Neither the primary nor secondary sources of Wg alone are able to promote ommatidial death, but together they suffice to drive the apoptosis. This represents an unusual gradient read-out process in which a morphogen induces its own expression in its target cells to generate a concentration spike required to push the local cellular responses to the next threshold response.
A recurring, significant theme in insect development is the subdivision of the embryo into ever greater numbers of compartments within segments. At the earliest stages of development segments are defined by pair rule genes, and subsequently, each segment is subdivided into anterior and posterior compartments by the action of segment polarity genes. wingless, as a segment polarity gene, has a role in the establishment of different cell fates, working within and between the anterior and posterior compartments of segments.
Normally, each thoracic and abdominal segment contains an anterior denticle band, and a more posterior region of naked cuticle. In wingless mutants, the naked cuticle is absent, replaced by a disordered array of denticles (Bejsovec, 1991).
The effects of wingless mutation on morphology are mirrored by events inside the embryonic cells. Wingless is secreted by cells in each of 14 posterior compartments of parasegments (embryonic segments). Wingless secretion is dependent on Hedgehog, produced in adjacent compartments. Lack of functional posterior parasegmental compartments (due to a failure to secrete Wingless) results in altered activity just underneath the outer cell membrane. There is an altered distribution of Armadillo, and altered expression of shaggy/zeste-white3. Armadillo is associated with adherens junctions, structures that bind one cell to another, and Shaggy is involved in the transmission of the wingless signal inside the cell. Mutation of wingless also alters the secretion of cuticle and the regulation of denticle production both in the posterior cells of each compartment, and in adjacent cells that would otherwise have responded to wingless signaling.
Wg influences two distinct cellular decisions in patterning the larval ventral epidermis. This segmentally repeating pattern consists of six rows of uniquely shaped denticles arranged in a belt at the anterior of the segment, anterior to the cells that secrete Wingless protein, and an expanse of smooth, naked cuticle form in the posterior portion of the segment. In the absence of wg both the generation of diverse denticle types and the specification of naked cuticle are disrupted, resulting in a lawn of uniform denticles. wg is expressed in one row of cells in each wild-type segment, roughly in the middle of the naked cuticle region. Thus Wg activity influences cell fate decisions many rows of cells away from its source. What then accounts for the two cell fate regulated by Wg signaling in the ectoderm (Moline, 1999)?
Proper pattern formation requires temporal as well as spatial control of Wg activity (Bejsovec, 1991). Analysis of a temperature-sensitive wg allele that is wild type at 18oC and null for function at 25oC has shown that Wg activity between 4 and 5.5 hours of development generates diverse denticle types and stabilizes the expression of engrailed. en is a segment polarity gene expressed in the two rows of cells just posterior to the wg domain, at the posterior boundary of each segment. After 6 hours, Wg activity no longer produces these cellular responses, but instead promotes the naked cuticle-secreting cell fate. Thus the population of cells responding to Wg activity changes during development (Moline, 1999 and references therein).
Wg and Wnt molecules tightly associate with membrane and extracellular matrix and appear not to be readily soluble. Thus, it is unlikely that these proteins freely diffuse through extracellular spaces. Rather, Wg appears to be transported via active cellular processes. This phenomenon was first demonstrated using the shibirets (shits) mutation to block endocytosis (Bejsovec, 1995). shi encodes the fly dynamin homologue, a GTPase required for clathrin-coated vesicle formation. Rather than the broad, punctate Wg protein distribution normally found over several cell diameters on either side of the wg-expressing cells, shi mutant embryos show high level accumulation of Wg around the wg-expressing cells (Moline, 1999).
Reducing endocytosis in defined domains within the segment, through moderate-level expression of a dominant negative form of Shibire, alters the normal distribution of Wg and changes the domain of cells that respond to Wg. When expressed using the prd-Gal4, shiD reduces both anterior and posterior movement of Wg protein, causing it to accumulate in and around the wg-expressing row of cells. Driving expression of shiD with the en-Gal4 reduces movement only in the posterior direction, since the en-expressing cells are a non-overlapping cell population just posterior to the wg-expressing row of cells (Moline, 1999).
