Toll‐7 promotes tumour growth and invasion in Drosophila

Abstract Objectives Drosophila melanogaster has become an excellent model organism to explore the genetic mechanisms underlying tumour progression. Here, by using well‐established Drosophila tumour models, we identified Toll‐7 as a novel regulator of tumour growth and invasion. Materials and methods Transgenic flies and genetic epistasis analysis were used. All flies were raised on a standard cornmeal and agar medium at 25°C unless otherwise indicated. Immunostaining and RT‐qPCR were performed by standard procedures. Images were taken by OLYMPUS BX51 microscope and Zeiss LSM 880 confocal microscope. Adobe Photoshop 2020 and Zeiss Zen were used to analyse the images. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0. Results Loss of Toll‐7 suppresses RasV12/lgl −/−‐induced tumour growth and invasion, as well as cell polarity disruption‐induced invasive cell migration, whereas expression of a constitutively active allele of Toll‐7 is sufficient to promote tumorous growth and cell migration. In addition, the Egr‐JNK signalling is necessary and sufficient for Toll‐7‐induced invasive cell migration. Mechanistically, Toll‐7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. Moreover, Toll‐7 activates the EGFR‐Ras signalling, which cooperates with the Egr‐JNK signalling to promote Yki‐mediated cell proliferation and tissue overgrowth. Finally, Toll‐7 is necessary and sufficient for the proper maintenance of EGFR protein level. Conclusions Our findings characterized Toll‐7 as a proto‐oncogene that promotes tumour growth and invasion in Drosophila, which shed light on the pro‐tumour function of mammalian Toll‐like receptors (TLRs).

several tumour models have been established and widely utilized in fly. For instance, cell polarity genes such as scribble (scrib) and lethal giant larvae (lgl) encode tumour suppressors, 3,11,12 whose mutation cooperates with oncogenic Ras to promote tumorous growth and metastasis in the larval eye discs. [13][14][15] In addition, depletion of tumour suppressor gene csk or cell polarity genes in the A/P boundary region of larval wing discs induces invasive cell migration. 12,16 Further researches indicate that the c-Jun Nterminal kinase (JNK) signalling plays a critical role in these tumour models. 17,18 The JNK pathway, which is evolutionarily conserved from fly to human, plays pivotal roles in a wide range of cellular processes including cell death, proliferation, differentiation and migration. 19,20 In Drosophila, JNK signalling is activated by Eiger (Egr), the orthologue of tumour necrosis factor (TNF). 21,22 Egr binds to its receptor Grindelwald and recruits the adaptor protein tumour necrosis factor receptor-associated factor 2 (dTRAF2), 23,24 which, in turn, activates the JNK cascade including JNKK Kinase dTAK1, JNK Kinase Hemipterous (Hep) and JNK Basket (Bsk). 25,26 Activation of JNK leads to phosphorylation and activation of the transcription factors Jun and Fos, which translocate to the nucleus and regulate target gene expression. 27,28 The Toll signal pathway was first identified as a regulator of dorsal-ventral axis formation in Drosophila embryos 29 and was subsequently found to play an important role in the innate immune response. 30 Besides the trans-membrane receptor Toll, other components of the canonical Toll pathway include Spätzle, Myd88, Tube, Pelle, Cactus, Dorsal and Dif. 31,32 Upon binding to the ligand Spätzle, Toll recruits Tube and Pelle through the adaptor protein Myd88. Activated Pelle phosphorylates Cactus and triggers its degradation, which releases the transcription factors Dorsal and Dif, allows their entry into the nucleus to activate the expression of target genes. [33][34][35] The Toll signal pathway is highly conserved from insect to mammal, with the mammalian counterparts of Toll being named Toll-like receptors (TLRs). 36 To date, nine Toll family members have been identified in Drosophila. While the roles of Toll in embryonic patterning and innate immunity have been well-documented, recent studies suggest Toll signalling is also involved in other biological processes including cell death, wound healing and cell competition. 35,[37][38][39] Furthermore, crosstalk between Toll signalling and other pathways, for example JNK or Hippo, has been reported to regulate cell death or immunity. 40,41 Yet, the functions of other Toll family members remain largely unexplored.
In this study, we identified Toll-7 as a proto-oncogene that promotes tumour growth and invasion by activating both Egr-JNK and EGFR-Ras signalling. The Egr-JNK signalling is necessary and sufficient to trigger invasive cell migration, while the EGFR-Ras signalling cooperates with the Egr-JNK signalling to promote Yki-mediated cell proliferation and tissue overgrowth. Mechanistically, Toll-7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. In addition, Toll-7 promotes EGFR expression post-transcriptionally. Therefore, these data provide in-vivo evidence and underlying genetic mechanism for the role of Toll-7 in promoting tumour growth and invasion, which shed light on the protumour function of mammalian TLRs.

