Ras oncogene at 85D


In Drosophila, the Ras1 gene is required downstream of receptor tyrosine kinases for correct eye development, embryonic patterning, wing vein formation, and border cell migration. A P-element allele of Ras1, Ras1(5703), affects viability, eye morphogenesis, and early and late stages of oogenesis. Flies transheterozgyous for Ras1(5703) and existing EMS-induced Ras1 alleles are viable and exhibit a range of eye and eggshell defects. Differences in the severity of these phenotypes in different tissues suggest that there are allele-specific effects of Ras1 in development. Analysis of rescue constructs demonstrates that these differential phenotypes are due to loss of function in Ras1 alone and not due to effects on neighboring genes. Females mutant at the Ras1 locus lay eggs with reduced or missing dorsal eggshell structures. Dominant interactions are observed between Ras1 mutants and other dorsoventral pathway mutants, including Egfr(Torpedo) and gurken. Ras1 is also epistatic to K10. Unlike Egfr and gurken mutants, Ras1 females are moderately fertile, laying eggs with ventralized eggshells that can hatch normal larvae. These results suggest that Ras1 may have a different requirement in the patterning of the eggshell axis than in the patterning of the embryonic axis during oogenesis (Schnorr, 1996).

In Drosophila, Drk (an SH2 adaptor protein), Sos (a putative activator of Ras1), Ras1, raf and rolled/MAP kinase have been shown to be required for signaling from Sevenless and the torso receptor tyrosine kinase. From these studies, it was unclear whether these components act in a single linear pathway as suggested by the genetic analysis or whether different components serve to integrate different signals. Removing each of these components during the development of the adult epidermal structures produces a very similar set of phenotypes. These phenotypes resemble those caused by loss-of-function mutations in the Drosophila EGF receptor homolog. It appears that these components form a signaling cassette, which mediates all aspects of DER signaling but that is not required for other signaling processes during epidermal development (Diaz-Benjumea, 1994).

Mutations of the Drosophila homeotic proboscipedia gene (pb, the Hox-A2/B2 homolog) provoke dose-sensitive defects. These effects were used to search for dose-sensitive dominant modifiers of pb function. Two identified interacting genes are the proto-oncogene Ras1 and its functional antagonist Gap1, prominent intermediaries in known signal transduction pathways. Ras1+ is a positive modifier of pb activity both in normal and ectopic cell contexts, while Gap1, the Ras1-antagonist, has an opposite effect. Ras1-modulated changes were observed in homeotic effects on cell identity (bristle to distal sex combs, wing trichomes to veins, veins to trichomes or veins to bristles). Only a small number of cell identities in precise contexts are changed by HSPB activity. This suggests that most cells are aware of their positions and their correctly associated fates, perhaps as a consequence of cell-cell communication. Ras1-dependent modifications of segmental identity are also observed. These occur in a concerted fashion on groups of adjacent cells, again suggesting cell communication. A general role for Ras1 in homeotic function is likely, since Ras1+ activity also modulates functions of the homeotic loci Sex combs reduced and Ultrabithorax. These data suggest that the modulation occurs by an independent mechanism for the transcriptional control of the homeotic loci themselves, or of the Ras1/Gap1 genes. Taken together the data support a role for Ras1-mediated cell signaling in the homeotic control of segmental differentiation (Boube, 1997).

The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine kinase (RTK) signaling. An extragenic suppressor of Raf, Su(Raf)1, has been isolated that encodes a Drosophila Src family gene (Src42A) identical to the previously cloned DSrc41. Characterization of Src42A mutations shows that Src42A acts independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Src42A negatively regulates Egfr signaling during oogenesis and negatively regulates receptor tyrosine kinase signaling in the eye. For example Src42A suppresses the rough eye phenotype caused by expression of hyperactive Ras or Raf. Src42A mutation also leads to defects in head and tail morphology, tracheal development and wing morphogenesis. This study provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the RTK signaling cascade. A possible model for Src42A function is discussed (Lu, 1999).

The functional status of Ras, Raf, Mek, or Mapk proteins does not appear to alter the ability of wild-type Src42A to repress receptor tyrosine kinase signaling: this would favor a model in which Src42A defines a branch pathway parallel to the main Ras/Mapk cascade with an integration point downstream of Mapk. This model is consistent with the reduction of maternal Src42A activity, which can still enhance Torso receptor tyrosine kinase signaling in the absence of Ras1 protein. The manner in which Src42A acts to modulate receptor tyrosine kinase signaling is similar in two ways to another branch pathway component, Kinase suppress of Ras-1 (ksr-1), of C. elegans. (1) Src42A does not appear to dramatically alter RTK-mediated processes when mutated alone. For example, mitotic clones of Src42A mutant cells in the eye do not produce extra photoreceptor cells. (2) The negative role of Src42A is only revealed when the Ras/Mapk cascade is compromised or hyperactived. Recently, Therrien (1998) reported the isolation of Src42A as a suppressor of a dominant negative form of fly ksr in the eye. In attempts to understand more about Src42A, a genetic screen was performed that isolated loss-of-function mutation in Egfr, rolled, and a new gene, semang, as suppressors of Src42A mutants (Zhang, 1999). It is suggested that Src42A works together with other branch pathway modulators such as Ksr to regulate signal transduction downstream of Egfr and other RTKs. If each branch pathway modulator takes over only a part of the total regulatory power, it would explain why Src42A or ksr-1 shows mild phenotypes when mutated alone. However, the phenotypes of Src42A do not overlap with two other Drosophila Src family members, Src64 and Tec29, both of which are involved in ring canal development during oogenesis. It is tempting to speculate that Src42A is activated following the activation of Egfr RTK via a Ras-1 independent mechanism. The signal from the activated Src42A would then integrate, in a negative fashion, with that from the main Ras/MAPK pathway to determine the final readout of an RTK pathway (Lu, 1999).

Connector enhancer of KSR (CNK) is a multidomain protein required for RAS signaling. Its C-terminal portion (CNKC-term) directly binds to RAF. The N-terminal portion of CNK (CNKN-term) strongly cooperates with RAS, whereas CNKC-term efficiently blocks RAS- and RAF-dependent signaling when overexpressed in the Drosophila eye. Two effector loop mutants of RASV12, S35 and C40, which selectively activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase pathways, respectively, do not cooperate with CNK. However, a strong cooperation is observed between CNK and RASV12G37, an effector loop mutant known in mammals to activate specifically the RAL pathway. Two domains in CNKN-term that are critical for cooperation with RAS have been identified. These results suggest that CNK functions in more than one pathway downstream of RAS. CNKc-term seems to regulate RAF, a component of the MAPK pathway, whereas CNKN-term seems to be involved in a MAPK-independent pathway (Therrien, 1999).

The ability of CNK or CNKN-term to enhance activated RAS but not activated RAF suggests that this effect occurs upstream of RAF or in a pathway parallel to RAF. Two independent sets of results presented in this paper are consistent with the idea that the CNK/RAS cooperation is mediated by a RAF/MAPK-independent pathway. The first is the fact that the coexpression of CNKN-term and RAS1V12 does not produce higher levels of activated MAPK. The second set of evidence is the striking observation that CNK strongly cooperates with RAS1V12G37, a RAS effector loop mutant known to stimulate the RAL pathway in mammals, but not with two other RAS effector loop mutants known to stimulate the MAPK and PI3-K pathways. The data cannot rule out the alternative hypothesis that the enhancement of RAS1V12G37 by CNK results from CNK-mediated stimulation of the MAPK pathway acting synergistically with the RAS1V12G37-stimulated pathway. This possibility would be analogous to the strong cooperation observed in mammalian cells between the MAPK pathway and the RAL pathway to transform cells. Consistent with this possibility, it has been found that a loss-of-function mutation in the rolled/mapk locus does not suppress the RAS1V12G37 mild rough-eye phenotype but suppresses the CNK/RAS1V12G37 cooperation. Moreover, the RAS1V12G37 mutant is leaky and stimulates MAPK when expressed in S2 cells to approximately 10% of the levels obtained with RAS1V12 or RAS1V12S35. It is thus formally possible that CNK functions in the MAPK pathway, which collaborates efficiently with another pathway also stimulated by RAS1V12G37. A strong argument against this model is the observation that CNK strongly cooperates with RAS1V12 but not with RAS1V12S35, a RAS effector loop mutant known to stimulate the RAF/MAPK pathway. It would be difficult to reconcile this observation with a model in which CNK is merely enhancing signaling through the MAPK pathway. More likely, the phenotypic effect of suppressing MAPK signaling might be due to the fact that MAPK is involved in secondary developmental defects resulting from the strong stimulation of the RAS1V12G37-specific pathway. Alternatively, it might reflect the fact that the CNK/RAS1V12G37-stimulated pathway also requires a basal level of MAPK signaling to mediate its effects. A similar dependency in basal MAPK activity has been suggested recently for the RAL pathway to induce the differentiation of F9 embryonal carcinoma cells (Therrien, 1999).