The effects on cuticular pattern elements indicate that Wg moving in an anterior direction from the row of wg-expressing cells defines the domain of cells destined to secrete naked cuticle, whereas posterior movement of Wg is required for correct specification of denticle types in the anterior of the adjacent segment. The patterning defects caused by shiD expression are reversed by co-expression with wg plus, suggesting that the primary effect of reducing endocytosis in the embryonic epidermis is a disruption of Wg protein transport. Moreover, en-Gal4-driven shiD reduces endocytosis in a non-wg-expressing group of cells, and causes patterning defects in the cell population posterior to the en domain. Thus, reducing Wg transit through the en cells casts a shadow, producing patterning anomalies in an otherwise wild-type cell population. This supports the idea that Wg ligand is moved by active cellular processes through cells to arrive at distant target cell populations in the embryo (Moline, 1999).
The results suggest that, during normal development, the temporal changes observed in directionality of Wg protein movement (Gonzalez, 1991) may correlate with the temporal changes in its apparent function (Bejsovec, 1991). In wild-type embryos prior to stage 10, Wg protein is detected over many cell diameters both anterior and posterior to the wg-expressing row of cells (Gonzalez, 1991). Disrupting posterior movement of Wg alters patterning of at least the first three rows of denticles in the segment posterior to the affected source of Wg. Thus, posterior movement of Wg is detectable during the early time period when Wg activity is required in these cells for the generation of diverse denticle types and for the stabilization of en expression (Bejsovec, 1991). At and after stage 10, Wg protein is no longer detected in cells posterior to the wg-expressing row, including the en-expressing cells of that segment, and shows an asymmetric distribution toward the anterior of the segment (Bejsovec, 1991; Gonzalez, 1991). The results reported here correlate this anterior movement with specification of the correct expanse of naked cuticle-secreting cells, presumably through Wg-mediated antagonism of the EGF pathway. This is consistent with previous reports that, after stage 10, Wg is no longer required for maintenance of en expression (Bejsovec, 1991) or for the generation of denticle diversity, and instead promotes specification of naked cuticle cell fate (Bejsovec, 1991, Moline, 1999).
It is unclear by what mechanism Wg is excluded from the posterior cells at stage 10. It is proposed that wild-type naked gene function may contribute to the change in direction of Wg protein movement. Reducing Wg movement through the en-expressing cells eliminates Wg-mediated specification of excess naked cuticle and substantially rescues the nkd mutant phenotype. Thus, posterior movement of Wg from the adjacent segment, and not anterior movement of Wg within the segment, appears to be responsible for the naked mutant phenotype. This observation suggests a role for nkd gene function in restricting posterior Wg transport (Moline, 1999).
Although some aspects of Wg transport appear to be independent of Wg signal transduction, the two processes cannot be completely separated. Overexpression of Dfz2, a Wg signaling receptor, appears to restrict the distribution of the Wg protein, suggesting that it has the capacity to sequester ligand. In contrast, Dfz2 overexpression in the imaginal disc has been shown to enhance the transport of Wg protein and consequently increase its range of activity. This dramatic change in the role of Dfz2 from embryo to imaginal disc suggests that mechanisms controlling Wg distribution may differ between these two developmental stages of Drosophila. For example, recent work has revealed that imaginal disc cells project cytoplasmic extensions, called cytonemes, toward the source of signaling molecules at the center of the discs. These extensions may assist in the broad distribution and long-range activity documented for Wg in the imaginal discs (Moline, 1999 and references therein).
Such cytoplasmic extensions have not been detected in vivo in embryonic epidermal cells. If embryonic cells do produce cytonemes, they may not be functionally relevant to the distribution of Wg signaling activity. Reducing endocytosis in the two rows of en-expressing cells produces Wg-related pattern disruptions in the cells posterior to the affected domain. This suggests that Wg must physically move through the en cells in order to influence cell fate decisions in the posterior cell population. Such an effect would not be predicted if the posterior population were able to extend cytoplasmic projections through the affected 2 cell diameters and directly contact the cells expressing wg (Moline, 1999).
