|Home | About | Journals | Submit | Contact Us | Français|
High levels of Src activity are found in a broad spectrum of cancers. The roles of Src and its negative regulator Csk have been extensively studied, although results have often proved contradictory or the relevance to whole organisms is unclear. In Drosophila, overexpression of either Src orthologue resulted in apoptotic cell death, but paradoxically, reducing dCsk activity led to over-proliferation and tissue overgrowth. Here, we show that in Drosophila epithelia in situ, the levels of Src signaling determine the cellular outcome of Src activation. Apoptotic cell death was triggered specifically at high Src signaling levels; lower levels directed antiapoptotic signals while promoting proliferation. Furthermore, our data indicate that expression of kinase-dead Src isoforms do not necessarily act as dominant-negative factors, but can instead increase Src pathway activity, most likely by titrating Csk activity away from endogenous Src. The importance of Src activity levels was emphasized when we examined oncogenic cooperation between Src and Ras: malignant overgrowth was observed specifically when high Src signaling levels were achieved. We propose a model in which low levels of Src signaling promote survival and proliferation during early stages of tumorigenesis, whereas strong Src signaling, coupled with antiapoptotic signals, directs invasive migration and metastasis during advanced tumor stages.
Src is the first kinase linked to cancer and one of the original identified oncogenes (1). It is a membrane-associated kinase that regulates a broad palette of developmental and homeostatic processes, including cell proliferation, apoptosis, and adhesion. Src family kinases (SFK) are activated in most major cancer types, including breast cancer, colon cancer, melanomas, etc. (2–6).
Nevertheless, the role of SFKs in oncogenesis is not fully understood. Cell culture studies have provided important but conflicting data: for example, expression of v-Src or c-Src (viral or cellular Src, respectively) can induce differentiation, proliferation, apoptosis, or transformation depending on the cell line used (7, 8). Results such as these suggest that SFKs can act differently in different cellular contexts. Also, although v-Src is strongly oncogenic, c-Src protein is only weakly transforming, and Src activity is linked to tumorigenesis primarily through its increase late in a tumor’s maturation (9). Mammals contain nine SFKs, and at least some act redundantly. c-src knock-out mice are viable and fertile, exhibiting minor bone defects (10). Redundancy is indicated by the observation that Src−/−;Fyn−/−;Yes−/− triple mutant embryos die early in development (11).
v-Src’s derives its strong activity in part due to a lack of the negative regulatory COOH-terminal domain, which contains a conserved tyrosine targeted for phosphorylation by COOH-terminal Src kinase (Csk) and its homologue Chk (12). Csk and Chk are cytoplasmic kinases that represent the major negative regulators of SFK activity, and the Csk/Src regulatory circuit is conserved from the early-diverging metazoan Hydra to humans (13). Knock-out of Csk activity in mice leads to strong hyper-activation of SFKs and early embryonic lethality (14). Csk and Chk have been linked to breast and brain cancer, presumably through the activation of Src (e.g., ref. 15).
Recent work has begun to characterize the sole Drosophila Csk/Chk orthologue dCsk. Reduction of dCsk activity led to increased cell proliferation, increased organ size, and organismal lethality (16, 17). By contrast, overexpression of the Drosophila Src orthologues dSrc42A or dSrc64B led to a small rough eye phenotype more reminiscent of proapoptotic genes than a reduction in dCsk activity (18–20). v-Src can induce apoptosis of cultured cells under some circumstances (21, 22), indicating that this outcome of Src overexpression can also be observed in mammalian cell lines.
Here, we use an in situ approach in Drosophila to examine more closely the nature of Src’s ability to direct an overgrowth phenotype within the context of simple polarized epithelia. We present evidence supporting a model that reconciles the different phenotypes resulting from dSrc overexpression versus dCsk reduction. Our data indicate that Src signaling is biphasic: lower levels of Src pathway activity result in antiapoptotic signals, whereas strong levels potently induce apoptosis. However, when coupled with other oncogenes such as Ras, high levels of Src activity can lead to malignant overgrowth and invasion. Src activity has been reported to increase progressively in most common solid tumor types (23), and these results suggest that the role of Src changes as tumorigenesis progresses.