It is expect that RAS1, like its mammalian homologues, controls the RAL pathway in Drosophila. Because the effector loop regions of Drosophila RAS1 and mammalian RAS proteins are identical in sequence and because the mammalian RALA and Drosophila RAL are nearly identical, it is likely that RAS1V12G37 also stimulates the RAL pathway in Drosophila. Although little is known regarding the RAL pathway in Drosophila, it has been reported recently that overexpression of activated RAL in the Drosophila eye disrupts the normal actin cytoskeleton assembly but does not interfere with cell differentiation. This result is consistent with studies conducted in mammalian cells that suggested that RAL controls the organization of the actin cytoskeleton. Based on these findings, it will be interesting to determine whether RAS1V12G37 has a similar effect on the actin cytoskeleton and whether CNK enhances this effect (Therrien, 1999).

The CNK/RAS cooperation clearly depends on the integrity of the SAM and the CRIC domains. SAM domains have been found in various types of proteins and seem to mediate homodimerization and/or heterodimerization with other SAM domain-containing proteins. The CRIC domain is a unique region shared by all CNK homologs identified thus far. The boundaries of this domain (~ 80 amino acids) have been arbitrarily defined based on sequence homology. Its functional relevance was initially suggested by a cnk loss-of-function allele, cnkXE-726, which has a 3-amino acid in-frame deletion within this region. The elucidation of the functions of the SAM and CRIC domains of CNK awaits the molecular characterization of their effect on RAS signaling and the identification of the proteins that interact with them (Therrien, 1999).

Mutations have been characterized in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions (Tapon, 2001).

The enhanced growth observed in the Tsc1 or Tsc2 mutants most resembles the results of inactivating PTEN or increasing Ras1 or dmyc activity. In each of these situations, there is a reduction in the length of the G1 phase. In contrast, increased growth driven by Cyclin D/cdk4 does not alter the distribution of cells in different phases of the cell cycle. The effects of the combined overexpression of Tsc1 and Tsc2 displays genetic interactions with multiple pathways. The phenotype is influenced by alterations in the levels of dS6K, PTEN, Ras1, dmyc, cyclin D, and cdk4. Thus, Tsc1 and Tsc2 may function downstream of the point of convergence of these pathways. Alternatively, Tsc1 and Tsc2 may primarily antagonize one of these pathways, but this effect could be overcome by increasing the activity of one of the others (Tapon, 2001).

The Ca2+-calmodulin-activated protein phosphatase calcineurin negatively regulates Egf receptor signaling in Drosophila development

Calcineurin is a Ca2+-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca2+ signals into changes in cell function and development. A dominant modifier screen was carried out in the Drosophila eye using an activated form of Calcineurin A1 (FlyBase name: Protein phosphatase 2B at 14D), the catalytic subunit, to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. A second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways have suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of these results, it is proposed that calcineurin, upon activation by Ca2+-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of Sprouty and the GTPase-activating protein Gap1 (Sullivan, 2002).

To examine the interaction between calcineurin and individual components of the Egfr pathway, the ability of mutations in these components to modify the activated calcineurin phenotype was tested. Hypomorphic mutations in Egfr, Ras, pnt, sty, Gap1, and small wing modified activated calcineurin, although this was not the case for most downstream components of the Egfr pathway. TCAGB (ectopically expressed activated calcineurin) is enhanced by removing one copy of Egfr, Ras, or pnt and was suppressed by Gap1 and small wing. Both TCAGB and TCAG (another form of ectopically expressed activated calcineurin) suppress the rough eye caused by hypermorphic Egfr alleles: flies that have one copy of EgfrE1 and TCAGB have a rough eye that closely resembles that of TCAGB alone. TCAG is not detectably modified by hypomorphic Egfr, Ras, or pnt alleles. Aside from CS3-3, none of the modifier groups corresponded to Egf receptor/Ras signaling components that genetically interact with TCAG. However, it is possible that these genes are present among the 61 single hits, which have not been characterized (Sullivan, 2002).

Analysis of Ras-induced overproliferation in Drosophila hemocytes

The Drosophila larval hematopoietic system has been used as an in vivo model for the genetic and functional genomic analysis of oncogenic cell overproliferation. Ras regulates cell proliferation and differentiation in multicellular eukaryotes. To further elucidate the role of activated Ras in cell overproliferation, a collagen promoter-Gal4 strain was generated to overexpress RasV12 (Ras-act) in Drosophila hemocytes. Activated Ras causes a dramatic increase in the number of circulating larval hemocytes (blood cells); this increase is caused by cellular overproliferation. This phenotype is mediated by the Raf/MAPK pathway. The mutant hemocytes retain the ability to phagocytose bacteria as well as to differentiate into lamellocytes. Microarray analysis of hemocytes overexpressing RasV12 vs. Ras+ identified 279 transcripts that are differentially expressed threefold or more in hemocytes expressing activated Ras. This work demonstrates that it will be feasible to combine genetic and functional genomic approaches in the Drosophila hematopoietic system to systematically identify oncogene-specific downstream targets (Asha, 2003).

One overall finding is that many of the genes that are upregulated in Ras-act cells include genes that function in cell cycle regulation and DNA replication. These genes include both positive and negative regulators of cell proliferation. The cyclin-dependent kinase inhibitor dacapo (which antagonizes the function of cyclin E/cdk2 complexes), as well as the wee1 kinase (which inactivates cdc2), are both induced. There is currently no known function for either gene in promoting cell cycle progression. Thus the induction of these genes may represent a negative feedback mechanism that attempts to reduce cell proliferation under conditions of excessive cell proliferation. Another possibility is that these two genes have currently unknown roles in promoting cell cycle progression. The microarray data also show that regulators that promote all stages of cell cycle progression are induced, not only those that promote the G1/S transition. These data therefore suggest that both the G1/S and G2/M cell cycle transitions may be influenced by an increase in Ras activity (Asha, 2003).

A second finding is that many of the transcriptional targets known to be induced by Ras1 in other tissues are not induced in Ras-act hemocytes. Therefore, although the RTK/Ras pathway induces the expression of phyllopod in the eye disc, mirror in the ovary, and blistered and ribbon in the tracheal cells, none of these genes are obviously induced in Ras-act hemocytes. This is consistent with tissue-specific factors acting together with Ras to determine which target genes are expressed in each cell type. Another gene whose expression is modulated by Ras activity is the pro-apoptotic gene, hid (also known as Wrinkled). It is believed that the anti-apoptotic effect of Ras in embryos is mediated in part by a reduction in hid transcription. The hid RNA level does not decrease in Ras-act cells, indicating that this mechanism may not be of importance in hemocytes. Ras may still inactivate hid in these cells via MAPK-mediated phosphorylation of the Hid protein. Other pro-apoptotic genes like reaper or grim are not expressed in either Ras-wt or Ras-act hemocytes (Asha, 2003).

Finally, the data indicate that the large overproliferation of hemocytes in response to activated Ras does not lead to a general activation of the immune response. Among the 134 Drosophila immune-regulated genes induced by septic injury and fungal infection, only 6 genes are upregulated and 4 genes are downregulated by a factor of 3 or more in Ras-act hemocytes. The 6 upregulated genes in Ras-act are Tep2 (complement like), alpha-2M receptor like (complement binding), a trypsin-like serine protease (phenol oxidase cascade), a serpin (serine protease inhibitor), spz (antifungal response), and Tl (antifungal response). The 4 downregulated genes include Tep1 (complement like), Rel (transcription factor), Metchnikowin (antimicrobial response), and a lipase (Asha, 2003).

It is concluded that activated versions of both Ras and the Hop Jak kinase induce leukemia-like phenotypes in Drosophila larvae. Further, it is possible to isolate sufficient quantities of larval hemocytes to conduct microarray expression studies. By comparing the expression profiles from different oncogene-induced leukemia cells, coupled with mutational analysis of the newly identified targets, it should be possible to systematically characterize the critical, oncogene-specific target genes. This approach could prove beneficial to the treatment of human cancers (Asha, 2003).

Involvment of Ras pathway in PDGF/VEGF receptor-controlled blood cell survival in Drosophila

The Drosophila PDGF/VEGF receptor (PVR) has known functions in the guidance of cell migration. It has been demonstrated that during embryonic hematopoiesis, PVR has a role in the control of antiapoptotic cell survival. In Pvr mutants, a large fraction of the embryonic hemocyte population undergoes apoptosis, and the remaining blood cells cannibalistically phagocytose their dying peers. Consequently, total hemocyte numbers drop dramatically during embryogenesis, and large aggregates of engorged macrophages carrying multiple apoptotic corpses form. Hemocyte-specific expression of the pan-caspase inhibitor p35 in Pvr mutants eliminates hemocyte aggregates and restores blood cell counts and morphology. Additional rescue experiments suggest involvement of the Ras pathway in PVR-mediated blood cell survival. In cell culture, PVR has been demonstrated to directly control survival of a hemocyte cell line. This function of PVR shows striking conservation with mammalian hematopoiesis and establishes Drosophila as a model to study hematopoietic cell survival in development and disease (Brückner, 2004).