Mutant Wg molecules that are secreted properly, but fail to signal, are transported as if by default (Bejsovec, 1995). Initially, these mutant embryos show a wild-type distribution of Wg protein, but over time they begin to accumulate Wg-containing vesicles in tissues that do not express the gene and in which the protein is not normally detected. This indicates that most, if not all, embryonic cells have the ability to internalize Wg, and that this process does not require signal transduction. Moreover, it suggests that the mutant Wg ligand is able to bind to a cell surface receptor that does not transduce signal. This is consistent with a multiple-receptor model for Wg, where some Wg-binding receptors are dedicated exclusively to the transport process. Thus the dynamic distribution of Wg during development may reflect an interplay between signaling receptors and other cell surface molecules essential for ligand transport (Moline, 1999). These results suggest that a single signaling molecule, in this case Wingless, can determine multiple cell fates. These alternate cell fates depend on cell autonomous temporal changes in responsiveness to the Wg ligand and on regulated transport across adjacent cell populations that facilitate or interfere with this transport differently.
The effects of wingless signaling in the margin of the wing are fairly well understood. Here decapentaplegic is not expressed adjacent to Wingless producing cells, as is the case in embryonic segmentation. Any possible compounding effects attributable to DPP are removed, due to its absence, thus demonstrating a pure wingless effect. In the case of the wing, wingless expression is independent of hedgehog while dpp expression remains dependent on hh. The anterior edge of the wing is marked by stout, slender, and chemosensory bristles, all three types of which are innervated. Bristles and epidermal hairs are not innervated. Thus in the wing margin one can more easily observe the effect of the presence or the absence of wingless on bristle cell production and innervation, without having to contend with the effects of dpp production.
Both achaete and cut are involved in the specification of sensory bristles, the peripheral sense organs of the wing margin. wingless is expressed in a narrow band of cells. Adjacent cells which do not produce wingless serve as precursors of both sensory and non-sensory elements. Cut protein is expressed in a wingless dependent fashion in cells expressing wingless; achaete is expressed in the adjacent cells, those not expressing wingless. Both cut and achaete expression are dependent on wingless. The wings of flies carrying conditional lethal mutations of wingless show an absense of bristles; mechanoreceptors are transformed into chemoreceptors and the arrangement of chemoreceptors is altered. Thus the wingless signal modifies the production of achaete and cut resulting in altered sensory cell and bristle production (Couso, 1994). In summary, wingless critically regulates the production of bristles and sensory cells on the wing margin. It does this as a secreted molecule acting locally on adjacent cells, modifying the production of Cut and Achaete, two proteins involved in neurogenesis.
It has been suggested that wingless expression at the dorsal-ventral boundary of the wing disc depends on a signal from dorsal to ventral cells mediated by Serrate and Notch. Wingless expression is lost from the wing margin and the size of the wing is significantly reduced when Notch activity is removed from the third instar larva using a temperature sensitive allele of Notch. Therefore, it is likely that wingless is regulated by the Notch pathway acting through Suppressor of Hairless (Diaz-Benjumea, 1995).
Wingless has an earlier role in specification of the wing. Wing discs arise during embryonic development from a region of the epidermis devoid of wg expression. Ten to thirteen cells in each wing primordium express engrailed but not wingless. Thus, the obligitory role of wingless in leg disc formation does not appear to hold for wing disc formation.
During the second larval instar wg expression is first detected in the anterior compartment of wing discs. wingless appears to have a primary role in specifying the wing primordium. This conclusion is based on the observation that ectopic expression of wg can induce supernumary wings in the portion of the disc normally fated to give rise to body wall. Thus WG protein can reprogram cells in the notum to wing pouch identity very early in wing development. An important target of WG in this function is the gene pdm-1 which is involved in specifying the proximal-distal axis of the wing (Ng, 1996).
Thus, two distinct roles for wingless in wing morphogenesis have been identified: a primary role in specifying the wing primordium, and subsequent role mediating the patterning activities of the dorso-ventral compartment boundary (Ng, 1996).
Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. This study describe a new tissue exhibiting planar cell polarity -- the denticles, hair-like projections of the Drosophila embryonic epidermis. the changes in the actin cytoskeleton that underlie denticle development are described in real time, and this is compared with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. An initial characterization is presented of the localization of several actin regulators during denticle development. Several core planar cell polarity proteins are asymmetrically localized during the process. Finally, roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity are described (Price, 2006).