GMR-hid, UAS-dSrc42A.CA, and UAS-dSrc64B were obtained from the Bloomington Drosophila Stock Center. The following stocks were kindly provided to us: hs-dSrc64B, hs-dSrc64BΔ540, and hs-dSrc64BKD by N. Perrimon, sev-dSrc42KD by K Saigo, UAS-RasV12 and latswtsXI by G. Halder, scribble1 by D. Bilder, and dSrc64BD17 and dCskQ156Stop by A. O’Reilly and M. Simon.
Cultures and incubations were done at 25 °C unless otherwise specified. The genetic suppression of GMR-hid by mildly expressed dSrc64B (hs-dSrc64B) was observed at 25°C (data not shown) and 29°C (Fig. 1).
To create whole eye clones of dCsk (EGUF, ref. 24), we generated flies with the genotype yw:ey-gal4,UAS-FLP/+;FRT82B,GMR-hid,l(3)CL-R/FRT82B, dCsk. To create eyeless-driven green fluorescent protein (GFP)-labeled clones, flies with the genotype yweyFLP1;act>y+>gal4,UAS-GFP;FRT82B,tub-gal80 or, alternatively, ey(3.5) FLP;act>y+ >gal4,UAS-GFP;FRT82B,tub-gal80 were crossed with flies with the following genotypes: (a) UAS-RasV12; FRT82B,dCskj1D8/TM6b;(b) UAS-RasV12;FRT82B, dCskQ156Stop/TM6b; (c) UAS-dSrc64B,UAS-RasV12;FRT82B,dCskj1D8/TM6b; and (d) UAS-RasV12;FRT82B, scrib1/TM6b.
Immunofluorescence was done as described previously (25). Antibodies used were rabbit anti-pY434 (activated dSrc64B, 1:100, ref. 26), mouse anti-armadillo N27A1 and rat anti-DE-cadherin (1:3 and 1:20, respectively, both from the Developmental Studies Hybridoma Bank), rabbit anti-cleaved caspase-3 (1:200, Cell Signaling Technology), and rat anti-laminin A (α-220, 1:1,000, ref. 27). Secondary antibodies were conjugated with red or green Alexa (Molecular Probes).
For scanning electron microscopy, adult flies were fixed in 95% ethanol, critical point dried, sputter coated, and viewed with a Hitachi S-2600H scanning electron microscope.
Light micrographs from adult eyes were analyzed using ImageJ software (NIH) to measure eye area in arbitrary pixel units. The eye sizes from each genotype (n = 6 in all cases) relative the control genotype were analyzed using Student’s t test, and the level of statistical significance was displayed in the graphs. Error bars in the graphs correspond to SEs.
Csk represents the major cytoplasmic negative regulator of SFKs, and mutations that reduce or eliminate Csk function should exhibit phenotypes that are similar to over-expressing SFKs. Strong overexpression of either of the two Drosophila SFK members, dSrc42A or dSrc64B, in the developing eye led to small rough eyes (e.g., Fig. 1A right). Each SFK gave rise to similar gain-of-function phenotypes, but Pedraza et al. (20) described phenotypes ranging from small rough eyes to complete eye ablation or even organism lethality due to disruption of the head capsule. This likely reflects different levels of expression achieved by different transgenic lines and culture temperatures. In contrast, others and we (16, 17) observed that mutations that reduce activity of the Drosophila Csk orthologue dCsk led to tissue overgrowth, a block in apoptotic cell death, and a resulting enlarged eye (e.g., Fig. 1A, center). Removal of a single genomic copy of the downstream Src target dSrc29 rescues this phenotype, indicating that overgrowth is due to hyperactivation of Src signaling (17). These results mirror studies in mammalian model systems indicating that Csk targets primarily SFKs. However, it leads to the question as to why overexpression of dSrc42A or dSrc64B does not phenocopy loss of dCsk function. This issue has important implications for disease, as dynamic changes in the activity of Csk and Src have each been observed in human tumors, but their precise roles in situ are unclear.