Pvr mutant rescue experiments demonstrate that activated Ras is sufficient to restore hemocyte survival. This result resembles findings from survival signaling by DER, which was shown to inhibit action of the proapoptotic protein HID by phosphorylation through Ras-activated MAPK. Since PVR signaling triggers MAPK activation in Schneider cells and may have the same effect in embryonic hemocytes, it is likely that the Ras/MAPK pathway is a route of antiapoptotic PVR signaling. Expression of dominant-negative RasN17 did not lead to large hemocyte aggregates but induced mild enlargement of hemocytes at a low penetrance. This mild phenotype points to weak defects in blood cell survival. The incomplete effect of RasN17 may be due to a number of reasons. RasN17may be too weak to fully block endogenous Ras signaling, or Ras signaling may be redundant with other signaling pathways that are active in PVR-dependent cell survival. In Pvr1 rescue experiments, activated RasV12 was used, which was shown to ectopically activate other signaling pathways such as the PI3K pathway. Therefore, rescue by RasV12 may involve a number of downstream pathways, but the Ras/MAPK pathway itself may still be central to the observed effect, consistent with a current model for apoptosis in Drosophila. Regardless of the upstream pathways involved, inhibition of caspases by the baculovirus inhibitor of apoptosis p35 was sufficient to rescue the Pvr mutant hemocyte death and aggregation phenotype. In these rescue experiments, p35 appeared slightly less potent than RasV12, which may be due to the inability of p35 to inhibit the upstream caspase Dronc, or partially insufficient expression levels, since p35 inhibits caspases by stoichiometric binding (Brückner, 2004).

The role of Drosophila PVR in trophic cell survival emphasizes the high degree of conservation between Drosophila and vertebrate PDGF/VEGF family receptor function. In vivo and cell culture work now provides the basis to study cell survival in a simple but highly conserved hematopoietic system. In vertebrates, control of cell survival is an important aspect of hematopoiesis and stem cell maintenance. Antiapoptotic cell survival and its aberrant prolongation are a major mechanism in the formation of human neoplasias. In many cases, connections to deregulated upstream signaling pathways remain unclear. Interestingly, in Acute Myeloid Leukemias (AML), more than one-third of cases are associated with specific activating mutations in the PDGF/VEGF receptors Flt3 and c-Kit, and activating fusions of PDGFßR replace the more common oncogenic BCR-ABL fusions in some cases of Chronic Myeloid Leukemia (CML). The contribution of these and other disease-associated genes to aspects of cell survival versus proliferation is still difficult to assess in vivo, yet their mechanism of action is important for the selection of molecularly targeted therapies. Drosophila embryonic hematopoiesis allows the in vivo study of blood cell survival independent of cell proliferation, and this work has demonstrated the antiapoptotic potential of the Drosophila PDGF/VEGF receptor and activated signaling components such as RasV12. It will be interesting to exploit the system further by testing disease-related genes of the same and particularly other families for their in vivo potential to rescue blood cell survival in Drosophila. Complementary to these in vivo findings, a Drosophila cell culture system was established for the study of PVR-dependent blood cell survival. Genome-wide RNAi screens will allow identification of modifiers of PVR-dependent blood cell survival (Brückner, 2004).

scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila

Cancer is a multistep process involving cooperation between oncogenic or tumor suppressor mutations and interactions between the tumor and surrounding normal tissue. This study is the first description of cooperative tumorigenesis in Drosophila, and uses a system that mimics the development of tumors in mammals. The MARCM system was used to generate mutant clones of the apical-basal cell polarity tumor suppressor gene, scribbled, in the context of normal tissue. scribbled mutant clones in the eye disc exhibit ectopic expression of cyclin E and ectopic cell cycles, but do not overgrow due to increased cell death mediated by the JNK pathway and the surrounding wild-type tissue. In contrast, when oncogenic Ras or Notch is expressed within the scribbled mutant clones, cell death is prevented and neoplastic tumors develop. This demonstrates that, in Drosophila, activated alleles of Ras and Notch can act as cooperating oncogenes in the development of epithelial tumors, and highlights the importance of epithelial polarity regulators in restraining oncogenes and preventing tumor formation (Brumby, 2003).

A clonal approach, more closely resembling the clonal nature of mammalian cancer, was used to analyze the effects of removing Scrib function on tumor formation. This analysis indicates that Drosophila scrib- tumors: (1) lose tissue architecture, including apical-basal cell polarity; (2) fail to differentiate properly; (3) exert non-cell-autonomous effects upon the surrounding wild-type tissue; (4) upregulate cyclin E and undergo excessive cell proliferation; (5) are restrained from overgrowing by the surrounding wild-type tissue via a JNK-dependent apoptotic response, and (6) show strong cooperation with oncogenic alleles of Ras and Notch to produce large amorphous tumors. These conclusions are summarized in a model for tumor development in Drosophila. It is suggested that the role of epithelial cell polarity regulators in restraining oncogenes is likely to be of general significance in mammalian tumorigenesis (Brumby, 2003).

The model suggests that the wild-type larval eye disc is a monolayered columnar epithelium, in which cell proliferation is tightly regulated. Cell architecture is maintained by the formation of adherens junctions, the apical localization of Scribbled, and adhesion to the basement membrane. Mutation of scrib results in loss of apical-basal polarity, leading to multilayering and rounding up of cells. scrib- tissue also shows impaired differentiation, and ectopic cyclin E expression (by an unknown mechanism) leads to ectopic cell proliferation. Unrestrained overgrowth and tumor formation of scrib- cells is held in check by compensatory JNK-mediated apoptosis, dependent upon the presence of surrounding wild-type cells. Secondary mutations are required to avoid this apoptotic fate. If JNK activity is blocked within scrib- cells, by expressing a dominant-negative form of JNK, apoptosis is prevented, resulting in tissue overgrowth and lethality. Even more aggressive overgrowth results from the addition of activating oncogenic alleles of Ras or Notch. In addition to promoting cell survival, these oncogenes must also promote tumor cell proliferation; however, it is proposed that other downstream effectors of these oncogenes are likely also to be important, since it was not possible to mimic the cooperative overgrowth effects of RasACT or NACT on scrib- tissue by simply blocking apoptosis and enhancing cell proliferation (Brumby, 2003).

In Drosophila, activated Ras exerts its oncogenic effects through Raf and the MAPK pathway. Downstream targets of MAPK in the eye disc promote differentiation, cell survival and cell proliferation. This work also demonstrates that Ras can increase cyclin E protein levels in the eye disc. In combination with scrib-, the differentiation output of RasACT signaling appears to be attenuated, and the proliferative and anti-apoptotic responses prevail (Brumby, 2003).

Activated Notch also cooperates with scrib-, resulting in neoplastic overgrowth, and although no anti-apoptotic role for Notch signaling in the eye has been described previously, NACT exerts hyperproliferative effects in flies, and Notch signaling is required for proliferation of eye disc cells. Although it is not known if NACT induces the same critical downstream targets as RasACT to cause overgrowth of scrib- tissue, removing ras function in scrib- cells overexpressing NACT rescues the overgrowth phenotype, suggesting that the effects of NACT are at least partially dependent on Ras (Brumby, 2003).

Initially it seemed likely that the cooperative effects of RasACT or NACT on scrib- tissue could be explained by the ability of these oncogenes to promote cell proliferation while blocking apoptosis. However, the expression of neither cyclin E nor E2F1/DP, in combination with the apoptosis inhibitor p35 (or with the inhibitor of JNK pathway activity, BskDN), was capable of phenocopying the effect of RasACT or NACT in scrib- clones. It is therefore suggested that other downstream effectors, apart from anti-apoptotic and cell cycle regulators, must be important in mediating the oncogenic effects of RasACT or NACT. In fact, in Drosophila, Ras has also been shown to be a potent inducer of cellular growth, while cyclin E and E2F1 mainly promote cell cycle progression. Whether NACT also promotes cell growth in Drosophila has not been examined in detail. If growth promotion targets downstream of RasACT or NACT are critical in promoting the overgrowth of scrib- tumors, these are likely to be independent of the PI3 kinase pathway since ectopic PI3 kinase signaling in scrib- clones does not induce synergistic overgrowth, and RafACT is able to induce overgrowth as equally extensive as RasACT (Brumby, 2003).

Finally, it is noted that in mammalian systems, evidence exists for a role for Ras signaling in modulating cell junction complexes and enhancing epithelial to mesenchymal transitions, and in Drosophila also, constitutive RasACT signaling in clones alters cell affinities and changes the levels of E-cadherin and ß-catenin. Whether RasACT or NACT signaling destabilizes adherens junctions in Drosophila and this potentiates scrib- neoplastic overgrowth or whether alterations in the structure of the adherens junction resulting from the absence of Scrib alters a cells response to constitutive activation of these oncogenes are important future questions (Brumby, 2003).

This study has described a novel multi-hit model of tumorigenesis in Drosophila. Furthermore, although it has been suspected that disruptions to cell polarity could potentiate tumor progression and metastasis, this work demonstrates for the first time how the oncogenic effects of activated Ras and Notch are unleashed in the absence of epithelial polarity regulators. It is predicted that in mammals also, defects in apical-basal polarity could cooperate with oncogenes during neoplastic development. This approach in Drosophila can now be used to screen for novel oncogenes that, when specifically overexpressed in scrib- clones, are capable of inducing cooperative tumorigenesis, and can also be extended to identify cooperative interactions between other tumor suppressors and oncogenes within a whole animal context (Brumby, 2003).