Among the hallmarks of PCP in structures as diverse as Drosophila wing hairs to stereocilia in the mammalian ear is polarization of the actin cytoskeleton. The polarized actin cytoskeleton underlying wing hair polarity has been described and defects in polarization in fz and dsh mutants have been documented. Microtubules (MTs) are also polarized in developing wing hairs, and disruption of either actin or MTs disrupts wing hair formation. The data suggest that basic features of cytoskeletal polarity in pupal wing hairs are also seen in denticles. Denticles, like wing hairs, arise from polarized actin accumulations in denticles this occurs along the posterior cell margin. Further, like wing hairs, denticles all elongate in the same direction. The less detailed analysis of dorsal hairs suggests that they also arise from polarized actin accumulations, but these are more complex; different cell rows accumulate actin either along the anterior or posterior cell margin (Price, 2006).
The effect of Wg and Hh on denticle development is mediated in part by their regional activation of the Shaven-baby transcription factor (Ovo), which is necessary and sufficient for cells to generate actin-based denticles. Therefore genes that are targets of Shaven-baby are likely to be triggers for actin accumulation and cytoskeletal rearrangements. Wg and Hh signaling may also trigger polarization of cellular machinery that is not typically thought to be involved in PCP e.g. the polarity of Arm that was observed. It will be useful in the future to examine whether proteins polarized during germband extension, such as Bazooka, are also polarized during denticle formation. Mutations in both hh and wg also affected the normal changes in cell shape accompanying denticle formation rather than elongating along the dorsal-ventral axis, cells remain columnar. A similar failure of cells to polarize during dorsal closure is observed in wg mutants. These effects may reflect alterations in cell polarization or cytoskeletal regulation. It will be of interest to determine whether changes in cell shape are coupled to the establishment of cytoskeletal polarity (Price, 2006).
Thus far the analysis of actin in wild-type and mutant pupal wings has been restricted to snapshots in fixed tissue. This was extended by examining F-actin in developing denticles in real time, revealing features of polarization that have not been noted previously; these features may be shared with wing hairs or other polarized structures. The initial cytoskeletal change observed was actin accumulation all across the apical surface of the cell. This actin gradually 'condenses', becoming more restricted to the posterior cell margin and forming distinct condensations, which then brighten and sometimes merge. They then elongate, all in the posterior direction. It will be interesting to learn whether the dynamic aspects of condensation involve de novo actin polymerization and/or collection of preexisting actin filaments (Price, 2006).
It is only in late condensations that enrichment was seen of any of the actin regulators that were examined. Arp3 and Dia are weakly enriched in late condensations, with enrichment increasing as denticles elongate, and Ena is enriched even later. Of course, the localization of these actin regulators to developing denticles does not by itself demonstrate that they play an important role there, but it is consistent with the possibility that they have a role in actin remodeling associated with denticle elongation. To test this hypothesis, genetic analyses will be necessary. This presents significant obstacles, since Arp2/3 and Dia are required for much earlier events (syncytial stages and cellularization), while maternal Ena plays a role in oogenesis, complicating analysis of loss-of-function mutants. Surprisingly, none of these actin regulators localizes in an informative fashion during the initial formation of actin condensations (though APC2 localizes there during this time). Thus additional regulators functioning during early denticle development need to be identified. Studies of cytoskeletal regulation in the larger adult sensory bristles may guide this. EM studies, the use of cytoskeletal inhibitors, and FRAP, which has proved informative in studies of wing hairs and bristles, may reveal how actin in denticles is assembled. Finally, it will be important to study in denticles additional actin regulators that regulate bristle development (Price, 2006).