One explanation for this paradox has been suggested by mouse knock-out studies, which suggest that Csk may act independent of SFKs (28). In Drosophila, dCsk has been linked functionally to the cytoplasmic kinase Lats/Warts. Mutations in lats lead to an increase in organ size (29), a phenotype similar to dCsk. Based on its ability interact genetically with lats in vivo and to phosphorylate Lats protein in vitro, dCsk has been proposed to phosphorylate Lats in situ and in a Src-independent fashion (20). Such Src-independent effects of dCsk could explain the phenotypic differences between Src overexpression and loss of dCsk function. However, removing a genomic copy of lats also led to a significant suppression of mildly misexpressed dSrc64BΔ540(Supplementary Fig. S1), an activated version of dSrc64B that is insensitive to Csk activity. That is, lats shows a functional link with dSrc64B in addition to dCsk. Based on these functional relationships, we propose that most or all dCsk activity that is dependent on Lats also requires Src. These data argue against Src-independent links between dCsk and Lats.
Interestingly, weak overexpression of dSrc64BΔ540 led to a mildly rough eye that was, upon closer examination, more reminiscent of phenotypes observed in dCsk mutants than those with strongly expressed Src isoforms. Pupal retinas that expressed dSrc64BΔ540 through mild induction of the heat-inducible hsp70 promoter displayed defects that included extranumerary interommatidial precursor cells (IPCs; Fig. 2A). Ectopic IPCs are a hallmark of insufficient programmed cell death (PCD) during development (30). They are also a characteristic of reduced dCsk activity, which leads to a block in Hid-dependent apoptotic removal of excess IPCs (17, 31). Furthermore, weakly expressed dSrc64B suppressed mildly but significantly the ectopic cell-death phenotype resulting from the overexpression of the proapoptotic gene hid (Fig. 1B). This antiapoptotic activity observed with low ectopic levels of Src signaling stands in contrast to the proapoptotic signals observed with high levels of ectopic Src.
These observations suggest a model that would explain the phenotypic differences between (a) a modest increase in Src activity, obtained through mild Src overexpression or partial reduction of Csk levels and (b) strong Src misexpression. In this view, low versus high levels of Src signaling differ in the pathways they invoke, with low levels favoring stable overgrowth and high levels favoring apoptotic cell death. Next, we tested predictions of this biphasic Src activity model.
If dCsk is the major negative regulator of Src activity, then a full loss of dCsk activity should lead to strong Src activity and an apoptosis phenotype. A previously described dCsk RNA-interference transgenic line (dCsk-IR, ref. 32) is likely a hypomorph, accounting for its overgrowth phenotype. Previously characterized dCsk mutant alleles, dCskflick (16), dCskSO30003, dCskSO17909 and dCskj1D8 (17), and dCskc04256 (32), are all P transposable element insertions within introns or in the carboxyl-terminus end of the gene and are likely hypomorphs as well.
Recently, a null dCsk allele was generated; dCskQ156Stop homozygotes show increased dSrc64B activation and defects in cell packaging in oogenesis (26) and are embryonic lethal (data not shown). Although whole eye clones of hypomorphic dCsk alleles led to an enlarged eye (Fig. 1A; refs. 17, 32), attempts to obtain adult whole eye clones (EGUFs, ref. 24) of dCskQ156Stop resulted in pupal lethality, presumably due to a failure to properly form the head capsule. The few dCskQ156Stop animals that progressed to mature pupal stages had eye regions that were replaced by melanotic scars suggestive of extensive tissue death (Fig. 2B). This pupal lethality and loss of eye tissue phenocopied the strongest phenotypes observed by strong overexpression of dSrc isoforms (Fig. 2B and data not shown). Taken together, these data indicate that whereas partial loss of dCsk activity results in mild SFK activation and tissue overgrowth, more complete loss directs strong SFK activation and tissue death.
A direct test on whether Csk signals primarily or exclusively through Src would be to block Src signaling in the context of a Csk loss of function. Mutation of a key lysine residue in the catalytic domain of Src abolishes kinase activity (33). Such kinase-dead versions of Src (SrcKD) have been extensively used as dominant-negative tools, with the assumption that they compete with the endogenous molecules for binding to upstream activators and downstream substrates. However, previous efforts at expressing these SrcKD isoforms yielded unexpected results, including phenotypic rescue of c-src mutations, leading to the view that the intrinsic kinase activity of Src is dispensable for a significant portion of its biological roles (34, 35).