Interaction between RasV12 and scribbled clones induces tumour growth and invasion

Human tumours have a large degree of cellular and genetic heterogeneity. Complex cell interactions in the tumour and its microenvironment are thought to have an important role in tumorigenesis and cancer progression. Furthermore, cooperation between oncogenic genetic lesions is required for tumour development; however, it is not known how cell interactions contribute to oncogenic cooperation. The genetic techniques available in the fruitfly Drosophila melanogaster allow analysis of the behaviour of cells with distinct mutations, making this the ideal model organism with which to study cell interactions and oncogenic cooperation. In Drosophila eye-antennal discs, cooperation between the oncogenic protein RasV12 and loss-of-function mutations in the conserved tumour suppressor scribbled (scrib) gives rise to metastatic tumours that display many characteristics observed in human cancers. This study shows that clones of cells bearing different mutations can cooperate to promote tumour growth and invasion in Drosophila. The RasV12 and scrib- mutations can also cause tumours when they affect different adjacent epithelial cells. This interaction between RasV12 and scrib- clones involves JNK signalling propagation and JNK-induced upregulation of JAK/STAT-activating cytokines, a compensatory growth mechanism for tissue homeostasis. The development of RasV12 tumours can also be triggered by tissue damage, a stress condition that activates JNK signalling. Given the conservation of the pathways examined in this study, similar cooperative mechanisms could have a role in the development of human cancers (Wu, 2010).

Clones of mutant cells marked with green fluorescent protein (GFP) can be generated in the eye-antennal imaginal discs of Drosophila larvae by mitotic recombination. Clones expressing RasV12, an oncogenic form of the Drosophila Ras85D protein, moderately overgrow. Clones mutant for scrib lose apico-basal polarity and die. In contrast, scrib clones simultaneously expressing RasV12 grow into large metastatic tumours. To understand better the cooperation between these two mutations, animals were produced in which cell division after a mitotic recombination event creates two daughter cells: one expressing RasV12 and the other mutant for scrib. Discs containing adjacent RasV12 (GFP-positive) and scrib- clones developed into large tumours, capable of invading the ventral nerve cord. This shows that RasV12 and scrib also cooperate for tumour induction when they occur in different cells. These tumours are referred to as RasV12//scrib- tumours, to denote interclonal oncogenic cooperation and distinguish them from RasV12scrib- tumours, in which cooperation occurs in the same cells intraclonally (Wu, 2010).

This study has used Drosophila to investigate how oncogenic cooperation between different cells can promote tumour growth and invasion. These experiments, addressed to understanding interclonal cooperation in RasV12//scrib- tumours, uncovered a two-tier mechanism by which scrib- cells promote neoplastic development of RasV12 cells: (1) propagation of stress-induced JNK activity from scrib- cells to RasV12 cells; and (2) expression of the JAK/STAT-activating Unpaired cytokines downstream of JNK. These findings, therefore, highlight the importance of cell interactions in oncogenic cooperation and tumour development. It was also shown that stress-induced JNK signalling and epigenetic factors such as tissue damage can contribute to tumour development in flies. Notably, tissue damage caused by conditions such as chronic inflammation has been linked to tumorigenesis in humans. Furthermore, expression of the Unpaired cytokines promotes tumour growth as well as an antitumoural immune response, which parallels the situation in mice and humans. Future research into phenomena such as compensatory growth and interclonal cooperation in Drosophila will provide valuable insights into the biology of cancer (Wu, 2010).

Interplay among Drosophila transcription factors Ets21c, Fos and Ftz-F1 drives JNK-mediated tumor malignancy

This study defines TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (RasV12) and loss of the tumor suppressor Scribble (scrib1). Malignant transformation of the rasV12scrib1 tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to rasV12scrib1 tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in rasV12scrib1 tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with Ras(V12) in inducing malignant clones that, like rasV12scrib1 tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While rasV12ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, this study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes. (Kulshammer, 2015).

Genome-wide transcriptome profiling in the Drosophila epithelial tumor model has generated a comprehensive view of gene expression changes induced by defined oncogenic lesions that cause tumors of an increasing degree of malignancy. These data allowed discovery of how a network of collaborating transcription factors confers malignancy to RasV12scrib1 tumors (Kulshammer, 2015).

This study revealed that the response of transformed RasV12scrib1 epithelial cells is more complex in comparison to those with activated RasV12 alone with respect to both the scope and the magnitude of expression of deregulated genes. Aberrant expression of more than half of the genes in RasV12scrib1 tumors requires JNK activity, highlighting the significance of JNK signaling in malignancy. Importantly, the tumor-associated, JNK-dependent transcripts cluster with biological functions and processes that tightly match the phenotypes of previously described tumor stages. Furthermore, the RasV12scrib1 transcriptome showed significant overlap (27% upregulated and 15% downregulated genes) with microarray data derived from mosaic EAD in which tumors were induced by overexpressing the BTB-zinc finger TF Abrupt (Ab) in scrib1 mutant clones as well as with a transcriptome of scrib1 mutant wing discs. It is proposed that 429 misregulated transcripts (e.g. cher, dilp8, ets21c, ftz-f1, mmp1, upd), shared among all the three data sets irrespective of epithelial type (EAD versus wing disc) or cooperating lesion (RasV12 or Ab), represent a 'polarity response transcriptional signature' that characterizes the response of epithelia to tumorigenic polarity loss. Genome-wide profiling and comparative transcriptome analyses thus provide a foundation to identify novel candidates that drive and/or contribute to tumor development and malignancy while unraveling their connection to loss of polarity and JNK signaling (Kulshammer, 2015).

In agreement with a notion of combinatorial control of gene expression by an interplay among multiple TFs, this study identified overrepresentation of cis-acting DNA elements for STAT, GATA, bHLH, ETS, BTB, bZIP factors and NRs in genes deregulated in RasV12scrib1 mosaic EAD, implying that transcriptome anomalies result from a cross-talk among TFs of different families. Many of the aberrantly expressed genes contained binding motifs for AP-1, Ets21c and Ftz-F1, indicating that these three TFs may regulate a common set of targets and thus cooperatively promote tumorigenesis. This is consistent with the occurrence of composite AP-1-NRRE (nuclear receptor response elements), ETS-NRRE and ETS-AP-1 DNA elements in the regulatory regions of numerous human cancer-related genes, such as genes for cytokines, MMPs (e.g., stromelysin, collagenase) and MMP inhibitors (e.g., TIMP) (Kulshammer, 2015).

Interestingly, Drosophila ets21c and ftz-f1 gene loci themselves contain AP-1 motifs and qualify as polarity response transcriptional signature transcripts. Indeed, this study has detected JNK- and Fos-dependent upregulation of ets21c and ftz-f1 mRNAs in RasV12scrib1 tumors. While JNK-mediated control of ftz-f1 transcription has not been reported previously, upregulation of ets21c in the current tumor model is consistent with JNK requirement for infection-induced expression of ets21c mRNA in Drosophila S2 cells and in vivo. Based on these data, it is proposed that Ftz-F1 and Ets21c are JNK-Fos-inducible TFs that together with AP-1 underlie combinatorial transcriptional regulation and orchestrate responses to cooperating oncogenes. Such an interplay between AP-1 and Ets21c is further supported by a recent discovery of physical interactions between Drosophila Ets21c and the AP-1 components Jun and Fos (Rhee, 2014). Whether regulatory interactions among AP-1, Ets21c and Ftz-F1 require their direct physical contact and/or the presence of composite DNA binding motifs of a particular arrangement to control the tumor-specific transcriptional program remains to be determined (Kulshammer, 2015).

Importantly, some of the corresponding DNA elements, namely AP-1 and STAT binding sites, have recently been found to be enriched in regions of chromatin that become increasingly accessible in RasV12scrib1 mosaic EAD relative to control. This demonstrates that comparative transcriptomics and open chromatin profiling using ATAC-seq and FAIRE-seq are suitable complementary approaches for mining the key regulatory TFs responsible for controlling complex in vivo processes, such as tumorigenesis (Kulshammer, 2015).

The prototypical form of AP-1 is a dimer comprising Jun and Fos proteins. In mammals, the Jun proteins occur as homo- or heterodimers, whereas the Fos proteins must interact with Jun in order to bind the AP-1 sites. In contrast to its mammalian orthologs, the Drosophila Fos protein has been shown to form a homodimer capable of binding to and activating transcription from an AP-1 element, at least in vitro (Kulshammer, 2015).

The role of individual AP-1 proteins in neoplastic transformation and their involvement in pathogenesis of human tumors remain somewhat elusive. While c-Jun, c-Fos and FosB efficiently transform mammalian cells in vitro, only c-Fos overexpression causes osteosarcoma formation, whereas c-Jun is required for development of chemically induced skin and liver tumors in mice. In contrast, JunB acts as a context-dependent tumor suppressor. Thus, cellular and genetic context as well as AP-1 dimer composition play essential roles in dictating the final outcome of AP-1 activity in tumors (Kulshammer, 2015).