| Immunostaining
Antibody staining of imaginal discs was performed by standard pro-

| RT-qPCR
To assess the knockdown efficiencies of Toll-7 RNAi lines, act-Gal4; tub-Gal80 ts driver was used. Animals were raised at 25°C for 2 day, and then shifted to 29°C for 3 day before dissecting the larvae.
Primers used were as follows:

| Statistical analysis
Adobe Photoshop 2020 was used to measure the tumour size and quantified as relative volume to that of wild-type. Adobe Photoshop 2020 was also used to count the migrating cell number. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0. A combination of One-way ANOVA with Bonferroni's multiple comparison test and the T-test were used to compute p-values, p-value < 0.05 was considered as significant. n.s means not significant. *, **, **** and **** represent p-value less than 0.05, 0.01, 0.001 and 0.0001, respectively.

| Loss of Toll-7 suppresses Ras V12 /lgl −/− induced tumour growth and invasion
It was shown previously that oncogenic cooperation of activated Ras (Ras V12 ) and lgl mutation (lgl −/− ) in Drosophila eye-antennal discs could induce massive tumour-like overgrowth and invasive metastasis to the ventral nerve cord (VNC). 13,42 Using this in-vivo tumour model, we have been performing a genetic screen for modulators of tumour growth and invasion. 26,43 We found knockdown of Toll-7 by two independent RNAi lines significantly inhibited Ras V12 /lgl −/−triggered tumour growth in the eye-antennal discs ( Figure 1A

| Toll-7 is required for cell polarity disruptioninduced invasive cell migration
Since the above tumour invasion phenotype also depends on early cell proliferation and tumorous growth, and Toll-7 depletion dramatically impedes Ras/lgl −/− -induced tumour overgrowth, the role of Toll-7 in tumour invasion needs further verification. To this end, we employed another well-established cell invasion model. In Drosophila wing discs, knockdown cell polarity gene scrib along the A/P compartment boundary driven by ptc-Gal4 induces invasive cell migration with no obvious overgrowth phenotype. 28 We counted the total number of migrating cells to quantify this phenotype and found that knockdown of Toll-7 resulted in decreased migrating cell number, while expression of Puc served as a positive control ( Figure 1H-L, P). 44 Meanwhile, loss-of-scrib also induced strong MMP1 expression, which is a biomarker for epithelial-mesenchymal transition (EMT). 26,28,45 Consistently, knockdown of Toll-7 suppressed scrib depletion-induced MMP1 expression, while expression of Puc served as a positive control ( Figure 1H'-L', Q). Again, knockdown of Toll-7 alone did not cause any obvious phenotype ( Figure 1M, N, M', N'). Together, these results indicate that Toll-7 is required for disrupted cell polarity-induced invasive cell migration.

| Overexpression of Toll-7 promotes tissue overgrowth and invasive migration
To test if Toll-7 is sufficient to induce tissue overgrowth and invasion, we expressed Toll-7 CY , a constitutively active form of Toll-7, 46 along the A/P boundary in the wing discs. Compared with the ptc>GFP control, expression of Toll-7 CY caused dramatic expansion of the GFP-positive stripe (Figure 2A we checked the Z-section of the wing discs and found that Toll-7 CY expressing cells were basally extruded and migrated from the A/P boundary ( Figure 2E-E'', F-F''), which has been characterized as a typical cell invasion phenotype. 12 Collectively, these results indicate that activated Toll-7 is sufficient to promote tissue overgrowth and invasive cell migration.

| Toll-7 promotes cell proliferation and migration through Egr-JNK signalling
The Egr-JNK signalling has been implicated in cell proliferation and invasion in fly tumour models. 17,50 To investigate the role of Egr-JNK signalling in Toll-7-induced tissue overgrowth and invasion, we first checked whether JNK signalling is activated by ectopic Toll-7. To this end, we examined the expression of two well-known JNK pathway reporters, TRE-RFP 51 and puc-lacZ, 52  Next, to investigate whether Egr-JNK signalling is required for Toll-7-induced tissue growth and invasive cell migration, we and also a JNK signalling reporter. 23 These results indicate that Egr-JNK signalling plays a critical role in Toll-7-induced tissue overgrowth and invasion and that Toll-7 acts upstream of or in parallel to Egr.

| Toll-7 regulates Egr-induced cell migration and JNK activation
The JNK signalling was reported to be both necessary and sufficient for cell migration in Drosophila. 26,53,54 Overexpression of Egr driven by ptc-Gal4 induced invasive cell migration and MMP1 upregulation. 28 Intriguingly, both phenotypes were considerably suppressed by Toll-7 depletion ( Figure 4A-H). Egr is known to activate JNK signalling and induce JNK-dependent cell migration. We found that