If kinase-dead Drosophila Src isoforms act to inhibit Src pathway activity, then they should suppress the phenotypes of dCsk-IR. Contrary to our expectations, however, the eye phenotype of GMR>dCsk-IR was strongly enhanced by coexpressing kinase-inactivated forms of either dSrc42A or dSrc64B (Fig. 3A and data not shown). GMR>dCsk-IR;sev-dSrc42AKD eyes were significantly smaller and mispatterned and displayed supernumerary, clustered bristles (Fig. 3A, inset, right). Together with altered cell proliferation/survival, this phenotype was also suggestive of cell fate changes and abnormal cell adhesion/migration, both previously associated with elevated Src signaling (17, 32).
We considered two possible explanations for the strong genetic enhancement observed between dSrc42AKD and dCsk-IR. First, perhaps dSrc42AKD has (kinase-independent) activity that is normally repressed by dCsk. Alternatively, kinase-dead Src isoforms may titrate away endogenous dCsk, releasing endogenous SFKs to increase pathway activity. Consistent with the latter model, sev-dSrc42AKD by itself led to the ectopic IPC phenotype characteristic of mild Src activation (Fig. 2A, right). Furthermore and similar to hs-dSrc64B and GMR>dCsk-IR (Fig. 3B; ref. 32), sev-dSrc42AKD significantly suppressed the small eye phenotype resulting from hid misexpression (Fig. 3B). Finally, GMMR>dCsk-IR;sev-dSrc42AKD eyes were reduced in size in a manner similar to eyes strongly overexpressing Src (Fig. 3A right).
These results are consistent with a role for dSrc42AKD in activating Src signaling in vivo: activation presumably occurs by sequestering the remaining dCsk activity in GMR>dCsk-IR eyes, leading to a switch from low to high Src activity and concomitant apoptosis. Interestingly, further reducing the remaining dCsk activity in dCsk-IR eyes by enhancing RNA interference-mediated efficiency also yielded a small rough eye phenotype,4 again mirroring the strong overexpression of Src.
Together, these experiments further support a model in which high levels of total Src activity lead to phenotypic outcomes that differ qualitatively from low Src levels.
Multiple potential mechanisms have been described to account for high Src activity in solid tumors: (a) COOH-terminal dephosphorylation by inactivation of Csk or its paralogue Chk (15) or by activation of the RPTP and PTP1B phosphatases (36); (b) loss of Src’s COOH-terminal phosphorylation site through mutation (35); (c) direct activation by receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and HER2 (37); and/or (d) a strong increase in Src expression levels (35). Notably, although Src overexpression has been frequently observed in progressing tumors (9), cytoplasmic kinases are not commonly activated by simple overexpression. Csk is a common negative regulator shared by all SFKs, and our results with dSrc42AKD further suggested that high expression levels of one SFK may activate another by titrating Csk activity.
We tested this possibility using antibodies that specifically detect activated dSrc64B (targeting pY434, ref. 26). In wild-type larvae, activated dSrc64B staining localized to the adherens junctions of epithelial cells from imaginal discs and included all cells of the eye anlage (Fig. 4), similar to reported localization of its paralogue dSrc42A (38). Such adherens junction-specific staining was abolished in dSrc64B mutant eye discs (Fig. 4), indicating that pY434 did not cross-react with any protein normally expressed in the eye. As expected, expression of ectopic dSrc64B with a sevenless promoter (sev-GAL4;UAS-dSrc64B) resulted in a strong increase in activated dScr64B staining that was specific to sevenless-expressing cells (Fig. 4; ref. 39). Strikingly, similar ectopic expression of activated dSrc42A also resulted in a modest but significant increase in the activation of dSrc64B (Fig. 4). To distinguish whether ectopic dSrc42A increased dSrc64B activity by direct phosphorylation, SFKs could undergo inter-molecular autophosphorylation (40), or alternatively, by titrating away dCsk, we next expressed a kinase-dead version of dSrc42A. Expression of a kinase-dead dSrc42A isoform (19) also resulted in increased dSrc64B activation (Fig. 4). Taken together, these experiments indicate that overexpression of a SFK family member can activate other SFKs and presumably itself as well, by titration of negative regulators such as Csk.