This study shows that, similar to blocking JNK with its dominant-negative form, Bsk, removal of Fos inhibits ets21c and ftz-f1 upregulation, suppresses invasiveness, improves epithelial organization and differentiation within RasV12scrib1 tumors and allows larvae to pupate. Strikingly, depletion of Jun had no such tumor-suppressing effects. It is therefore concluded that in the malignant RasV12scrib1 tumors, Fos acts independently of Jun, either as a homodimer or in complex with another, yet unknown partner. A Jun-independent role for Fos is further supported by additional genetic evidence. Fos, but not Jun, is involved in patterning of the Drosophila endoderm ┬čand is required for expression of specific targets, e.g., misshapen (msn) and dopa decarboxylase (ddc), during wound healing. Future studies should establish whether the JNK-responsive genes containing AP-1 motifs, identified in this study, are indeed regulated by Fos without its 'canonical' partner (Kulshammer, 2015).

The current data identify Fos as a key mediator of JNK-induced MMP1 expression and differentiation defects in RasV12scrib1 tumors. Only Fos inhibition caused clear suppression of MMP1 levels and restoration of neurogenesis within clonal EAD tissue, thus mimicking effects of JNK inhibition. Improved differentiation and reduced invasiveness are, however, not sufficient for survival of animals to adulthood, because interfering with Fos function in RasV12scrib1 clones always resulted in pupal lethality (Kulshammer, 2015).

The systems approach of this paper, followed by genetic experiments, identified Ets21c and Ftz-F1 as being essential for RasV12scrib1-driven tumorigenesis. It was further shown that mutual cooperation of both of these TFs with Fos is required to unleash the full malignancy of RasV12scrib1 tumors (Kulshammer, 2015).

TFs of the ETS-domain family are key regulators of development and homeostasis in all metazoans, whereas their aberrant activity has been linked with cancer. ets21c encodes the single ortholog of human Friend leukemia insertion1 (FLI1) and ETS-related gene (ERG) that are commonly overexpressed or translocated in various tumor types. While FLI1 is considered pivotal to development of Ewing's sarcoma, ERG has been linked to leukemia and prostate cancer. As for Ftz-F1 orthologs, the human liver receptor homolog-1 (LRH-1) has been associated with colonic, gastric, breast and pancreatic cancer, whereas steroidogenic factor 1 (SF-1) has been implicated in prostate and testicular cancers and in adrenocortical carcinoma. However, the molecular mechanisms underlying oncogenic activities of either the ERG/FLI1 or the SF-1/LRH-1 proteins are not well understood (Kulshammer, 2015).

This study shows that removal of Ftz-F1 markedly suppressed invasiveness of RasV12scrib1 tumors, restoring the ability of tumor-bearing larvae to pupate. Additionally, and in contrast to Fos, Ftz-F1 inhibition also partly reduced tumor growth in the third-instar EAD and allowed emergence of adults with enlarged, rough eyes composed predominantly of non-clonal tissue. The reduced clonal growth coincided with downregulation of the well-established Yki target, expanded, implicating Ftz-F1 as a potential novel growth regulator acting on the Hpo/Yki pathway. It is further speculated that reduced viability of RasV12scrib1ftz-f1RNAi clones and induction of non-autonomous compensatory proliferation by apoptotic cells during the pupal stage could explain the enlargement of the adult eyes. The precise mechanism underlying compromised growth and invasiveness of RasV12scrib1ftz-f1RNAi tumors and improved survival of the host remains to be identified (Kulshammer, 2015).

In contrast, effects of Ets21cLONG knockdown in RasV12scrib1 tumors appeared moderate relative to the clear improvement conferred by either Fos or Ftz-F1 elimination. ets21cLONG RNAi neither reduced tumor mass nor suppressed invasiveness, and pupation was rescued only partly. However, unlike ftz-f1RNAi, ets21cLONG RNAi significantly reduced expression of dilp8 mRNA. Based on abundance of Ets21c binding motifs in the regulatory regions of tumor-associated genes and the normalized expression of >20% of those genes upon removal of Ets21c, it is further suggested that Ets21c acts in RasV12scrib1 tumors to fine-tune the tumor gene-expression signature (Kulshammer, 2015).

Dilp8 is known to be secreted by damaged, wounded or tumor-like tissues to delay the larval-to-pupal transition. This study has corroborated the role of JNK in stimulating dilp8 expression in RasV12scrib1 tumor tissue, and has further implicated Ets21c and Fos as novel regulators of dilp8 downstream of JNK. However, the data also show that elevated dilp8 transcription per se is not sufficient to delay metamorphosis. Unlike the permanent larvae bearing RasV12scrib1 tumors, those with RasV12scrib1ftz-f1RNAi tumors pupated despite the excessive dilp8 mRNA. Likewise, pupation was not blocked by high dilp8 levels in larvae bearing EAD clones overexpressing Abrupt. As Dilp8 secretion appears critical for its function, it is proposed that loss of Ftz-F1 might interfere with Dilp8 translation, post-translational processing or secretion (Kulshammer, 2015).

Consistent with the individual TFs having unique as well as overlapping functions in specifying properties of RasV12scrib1 tumors, knocking down pairwise combinations of the TFs had synergistic effects on tumor suppression compared with removal of single TF. This evidence supports the view that malignancy is driven by a network of cooperating TFs, and elimination of several tumor hallmarks dictated by this network is key to animal survival. An interplay between AP-1, ETS-domain TFs and NRs is vital for development. For example, the ETS-factor Pointed has been shown to cooperate with Jun to promote R7 photoreceptor formation in the Drosophila adult eye. In mosquitoes, synergistic activity of another ETS-factor, E74B, with the ecdysone receptor (EcR/USP) promotes vitellogenesis. It is thus proposed that tumors become malignant by hijacking the developmental mechanism of combinatorial control of gene activity by distinct TFs (Kulshammer, 2015).

Despite the minor impact of ets21cLONG knockdown on suppressing RasV12scrib1 tumors, Ets21cLONG is the only one of the tested TFs that was capable of substituting for loss of scrib in inducing malignant clonal overgrowth when overexpressed with oncogenic RasV12 in EAD. While invasiveness of such RasV12ets21cLONG tumors required JNK activity, JNK signaling appeared dispensable for tumor growth. Importantly, the overgrowth of RasV12ets21cLONG tumors was primarily independent of a prolonged larval stage, because dramatic tumor mass expansion was detected already on day 6 AEL. How cooperativity between Ets21cLONG and RasV12 ensures sufficient JNK activity and the nature of the downstream effectors driving tumor overgrowth remain to be determined. In contrast, co-expression of either Ftz-F1 or Fos with RasV12 resulted in a non-invasive, RasV12-like hyperplastic phenotype (Kulshammer, 2015).

Why does Ets21cLONG exert its oncogenic potential while Fos and Ftz-F1 do not? Simple overexpression of a TF may not be sufficient, because many TFs require activation by a post-translational modification (e.g., phosphorylation), interaction with a partner protein and/or binding of a specific ligand. Full activation of Fos in response to a range of stimuli is achieved through hyperphosphorylation by mitogen-activated protein kinases (MAPKs), including ERK and JNK. Indeed, overexpression of a FosN-Ala mutated form that cannot be phosphorylated by JNK was sufficient to phenocopy fos deficiency, indicating that Fos must be phosphorylated by JNK in order to exert its oncogenic function. Consistent with the current data, overexpression of FosN-Ala partly restored polarity of lgl mutant EAD cells. It is therefore conclude that the tumorigenic effect of Fos requires a certain level of JNK activation, which is lacking in EAD co-expressing Fos with RasV12. Nevertheless, the absence of an unknown Fos-interacting partner cannot be excluded (Kulshammer, 2015).

Interestingly, MAPK-mediated phosphorylation also greatly enhances the ability of SF-1 and ETS proteins to activate transcription. Two potential MAPK sites can be identified in the hinge region of Ftz-F1, although their functional significance is unknown. Whether Ets21c or Ftz-F1 requires phosphorylation and how this would impact their activity in the tumor context remains to be determined. Genetic experiments demonstrate that at least the overgrowth of RasV12ets21cLONG tumors does not require Ets21c phosphorylation by JNK (Kulshammer, 2015).

In addition, previous crystallography studies revealed the presence of phosphoinositides in the ligand binding pocket of LHR-1 and SF-1 and showed their requirement for the NR transcriptional activity. Although developmental functions of Drosophila Ftz-F1 seem to be ligand independent, it is still possible that Ftz-F1 activity in the tumor context is regulated by a specific ligand. An effect of Ftz-F1 SUMOylation cannot be ruled out (Kulshammer, 2015).

In summary, this work demonstrates that malignant transformation mediated by RasV12 and scrib loss depends on MAPK signaling and at least three TFs of different families, Fos, Ftz-F1 and Ets21c. While their coordinated action ensures precise transcriptional control during development, their aberrant transcriptional (Ets21c, Ftz-F1) and/or post-translational (Fos, Ftz-F1, Ets21c) regulation downstream of the cooperating oncogenes contributes to a full transformation state. The data implicate Fos as a primary nuclear effector of ectopic JNK activity downstream of disturbed polarity that controls ets21c and ftz-f1 expression. Through combinatorial interactions on overlapping sets of target genes and acting on unique promoters, Fos, Ftz-F1 and Ets21c dictate aberrant behavior of RasV12scrib1 tumors. Although originally described in Drosophila, detrimental effects of cooperation between loss of Scrib and oncogenic Ras has recently been demonstrated in mammalian tumor models of prostate and lung cancer. This study and further functional characterization of complex TF interactions in the accessible Drosophila model are therefore apt to provide important insight into processes that govern cancer development and progression in mammals (Kulshammer, 2015).