What signals regulate denticle polarity? As examples of PCP have proliferated, understanding of the signals that instruct cells about their orientation in epithelial sheets has evolved. Certain features are shared in many, if not all, tissues. Fz receptors play a key role. Other core polarity proteins including Dsh, Fmi, Van Gogh/Strabismus and Prickle act in many if not all places. The current data extend this analysis to the denticles. Intriguing differences were found between the phenotypes of loss of Wg or Hh signaling, in which polarity was severely altered or abolished and loss of proteins that play dedicated roles in PCP, such as embryos null for either fz or stbm, which exhibit more subtle defects. A strong polarity bias was retained in these latter mutants, with cells in the posterior denticle rows correctly polarized and only cells in the anterior two rows making frequent mistakes. Interestingly, occasional mistakes are also observed in wild-type embryos (albeit at much lower frequency) and these are also restricted to the anterior most rows. This is in strong contrast to the effects of these mutants in the wing disc, where they globally disrupt polarity (Price, 2006).
One possible reason for this difference is the different scales of the tissues. The embryonic segment is only 12 cells across, while the wing disc encompasses hundreds of cells. Many core polarity proteins help mediate a feedback loop that amplifies an initially small difference in signal strength between the two sides of a wing cell. Perhaps the small scale of the embryonic segment makes this reinforcement less essential. It is also intriguing that the polarity is most sensitive to disruption in the anterior two denticle rows. If signal emanated from the posterior, signal strength might be lower in the anteriormost cells, rendering the reinforcement process more important. The lower frequency of defects in pk1 mutants may also reflect the reduced role of the feedback loop, but this is subject to the caveat that pk is a complex locus with different mutations having different consequences. Future work will be needed to test these possibilities (Price, 2006).
Significant questions also remain about the signal(s) activating Fz receptors during PCP. Wnts were initial candidates, since Fz proteins are Wnt receptors. In vertebrates, this may be the case Wnt11 regulates convergent extension and Wnt proteins can regulate PCP in the inner ear. By contrast, Drosophila Wnt proteins may not play a direct role. The Wg expression pattern in the eye and wing discs is not consistent with a role as the PCP ligand. Detailed studies of PCP in the eye and abdomen are most consistent with the idea that neither Wg nor other Wnt proteins are polarizing signals, but suggest that Wg regulates production of a secondary signal [dubbed `X'). Recent work suggests that Fj, Ds and Fat may be this elusive signal, with Drosophila Wg acting as an indirect cue of polarity. In fact, one cannot rule out the possibility Wnt11's role in vertebrate convergent extension is also indirect (Price, 2006).
Roles were found for Wg, Dsh and Arm in establishing denticle polarity. At face value, Arm's role is surprising, since the current view is that the Wg pathway diverges at Dsh, with a non-canonical branch (see Eisenmann's Wnt Signaling) mediating PCP and the canonical pathway playing no role in this. However, the data do not imply that Arm is required in denticle PCP per se. Wg acts in a paracrine feedback loop to maintain its own expression. In embryos maternally and zygotically mutant for arm alleles that cannot transduce Wg, Wg expression is lost by late stage 9. Thus, even though Arm is not in the non-canonical pathway, loss of Arm could still disrupt PCP indirectly due to the loss of Wg expression (Price, 2006).
While the data demonstrate that Wg is required for denticle PCP, two things suggest its role is indirect. wg mutants retain segmental periodicity in denticle orientation, suggesting that polarity is not totally disrupted, while in hh mutants there is no segmental periodicity. Second, when Wg signaling was reduced but did not eliminated, many cells retained normal polarity and there was segmental periodicity to which cells lost polarity or exhibited polarity reversals. This is consistent with the idea that Wg regulates production of another ligand. In fact, Wg's role may be even more indirect given the more dramatic effect of hh, Wg's primary role in polarity may be to maintain Hh expression (this is also consistent with a requirement for canonical pathway components like Arm). Global activation of Hh signaling in the ptc mutant also disrupts polarity. Hh thus remains a possible directional cue. In the abdomen, Hh also plays an important role in polarity, but it does not seem to be the directional cue either but rather regulates its production; this may also be the case in the embryo. Thus the precise roles for canonical Wg and Hh signaling in denticle polarization must be addressed by future experiments. If neither Wnts nor Hh are directional signals, what is? Data from the eye, wing and abdomen suggest roles for Ds, Fj, Fat and Fmi but details differ in different tissues. It thus will also be useful to examine Ds, Fj and Fat's roles in embryonic PCP (Price, 2006).