Finally, we asked whether the importance of differing Src/Csk levels might prove relevant to models of oncogenesis. Oncogenic cooperation has been observed in most tumor paradigms: typically, two or more loci participate in aggressive tumors. For example, Src synergizes with EGFR to direct oncogenic growth in fibroblasts (5). In Drosophila, expression of the oncogenic Ras isoform dRasV12 alone led to epithelial outgrowths described as “benign tumors,” but combining dRasV12 with loss of Scribble (scrib) led to aggressive tumors that exhibited metastatic-like behavior (41, 42). Because c-jun-NH2-kinase activation plays a role in this oncogenic cooperation (43, 44) and is also a common effector of dCsk-mediated phenotypes (17, 32), we asked whether dCsk/Src could cooperate oncogenically with dRasV12.
Using the MARCM system (45), we created early eye clones of cells that expressed dRasV12, together with mutations expected to drive differing levels of Src activation. Combining the hypomorphic allele dCskJ1DS together with dRasV12 within the developing eye resulted in phenotypes similar to the ones described for dRasV12 alone (41): most animals were viable with discrete outgrowths present in the heads of adult flies (Fig. 5B). Pairing dRasV12 with high levels of Src activity led to a different outcome. When dRasV12 was combined with (a) overexpression of dSrc64B plus the hypomorphic mutant dCskj1D8 or (b) the null allele dCskQ156Stop,we observed a wide palette of phenotypes that ranged from discrete outgrowths in the adult heads to larval or pupal lethality. In the most severely affected animals, strong malignant overgrowth was observed in the larva; this led to an extended larval life in which the animals continued to grow and delayed pupariation. These severely affected animals displayed an abnormally large body size, and the eye clones of cells with activated Ras and Src activities expanded dramatically, overtaking the entire cephalic complex and resulting eventually in lethality of the giant larvae (Fig. 5A and C). This developmental delay was never observed in dRasV12;dCskJ1D8 animals. Penetrance of each of these phenotypes is quantified in Fig. 5D.
These results indicate that (a) Src can cooperate with Ras to drive malignant overgrowth, and (b) this malignant overgrowth requires high rather than low levels of Src activation.
These experiments suggested two additional points of note. First, the strong phenotypes we observed were strikingly similar to those reported for dRasV12 and scrib1 oncogenic cooperation (41, 42), and further experiments were done to determine whether scrib and src act through similar mechanisms. Our results indicate that (a) dRasV12;dCskQ156Stop cells displayed rounded morphology and reduced or delocalized DE-cadherin expression, suggestive of altered epithelial polarity (Fig. 6A); (b) dRasV12;dCskQ156Stop cells were found occasionally embedded within the basal extracellular matrix (ECM; Fig. 6B); and (c) dRasV12;dCskQ156Stop eye-antenna clones eventually extended actin-rich processes to invade distant regions of the brain (Supplementary Fig. S3). Together, these observations indicate that scribble and dCsk/Src cooperate oncogenically with RasV12 through similar mechanisms.
Second, in some experimental animals, we observed tumors distant from the main eye tumors primarily in the region of the gonads (e.g., Fig. 5A). This suggested that secondary tumors emerged by metastasis of cells from the primary eye tumors to distant sites as previously reported (41). However, our FLPase-mediated clones were generated with an eyeless promoter that could have weak expression in other tissues such as the optic lobes of the brain and the gonads (41, 46). Interestingly, we observed posterior tumors almost exclusively in males; closer examination indicated that early-stage tumors were localized to cells within the posterior testes (Supplementary Fig. S2). To clarify this issue, we used the more restricted eyeless-driven Flipase ey(3.5) FLP, which does not drive expression in the testes or brain.5 We observed no secondary tumors, although we still observed invasion from eye clones into the brain (Supplementary Fig. S3).
This study provides evidence that different levels of Src signaling lead to different biological outcomes. We speculate that Src activity plays two important but separable roles during tumor maturation: early low levels of Src contribute to tumor overgrowth, whereas later high levels of Src, coupled with other oncogenes such as Ras, lead to invasive migration. Our previous work emphasized the importance of Csk/Src-dependent signals present at tumor boundaries that can provoke metastatic-like behavior (32). In addition, our current data reconcile the contrasting phenotypes observed between partial reduction of Csk activity versus a strong increase in Src activity by demonstrating the importance of low versus high levels of Src pathway activation, respectively.