Oncogenic mutations produce similar phenotypes in Drosophila tissues of diverse origins

An emerging interest in oncology is to tailor treatment to particular cancer genotypes, i.e. oncogenic mutations present in the tumor, and not the tissue of cancer incidence. Integral to such a practice is the idea that the same oncogenic mutation(s) produces similar outcomes in different tissues. To test this idea experimentally, tumors were studied driven by a combination of RasV12 and scrib1 mutations in Drosophila larvae. Tumors induced in tissues of neural ectodermal and mesodermal origins were found to behave similarly in every manner examined: cell cycle checkpoints, apoptosis, cellular morphology, increased aneuploidy and response to Taxol. It is concluded that oncogenic effects override tissue-specific differences, at least for the mutations, tissues, and phenotypes in this study (Stickel, 2014).

p53 activity is selectively licensed in the Drosophila stem cell compartment

Oncogenic stress provokes tumor suppression by p53 but the extent to which this regulatory axis is conserved remains unknown. Using a biosensor to visualize p53 action, this study found that Drosophila p53 is selectively active in gonadal stem cells after exposure to stressors that destabilize the genome. Similar p53 activity occurred in hyperplastic growths that were triggered either by the Ras(V12) oncoprotein or by failed differentiation programs. In a model of transient sterility, p53 was required for the recovery of fertility after stress, and entry into the cell cycle was delayed in p53(-) stem cells. Together, these observations establish that the stem cell compartment of the Drosophila germline is selectively licensed for stress-induced activation of the p53 regulatory network. Furthermore, the findings uncover ancestral links between p53 and aberrant proliferation that are independent of DNA breaks and predate evolution of the ARF/Mdm2 axis (Wylie, 2014).

Loss of PI3K blocks cell-cycle progression in a Drosophila tumor model

Tumorigenesis is a complex process, which requires alterations in several tumor suppressor or oncogenes. This study used a Drosophila tumor model to identify genes, which are specifically required for tumor growth. Reduction of phosphoinositide 3-kinase (PI3K) activity was found to result in very small tumors while only slightly affecting growth of wild-type tissue. The observed inhibition on tumor growth occurred at the level of cell-cycle progression. It is concluded that tumor cells become dependent on PI3K function and that reduction of PI3K activity synthetically interferes with tumor growth. The results of this study broaden insights into the intricate mechanisms underling tumorigenesis and illustrate the power of Drosophila genetics in revealing weak points of tumor progression (Willecke, 2011).

This study employed a genetic approach to identify genes required for the neoplastic growth phenotype of RasV12, DlgRNAi tumors. It was found that RasV12, DlgRNAi tumors are highly sensitive to reductions of the PI3K pathway and that changes in PI3K activity block cell-cycle progression (Willecke, 2011).

In agreement with previous reports, this study found that RasV12 induces the PI3K pathway in Drosophila. Yet curiously, RasV12, DlgRNAi tumors exhibit low levels of PI3K signaling. A possible reason for the paradoxical result may be derived from the cell polarity defects of RasV12, DlgRNAi tumors, which are not induced when RasV12 is expressed in otherwise wild-type cells or in combination with Upd. This interpretation implies that the activation of PI3K through RasV12 depends on proper cell polarity. In support of this explanation, it has been reported that RasV12 activates the PIP3 reporter only at the apical side of cells. Additionally, studies have shown that PTEN directly binds to the polarity gene Bazooka and that the activity of the PI3K pathway is polarized in Drosophila oocyte cells. The activation of the PI3K pathway might, therefore, require the preservation of cell polarity, which could explain why RasV12 does not induce the PI3K pathway if Dlg is lost (Willecke, 2011).

Why are RasV12, DlgRNAi tumors sensitive to changes in PI3K signaling? RasV12, DlgRNAi tumor cells receive mitogenic signals from the JAK-STAT and MAPK pathways, which promote extensive tumor growth. RasV12, DlgRNAi tumors require a higher metabolic rate compared with wild-type cells, without gaining extra activation of PI3K through RasV12. As a result, PI3K signaling might be absolutely limiting for the growth of RasV12, DlgRNAi tumors. Expression of PI3KRNAi then causes PI3K activity to drop below the threshold for such cells, triggering a block in cell-cycle progression. Tumors which express RasV12 together with Upd are not sensitive to changes in PI3K levels even though they overgrow as much as RasV12, DlgRNAi tumors. An pAkt western blot shows, however, that these tumors have high levels of PI3K signaling. Expression of PI3KRNAi in this background might not cause PI3K activity to drop below a threshold for cell-cycle progression (Willecke, 2011).

Compounds that block PI3K pathway activity are known to be potent inhibitors of mammalian tumor growth and inhibitors that target PI3K, and other members of the pathway are currently being tested in clinical trials. Preclinical and clinical trials focus mainly on tumors that carry mutations in PI3K pathway components or that display abnormal levels of the biomarkers pAKT and PS6k1. The current results are, however, an example for a case where tumors with no genetic alterations in PI3K signaling components are also highly susceptible to reduction of PI3K levels. Understanding the molecular networks that create such PI3K dependency is a central topic in cancer research as it is highly relevant to identify PI3K-dependent tumors to predict the potential effectiveness of PI3K inhibitors. Genetic studies in Drosophila may therefore complement mammalian studies to more precisely determine which tumor-initiating pathways create PI3K sensitivity (Willecke, 2011).

In conclusion, this study has uncovered a synthetic interaction between the Drosophila PI3K signaling and RasV12, DlgRNAi tumor-initiating pathways. The results provide insights into the complex mechanisms underlying tumorigenesis and illustrate the power of Drosophila genetics in revealing the vulnerabilities of tumors. Identification of additional synthetic interactions through genetic screening in Drosophila may serve as a valuable resource for identifying potential drug targets in cancer therapy (Willecke, 2011).

Src42A modulates tumor invasion and cell death via Ben/dUev1a-mediated JNK activation in Drosophila

Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, this study identified Src42A, the ortholog of mammalian Src, as a key modulator of both RasV12/lgl -/-triggered tumor invasion and loss of cell polarity gene-induced cell migration. The genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, this study showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with RasV12 to promote tumor overgrowth and invasion. In addition, it was found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. These data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a (Ma, 2013).

BTB-zinc finger oncogenes are required for Ras and Notch-driven tumorigenesis in Drosophila

During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers, Drosophila epithelial tumor models were used, that are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors a comparative microarray analysis was used to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, this work suggests that EMT-promoting signals in human cancers could similarly utilize networks of these proteins to promote cancer stem cell states (Doggett, 2015).

This report has defined the transcriptional changes induced by JNK signaling within both scrib>RasACT and scrib>NACT tumors by carrying out comparative microarray expression arrays. This analysis that JNK exerts a profound effect upon the transcriptional profile of both Ras and Notch-driven tumor types. The expression of nearly 1000 genes was altered by the expression of bskDN in either Ras or Notch-driven tumors, and less than half of these changes were shared between the two tumor types, indicating that JNK signaling elicits unique tumorigenic expression profiles depending upon the cooperating oncogenic signal. Nevertheless, of the 399 JNK-regulated probe sets shared between Ras and Notch-driven tumors, it is hypothesized that these had the potential to provide key insights into JNK's oncogenic activity, and to prioritize these targets, it was considered that the expression of the critical oncogenic regulators would not just be altered by bskDN, but would be normalized to close to wild type levels. This subset of the 399 probe set was identified by comparing the expression profile of each genotype back to control tissue, thereby producing a more focussed JNK signature of 103 genes. Notably, this included previously characterized targets of JNK in the tumors, such as Mmp1,cherand Pax, thereby providing validation of the approach. Also amongst these candidates were 4 BTB-ZF genes; two of which were upregulated by JNK in the tumors (chinmo and fru), and two downregulated (br and ttk) (Doggett, 2015).

Focussing upon chinmo, chinmo overexpression was shown to be sufficient to prime epithelial cells for cooperation with RasACT in both the eye antennal disc and in the adult midgut epithelium, and that chinmo is required for cooperative RasACTor NACT-driven tumor overgrowth, although its function was only exposed when its knockdown was combined with knockdown of a functionally similar BTB-ZF transcription factor, abrupt. This family of proteins is highly oncogenic in Drosophila, since previous work has shown that ab overexpression can cooperate with loss of scrib to promote neoplastic overgrowth, and in these studies, it was also shown that overexpression of a fru isoform normally expressed in the eye disc is capable of promoting cooperation with RasACT and NACT in the eye-antennal disc, in a similar manner to chinmo overexpression. Thus, whether fru also plays a role in driving Ras or Notch-driven tumorigenesis warrants further investigation. Indeed, a deeper understanding of the oncogenic activity of these genes is likely to be highly relevant to human tumors, since of the over 40 human BTB-ZF family members, many are implicated in both haematopoietic and epithelial cancers, functioning as either oncogenes (eg., Bcl6, BTB7) or tumor suppressors (eg., PLZF, HIC1). Furthermore, over-expression of BTB7, can also cooperate with activated Ras in transforming primary cells, and its loss makes MEFs refractory to transformation by various key oncogenes such as Myc, H-rasV12 and T-Ag, suggesting that cooperating mechanisms between BTB-ZF proteins and additional oncogenic stimuli might be conserved (Doggett, 2015).