| Toll-7 is required for endocytosis-mediated early endosomal localization of Egr
The above genetic data imply that Toll-7 regulates JNK signalling in parallel to Egr. Previous study suggests that Egr is translocated from plasma membrane to endosomes through endocytosis and activates JNK signalling in the early endosomes. 55 In addition, endocytosis is required for activation of Toll signalling and generation of the NF-kB gradient during Drosophila embryogenesis. 56 Based on these observations, we proposed that Toll-7 might regulate JNK signalling via influencing Egr endocytosis. To test this hypothesis, we first checked the subcellular localization of Egr. Using Dlg antibody to mark the plasma membrane, we found Egr (red) was mainly co-localized with Dlg (green) on the plasma membrane, while some punctate dots of Egr (red only) also appeared in the cytoplasm ( Figure 5A-A'). Loss These data indicate that Toll-7 is sufficient to induce Yki activation.  Figure S6C-C'). Next, to investigate whether Toll-7-induced overgrowth depends on Hippo-Yki pathway, we knocked down yki in the ptc > Toll-7 CY background, and noted that Toll-7-induced overgrowth phenotype was suppressed by yki depletion (Figure 6G-K). These results indicate that Toll-7 promotes tissue overgrowth through JNKdependent Yki activation.

| Toll-7 promotes EGFR signalling-dependent tissue overgrowth
c-Jun N-terminal kinase signalling executes either anti-or protumour activity through differential regulation of Hippo-Yki pathway. Activated Ras converts JNK signalling from an inhibitor to an activator of Yki, which promotes cell proliferation and tissue overgrowth. 63,64 Therefore, we proposed that Toll-7 might activate two parallel pathways, Egr-JNK and EGFR-Ras. While Egr-JNK signalling is sufficient to induce cell migration, it needs to cooperate with EGFR-Ras pathway to activate Yki-mediated cell proliferation and tissue overgrowth ( Figure 7F). In support of this hypothesis, ptc-Gal4 driven Toll-7 CY overexpression elevated the expression of aos-LacZ ( Figure 7A-A', B-B'), a reporter of EGFR pathway, 65 confirming that Toll-7 induces activation of EGFR-Ras signalling. Furthermore, depletion of EGFR suppressed Toll-7-induced overgrowth phenotype ( Figure 7C-E), indicating EGFR is required for Toll-7-induced tumorous growth. Intriguingly, ectopic EGFR protein level, produced by ptc > EGFR, was decreased upon Toll-7 depletion ( Figure 7G-J), while endogenous EGFR level was enhanced upon Toll-7 CY overexpression ( Figure S8). Together, these data suggest Toll-7 regulates EGFR expression and promotes EGFR signalling-dependent tissue growth.

| DISCUSS ION
The c-Jun N-terminal kinase signalling has been implicated in a wide range of biological processes and has been associated with Toll signalling in apoptosis. 35,40 We found Toll-7-induced tumour growth and invasion depend on Egr-JNK signalling. Meanwhile, Toll-7 is also required for Egr-induced JNK activation and cell migration, suggesting Toll-7 regulates JNK signalling by targeting Egr. Egr was reported to undergo endocytosis, and it activates JNK signalling in the early endosomes. 55 We found loss of Toll-7 impedes endocytosis and Egr  On the other hand, Toll-7-induced Egr-JNK signalling suffices to promote invasive cell migration ( Figure 7F). Consistent with our findings, Toll-7 has been reported to activate pERK, a marker of EGFR signalling. 47 We also provided evidence that Toll-7 is necessary and sufficient for maintaining proper EGFR expression, most likely at the post-transcriptional level, since ptc-Gal4-induced EGFR (ptc > EGFR) expression was significantly reduced upon Toll-7 depletion (Figure 7G-J). It remains to be elucidated how Toll-7 regulates EGFR expression. Since our data suggest Toll-7 regulates endocytosis, and endocytosis is known to be involved in EGFR recycling, 65 Toll-7 might regulate EGFR protein recycling through endocytosis.
Malignant tumour development requires multiple factors and signal transduction pathways, which have not been fully unveiled.
Identifying novel regulators of tumour progression provides potential drug targets for cancer prevention and therapy. Toll/TLR signal pathway, which has conserved functions in innate immunity, is also considered to play a role in cancer inflammation. 68  TLR7 are upregulated in certain types of tumour cells, and their expression patterns are associated with tumour progression, 69 suggesting that the innate immunity-related factors may function as tumour promotors rather than suppressor. Yet, there is no in-vivo report to back this notion, let alone the mechanism by which Tollrelated genes promote tumorigenesis+. Therefore, our results provide the first in-vivo evidence for the pro-tumour function of Toll family receptors, which may shed light on the development of novel therapeutic strategies and clinical treatments of related cancers.

ACK N OWLED G EM ENT
We thank Drs. A Hidalgo, M Miura and J Zhu, Bloomington Drosophila Stock Center, Vienna Drosophila Research Center and NIG-FLY center for fly stocks and reagents.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interest.

AUTH O R CO NTR I B UTI O N S
X.D., Z.L. W.L. and L.X. conceived and designed the experiments.
X.D., Z.L. and W.L. performed the experiments and analysed the data, G. L. and L.X. supervised the study, X.D., Z.L. and L.X. wrote the manuscript. All authors approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.