We also considered what precisely is being modeled with the use of altered Src isoforms, a common approach in the study of signaling pathways including Src. Unexpectedly, we observed that kinase-dead versions of the two Drosophila SFKs did not behave as expected for dominant-negative isoforms. Furthermore, ectopic expression of dSrc42A, including as a kinase-dead isoform, led to the activation of its sole paralogue, dSrc64B. Our data suggest an alternative explanation to paradoxical and controversial observations reported for vertebrate Src. The primary phenotype of src−/− knock-out mice, osteopetrosis caused by defective osteoclasts (10), was rescued by introducing a kinase-dead Src isoform (SrcKD). In addition, SrcKD rescued the reduced levels of phosphorylated tyrosine observed in src−/− osteoclasts (34). This led to the suggestion that the essential activities of Src are mediated exclusively through protein-protein interactions and not through kinase activity. Our work suggests an alternative model in which endogenous SFKs are ectopically activated in SrcKD-rescued osteoclasts through the titration of Csk activity; this ectopic activation can then compensate for the loss of any individual SFK. In this view, the ability of SrcKD to rescue Src still invokes kinase activity, albeit indirectly.
Our results indicate that high levels of Src signaling can show oncogenic cooperation with Ras to direct tumoral growth and organismal lethality. Similarly to Ras/Scrib, Ras/dCsk cells displayed overgrowth, loss of epithelial polarity, migration, and invasion into the ECM. Our data indicate that, although cells did not metastasize into distant tissues, they were capable of invading nearby tissues within the cephalic complex such as the brain. In mammals, the ability to invade a local blood or lymph vessel could provide a malignant cell the ability to reach and colonize most tissues of the organism. Therefore, current Drosophila models could model some (albeit not all) fundamental aspects of the metastatic process.
Increasing levels of Src activation correlate with advancing tumorigenic stages in a variety of human cancers, including lung, breast, pancreatic, ovarian, and colorectal cancer (e.g., ref. 23). Strikingly, we observed a similar correlation with Src-driven oncogenesis in Drosophila: high (but not low) levels of Src signaling cooperated with oncogenic Ras to produce invasive overgrowth and organismal lethality. One question in the cancer field is whether ectopic expression studies truly model aspects of human tumors. Our results indicate that they can; however, attention must be paid to levels of activity and the specific aspects of tumorigenesis that each level emulates. Emerging models for tumorigenesis should consider not only the activation of multiple and specific pathways, but should also calibrate these levels to explore, e.g., benign overgrowth versus metastasis versus cell death.
Although Src activity can affect cell proliferation and survival (refs. 16, 17, this work), a growing consensus in the field is that a major role for Src is to promote metastasis by stimulating migratory behavior in transformed cells (reviewed in ref. 35). Our study suggests that the proliferative and survival signals from low Src activation contributes to tumor growth in early stages. In line with this, a recent study shows that mice with keratinocyte-restricted deletion of Csk have mild SFK activation and develop epidermal hyperplasia, but without malignant transformation (47).
In advanced stages, increased Src signaling, together with the acquisition of strong antiapoptotic signals that protect cells from Src-induced apoptosis such as oncogenic Ras, may reveal the ability of Src activation to drive invasive migration and, potentially, metastasis. For example, we note that pancreatic ductal adenocarcinoma involves the invasion of exocrine cells through intrapancreatic nerves, leading to severe damage and pain, promoting cancer spread, and precluding resection. Src, expressed at high levels in the majority of these tumors, is likely required for tumor progression (48, 49), and cell culture studies suggest that invasion requires Ras signaling for pancreatic tumor targeting (50). Finally, Src-specific kinase inhibitors have entered clinical trials (35). Our results with dominant-negative Src isoforms suggest caution, however, as the potential of a Src:drug complex to titrate Csk/Chk activity may yield unpredictable results in the context of Src-overexpressing tumors.
Grant support: NIH grants R01CA109730 and R01CA84309.
We thank A. O’Reilly, M. Simon, M. Uhlirova, D. Bohmann, G. Halder, D. Montell, N. Perrimon, T. Miura, and D. Van Vactor for generously providing reagents that were critical for this study. We also thank current and former Cagan lab members for their advice and support and M. Encinas and G. Halder for helpful discussions.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
4R.W. Carthew, personal communication.
5G. Halder, personal communication, and data not shown.