JNK signaling in Drosophila tumors is known to promote tumor overgrowth through both the STAT and Hippo pathways. Deregulation of the STAT pathway was evident in the arrays through the upregulation of Upd ligands by JNK in both Ras and Notch-driven tumors. In contrast, although cher was identified in the arrays as being upregulated in both tumor types and previous studies have shown that cher is partly required for the deregulation of the Hippo pathway in scrib>RasACT tumors, more direct evidence for Hippo pathway deregulation amongst the JNK signature genes was lacking. In part, this could be due to JNK regulating the pathway through post-transcriptional mechanisms involving direct phosphorylation of pathway components. However, the failure to identify known Hippo pathway target genes, and proliferation response genes in general, may simply highlight limitations in the sensitivity of the array assay and the cut-offs used for determining significance, despite its obvious success in correctly identifying many known JNK targets (Doggett, 2015).

Whether tumor overgrowth through STAT and Yki activity is somehow associated with a stem cell or progenitor-like state remains uncertain. Although imaginal discs exhibit developmental plasticity and regeneration potential, and JNK signaling is required for both of these stem-like properties, there is no positive evidence for the existence of a population of asymmetrically dividing stem cells within imaginal discs. Instead, symmetrical divisions of progenitor cells may be the means by which imaginal discs can rapidly generate enough cells to form the differentiated structures of the adult fly. To date, progenitor cells have only been characterized in the eye disc neuroepithelium. These cells have a pseudostratified columnar epithelial morphology and express the MEIS family transcription factor, Hth, which is downregulated as cells initiate differentiation and begin expressing Dac and Eya. Interestingly, they also require Yki for their proliferation, and can be induced to overproliferate in response to increased STAT activity. However, analysis of cell fate markers indicated that tumor overgrowth was not likley to be solely due to the overproliferation of these undifferentiated progenitor cells. Although scrib>RasACT/NACT tumors, were characterized by the failure to transition to Dac/Eya expression in the eye disc, blocking JNK in scrib > RasACT/NACT tumors did not restore tumor cell differentiation, despite overgrowth being curtailed, and Hth expression was not maintained in the tumors in a JNK-dependent manner. Nevertheless, a JNK-induced gene such as chinmo is likely to be associated with promoting a progenitor-like state, since it is a potential STAT target gene required for adult eye development that is expressed in eye disc progenitor cells in response to increased Upd activity and its overexpression alone is sufficient to block Dac/Eya expression. Furthermore, chinmo is also required for cyst stem cell maintenance in the Drosophila testis, and the current work has shown that chinmo overexpression promotes increased numbers of esgGFP expressing stem cells or enteroblasts in the adult midgut. As the BTB-ZF protein Ab is also highly oncogenic and expressed in the eye disc progenitor cells, it is hypothesize that the JNK-induced expression ofchinmo in scrib>RasACT/NACT tumors could cooperate with Ab to maintain a progenitor-like cell state in the eye disc, and that this is required for scrib->RasACT/NACT tumor overgrowth. However, although Ab was expressed in chinmo-expressing, JNK positive tumor cells, Ab does not appear to be a JNK-induced gene. What JNK-independent mechanisms control ab expression will therefore require further analysis (Doggett, 2015).

Interestingly, previous studies have observed that ab overexpression in eye disc clones upregulates chinmo expression and although the effect of chinmo expression upon ab is yet to be described, the data at least suggest that the control of their expression is interlinked in a yet to be defined manner (Doggett, 2015).

Consistent with Chinmo being important for scrib->RasACT/NACTv tumor overgrowth, chinmo overexpression itself is also highly oncogenic. Over-expression of chinmo with RasACT or NACT drives tumorigenesis in the eye-antennal disc, and also resulted in enlarged brain lobes, presumably due to the generation of overexpressing clones within the neuroepithelium of the optic lobes. In the adult midgut, the overexpression of chinmo with RasACT in the stem cell and its immediate progeny, the enteroblast, promoted massive tumor overgrowth, resulting in esgGFP expressing cells completely filling the lumen of the gut, and eventual host lethality. The luminal filling of esgGFP cells is reminiscent of the effects of RasACT expression in larval adult midgut progenitor cells. Together with the data linking Chinmo function to stem or progenitor cells, these data reinforce the idea that epithelial tumorigenesis can be primed by signals, such as chinmo over-expression, that promote a stem or progenitor cell state (Doggett, 2015).

The function of some Drosophila BTB-ZF proteins including Chinmo and Ab, has also been linked to heterochronic roles involving the conserved let-7 miRNA pathway and hormone signals, to regulate the timing of differentiation. Indeed, Ab can directly bind to the steroid hormone receptor co-activator Taiman (Tai or AIB1/SRC3 in humans), to represses the transcriptional response to ecdysone signaling. Thus, the capacity of BTB-ZF proteins to influence the timing of developmental transitions, particularly if they impede developmental transitions within stem or progenitor cells, could help account for their potent oncogenic activity. Indeed, ecdysone-response genes were repressed by JNK in the tumorigenic state, consistent with the failure of the larvae to pupate and a delay in developmental timing. Whether repressing the ecdysone response cell autonomously might contribute to tumor overgrowth and/or invasion will be an interesting area of future investigation, given the complex role of hormone signaling in mammalian stem cell biology and cancers (Doggett, 2015).

Previous studies have suggested that JNK-dependent tumor cell invasion is developmentally similar to the JNK-induced EMT-like events occurring during imaginal disc eversion. Thus the capacity of JNK to also promote tumor overgrowth is reminiscent of how EMT inducers such as Twist (Twi) and Snail (Sna) are associated with the acquisition of cancer stem cell properties. In Drosophila, however, twi and snawere not induced by JNK in the tumors, although transcription factors involved in mesoderm specification, including the NF-kappaB homologue, dl (a member of the 103 JNK signature), and Mef2 (a member of the 399 JNK signature), were amongst the up-regulated JNK targets. Mesoderm specification is not necessarily associated with a mesenchymal-like cell morphology, however, dl is involved in the induction of EMT during embryonic development, and both dl and Mef2 act with Twi and Sna to coordinate mesoderm formation. Interestingly, recent studies have identified dl in an overexpression screen for genes capable of cooperating with scrib > in Drosophila tumorigenesis, and Mef2 has been identified as a cooperating oncogene in Drosophila, and possibly also in humans, where a correlation exists between the expression of Notch and Mef2 paralogues in human breast tumor samples. It is therefore possible that dl and Mef2 either act in combination with Twi or Sna, or independently of them but in a similar oncogenic capacity, to promote a mesodermal cell fate in scrib > RasACT/NACT tumors. The potential relevance of this to the mesenchymal cell morphology associated with tumor cell invasion, as well as the acquisition of progenitor states is worthy of further investigation (Doggett, 2015).

In mef2-driven tumors both overgrowth and invasion depend upon activation of JNK signaling, suggesting that Mef2 is not capable of promoting invasive capabilities independent of JNK. In contrast, chinmo+RasACT/NACT tumors appeared non-invasive and retained epithelial morphology despite the massive overgrowth, although closer examination of cell polarity markers will be required to confirm this. Furthermore, the overgrowth of chinmo+RasACT/NACT tumors was not dependent upon JNK signaling, suggesting that the maintenance of a progenitor-like state could be uncoupled from JNK-induced EMT-effectors associated with invasion. Whether clear divisions between mesenchymal behaviour and progenitor states in tumors can be clearly separated in this manner is not yet clear, however, overall, it is likely that multiple JNK-regulated genes will participate in both promoting tumor overgrowth as well as migration/invasion. Although this study used the 103 JNK signature as a means to focus upon potential key candidates, an analysis of the 399 JNK-regulated probe sets common to both Ras and Notch-driven tumours has the potential to provide deeper insights into the multiple effectors of JNK signaling during tumorigenesis. Whilst the individual role of these genes can be probed with knockdowns, the complexity of the response, potentially with multiple redundancies and cross-talk, will ultimately need a network level of understanding to more fully expose key nodes participating in overgrowth and invasion. This approach has considerable potential to further expose core principles and mechanisms that drive human tumorigenesis, since it is clear that many fundamental commonalities underlie the development of tumors in Drosophila and mammals (Doggett, 2015).

Ras pathway and terminal development

Determination of cell fate at the posterior termini of the Drosophila embryo is specified by the activation of the Torso receptor tyrosine kinase. This signaling pathway is mediated by the serine/threonine kinase D-raf and a protein tyrosine phosphatase corkscrew. Expression of an activated form of Ras1 during oogenesis resulted in embryos with tor gain-of-function phenotypes. Mammalian p21ras variants were injected into early Drosophila embryos. The injection of activated mammalian p21v-ras rescues the maternal-effect phenotypes of both tor and csw null mutations. These rescuing effects of p21v-ras are dependent on the presence of maternally derived D-raf activity. Wild-type embryos show a terminal-class phenotype resembling csw mutation when injected with p21rasN17, a dominant-negative form of p21ras. The maternal-effect phenotype embryos lacking Son of sevenless (Sos) exhibit a terminal-class phenotype. The Drosophila p21ras, encoded by Ras1, is an intrinsic component of the tor signaling pathway, where it is both necessary and sufficient in specifying posterior terminal cell fates. p21ras/Ras1 operates upstream of the D-raf kinase in this signaling pathway (Lu, 1993).