Cancer cells demand excessive nutrients to support their proliferation but how cancer cells sense and promote growth in the nutrient favorable conditions remain incompletely understood. Epidemiological studies have indicated that obesity is a risk factor for various types of cancers. Feeding Drosophila a high dietary sugar was previously demonstrated to not only direct metabolic defects including obesity and organismal insulin resistance, but also transform Ras/Src-activated cells into aggressive tumors. This study demonstrates that Ras/Src-activated cells are sensitive to perturbations in the Hippo signaling pathway. Evidence that nutritional cues activate Salt-inducible kinase, leading to Hippo pathway downregulation in Ras/Src-activated cells. The result is Yorkie-dependent increase in Wingless signaling, a key mediator that promotes diet-enhanced Ras/Src-tumorigenesis in an otherwise insulin-resistant environment. Through this mechanism, Ras/Src-activated cells are positioned to efficiently respond to nutritional signals and ensure tumor growth upon nutrient rich condition including obesity (Hirabayashi, 2015).
The prevalence of obesity is increasing globally. Obesity impacts whole-body homeostasis and is a risk factor for severe health complications including type 2 diabetes and cardiovascular disease. Accumulating epidemiological evidence indicates that obesity also leads to elevated risk of developing several types of cancers. However, the mechanisms that link obesity and cancer remain incompletely understood. Using Drosophila, a whole-animal model system has been developed to study the link between diet-induced obesity and cancer: this model has provided a potential explanation for how obese and insulin resistant animals are at increased risk for tumor progression (Hirabayashi, 2015).
Drosophila fed a diet containing high levels of sucrose (high dietary sucrose or ‘HDS') developed sugar-dependent metabolic defects including accumulation of fat (obesity), organismal insulin resistance, hyperglycemia, hyperinsulinemia, heart defects and liver (fat body) dysfunctions. Inducing activation of oncogenic Ras and Src together in the Drosophila eye epithelia led to development of small benign tumors within the eye epithelia. Feeding animals HDS transformed Ras/Src-activated cells from benign tumor growths to aggressive tumor overgrowth with tumors spread into other regions of the body (Hirabayashi, 2013). While most tissues of animals fed HDS displayed insulin resistance, Ras/Src-activated tumors retained insulin pathway sensitivity and exhibited an increased ability to import glucose. This is reflected by increased expression of the Insulin Receptor (InR), which was activated through an increase in canonical Wingless (Wg)/dWnt signaling that resulted in evasion of diet-mediated insulin resistance in Ras/Src-activated cells. Conversely, expressing a constitutively active isoform of the Insulin Receptor in Ras/Src-activated cells (InR/Ras/Src) was sufficient to elevate Wg signaling, promoting tumor overgrowth in animals fed a control diet. These results revealed a circuit with a feed-forward mechanism that directs elevated Wg signaling and InR expression specifically in Ras/Src-activated cells. Through this circuit, mitogenic effects of insulin are not only preserved but are enhanced in Ras/Src-activated cells in the presence of organismal insulin resistance (Hirabayashi, 2015).
These studies provide an outline for a new mechanism by which tumors evade insulin resistance, but several questions remain: (1) how Ras/Src-activated cells sense the organism's increased insulin levels, (2) how nutrient availability is converted into growth signals, and (3) the trigger for increased Wg protein levels, a key mediator that promotes evasion of insulin resistance and enhanced Ras/Src-tumorigenesis consequent to HDS. This study identifies the Hippo pathway effector Yorkie (Yki) as a primary source of increased Wg expression in diet-enhanced Ras/Src-tumors. Ras/Src-activated cells are sensitized to Hippo signaling, and even a mild perturbation in upstream Hippo pathway is sufficient to dominantly promote Ras/Src-tumor growth. Functional evidence is provided that increased insulin signaling promotes Salt-inducible kinases (SIKs) activity in Ras/Src-activated cells, revealing a SIKs-Yki-Wg axis as a key mediator of diet-enhanced Ras/Src-tumorigenesis. Through this pathway, Hippo-sensitized Ras/Src-activated cells are positioned to efficiently respond to insulin signals and promote tumor overgrowth. These mechanisms act as a feed-forward cassette that promotes tumor progression in dietary rich conditions, evading an otherwise insulin resistant state (Hirabayashi, 2015).