Activation of the receptor tyrosine kinase (RTK) Torso defines the spatial domains of expression of the transcription factors Tailless and Huckebein. Previous analyses have demonstrated that Ras1 (p21ras) operates upstream of the D-Raf (Raf1) serine/threonine kinase in this signaling pathway. D-Raf can be activated by Torso in the complete absence of Ras1. This result is supported by analysis of D-Raf activation in the absence of either the exchange factor Son of sevenless (Sos) or the adaptor protein drk (Grb2), as well as by the phenotype of a D-Raf mutation that abolishes binding of Ras1 to D-Raf. This study provides in vivo evidence that Raf can be activated by an RTK in a Ras-independent pathway (Hou, 1995).

The terminal portions of the Drosophila body pattern are specified by the localized activity of the receptor tyrosine kinase Torso (Tor) at each pole of the early embryo. Tor activity elicits the transcription of two 'gap' genes, tailless (tll) and huckebein (hkb), in overlapping but distinct domains by stimulating the Ras signal transduction pathway. Quantitative variations in the level of Ras activity can specify qualitatively distinct transcriptional and morphological responses. Low levels of Ras activity at the posterior pole direct tll but not hkb transcription; higher levels drive transcription of both genes. Correspondingly, low levels of Ras activity specify a limited subset of posterior terminal structures, whereas higher levels specify a larger subset. When a constitutively active 1X RasV12 gene is expressed in torso mutant embryos, brachyenteron (byn) is expressed in a small terminal cap, whereas the domain of expression in 2X RasV12 is much broader. Because both the activation and repression of terminal byn expression is known to depend, respectively, on tll and hkb, it is surmised that higher levels of Ras activity are required at the posterior of wild-type (ras+) embryos to drive sufficiently high levels of Hkb expression to repress byn expression (a phenomenon not observed with even 2X RasV12 ectopic expression). 1X RasV12 forms the least terminal of the posterior terminal structures: the eighth abdominal dentical band and the posterior spiracles. The extent of restoration is considerably greater in 2X RasV12 embryos: these form additional terminal structures such as the anal tuft and anal pads.The response to Ras activity is not uniform along the body. Instead, levels of Ras activity that suffice to drive tll and hkb transcription at the posterior pole fail to drive their expression in more central portions of the body, apparently due to repression by other gap gene products. The levels of Huckebein and/or Kruppel through the embryo might be responsible for a failure to express hkb in response to moderate RasV12 activity. It is concluded that tll and hkb transcription, as well as the terminal structures, are specified by two inputs: a gradient of Ras activity, which emanates from the pole, and the opposing influence of more centrally deployed gap genes, which repress the response to Ras (Greenwood, 1997).

14-3-3 proteins have been shown to interact with Raf-1 and cause its activation when overexpressed. However, their precise role in Raf-1 activation is still enigmatic, as they are ubiquitously present in cells and found to associate with Raf-1 in vivo regardless of Raf's activation state. The function of the Drosophila 14-3-3 gene leonardo (leo) has been analyzed in the Torso (Tor) receptor tyrosine kinase (RTK) pathway. In the syncytial blastoderm embryo, activation of Tor triggers the Ras/Raf/MEK pathway that controls the transcription of tailless (tll). In the absence of Tor, overexpression of leo is sufficient to activate tll expression. The effect of leo requires D-Raf and Ras1 activities but not KSR or DOS, two recently identified essential components of Drosophila RTK signaling pathways. Tor signaling is impaired in embryos derived from females lacking maternal expression of leo. It is proposed that binding to 14-3-3 by Raf is necessary but not sufficient for the activation of Raf and that overexpressed Drosophila 14-3-3 requires Ras1 to activate D-Raf (Li, 1997).

Coactivation of STAT and Ras by Torso is required for germ cell proliferation and invasive migration in Drosophila

Primordial germ cells (PGCs) undergo proliferation, invasion, guided migration, and aggregation to form the gonad. In Drosophila, the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development, and coactivation of STAT and Ras is required for PGC proliferation and invasive migration. Embryos mutant for stat92E or Ras1 have fewer PGCs, and these cells migrate slowly, errantly, and fail to coalesce. Conversely, overactivation of these molecules causes supernumerary PGCs, their premature transit through the gut epithelium, and ectopic colonization. A requirement for RTK in Drosophila PGC development is analogous to the mouse, in which the RTK c-kit is required, suggesting a conserved molecular mechanism governing PGC behavior in flies and mammals (Li, 2003).

STAT92E plays an essential role in mediating the phenotypic effects of gain-of-function mutations of Torso, TorGOF, but is only minimally required for wild-type Tor function in patterning the terminal structures of the Drosophila embryo. To investigate whether wild-type Tor nevertheless activates STAT92E, an antibody was used that recognizes the phosphorylated, or active form of STAT92E (pSTAT92E) to examine the activation status of STAT92E in different genetic backgrounds. In early embryos, pSTAT92E is detected in the anterior and posterior terminal regions in a pattern reminiscent of Tor activation. By analyzing embryos mutant for loss- or gain-of-function mutations of tor as well as those lacking JAK, encoded by hopscotch (hop), it was concluded that the early STAT92E activation is dependent on Tor but not Hop, suggesting that Tor may activate STAT92E independent of Hop. Because STAT92E contributes only marginally to the expression of the Tor target gene tailless (tll), it was of interest to find whether the early activation of STAT92E by Tor had any other biological functions. It was evident that Tor activation correlates temporally and spatially with the formation of PGCs, which are localized at the posterior pole of the early embryo. Tor-dependent activation of STAT92E as well as that of the Ras-MAPK signaling cassette, as detected by an antibody against activated ERK/MAPK (diphospho-ERK), persists in pole cells at this stage. STAT92E activation was detected in PGCs during their migration and in the gonads of late embryos, that are formed following the migration of pole cells through a complex route. These observations indicate that STAT92E and Ras1/Draf activation may play a role in PGC development (Li, 2003).

So far the only known function of Tor has been in pattern formation, since Tor protein is present only transiently in early embryos. Therefore, the finding that Tor is involved in germ cell migration was initially unexpected. However, there is a precedent for the requirement of an RTK in germ cell migration in the mouse. Mutations in the mouse genes dominant white-spotting (W) cause migration and proliferation defects in germ cells as well as a few other cell types. W encodes the protooncoprotein c-kit, an RTK that is expressed on the membrane of mouse PGCs. Sl encodes the c-kit ligand termed stem cell factor (SCF), which is localized on the membrane of somatic cells associated with PGC migratory pathways. Interestingly, c-kit and Tor share structural similarities and both are structurally similar to the platelet derived growth factor (PDGF) receptor, in which an insert region separates the intracellular kinase domain. Moreover, similar to Tor and the PDGF receptor, c-kit is able to activate STAT molecules as well as the Ras-MAPK cascade. Although true molecular homologs of c-kit and SCF are not yet found in the Drosophila genome, the functional and structural similarities between Tor and c-kit suggest that flies and mice share molecular mechanisms for regulating primordial germ cell proliferation and migration (Li, 2003).

In addition to germ cells, the ovarian border cells of Drosophila are also capable of invasive and guided migration. Border cells of the Drosophila ovary are follicle cells that, during oogenesis, delaminate as a cluster six to ten cells from the anterior follicle epithelium, invade the nurse cells, and migrate toward the oocyte. Interestingly, it has been shown that the detachment and guided migration of these cells require STAT92E activation. Mutations in components of the Hop/STAT92E pathway cause border cell migration defects. In addition, border cell migration also requires RTK signaling. An RTK related to mammalian PDGF and VEGF receptors, PVR, is required in border cells for their guided migration toward the oocyte. PVR appears functionally redundant with another fly RTK, EGFR, in guiding border cells. Taken together, these results indicate that the invasive behavior and guided migration of Drosophila ovarian border cells require both STAT92E and RTK activation. In light of the results from analyzing PGC migration, it is proposed that activation of both STAT and components downstream of RTK signaling may serve as a general mechanism for invasive and guided cell migration (Li, 2003).

It has been shown that actin-based cytoskeletal reorganization plays a crucial role in cell shape changes and movements. The identification of STAT and Ras coactivation as an essential requirement for germ cell migration raises an interesting question of how activated STAT and Ras coordinate the cytoskeletal reorganization required for germ cell migration. STAT92E has been shown to be involved in the transcriptional activation of many signaling molecules as well as key transcription factors. A recent systematic search for STAT92E target genes has revealed a plethora of genes that might be directly activated by STAT92E, among which are those involved in the regulation of cytoskeletal movements and actin reorganization. Upregulation of such genes in response to spatial cues should facilitate cell movements. In addition, Ras and other small GTP proteins have been implicated in multiple cellular processes that require cytoskeletal reorganization. It remains to be determined how these two signaling pathways coordinate germ cell movements in response to guidance cues from surrounding somatic tissues (Li, 2003).

Ras and glia

Continued Ras85 Effects of mutation: part 2/3 | part 3/3

Ras85D: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | References

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