Previously work has demonstrated that Ras/Src-activated cells preserve mitogenic effects of insulin under the systemic insulin resistance induced by HDS-feeding of Drosophila (Hirabayashi, 2013). Evasion of insulin resistance in Ras/Src-activated cells is a consequence of a Wg-dependent increase in InR gene expression (Hirabayashi, 2013). This study identified the Hippo pathway effector Yki as a primary source of the Wnt ortholog Wg in diet-enhanced Ras/Src-tumors. Mechanistically, functional evidence is provided that activation of SIKs promotes Yki-dependent Wg-activation and reveal a SIK-Yki-Wg-InR axis as a key feed-forward signaling pathway that underlies evasion of insulin resistance and promotion of tumor growth in diet-enhanced Ras/Src-tumors (Hirabayashi, 2015).
In animals fed a control diet, at most a mild increase was observed in Yki reporter activity within ras1G12V;csk-/- cells. A previous report indicates that activation of oncogenic Ras (ras1G12V) led to slight activation of Yki in eye tissue. Activation of Src through over-expression of the Drosophila Src ortholog Src64B has been shown to induce autonomous and non-autonomous activation of Yki. In contrast, inducing activation of Src through loss of csk (csk-/-) failed to elevate diap1 expression. The results indicate that activation of Yki is an emergent property of activating Ras plus Src (ras1G12V;csk-/-). However, this level of Yki-activation was not sufficient to promote stable tumor growth of Ras/Src-activated cells in the context of a control diet: Ras/Src-activated cells were progressively eliminated from the eye tissue (Hirabayashi, 2013). It was, however, sufficient to sensitize Ras/Src-activated cells to upstream Hippo pathway signals: loss of a genetic copy of ex-which was not sufficient to promote growth by itself-dominantly promoted tumor growth of Ras/Src-activated cells even in animals fed a control diet. These data provide compelling evidence that Ras/Src-transformed cells are sensitive to upstream Hippo signals (Hirabayashi, 2015).
SIK was recently demonstrated to phosphorylate Sav at Serine-413, resulting in dissociation of the Hippo complex and activation of Yki (Wehr, 2013). SIKs are required for diet-enhanced Ras/Src-tumor growth in HDS. Conversely, expression of a constitutively activated isoform of SIK was sufficient to promote Ras/Src-tumor overgrowth even in a control diet. Mammalian SIKs are regulated by glucose and by insulin signaling. However, a recent report indicated that glucagon but not insulin regulates SIK2 activity in the liver. The current data demonstrate that increased insulin signaling is sufficient to promote SIK activity through Akt in Ras/Src-activated cells. It is concluded that SIKs couple nutrient (insulin) availability to Yki-mediated evasion of insulin resistance and tumor growth, ensuring Ras/Src-tumor growth under nutrient favorable conditions (Hirabayashi, 2015).
The results place SIKs as key sensors of nutrient and energy availability in Ras/Src-tumors through increased insulin signaling and, hence, increased glucose availability. SIK activity promotes Ras/Src-activated cells to efficiently respond to upstream Hippo signals, ensuring tumor overgrowth in organisms that are otherwise insulin resistant. One interesting question is whether this mechanism is relevant beyond the context of an obesity-cancer connection: both Ras and Src have pleiotropic effects on developmental processes including survival, proliferation, morphogenesis, differentiation, and invasion, and these mechanisms may facilitate these processes under nutrient favorable conditions. From a treatment perspective the current data highlight SIKs as potential therapeutic targets. Limiting SIK activity through compounds such as HG-9-91-01 may break the connection between oncogenes and diet, targeting key aspects of tumor progression that are enhanced in obese individuals (Hirabayashi, 2015).
Bases in 5' UTR - 417
Exons - five
Bases in 3' UTR - 1085
The WG protein has an N-terminal hydrophobic region characteristic of a signal sequence whose function is to expedite secretion. There is one potential N-linked glycosylation site. The protein is rich in conserved cysteine residues (Rijsewijk, 1987).
date revised: 2 January 2001
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